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Apoptosis Contributes to Amphotericin B Induced Nephrotoxicity
The aim of this study was to investigate whether apoptosis contributes to nephrotoxicity caused by ampho- tericin B (AmB). By detecting apoptosis-specific DNA fragmentation, it is demonstrated that proximal tubular cells (LLC-PK1) and medullary interstitial cells (RMIC) respond with programmed cell death when treated with therapeutic doses of AmB. Concomitant application of AmB and recombinant human insulin-like growth factor-1 (rhIGF-1),
Carboxyfullerenes protect human keratinocytes from ultraviolet-B-induced apoptosis.
Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2. PMID:11069621
Fumelli, C; Marconi, A; Salvioli, S; Straface, E; Malorni, W; Offidani, A M; Pellicciari, R; Schettini, G; Giannetti, A; Monti, D; Franceschi, C; Pincelli, C
The Signaling Cascades of Ginkgolide B-Induced Apoptosis in MCF-7 Breast Cancer Cells
Ginkgolide B, the major active component of Ginkgo biloba extracts, can both stimulate and inhibit apoptotic signaling. Here, we demonstrate that ginkgolide B can induce the production of reactive oxygen species in MCF-7 breast cancer cells, leading to an increase in the intracellular concentrations of cytoplasmic free Ca2+ and nitric oxide (NO), loss of mitochondrial membrane potential (MMP), activation of caspase-9 and -3, and increase the mRNA expression levels of p53 and p21, which are known to be involved in apoptotic signaling. In addition, prevention of ROS generation by pretreatment with N-acetyl cysteine (NAC) could effectively block intracellular Ca2+ concentrations increases and apoptosis in ginkgolide B-treated MCF-7 cells. Moreover, pretreatment with nitric oxide (NO) scavengers could inhibit ginkgolide B-induced MMP change and sequent apoptotic processes. Overall, our results signify that both ROS and NO played important roles in ginkgolide B-induced apoptosis of MCF-7 cells. Based on these study results, we propose a model for ginkgolide B-induced cell apoptosis signaling cascades in MCF-7 cells.
Chan, Wen-Hsiung
The Marine Fungal Metabolite, Dicitrinone B, Induces A375 Cell Apoptosis through the ROS-Related Caspase Pathway
Dicitrinone B, a rare carbon-bridged citrinin dimer, was isolated from the marine-derived fungus, Penicillium citrinum. It was reported to have antitumor effects on tumor cells previously; however, the details of the mechanism remain unclear. In this study, we found that dicitrinone B inhibited the proliferation of multiple tumor types. Among them, the human malignant melanoma cell, A375, was confirmed to be the most sensitive. Morphologic evaluation, cell cycle arrest and apoptosis rate analysis results showed that dicitrinone B significantly induced A375 cell apoptosis. Subsequent observation of reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) reduction revealed that the apoptosis induced by dicitrinone B may be triggered by over-producing ROS. Further studies indicated that the apoptosis was associated with both intrinsic and extrinsic apoptosis pathways under the regulation of Bcl-2 family proteins. Caspase-9, caspase-8 and caspase-3 were activated during the process, leading to PARP cleavage. The pan-caspase inhibitor, Z-VAD-FMK, could reverse dicitrinone B-induced apoptosis, suggesting that it is a caspase-dependent pathway. Our data for the first time showed that dicitrinone B inhibits the proliferation of tumor cells by inducing cell apoptosis. Moreover, compared with the first-line chemotherapy drug, 5-fluorouracil (5-Fu), dicitrinone B showed much more potent anticancer efficacy, suggesting that it might serve as a potential antitumor agent.
Chen, Li; Gong, Mei-Wei; Peng, Zhen-Fei; Zhou, Tong; Ying, Min-Gang; Zheng, Qiu-Hong; Liu, Qin-Ying; Zhang, Qi-Qing
Time-dependent onset of Interferon-alpha2b-induced apoptosis in isolated hepatocytes from preneoplastic rat livers.
We have already demonstrated that interferon alfa-2b (IFN-alpha2b) induces apoptosis in isolated hepatocytes from preneoplastic rat livers via the secretion of transforming growth factor beta(1) (TGF-beta(1)), and this process is accompanied by caspase-3 activation. The aim of this study was to further investigate the mechanism of this activation. Isolated hepatocytes from preneoplastic livers induced DNA fragmentation in response to IFN-alpha2b, which was completely blocked when anti-TGF-beta(1) was added to the culture media. IFN-alpha2b mediated radical oxygen species (ROS) production that preceded the loss of mitochondrial transmembrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-3. Bax levels increased in a time-dependent fashion, and Bcl-x(L) was down-regulated in the early hours of IFN-alpha2b treatment. The delayed translocation of Bid into the mitochondria was in concordance with late caspase-8 activation. In conclusion, endogenous TGF-beta(1) secreted under IFN-alpha2b stimulus seems to induce cytochrome c release through a mechanism related to Bcl-2 family members and loss of mitochondrial DeltaPsi. Bax protein could be responsible of the release of cytochrome c during the initial hours of IFN-alpha2b-induced apoptosis via TGF-beta(1). Activated Bid by caspases could amplificate the mitochondrial events, enhancing the release of cytochrome c. PMID:17376698
Alvarez, Mar�a de Luj�n; Quiroga, Ariel D; Ronco, Mar�a Teresa; Parody, Juan Pablo; Ochoa, J Elena; Monti, Juan A; Carnovale, Cristina E; Carrillo, Mar�a Cristina
Inhibition of erbB Receptor Family Members Protects HaCaT Keratinocytes from Ultraviolet-B-Induced Apoptosis
In the human epidermis, the cells most at risk for the development of cancer due to sunlight exposure are the keratinocytes. In animal models, ultraviolet-B is a complete carcinogen, capable of inducing and promoting the development of malignant cells. A key element of ultraviolet-B-induced carcinogenesis is the ability of ultraviolet-B to induce the expression of a number of cellular proteins
Davina A. Lewis; Bryan Zweig; Steven A. Hurwitz; Dan F. Spandau
The scaffold protein JLP plays a key role in regulating ultraviolet B-induced apoptosis in mice.
The ultraviolet B (UVB) component of sunlight can cause severe damage to skin cells and even induce skin cancer. Growing evidence indicates that the UVB-induced signaling network is complex and involves diverse cellular processes. In this study, we investigated the role of c-Jun NH2 -terminal kinase-associated leucine zipper protein (JLP), a scaffold protein for mitogen-activated protein kinase (MAPK) signaling cascades, in UVB-induced apoptosis. We found that UVB-induced skin epidermal apoptosis was prevented in Jlp knockout (KO) as well as in keratinocyte-specific Jlp KO mice. Analysis of the repair of UVB-induced DNA damage over time showed no evidence for the involvement of JLP in this process. In contrast, UVB-stimulated p38 MAPK activation in the skin was impaired in both Jlp KO and keratinocyte-specific Jlp KO mice. Moreover, topical treatment of UVB-irradiated mouse skin with a p38 inhibitor significantly suppressed the epidermal apoptosis in wild-type mice, but not in Jlp KO mice. Our findings suggest that JLP in skin basal keratinocytes plays an important role in UVB-induced apoptosis by modulating p38 MAPK signaling pathways. This is the first study to show a critical role for JLP in an in vivo response to environmental stimulation. PMID:24520900
Enkhtuya, Radnaa; Sato, Tokiharu; Wakasugi, Mitsuo; Tuvshintugs, Baljinnyam; Miyata, Hirofumi; Sakurai, Takeshi; Matsunaga, Tsukasa; Yoshioka, Katsuji
Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways
Pancreatic cancer is difficult to detect early and responds poorly to chemotherapy. A breakthrough in the development of new therapeutic agents is urgently needed. Eriocalyxin B (EriB), isolated from the Isodon eriocalyx plant, is an ent-kaurane diterpenoid with promise as a broad-spectrum anti-cancer agent. The anti-leukemic activity of EriB, including the underlying mechanisms involved, has been particularly well documented. In this study, we demonstrated for the first time EriB's potent cytotoxicity against four pancreatic adenocarcinoma cell lines, namely PANC-1, SW1990, CAPAN-1, and CAPAN-2. The effects were comparable to that of the chemotherapeutic camptothecin (CAM), but with much lower toxicity against normal human liver WRL68 cells. EriB's cytoxicity against CAPAN-2 cells was found to involve caspase-dependent apoptosis and cell cycle arrest at the G2/M phase. Moreover, the p53 pathway was found to be activated by EriB in these cells. Furthermore, in vivo studies showed that EriB inhibited the growth of human pancreatic tumor xenografts in BALB/c nude mice without significant secondary adverse effects. These results suggest that EriB should be considered a candidate for pancreatic cancer treatment. -- Highlights: ? We study Eriocalyxin B (EriB)'s cytotoxic effects on pancreatic cancer cell lines. ? EriB inhibits cell proliferation via mediation of apoptosis and cell cycle arrest. ? The effects are involved in caspase-dependent apoptosis and p53 pathway. ? In vivo study also shows EriB inhibits the growth of human pancreatic tumor. ? EriB can be a good candidate for chemotherapy in pancreatic cancer.
Li, Lin [School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (China) [School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (China); Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); Yue, Grace G.L. [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China) [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); Lau, Clara B.S. [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China) [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); Sun, Handong [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, CAS, Yunnan (China)] [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, CAS, Yunnan (China); Fung, Kwok Pui [School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (China) [School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (China); Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); Leung, Ping Chung [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China) [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); Han, Quanbin, E-mail: email@example.com [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China) [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); School of Chinese Medicine, The Hong Kong Baptist University, Hong Kong (China); Leung, Po Sing, E-mail: firstname.lastname@example.com [School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (China)
Combination of Salermide and Cholera Toxin B Induce Apoptosis in MCF-7 but Not in MRC-5 Cell Lines
Background: Sirtuin1 is an enzyme that deacetylates histones and several non-histone proteins including P53 during the stress. P300 is a member of the histone acetyl transferase family and enzyme that acetylates histones. Hereby, this study describes the potency combination of Salermide as a Sirtuin1 inhibitor and cholera toxin B (CTB) as a P300 activator to induce apoptosis Michigan Cancer Foundation-7 (MCF-7) and MRC-5. Methods: Cells were cultured and treated with a combination of Salermide and CTB respectively at concentrations of 80.56 and 85.43 ?mol/L based on inhibitory concentration 50 indexes at different times. The percentage of apoptotic cells were measured by flow cytometry. Real-time polymerase chain reaction was performed to estimate the messenger ribonucleic acid expression of Sirtuin1 and P300 in cells. Enzyme linked immunosorbent assay and Bradford protein techniques were used to detect the endogenous levels of total and acetylated P53 protein generated in both cell lines. Results: Our findings indicated that the combination of two drugs could effectively induced apoptosis in MCF-7 significantly higher than MRC-5. We showed that expression of Sirtuin1 and P300 was dramatically down-regulated with increasing time by the combination of Salermide and CTB treatment in MCF-7, but not MRC-5. The acetylated and total P53 protein levels were increased more in MCF-7 than MRC-5 with incubated combination of drugs at different times. Combination of CTB and Salermide in 72 h through decreasing expression of Sirtuin1 and P300 genes induced acetylation of P53 protein and consequently showed the most apoptosis in MCF-7 cells, but it could be well-tolerated in MRC-5. Conclusion: Therefore, combination of drugs could be used as an anticancer agent.
Salahshoor, Mohammad Reza; Dastjerdi, Mehdi Nikbakht; Jalili, Cyrus; Mardani, Mohammad; Khazaei, Mozafar; Darehdor, Ahmad Shabanizadeh; Valiani, Ali; Roshankhah, Shiva
Photoprotective effects of two natural products on ultraviolet B-induced oxidative stress and apoptosis in SKH-1 mouse skin.
Solar ultraviolet radiation (UV) is the major cause of nonmelanoma skin cancer in humans. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. We studied the photoprotective activity of Calluna vulgaris and red grape seed (Vitis vinifera L, Burgund Mare variety [BM]) extracts in vivo in an SKH-1 hairless mice skin model. Fifty 8-week-old female SKH-1 hairless mice were randomly divided into 5 groups (n?=?10 each): controls, UVB-irradiated, C. vulgaris plus UVB-irradiated, BM plus UVB-irradiated, and epigallocatechin gallate (EGCG) plus UVB-irradiated. A dose of 4 mg/mouse per cm� of skin area for both extracts was topically applied to the mice 30 minutes before a single-dose (240 mJ/cm�) UVB exposure. EGCG dissolved in phosphate-buffered saline (pH 6.6; 0.067 M) was administered at 2 mg/mouse per cm�. Glutathione peroxidase and catalase activities, reduced glutathione (GSH), malondialdehyde, nitric oxide, and caspase 3 activity were determined in skin homogenates 24 hours after irradiation. A single dose of UVB increased GSH levels and glutathione peroxidase activity in the exposed skin. C. vulgaris and BM pretreatment significantly decreased GSH formation and glutathione peroxidase activity (P?.001) and inhibited UVB-induced lipid peroxidation (P?.0001) and nitric oxide production (C. vulgaris: P?.06). Topical treatments with C. vulgaris and particularly BM extracts (P?.002) significantly reduced caspase 3 activity, indicating that the cells were protected against apoptosis. These results suggest that C. vulgaris and BM extracts might be chemopreventive candidates for reducing UV-induced risk for skin cancer. PMID:21470043
Filip, Adriana; Daicoviciu, Doina; Clichici, Simona; Mocan, Teodora; Muresan, Adriana; Postescu, Ion Dan
Interferon-alpha2b (IFN-alpha2b)-induced apoptosis is mediated by p38 MAPK in hepatocytes from rat preneoplastic liver via activation of NADPH oxidase.
It is still unclear how Interferon-alfa (IFN-alpha) acts on preventing the appearance of hepatocarcinogenesis. We have demonstrated that IFN-alpha2b induces hepatocytic transforming growth factor-beta1 (TGF-beta(1)) production and secretion by inducing reactive oxygen species (ROS) formation through the activation of NADPH oxidase. This TGF-beta(1), alters antioxidant defences and induces programmed cell death. Since it was demonstrated that IFN-alpha induces apoptosis through the activation of p38 mitogen-activated protein kinase (p38 MAPK), this study was aimed to assess the role of this kinase in the IFN-alpha2b-induced apoptosis in rat liver preneoplasia; and to further evaluate the participation of NADPH oxidase. p38 MAPK pathway was activated during the IFN-alpha2b-induced apoptosis in rat liver preneoplasia. This activation was accompanied with phosphorylation of different transcription factors, depending on the time of IFN-alpha2b stimulus. Our data suggest that NADPH oxidase is activated by IFN-alpha2b through p38 MAPK. p38 MAPK-induced activation of NADPH oxidase is accomplished by a two-step pathway: first, ROS-independent and second ROS- and TGF-beta(1)-dependent. PMID:19455458
Quiroga, Ariel D; de Lujan Alvarez, Maria; Parody, Juan P; Ronco, Maria T; Carnovale, Cristina E; Carrillo, Maria Cristina
Down-modulation of heat shock protein 70 and up-modulation of Caspase3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells
AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC- 7721 cells in vitro and regulation of Hsp70 and Caspases- 3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell
Yi-Feng Wu; Ming-Fu Cao; Yan-Ping Gao; Fei Chen; Tao Wang; Edward P. Zumbika; Kai-Xian Qian
Wentilactone B induces G2/M phase arrest and apoptosis via the Ras/Raf/MAPK signaling pathway in human hepatoma SMMC-7721 cells.
Hepatocellular carcinoma (HCC) is generally acknowledged as the most common primary malignant tumor, and it is known to be resistant to conventional chemotherapy. Wentilactone B (WB), a tetranorditerpenoid derivative extracted from the marine algae-derived endophytic fungus Aspergillus wentii EN-48, has been shown to trigger apoptosis and inhibit metastasis in HCC cell lines. However, the mechanisms of its antitumor activity remain to be elucidated. We report here that WB could significantly induce cell cycle arrest at G2 phase and mitochondrial-related apoptosis, accompanying the accumulation of reactive oxygen species (ROS). Additionally, treatment with WB induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), but not p38 MAP kinase. Among the pathway inhibitors examined, only SP600125 (JNK inhibitor) markedly reversedWB-induced apoptosis, and only U0126 (ERK inhibitor) significantly blocked WB-triggered G2 phase arrest. We also found that WB treatment increased both Ras and Raf activation, and transfection of cells with dominant-negative Ras (RasN17) abolishedWB-induced apoptosis and G2 phase arrest in SMMC-7721 cells. Furthermore, the results of inverse docking (INVDOCK) analysis suggested that WB could bind to Ras-GTP, and the direct binding affinity was also confirmed by surface plasmon resonance (SPR). Finally, in vivo, WB suppressed tumor growth in mouse xenograft models. Taken together, these results indicate that WB induced G2/M phase arrest and apoptosis in human hepatoma SMMC-7721 cells via the Ras/Raf/ERK and Ras/Raf/JNK signaling pathways, and this agent may be a potentially useful compound for developing anticancer agents for HCC. PMID:23744357
Zhang, Z; Miao, L; Lv, C; Sun, H; Wei, S; Wang, B; Huang, C; Jiao, B
Wentilactone B induces G2/M phase arrest and apoptosis via the Ras/Raf/MAPK signaling pathway in human hepatoma SMMC-7721 cells
Hepatocellular carcinoma (HCC) is generally acknowledged as the most common primary malignant tumor, and it is known to be resistant to conventional chemotherapy. Wentilactone B (WB), a tetranorditerpenoid derivative extracted from the marine algae-derived endophytic fungus Aspergillus wentii EN-48, has been shown to trigger apoptosis and inhibit metastasis in HCC cell lines. However, the mechanisms of its antitumor activity remain to be elucidated. We report here that WB could significantly induce cell cycle arrest at G2 phase and mitochondrial-related apoptosis, accompanying the accumulation of reactive oxygen species (ROS). Additionally, treatment with WB induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), but not p38 MAP kinase. Among the pathway inhibitors examined, only SP600125 (JNK inhibitor) markedly reversedWB-induced apoptosis, and only U0126 (ERK inhibitor) significantly blocked WB-triggered G2 phase arrest. We also found that WB treatment increased both Ras and Raf activation, and transfection of cells with dominant-negative Ras (RasN17) abolishedWB-induced apoptosis and G2 phase arrest in SMMC-7721 cells. Furthermore, the results of inverse docking (INVDOCK) analysis suggested that WB could bind to Ras�GTP, and the direct binding affinity was also confirmed by surface plasmon resonance (SPR). Finally, in vivo, WB suppressed tumor growth in mouse xenograft models. Taken together, these results indicate that WB induced G2/M phase arrest and apoptosis in human hepatoma SMMC-7721 cells via the Ras/Raf/ERK and Ras/Raf/JNK signaling pathways, and this agent may be a potentially useful compound for developing anticancer agents for HCC.
Zhang, Z; Miao, L; Lv, C; Sun, H; Wei, S; Wang, B; Huang, C; Jiao, B
A PEM/SPE fuel cell is described including a membrane-electrode assembly (MEA) having a plurality of oriented filament embedded the face thereof for supporting the MEA and conducting current therefrom to contiguous electrode plates. 4 figs.
Grot, S.A.
NSDL National Science Digital Library
Apoptosis is a highly orchestrated form of cell death in which cells neatly commit suicide by chopping themselves into membrane-packaged bits. Apoptosis, also known as programmed cell death, has caught the imagination of researchers worldwide. This article introduces a special issue on apoptosis.
Linda J. Miller (AAAS;); Jean Marx (AAAS;)
Biological Response to SPE Exposures
NASA Technical Reports Server (NTRS)
It has long been recognized that a single solar particle event (SPE) can produce, over a short period of time, exposures on the order of LD50 for humans under normal physiological conditions. It is further recognized that recovery from injury over the period of exposure would greatly increase the chances of survival (dose rate effects) although such effects were left unquantified. In the present report we use the bioresponse model derived from a broad range of animal and human exposure data for evaluation of troop readiness in tactical nuclear warfare to evaluate the biological risk posed by the solar event of 4 August 1972. The astronaut blood forming organ (BFO) exposure in deep space would have been 2.2 Sv (1.6 Gy) in a space suit, 1.8 Sv (1.3 Gy) in an aluminum pressure vessel, and 0.7 Sv (0.5 Gy) in an equipment room compared to an X-ray mortality threshold of 1.5 Gy (assuming high dose rate). We find BFO dose rate effectiveness factors for this SPE on the order of 3 to 4, greatly reducing the mortality risks for this event. There is an approximate 3 percent chance that an even larger event may occur for which exposures could be 2-4 times higher. Assured survival of the astronaut requires added shelter shielding and a warning system for this event. The required mass of the shelter shield can be greatly reduced by using hydrogenous materials such as polymers, water, food, and other biological materials in its construction. Limitations of the current bioresponse model arise from the exposures taking place in the microgravity environment wherein the immune system is already challenged and the effective mortality threshold may be reduced by a factor of two. Such microgravity effects could greatly affect astronaut risks.
Wilson, J. W.; Cucinotta, F. A.; Kim, M.; Shinn, J. L.; Jones, T. D.; Chang, C. K.
Current SPE Hydrodynamic Modeling and Path Forward
Extensive work has been conducted on SPE analysis efforts: Fault effects Non-uniform weathered layer analysis MUNROU: material library incorporation, parallelization, and development of non-locking tets Development of a unique continuum-based-visco-plastic strain-rate-dependent material model With corrected SPE data path is now set for a multipronged approach to fully understand experimental series shot effects.
Knight, Earl E. [Los Alamos National Laboratory; Rougier, Esteban [Los Alamos National Laboratory
Seismic Observations of SPE1, SPE2, and SPE3 and Integration with Multiple Data Sets
NASA Astrophysics Data System (ADS)
Here we review the seismic data collected during the three SPE experiments. The focus is on first-order phenomena observed in the surface seismic data in comparison with other datasets and with modeling. Results from a seismic moment tensor inversion using a 1D model, which displays significant non-isotropic components, are presented and compared with data from surface accelerometers, LIDAR measurements, and known geology (surface and borehole) to provide constraints on extent of spall and the non-isotropic components of the moment tensor. 3D seismic waveforms are computed using both a geologic-based model and a model refined using seismic interferometry, with the results compared with observed waveforms.
Mellors, R. J.; Matzel, E.; Ford, S. R.; Walter, W. R.; Pitarka, A.; Myers, S. C.; Hauk, T. F.; Rodgers, A.; Wagoner, J. L.
Recent advances in SPE (tm) water electrolyzer
NASA Technical Reports Server (NTRS)
A new cell structure has been introduced into the SPE Water Electrolyzer which has improved overall characteristics significantly. Weight, reliability, and efficiency are the characteristics that are improved the most, with volume having a second order improvement. This paper discusses the capabilities of the new cell structure and the impact it would have in various space applications.
Mcelroy, James F.
SPE water electrolyzers in support of the lunar outpost
NASA Technical Reports Server (NTRS)
During the 1970s, the SPE water electrolyzer, which uses ion exchange membranes as its sole electrolyte, was developed for nuclear submarine metabolic oxygen production. These developments included SPE water electrolyzer operation at up to 3,000 psia and at current densities in excess of 1,000 amps per square foot. The SPE water electrolyzer system is now fully qualified for both the U.S. and U.K. Navies with tens of thousands of system hours accumulated at sea. During the 1980s, the basic SPE water electrolyzer cell structure developed for the Navies was incorporated into several demonstrations for NASA's Space Station Program. Among these were: the SPE regenerative fuel cell for electrical energy storage; the SPE water electrolyzer for metabolic oxygen production; and the high pressure SPE water electrolyzer for reboost propulsion reactant production. In the 1990s, one emphasis will be the development of SPE water electrolyzers for the Lunar Outposts Currently defined potential Lunar Outpost applications for the SPE water electrolyzer include: SPE water electrolyzers for metabolic oxygen and potable water production from reclaimed water; and SPE water electrolyzers operating at high pressure as part of stationary and mobile surface energy storage systems.
Mcelroy, J. F.
SPE dose prediction using locally weighted regression.
When astronauts are outside earth's protective magnetosphere, they are subject to large radiation doses resulting from solar particle events (SPEs). The total dose received from a major SPE in deep space could cause severe radiation poisoning. The dose is usually received over a 20-40 h time interval but the event's effects may be mitigated with an early warning system. This paper presents a method to predict the total dose early in the event. It uses a locally weighted regression model, which is easier to train and provides predictions as accurate as neural network models previously used. PMID:16604634
Hines, J W; Townsend, L W; Nichols, T F
SPE propulsion electrolyzer for NASA's integrated propulsion test article
NASA Technical Reports Server (NTRS)
Hamilton Standard has delivered a 3000 PSI SPE Propulsion Electrolyzer Stack and Special Test Fixture to the NASA Lyndon B. Johnson Space Center (JSC) Integrated Propulsion Test Article (IPTA) program in June 1990, per contract NAS9-18030. This prototype unit demonstrates the feasibility of SPE-high pressure water electrolysis for future space applications such as Space Station propulsion and Lunar/Mars energy storage. The SPE-Propulsion Electrolyzer has met or exceeded all IPTA program goals. It continues to function as the primary hydrogen and oxygen source for the IPTA test bed at the NASA/JSC Propulsion and Power Division Thermochemical Test Branch.
SPE-HPLC determination of catecholamines using an affinity principle.
Solid-phase extraction (SPE) with the affinity chromatography principle was applied for high performance liquid chromatography (HPLC) determination of catecholamines in patients urine samples. Electrochemical amperometric detection (ED) was used for all HPLC analyses and both analytical and microbore columns were tested for optimal chromatographic resolution of all analyzed catecholamines. Capacity ratio k' and detection limits were evaluated for all columns used. Precolumn affinity chromatographic procedure of catecholamines with diphenylboronic acid (DPBA) in alkaline medium improved their retention on different commercial SPE reversed-phase cartridges. After clean-up and preconcentration step the complex catecholamine-diphenylboronic acid was degraded by acidic pH and eluted from SPE cartridge. After SPE affinity step catecholamines were analyzed by HPLC-ED. The optimal elution solutions was chosen also as suitable SPE cartridges. Extraction recoveries of catecholamines were 89.6-93.3% with relative standard deviation RSD = 3.8-4.3%. Total analysis time was 30 min including 15 min for the preseparation procedure. PMID:8843972
Brandsteterov�, E; Kubalec, P; Kraj?�k, K; Skac�ni, I
Fiber-packed SPE tips based on electrospun fibers.
A novel fiber-packed solid-phase extraction (SPE) tip was designed based on electrospun nanofibers. The tip was used to investigate the extraction of hydrocortisone (HC), cortisone acetate (CA), ethinylestradiol (EE), and estradiol (E2). The effects of diameters, porous figurations, and functional groups of the electrospun fibers on the selectivity and efficiency were studied. The experimental results indicated that the detection limit of cortisol in water sample could be as low as 0.75 ng/mL. When the tip is used for detection of cortisol in human hair the efficiency of biological sample pretreatment is better than the traditional SPE method. Our method could significantly simplify the traditional SPE process and lower the cost. Industrial application of the tip is anticipated. PMID:18496677
Zhang, Yiyun; Kang, Xuejun; Chen, Liqin; Pan, Chao; Yao, Yingfang; Gu, Zhong-Ze
Theoretical performance of hydrogen-bromine rechargeable SPE fuel cell
NASA Technical Reports Server (NTRS)
A mathematical model was formulated to describe the performance of a hydrogen-bromine fuel cell. Porous electrode theory was applied to the carbon felt flow-by electrode and was coupled to theory describing the solid polymer electrolyte (SPE) system. Parametric studies using the numerical solution to this model were performed to determine the effect of kinetic, mass transfer, and design parameters on the performance of the fuel cell. The results indicate that the cell performance is most sensitive to the transport properties of the SPE membrane. The model was also shown to be a useful tool for scale-up studies.
Savinell, Robert F.; Fritts, S. D.
Spermiogenesis initiation in Caenorhabditis elegans involves a casein kinase 1 encoded by the spe-6 gene.
Immature spermatids from Caenorhabditis elegans are stimulated by an external activation signal to reorganize their membranes and cytoskeleton to form crawling spermatozoa. This rapid maturation, termed spermiogenesis, occurs without any new gene expression. To better understand this signal transduction pathway, we isolated suppressors of a mutation in the spe-27 gene, which is part of the pathway. The suppressors bypass the requirement for spe-27, as well as three other genes that act in this pathway, spe-8, spe-12, and spe-29. Eighteen of the suppressor mutations are new alleles of spe-6, a previously identified gene required for an early stage of spermatogenesis. The original spe-6 mutations are loss-of-function alleles that prevent major sperm protein (MSP) assembly in the fibrous bodies of spermatocytes and arrest development in meiosis. We have isolated the spe-6 gene and find that it encodes a predicted protein-serine/threonine kinase in the casein kinase 1 family. The suppressor mutations appear to be reduction-of-function alleles. We propose a model whereby SPE-6, in addition to its early role in spermatocyte development, inhibits spermiogenesis until the activation signal is received. The activation signal is transduced through SPE-8, SPE-12, SPE-27, and SPE-29 to relieve SPE-6 repression, thus triggering the formation of crawling spermatozoa.
Muhlrad, Paul J; Ward, Samuel
A simple model for solid polymer electrolyte (SPE) water electrolysis
Solid polymer electrolyte (SPE) water electrolysis is analyzed by a simple model based on Butler�Volmer kinetics for electrodes and transport resistance in the polymer electrolyte. An equivalent electrical circuit analogy is provided for the sequential kinetic and transport resistances. The model provides a relation between applied terminal voltage of the electrolysis cell and current density in terms of Nernst potential,
Pyoungho Choi; Dmitri G. Bessarabov; Ravindra Datta
Supporting flexible collaborative software development with SPE-Serendipity
Collaborative software development environments are large cooperative work systems. To effectively support collaborative development, such environments should support software process modelling and enactment, work coordination, and fully integrated software development tools. We describe the facilitation of collaborative software development using the Serendipity process modelling environment and SPE integrated software development environment. Serendipity provides flexible, graphical process modelling tools which are
John C. Grundy
SPE (tm) water electrolyzers in support of mission from planet Earth
NASA Technical Reports Server (NTRS)
During the 1970's, the Solid Polymer Electrolyte (SPE) water electrolyzer, which uses ion exchange membranes as its sole electrolyte, was developed for nuclear submarine metabolic oxygen production. SPE water electrolyzer developments included operation at up to 3,000 psia and at current densities in excess of 1,000 amps per square foot. The SPE water electrolyzer system has accumulated tens of thousands of system hours with the Navies of both the United States and the United Kingdom. During the 1980's, the basic SPE water electrolyzer cell structure developed for the Navies was incorporated into several demonstrators for NASA's Space Station Program. Among these were: (1) the SPE regenerative fuel cell for electrical energy storage; (2) the SPE water electrolyzer for metabolic oxygen production; and (3) the high pressure SPE water electrolyzer for reboost propellant production. In the 1990's, emphasis will be the development of SPE water electrolyzers for Mission from Planet Earth. Currently defined potential applications for the SPE water electrolyzer include: (1) SPE water electrolyzers operating at high pressure as part of a regenerative fuel cell extraterrestrial surface energy storage system; (2) SPE water electrolyzers for propellant production from extraterrestrial indigenous materials; and (3) SPE water electrolyzers for metabolic oxygen and potable water production from reclaimed water.
Mcelroy, J. F.
SPE (tm) water electrolyzers in support of mission from planet Earth
NASA Astrophysics Data System (ADS)
During the 1970's, the Solid Polymer Electrolyte (SPE) water electrolyzer, which uses ion exchange membranes as its sole electrolyte, was developed for nuclear submarine metabolic oxygen production. SPE water electrolyzer developments included operation at up to 3,000 psia and at current densities in excess of 1,000 amps per square foot. The SPE water electrolyzer system has accumulated tens of thousands of system hours with the Navies of both the United States and the United Kingdom. During the 1980's, the basic SPE water electrolyzer cell structure developed for the Navies was incorporated into several demonstrators for NASA's Space Station Program. Among these were: (1) the SPE regenerative fuel cell for electrical energy storage; (2) the SPE water electrolyzer for metabolic oxygen production; and (3) the high pressure SPE water electrolyzer for reboost propellant production. In the 1990's, emphasis will be the development of SPE water electrolyzers for Mission from Planet Earth. Currently defined potential applications for the SPE water electrolyzer include: (1) SPE water electrolyzers operating at high pressure as part of a regenerative fuel cell extraterrestrial surface energy storage system; (2) SPE water electrolyzers for propellant production from extraterrestrial indigenous materials; and (3) SPE water electrolyzers for metabolic oxygen and potable water production from reclaimed water.
McElroy, J. F.
[Effect of flavin adenine dinucleotide on ultraviolet B induced damage in cultured human corneal epithelial cells].
This study evaluated the effects of flavin adenine dinucleotide (FAD) on ultraviolet B (UV-B)-induced damage in cultured human corneal epithelial (HCE-T) cells. The cultured HCE-T cells were treated with 0.003125-0.05% FAD before exposure to 80 mJ/cm2 UV-B. Cell viability was measured 24 h after UV-B irradiation using the MTS assay. Reactive oxygen species (ROS) were detected 30 min after UV-B irradiation using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester. Apoptosis was evaluated 4 h after UV-B irradiation in the caspase-3/7 activity assay. UV-B irradiation reduced cell viability and stimulated ROS production and caspase-3/7 activity in HCE-T cells. Pretreatment of UV-B irradiated HCE-T cells with FAD significantly attenuated cell viability reduction and inhibited the stimulation of both ROS production and caspase-3/7 activity due to UV-B exposure compared with those with vehicle (0% FAD). These results clarified that FAD inhibits ROS-mediated apoptosis by UV-B irradiation in HCE-T cells and suggest that FAD may be effective as a radical scavenger in UV-B-induced corneal damage. PMID:22864352
Sakamoto, Asuka; Nakamura, Masatsugu
Dynamics of speB mRNA Transcripts in Streptococcus pyogenes
Streptococcus pyogenes (group A streptococcus [GAS]) is a human-specific pathogen that causes a variety of diseases ranging from superficial infections to life-threatening diseases. SpeB, a potent extracellular cysteine proteinase, plays an important role in the pathogenesis of GAS infections. Previous studies show that SpeB expression and activity are controlled at the transcriptional and posttranslational levels, though it had been unclear whether speB was also regulated at the posttranscriptional level. In this study, we examined the growth phase-dependent speB mRNA level and decay using quantitative reverse transcription-PCR (qRT-PCR) and Northern blot analyses. We observed that speB mRNA accumulated rapidly during exponential growth, which occurred concomitantly with an increase in speB mRNA stability. A closer observation revealed that the increased speB mRNA stability was mainly due to progressive acidification. Inactivation of RNase Y, a recently identified endoribonuclease, revealed a role in processing and degradation of speB mRNA. We conclude that the increased speB mRNA stability contributes to the rapid accumulation of speB transcript during growth.
Itzek, Andreas; Malke, Horst; Ferretti, Joseph J.
Novel Chimeric Spermidine Synthase-Saccharopine Dehydrogenase Gene (SPE3LYS9) in the Human Pathogen Cryptococcus neoformans
The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase). spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively. Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydro- genase gene products. In contrast to Saccharomyces
Joanne M. Kingsbury; Zhonghui Yang; Tonya M. Ganous; Gary M. Cox; John H. McCusker
Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages
The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte�macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalize receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1beta (IL-1?) in cells primed with lipopolysaccharide (LPS), and this response was reduced by both ZYVAD-FMK and the cathepsin B inhibitor CA-074Me. In conclusion, EnnB was found to induce cell cycle arrest, cell death and inflammation. Caspase-1 appeared to be involved in the apoptosis and release of IL-1? and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B. -- Highlights: ? The mycotoxin EnnB induced cell cycle arrest, cell death and inflammation. ? The G0/G1-arrest was linked to a reduced ability to internalize receptors. ? EnnB caused lysosomal damage, leakage of cathepsin B and caspase-1 cleavage. ? Caspase-1 was partly involved in both apoptosis and release of IL-1?. ? There was a synergistic action between EnnB and bacterial LPS.
Gammelsrud, A. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway) [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Solhaug, A. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway)] [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Dendel�, B. [EA 4427 SeRAIC, IRSET, Universit� de Rennes 1, IFR 140, Rennes (France)] [EA 4427 SeRAIC, IRSET, Universit� de Rennes 1, IFR 140, Rennes (France); Sandberg, W.J. [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)] [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Ivanova, L. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway)] [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Kocbach B�lling, A. [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)] [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Lagadic-Gossmann, D. [EA 4427 SeRAIC, IRSET, Universit� de Rennes 1, IFR 140, Rennes (France)] [EA 4427 SeRAIC, IRSET, Universit� de Rennes 1, IFR 140, Rennes (France); Refsnes, M.; Becher, R. [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)] [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Eriksen, G. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway)] [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Holme, J.A., E-mail: firstname.lastname@example.com [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)
Observing Silicate-Rich Asteroids in Technicolor: Detailed Compositional Constraints from SpeX
We have recently begun a new survey of sili-cate-rich asteroids using SpeX, a low- to medium-resolution infrared spectrograph, at the Infrared Telescope Facility (IRTF) on Mauna Kea, Hawaii [1]. In its low-resolution mode (R approx.100), SpeX can produce spectra of faint asteroids from 0.8 to 2.5 microns with S\\/N comparable to data typically collected with visible wavelength CCDs. The SpeX
J. M. Sunshine; S. J. Bus; T. H. Burbine; T. J. McCoy; R. P. Binzel
SPE-8, a protein-tyrosine kinase, localizes to the spermatid cell membrane through interaction with other members of the SPE-8 group spermatid activation signaling pathway in C. elegans
Background The SPE-8 group gene products transduce the signal for spermatid activation initiated by extracellular zinc in C. elegans. Mutations in the spe-8 group genes result in hermaphrodite-derived spermatids that cannot activate to crawling spermatozoa, although spermatids from mutant males activate through a pathway induced by extracellular TRY-5 protease present in male seminal fluid. Results Here, we identify SPE-8 as a member of a large family of sperm-expressed non-receptor-like protein-tyrosine kinases. A rescuing SPE-8::GFP translational fusion reporter localizes to the plasma membrane in all spermatogenic cells from the primary spermatocyte stage through spermatids. Once spermatids become activated to spermatozoa, the reporter moves from the plasma membrane to the cytoplasm. Mutations in the spe-8 group genes spe-12, spe-19, and spe-27 disrupt localization of the reporter to the plasma membrane, while localization appears near normal in a spe-29 mutant background. Conclusions These results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid.
Premature Sperm Activation and Defective Spermatogenesis Caused by Loss of spe-46 Function in Caenorhabditis elegans
Given limited resources for motility, sperm cell activation must be precisely timed to ensure the greatest likelihood of fertilization. Like those of most species, the sperm of C. elegans become active only after encountering an external signaling molecule. Activation coincides with spermiogenesis, the final step in spermatogenesis, when the spherical spermatid undergoes wholesale reorganization to produce a pseudopod. Here, we describe a gene involved in sperm activation, spe-46. This gene was identified in a suppressor screen of spe-27(it132ts), a sperm-expressed gene whose product functions in the transduction of the spermatid activation signal. While spe-27(it132ts) worms are sterile at 25�C, the spe-46(hc197)I; spe-27(it132ts)IV double mutants regain partial fertility. Single nucleotide polymorphism mapping, whole genome sequencing, and transformation rescue were employed to identify the spe-46 coding sequence. It encodes a protein with seven predicted transmembrane domains but with no other predicted functional domains or homology outside of nematodes. Expression is limited to spermatogenic tissue, and a transcriptional GFP fusion shows expression corresponds with the onset of the pachytene stage of meiosis. The spe-46(hc197) mutation bypasses the need for the activation signal; mutant sperm activate prematurely without an activation signal in males, and mutant males are sterile. In an otherwise wild-type genome, the spe-46(hc197) mutation induces a sperm defective phenotype. In addition to premature activation, spe-46(hc197) sperm exhibit numerous defects including aneuploidy, vacuolization, protruding spikes, and precocious fusion of membranous organelles. Hemizygous worms [spe-46(hc197)/mnDf111] are effectively sterile. Thus, spe-46 appears to be involved in the regulation of spermatid activation during spermiogenesis, with the null phenotype being an absence of functional sperm and hypomorphic phenotypes being premature spermatid activation and numerous sperm cell defects.
Liau, Wei-Siang; Nasri, Ubaydah; Elmatari, Daniel; Rothman, Jason; LaMunyon, Craig W.
spe-12 encodes a sperm cell surface protein that promotes spermiogenesis in Caenorhabditis elegans.
During spermiogenesis, Caenorhabditis elegans spermatids activate and mature into crawling spermatozoa without synthesizing new proteins. Mutations in the spe-12 gene block spermatid activation, rendering normally self-fertile hermaphrodites sterile. Mutant males, however, are fertile. Surprisingly, when mutant hermaphrodites mate with a male, their self-spermatids activate and form functional spermatozoa, presumably due to contact with male seminal fluid. Here we show that, in addition to its essential role in normal activation of hermaphrodite-derived spermatids, SPE-12 also plays a supplementary but nonessential role in mating-induced activation. We have identified the spe-12 gene, which encodes a novel protein containing a single transmembrane domain. spe-12 mRNA is expressed in the sperm-producing germ line and the protein localizes to the spermatid cell surface. We propose that SPE-12 functions downstream of both hermaphrodite- and male-derived activation signals in a spermatid signaling pathway that initiates spermiogenesis.
Nance, J; Minniti, A N; Sadler, C; Ward, S
Development of an SPE/CE method for analyzing HAAs
The haloacetic acid (HAA) analysis methods approved by the US Environmental Protection Agency involve extraction and derivatization of HAAs (typically to their methyl ester form) and analysis by gas chromatography (GC) with electron capture detection (ECD). Concerns associated with these methods include the time and effort of the derivatization process, use of potentially hazardous chemicals or conditions during methylation, poor recoveries because of low extraction efficiencies for some HAAs or matrix effects from sulfate, and loss of tribromoacetic acid because of decarboxylation. The HAA analysis method introduced here uses solid-phase extraction (SPE) followed by capillary electrophoresis (CE) analysis. The method is accurate, reproducible, sensitive, relatively safe, and easy to perform, and avoids the use of large amounts of solvent for liquid-liquid extraction and the potential hazards and hassles of derivatization. The cost of analyzing HAAs using this method should be lower than the currently approved methods, and utilities with a GC/ECD can perform the analysis in-house.
Zhang, L.; Capel, P. D.; Hozalski, R. M.
SPE-39 Family Proteins Interact with the HOPS Complex and Function in Lysosomal Delivery
Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39�like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis.
Zhu, Guang-dan; Salazar, Gloria; Zlatic, Stephanie A.; Fiza, Babar; Doucette, Michele M.; Heilman, Craig J.; Levey, Allan I.
SPE-39 family proteins interact with the HOPS complex and function in lysosomal delivery.
Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39-like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis. PMID:19109425
Zhu, Guang-dan; Salazar, Gloria; Zlatic, Stephanie A; Fiza, Babar; Doucette, Michele M; Heilman, Craig J; Levey, Allan I; Faundez, Victor; L'hernault, Steven W
Method and Agent for Inducing Apoptosis/Cell Death in Leukemia Cells
National Technical Information Service (NTIS)
A method for inducing apoptosis or cell death in leukemia cells includesinhibiting the production of nitric oxide (NO) by using a nitric oxide synthase (NOS) inhibitor, or the actions of NO by a NO quencher/scavenger. The NOS inhibitor includes a NOS1-spe...
J. B. Weinberg M. C. Levesque D. K. Ghosh
Pre-shot simulations of far-field ground motion for the Source Physics Experiment (SPE) Explosions at the Climax Stock, Nevada National Security Site: SPE2
The Source Physics Experiment (SPE) is planning a 1000 kg (TNT equivalent) shot (SPE2) at the Nevada National Security Site (NNSS) in a granite borehole at a depth (canister centroid) of 45 meters. This shot follows an earlier shot of 100 kg in the same borehole at a depth 60 m. Surrounding the shotpoint is an extensive array of seismic sensors arrayed in 5 radial lines extending out 2 km to the north and east and approximately 10-15 to the south and west. Prior to SPE1, simulations using a finite difference code and a 3D numerical model based on the geologic setting were conducted, which predicted higher amplitudes to the south and east in the alluvium of Yucca Flat along with significant energy on the transverse components caused by scattering within the 3D volume along with some contribution by topographic scattering. Observations from the SPE1 shot largely confirmed these predictions although the ratio of transverse energy relative to the vertical and radial components was in general larger than predicted. A new set of simulations has been conducted for the upcoming SPE2 shot. These include improvements to the velocity model based on SPE1 observations as well as new capabilities added to the simulation code. The most significant is the addition of a new source model within the finite difference code by using the predicted ground velocities from a hydrodynamic code (GEODYN) as driving condition on the boundaries of a cube embedded within WPP which provides a more sophisticated source modeling capability linked directly to source site materials (e.g. granite) and type and size of source. Two sets of SPE2 simulations are conducted, one with a GEODYN source and 3D complex media (no topography node spacing of 5 m) and one with a standard isotropic pre-defined time function (3D complex media with topography, node spacing of 5 m). Results were provided as time series at specific points corresponding to sensor locations for both translational (x,y,z) and rotational components. Estimates of spectral scaling for SPE2 are provided using a modified version of the Mueller-Murphy model. An estimate of expected aftershock probabilities were also provided, based on the methodology of Ford and Walter, [2010].
Mellors, R J; Rodgers, A; Walter, W; Ford, S; Xu, H; Matzel, E; Myers, S; Petersson, N A; Sjogreen, B; Hauk, T; Wagoner, J
SPE (trademark) Oxygen Generator Assembly (OGA). (Refurbishment of the technology demonstrator LFSPE oxygen generation subsystem)
NASA Technical Reports Server (NTRS)
The SPE Oxygen Generator Assembly (OGA) has been modified to correct operational deficiencies present in the original system, and to effect changes to the system hardware and software such that its operating conditions are consistent with the latest configuration requirements for the International Space Station Alpha (ISSA). The effectiveness of these changes has recently been verified through a comprehensive test program which saw the SPE OGA operate for over 740 hours at various test conditions, including over 690 hours, or approximately 460 cycles, simulating the orbit of the space station. This report documents the changes made to the SPE OGA, presents and discusses the test results from the acceptance test program, and provides recommendations for additional development activities pertinent to evolution of the SPE OGA to a flight configuration. Copies of the test data from the acceptance test program are provided with this report on 3.5 inch diskettes in self-extracting archive files.
Roy, Robert J.
Determination of ethyl glucuronide in human hair by SPE and LC�MS\\/MS
A method for the sensitive and selective determination of ethyl glucuronide (EtG) in hair has been developed using solid-phase extraction (SPE) and liquid chromatography�tandem mass spectrometry (LC�MS\\/MS). Washed and cut hair segments were extracted by ultrasonication (3h, 50�C) and the extracts were cleaned-up with aminopropyl SPE columns. LC�MS\\/MS analysis was performed using a polar-endcapped phenyl-hexyl-RP-phase with negative mode electrospray ionisation
Ines Janda; Wolfgang Weinmann; Thorsten Kuehnle; Martina Lahode; Andreas Alt
SPE�GC\\/FTD determination of N-methyl-2-pyrrolidone and its metabolites in urine
An analytical method using a combination of solid-phase extraction (SPE) and gas chromatography with a flame thermionic detector (GC\\/FTD) was developed for determination of N-methyl-2-pyrrolidone (NMP), N-methylsuccinimide (MSI), and 2-hydroxy-N-methylsuccinimide (2-HMSI) in human urine. The SPE cartridge of poly(divinylbenzene\\/hydroxymethacrylate) used was directly loaded with urine sample, followed by elution with methyl isobutyl ketone (MIBK) and subsequent centrifugation, and the supernatant
Ryuichi Kubota; Yoko Endo; Akito Takeuchi; Yoshinori Inoue; Hiroko Ogata; Masanori Ogawa; Tomoo Nakagawa; Nobuhiko Onda; Ginji Endo
Mineralogical Constraints on Silicate-Rich Asteroids from SpeX
We have recently begun a new survey of silicate-rich asteroids using SpeX, a low- to medium-resolution infrared spectrograph, at the Infrared Telescope Facility (IRTF) on Mauna Kea, Hawaii. In its low-resolution mode (R 100), SpeX can produce spectra of faint asteroids from 0.8 to 2.5 mum with S\\/N comparable to data typically collected with visible wavelength CCDs. This rich dataset
J. M. Sunshine; S. J. Bus; T. H. Burbine; T. J. McCoy; R. P. Binzel
Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes.
Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome-lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion. PMID:23918659
Tornieri, Karine; Zlatic, Stephanie A; Mullin, Ariana P; Werner, Erica; Harrison, Robert; L'hernault, Steven W; Faundez, Victor
NAD-Glycohydrolase Production and speA and speC Distribution in Group A Streptococcus (GAS) Isolates Do Not Correlate with Severe GAS Diseases in the Australian Population
nous population is five times that among the general popula- tion, and no single S. pyogenes clone has been shown to be dominant (5). We now report on the distribution of the genes for three erythrogenic toxins (speA, speB, and speC) and NA- Dase activity in GAS strains from the NT and from southwest- ern Sydney (SWS), a subtropical region.
Armando DelVecchio; Michael Maley; Bart J. Currie; K. S. Sriprakash
Determination of organophosphorus pesticides in underground water by SPE-GC-MS.
A rapid and effective method is developed for the determination of organophosphorus pesticides (dichlorovos, methyl parathion, malathion, and parathion) in underground water by solid-phase extraction (SPE)-gas chromatography-mass spectrometry. Some important extraction parameters including types of SPE adsorbents, elution solvents, and injection volume of water samples are optimized. The use of Cleanert-PEP polymer SPE column improved higher extraction efficiencies than the C18 SPE column commonly used. Water samples are extracted using Cleanert-PEP as SPE adsorbent and ethyl acetate as elution solvent. Precision values expressed as relative standard deviation for 1 microg/L of spiked water sample are in the range of 1.6-4.0%. Dichlorvos, methyl parathion, malathion, and parathion are linear in the range of 0.1-1.0 microg/L (r2=0.9976), 0.1-2.0 microg/L (r2=0.9883), 0.1-2.0 microg/L (r2=0.9798), and 0.055-1.1 microg/L (r2=0.9790), respectively. The limits of detection for spiked water samples are in the range of 4-10 ng/L. The optimized method is applied to the determination of underground water samples. Recoveries are between 59.5% and 94.6% for spiked underground water samples. The benefit of the method developed is rapid, simple, and has good repeatability. PMID:19222918
Ma, Jiping; Xiao, Ronghui; Li, Jie; Zhao, Xiuhua; Shi, Benzhang; Li, Shuqing
Preliminary X-ray crystallographic studies of Bacillus subtilis SpeA protein
The speA gene in Bacillus subtilis encodes arginine decarboxylase, which catalyzes the conversion of arginine to agmatine. Arginine decarboxylase is an important enzyme in polyamine metabolism in B. subtilis. In order to further illustrate the catalytic mechanism of arginine decarboxylase by determining the three-dimensional structure of the enzyme, the speA gene was amplified from B.�subtilis genomic DNA and cloned into the expression vector pET-28a(+). SpeA was expressed in Escherichia coli and purified to homogeneity by nickel-chelation chromatography followed by size-exclusion chromatography. High-quality crystals were obtained using the hanging-drop vapour-diffusion method at 289?K. The best crystal diffracted to 2.0?� resolution and belonged to space group P21, with unit-cell parameters a = 86.4, b = 63.3 c�=�103.3?�, ?�=�113.9�.
Liu, Xiao-Yan; Lei, Jian; Liu, Xiang; Su, Xiao-Dong; Li, Lanfen
Apoptosis is the fate of most thymocytes. Many molecules participate in the decision of whether a thymocyte is to live or to die, including cell surface receptors, such as the T cell receptor for antigen, Notch-1, and costimulatory receptors, ligand-regulated nuclear transcription factors such as the glucocorticoid receptor, signaling, and effector proteases, and direct regulators of the apoptotic machinery such
Yili Yang; Jonathan D. Ashwell
Glaucocalyxin A and B-induced cell death is related to GSH perturbation in human leukemia HL-60 cells.
Glaucocalyxin (Gla) A-C are major ent-kauranoid diterpenoids isolated from Rabdosia japonica var. glaucocalyx, a plant used in Chinese traditional medicine as an antitumor and anti-inflammatory agent. The present investigation was carried out to observe whether cellular reduced glutathione (GSH) plays important roles in Gla -induced cytotoxicity. Among major ent-kauranoid diterpenoids isolated, Gla A and B dose-dependently decreased the growth of HL-60 cells with an IC50 of approximately 6.15 and 5.86 �M at 24 h, respectively. Both Gla A and B could induce apoptosis, G2/M-phase cycle arrest, DNA damage and the accumulation of reactive oxygen species (ROS) in HL-60 cells. Moreover, Gla A, B caused rapid decrease of the intracellular GSH content, while inhibition of cellular GSH synthesis by buthionine sulfoximine (BSO) augmented the induced cytotoxicity and apoptosis in HL-60 cells. On the other hand, the administration of GSH or GSH precursor N-acetyl-cysteine (NAC) could rescue Gla A, B-depleted cellular GSH, and abrogate the induced cytotoxicity, G2/M-phase cycle arrest, DNA damage and ROS accumulation in HL-60 cells. Furthermore, Gla A, B decreased the activity of the GSH-related enzymes including glutathione reductase (GR) and glutathione peroxidase (GPX). These data suggest that the intracellular GSH redox system plays important roles in regulating the Gla A, B-induced cytotoxicity on HL-60 cells. PMID:23796245
Yang, Wen Hua; Zhang, Zhen; Sima, Yang Hu; Zhang, Jian; Wang, Jian Wen
Mass distribution in 11B induced fission of 232Th
NASA Astrophysics Data System (ADS)
Formation cross sections of several fission products have been determined using recoil catcher technique followed by ?-ray spectrometry in 11B induced fission of 232Th at Elab=72, 60, and 55 MeV. The data show significant admixture of fission from compound nucleus formed by complete fusion as well as targetlike nuclei formed by transfer reaction. Mass distributions for both the fissioning systems have been obtained using the systematics of charge distribution in low and medium energy fission. The mass distribution for complete fusion fission is broad, Gaussian whereas it is asymmetric for transfer induced fission. The evaporation residue cross sections of targetlike nuclei formed in 232Th(11B, 10Be)233Pa reaction were also measured. The measured evaporation residue cross sections and the decay probabilities of targetlike nucleus 233Pa, calculated by the PACE2 code have been used to estimate the proton transfer fission cross sections which were found to be negligible compared to the total transfer cross section for all the projectile energies used. The transfer fission cross section is dominated by alpha transfer fission as inferred by measured forward to backward ratios for several fission products as well as Qgg systematics for the probable transfer reactions. The proportion of transfer fission cross section to the total fission cross section was 15, 17, and 22 % at projectile energies of 72, 60, and 55 MeV, respectively.
Gubbi, G. K.; Goswami, A.; Tomar, B. S.; Ramaswami, A.; Reddy, A. V. R.; Burte, P. P.; Manohar, S. B.; John, B.
An Overview of the Source Physics Experiments (SPE) at the Nevada National Security Site (NNSS)
NASA Astrophysics Data System (ADS)
Modeling of explosion phenomenology has been primarily empirically based when looking at the seismic, infrasound, and acoustic signals. In order to detect low-yield nuclear explosions under the Comprehensive Nuclear Test-Ban Treaty (CTBT), we must be able to understand and model the explosive source in settings beyond where we have empirical data. The Source Physics Experiments (SPE) at the Nevada National Security Site are the first step in this endeavor to link the empirically based with the physics-based modeling to develop this predictive capability. The current series of tests is being conducted in a granite body called the Climax Stock. This location was chosen for several reasons, including the site's expected "simple geology"-the granite is a fairly homogeneous body. In addition, data are available from underground nuclear tests that were conducted in the same rock body, and the nature of the geology has been well-documented. Among the project goals for the SPE is to provide fully coupled seismic energy to the seismic and acoustic seismic arrays so that the transition between the near and far-field data can be modeled and our scientists can begin to understand how non-linear effects and anisotropy control seismic energy transmission and partitioning. The first shot for the SPE was conducted in May 2011 as a calibration shot (SPE1) with 220 lb (100 kg) of chemical explosives set at a depth of 180 ft (55 m). An array of sensors and diagnostics recorded the shot data, including accelerometers, geophones, rotational sensors, short-period and broadband seismic sensors, Continuous Reflectometry for Radius vs. Time Experiment (CORRTEX), Time of Arrival (TOA), Velocity of Detonation (VOD) as well as infrasound sensors. The three-component accelerometer packages were set at depths of 180 ft (55 m), 150 ft (46 m), and 50 ft (15 m) in two rings around ground zero (GZ); the inner ring was at 10 m and the outer ring was 20 m from GZ. Six sets of surface accelerometers (100 and 500 g) were placed along in an azimuth of SW from GZ every 10 m. Seven infrasound sensors were placed in an array around the GZ, extending from tens of meters to kilometers. Over 100 seismic stations were positioned, most of which were in five radial lines from GZ out to 2 km. Over 400 data channels were recorded for SPE1, and data recovery was about 95% with high signal to noise ratio. Future tests will be conducted in the same shot hole as SPE1. The SPE2 experiment will consist of 2200 lb (1000 kg) of chemical explosives shot at 150 ft (46 m) depth utilizing the above-described instrumentation. Subsequent SPE shots will be the same size, within the same shot hole, and within the damage zone. The ultimate goal of the SPE Project is to develop predictive capability for using seismic energy as a tool for CTBT issues. This work was done by National Security Technologies, LLC, under Contract No. DE AC52 06NA25946 with the U.S. Department of Energy.
Snelson, C. M.; Barker, D. L.; White, R. L.; Emmitt, R. F.; Townsend, M. J.; Graves, T. E.; Becker, S. A.; Teel, M. G.; Lee, P.; Antoun, T. H.; Rodgers, A.; Walter, W. R.; Mellors, R. J.; Brunish, W. M.; Bradley, C. R.; Patton, H. J.; Hawkins, W. L.; Corbell, B. H.; Abbott, R. E.; SPE Working Group
From transcription to activation: how group A streptococcus, the flesh-eating pathogen, regulates SpeB cysteine protease production.
Streptococcal pyrogenic exotoxin B (SpeB) is a protease secreted by group A streptococci and known to degrade a wide range of host and GAS proteins in vitro. Although the role of SpeB in GAS infection is debated, recent evidence has conclusively demonstrated that SpeB is critical for the pathogenesis of severe invasive disease caused by GAS. Genetic inactivation of the speB gene results in significantly decreased virulence in a necrotizing fasciitis model of infection. Production of fully active SpeB by GAS is extremely complex. Following transcription and translation the SpeB protein is secreted as an inactive zymogen, which is autocatalytically processed through a series of intermediates to form an active protease. Each step from transcription to protease activation is tightly controlled and regulated by the bacterial cell reflecting the critical role played by this virulence factor in GAS infection. Here we review the molecular aspects of SpeB production by GAS from transcription to activation and the multiple layers of control involved. PMID:21707787
Carroll, Ronan K; Musser, James M
Neuropeptide B Induces Slow Wave Sleep in Mice
Study Objective: Neuropeptide B (NPB) and neuropeptide W (NPW) are two recently identified neuropeptides that act as endogenous ligands to orphan G protein coupled receptors, GPR7 and GPR8. In rodents, the GPR8 ortholog is absent and both NPB and NPW function exclusively through GPR7. Although NPB and NPW are implicated in the regulation of feeding behavior, endocrine function, and pain sensation, their physiological role is incompletely understood. Design: NPB or saline was administered into the lateral ventricle of mice during both the light and dark periods. In separate experiments, spontaneous locomotor activity or EEG and EMG were recorded after intracerebroventricular (i.c.v). injection. To confirm the involvement of GPR7 in NPB-induced responses, GPR7 knockout mice were also subjected to i.c.v. injections. Measurements and Results: NPB injections reduced locomotor activity during the dark period, but not during the light period. EEG and EMG recordings in freely moving mice revealed that NPB injection decreased the time spent in the waking state and increased the time spent in slow wave sleep (SWS) during the dark period. The time spent in paradoxical sleep was unaffected. The spectral power of NPB-induced SWS was indistinguishable from that of physiological SWS. The NPB-induced increase in SWS was not observed in GPR7 knockout mice. Conclusion: These results suggest that NPB induced physiological SWS through GPR7 and that NPB and GPR7 may have a role in modulating the occurrence of sleep and wakefulness. Citation: Hirashima N; Tsunematsu T; Ichiki K; Tanaka H; Kilduff TS; Yamanaka A. Neuropeptide B induces slow wave sleep in mice. SLEEP 2011;34(1):31-37.
Hirashima, Noriko; Tsunematsu, Tomomi; Ichiki, Kanako; Tanaka, Hirokazu; Kilduff, Thomas S.; Yamanaka, Akihiro
A New Method of Facial Expression Recognition Based on SPE Plus SVM
NASA Astrophysics Data System (ADS)
A novel method of facial expression recognition (FER) is presented, which uses stochastic proximity embedding (SPE) for data dimension reduction, and support vector machine (SVM) for expression classification. The proposed algorithm is applied to Japanese Female Facial Expression (JAFFE) database for FER, better performance is obtained compared with some traditional algorithms, such as PCA and LDA etc.. The result have further proved the effectiveness of the proposed algorithm.
Ying, Zilu; Huang, Mingwei; Wang, Zhen; Wang, Zhewei
Determination of antibiotic compounds in water by on-line SPE-LC\\/MSD
This study attempts to provide an improved approach for the analysis of antibiotics, which normally exist at low concentration in complex matrices such as receiving streams of wastewater treatment plant discharge. The analytical method developed in this study combines an existing pretreatment technique of solid-phase extraction (SPE) with liquid chromatography mass spectrometry (LC\\/MSD) through on-line connection. The on-line connection suppressed
Keun-Joo Choi; Sang-Goo Kim; Chang-won Kim; Seung-Hyun Kim
A validated SPE�LCMS\\/MS assay for Eplerenone and its hydrolyzed metabolite in human urine
An automated LC-MS\\/MS assay was validated to quantitate the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human urine. After the addition of the stable isotope labeled internal standards, human urine samples were extracted on a C18 solid phase extraction (SPE) cartridge using a Zymark RapidTrace� automation system. The extraction eluates were diluted with 20 mM
Ji Y Zhang; Douglas M Fast; Alan P Breau
Comparison of Radiation Transport Codes, HZETRN, HETC and FLUKA, Using the 1956 Webber SPE Spectrum
NASA Technical Reports Server (NTRS)
Protection of astronauts and instrumentation from galactic cosmic rays (GCR) and solar particle events (SPE) in the harsh environment of space is of prime importance in the design of personal shielding, spacec raft, and mission planning. Early entry of radiation constraints into the design process enables optimal shielding strategies, but demands efficient and accurate tools that can be used by design engineers in every phase of an evolving space project. The radiation transport code , HZETRN, is an efficient tool for analyzing the shielding effectiveness of materials exposed to space radiation. In this paper, HZETRN is compared to the Monte Carlo codes HETC-HEDS and FLUKA, for a shield/target configuration comprised of a 20 g/sq cm Aluminum slab in front of a 30 g/cm^2 slab of water exposed to the February 1956 SPE, as mode led by the Webber spectrum. Neutron and proton fluence spectra, as well as dose and dose equivalent values, are compared at various depths in the water target. This study shows that there are many regions where HZETRN agrees with both HETC-HEDS and FLUKA for this shield/target configuration and the SPE environment. However, there are also regions where there are appreciable differences between the three computer c odes.
Heinbockel, John H.; Slaba, Tony C.; Blattnig, Steve R.; Tripathi, Ram K.; Townsend, Lawrence W.; Handler, Thomas; Gabriel, Tony A.; Pinsky, Lawrence S.; Reddell, Brandon; Clowdsley, Martha S.; Singleterry, Robert C.; Norbury, John W.; Badavi, Francis F.; Aghara, Sukesh K.
SPE analysis of high efficiency PMTs for the DEAP-3600 dark matter detector
NASA Astrophysics Data System (ADS)
The Dark matter Experiment using Argon Pulse-shape discrimination is a collaborative effort to develop a next-generation, tonne-scale dark matter detector at SNOLAB. The detector will feature a single-phase liquid argon (LAr) target surrounded by an array of 266 photomultiplier tubes (PMTs). A new high-efficiency Hamamatsu R877-100 PMT has been delivered to the University of Alberta for evaluation by the DEAP collaboration. The increase in efficiency could lead to a much greater light yield, but other experiments have reported a slower rise time [1],[2]. We have placed the PMT in a small dark box and had a base and preamplifier designed to be used with either an oscilloscope or a multi-channel analyzer. With this setup we have demonstrated the PMT's ability to distinguish single photo-electrons (SPE) and characterized the PMT by measuring the SPE pulse height spectrum, the peak-to-valley ratio, the dark pulse rate, the baseline, time resolution and SPE efficiency for varying the high voltage supplied to the PMT.
Olsen, Kevin; Hallin, Aksel; DEAP/CLEAN Collaboration
SolidPhase Extraction (SPE) and HPLC Analysis of Toxic Compounds and Comparison of SPE and Liquid-Liquid Extraction. I. Analysis of 4, 4?-Methyl-Enedianiline in Serum. II. Analysis of the Components of Dental Materials
In this paper, we discuss two subjects concerned with solid phase extraction (SPE) and HPLC analysis of toxic compounds eluted to serum from materials used for medical devices. SPE and liquid-liquid extraction for serum toxic compounds were compared. Firstly, an analysis of a toxic and carcinogenic compound, 4,4?-methylenedianiline (MDA), from polyurethane sterilized by gamma-ray irradiation was studied. Serum MDA was
H. Shintani
The Crystal Structure of Escherichia coli Spermidine Synthase SpeE Reveals a Unique Substrate-binding Pocket
Polyamines are essential in all branches of life. Biosynthesis of spermidine, one of the most ubiquitous polyamines, is catalyzed by spermidine synthase (SpeE). Although the function of this enzyme from Escherichia coli has been thoroughly characterized, its structural details remain unknown. Here, we report the crystal structure of E. coli SpeE and study its interaction with the ligands by isothermal titration calorimetry and computational modelling. SpeE consists of two domains - a small N-terminal {beta}-strand domain, and a C-terminal catalytic domain that adopts a canonical methyltransferase (MTase) Rossmann fold. The protein forms a dimer in the crystal and in solution. Structural comparison of E. coli SpeE to its homologs reveals that it has a large and unique substrate-binding cleft that may account for its lower amine substrate specificity.
Zhou, X.; Chua, T; Tkaczuk, K; Bujnicki, J; Sivaraman, J
extraction (SPE) ??????????? ????????????????????
????????? High performance liquid chromatography (HPLC) ??? % Recovery ????????????????? 29-100% ??????????????? Linear working range ?????????? 0.05-10.4 mg\\/L ????????? Correlation coefficient (r) ?????? 0.9962-0.9999 ????????????????? dimethylphosphate ???????????????????? liquid-liquid extraction ?????? Shaker ??? Centrifuge ?????????????????????????????? Gas liquid chromatography (GC) ??? Flame photometric detector (FPD) ??? % Recovery ????????????????? 26-114 % ??????????????? Linear working range ?????????? 0.02-7.74 mg\\/L ?????? Correlation coefficient (r)
Tawatchai Hongtrakul; Sakson Suwannabutra
Development of colorimetric solid phase extraction (C-SPE) for in-flight monitoring of spacecraft water supplies
Colorimetric solid phase extraction (C-SPE) is a sorption-spectrophotometric technique that combines colorimetric reagents, solid phase extraction, and diffuse reflectance spectroscopy to quantify trace analytes in water samples. In C-SPE, a syringe is used to meter a known volume of sample through a membrane impregnated with a selective colorimetric reagent along with any additives required to optimize the complexation of the
Daniel Bryan Gazda
Characterization of a paracetamol metabolite using on-line LC-SPE-NMR-MS and a cryogenic NMR probe
In this study, the hyphenation of LC-SPE-NMR-MS at 500MHz was applied to the structural elucidation of a low concentrated paracetamol metabolite present in human urine. Single or multiple peak trapping of the mass detected metabolite on SPE cartridges was employed to increase the sensitivity and quality NMR measurement over the conventional LC-NMR method. After the elution of the metabolite from
Markus Godejohann; Li-Hong Tseng; Ulrich Braumann; Jens Fuchser; Manfred Spraul
Mineralogical Constraints on Silicate-Rich Asteroids from SpeX
NASA Astrophysics Data System (ADS)
We have recently begun a new survey of silicate-rich asteroids using SpeX, a low- to medium-resolution infrared spectrograph, at the Infrared Telescope Facility (IRTF) on Mauna Kea, Hawaii. In its low-resolution mode (R 100), SpeX can produce spectra of faint asteroids from 0.8 to 2.5 ?m with S/N comparable to data typically collected with visible wavelength CCDs. This rich dataset of near-Earth and main-belt objects contain subtleties in their spectral signatures that are well suited to analysis with the Modified Gaussian absorption band model. Among our results are clear unambiguous evidence for the presence of both high- and low-calcium pyroxene (HCP and LCP) as well as olivine and plagioclase. The quality of these data allow us to use the proportion of HCP relative to LCP, to constrain the petrologic history of these bodies. Very primitive bodies (e.g. primitive achondrites) have < 10% HCP, while moderately evolved bodies (e.g. ordinary chondrites) have 15-20% HCP, and highly evolved bodies (e.g. basaltic achondrites) have >25% of their pyroxene as HCP. Our ability to infer the presence (or absence) of HCP with SpeX data is a new tool for examining and mapping igneous processes on asteroids. For example, our current dataset contains several asteroids, including members of the Merxia and Agnia families, with spectra that show little to no evidence for olivine, but instead are dominated by pyroxene absorptions and include significant (>40%) proportions of HCP. This HCP content implies a history of partial melting and silicate differentiation. As such we are actively examining smaller members of the Agnia and Merxia families to further constrain the petrologic history of these bodies.
Sunshine, J. M.; Bus, S. J.; Burbine, T. H.; McCoy, T. J.; Binzel, R. P.
spe-10 Encodes a DHHC-CRD Zinc-Finger Membrane Protein Required for Endoplasmic Reticulum/Golgi Membrane Morphogenesis During Caenorhabditis elegans Spermatogenesis
C. elegans spermatogenesis employs lysosome-related fibrous body�membranous organelles (FB�MOs) for transport of many cellular components. Previous work showed that spe-10 mutants contain FB�MOs that prematurely disassemble, resulting in defective transport of FB components into developing spermatids. Consequently, spe-10 spermatids are smaller than wild type and contain defective FB�MO derivatives. In this article, we show that spe-10 encodes a four-pass integral membrane protein that has a DHHC�CRD zinc-finger motif. The DHHC�CRD motif is found in a large, diverse family of proteins that have been implicated in palmitoyl transfer during protein lipidation. Seven spe-10 mutants were analyzed, including missense, nonsense, and deletion mutants. An antiserum to SPE-10 showed significant colocalization with a known marker for the FB�MOs during wild-type spermatogenesis. In contrast, the spe-10(ok1149) deletion mutant lacked detectable SPE-10 staining; this mutant lacks a spe-10 promoter and most coding sequence. The spe-10(eb64) missense mutation, which changes a conserved residue within the DHHC�CRD domain in all homologues, behaves as a null mutant. These results suggest that wild-type SPE-10 is required for the MO to properly deliver the FB to the C. elegans spermatid and the DHHC�CRD domain is essential for this function.
Gleason, Elizabeth J.; Lindsey, Wesley C.; Kroft, Tim L.; Singson, Andrew W.; L'Hernault, Steven W.
Simultaneous determination of seven pesticides in waters using multi-walled carbon nanotube SPE and NACE.
In this work, NACE with UV detection is combined with SPE using multi-walled carbon nanotubes (MWCNT) as stationary phase to determine a group of seven pesticides (pirimicarb, pyrifenox, penconazol, carbendazim, cyromazine, pyrimethanil and cyprodinil) in mineral water samples using ametryn as internal standard. The optimized BGE, consisting of a mixture of MeOH and ACN (1:2 v/v) with 90 mM SDS and 20.5 mM HClO(4), was satisfactory to get a good resolution of the seven compounds in less than 13 min. On-line preconcentration was carried out by electrokinetic injection of the sample dissolved in 78:22 v/v MeOH/ACN, 1.11 mM HClO(4). Repeatability was studied for the same day (n=4), for nine different days (n=36) and for four different capillaries. RSD values were appropriate in all cases, i.e. in the range 4.3-9.4% between different capillaries. MWCNT of 10-15 nm od, 2-6 nm id and 0.1-10 mum length were used as SPE materials for the preconcentration of these pesticides from water samples. SPE parameters influencing the enrichment were optimized and the most favorable conditions were as follows: the amount of stationary phase, eluent, sample pH and sample volume were 40 mg MWCNT, 10 mL ACN and 10 mL dichloromethane containing 5% v/v formic acid, pH 8.0, and 750 mL, respectively. Mean recovery values ranged between 53 and 94% for Milli-Q water and between 47 and 93% for mineral waters (RSD values were in the range 2-16%). The method allowed the determination of these pesticides at concentrations below the maximum residue limits established by the European Union legislation (LOD in the range 27-58 ng/L). When the cost, amount and type of the carbon nanotubes used in this work are compared with those carbon nanotubes previously used in the literature it is clear that the proposed materials can be used as economical stationary phases, even cheaper than conventional SPE cartridges. PMID:18956435
Asensio-Ramos, Mar�a; Hern�ndez-Borges, Javier; Ravelo-P�rez, Lidia M; Rodr�guez-Delgado, Miguel Angel
Development of colorimetric solid phase extraction (C-SPE) for in-flight monitoring of spacecraft water supplies
NASA Astrophysics Data System (ADS)
Colorimetric solid phase extraction (C-SPE) is a sorption-spectrophotometric technique that combines colorimetric reagents, solid phase extraction, and diffuse reflectance spectroscopy to quantify trace analytes in water samples. In C-SPE, a syringe is used to meter a known volume of sample through a membrane impregnated with a selective colorimetric reagent along with any additives required to optimize the complexation of the reagent and analyte. As the sample is passed through the membrane, analytes are extracted and complexed, leading to a detectable change in the optical characteristics of the membrane. The analyte-reagent complex is then quantified directly on the membrane, using a hand-held diffuse reflectance spectrophotometer. This dissertation focuses on the development, ground testing, and microgravity flight demonstration of C-SPE methods to meet the near- and long-term water quality monitoring needs of NASA. To this end, the ability of C-SPE to function in a microgravity environment was tested through performance evaluations of methods for the determination of the biocidal agents silver(I) and iodine on the KC-135 microgravity simulator. The biocidal iodine platform was investigated further to determine which iodine species is responsible for the C-SPE signal. Through systematic comparisons of C-SPE results and UV-Visible absorbance studies it was determined that biocidally active I2 is the iodine species complexed by poly(vinylpyrrolidone). The application of C-SPE to additional target water quality parameters is demonstrated through the determination of nickel(II), a metal leachate found in archived water samples from the International Space Station, using dimethylglyoxime. This method introduced a new variation of C-SPE, the quantification of trace analytes based on the collection of an insoluble, colored precipitate. The nickel(II) method was then combined with the method for biocidal silver(I) and a new method to measure sample pH to create a multiplexed C-SPE platform. This invention is presented as an approach to increase the collection of on-orbit water quality data collected without requiring additional crew time. The dissertation is concluded with a summation of the current work and a look at future directions.
Gazda, Daniel Bryan
Recent improvements in SPE3D: a VR-based surgery planning environment
NASA Astrophysics Data System (ADS)
SPE3D is a surgery planning environment developed within TLEMsafe project [1] (funded by the European Commission FP7). It enables the operator to plan a surgical procedure on the customized musculoskeletal (MS) model of the patient's lower limbs, send the modified model to the biomechanical analysis module, and export the scenario's parameters to the surgical navigation system. The personalized patient-specific three-dimensional (3-D) MS model is registered with 3-D MRI dataset of lower limbs and the two modalities may be visualized simultaneously. Apart from main planes, any arbitrary MRI cross-section can be rendered on the 3-D MS model in real time. The interface provides tools for: bone cutting, manipulating and removal, repositioning muscle insertion points, modifying muscle force, removing muscles and placing implants stored in the implant library. SPE3D supports stereoscopic viewing as well as natural inspection/manipulation with use of haptic devices. Alternatively, it may be controlled with use of a standard computer keyboard, mouse and 2D display or a touch screen (e.g. in an operating room). The interface may be utilized in two main fields. Experienced surgeons may use it to simulate their operative plans and prepare input data for a surgical navigation system while student or novice surgeons can use it for training.
Witkowski, Marcin; Sitnik, Robert; Verdonschot, Nico
Determination of chlorotriazines, methylthiotriazines and one methoxytriazine by SPE-LC-UV in water samples.
Triazine herbicides form a wide group of substances that belong among the most common agrochemicals applied for pre- and post-emergence weed control. So, they can be found in the environment at trace level. In order to determine their concentrations in water samples by the usual analytical techniques, a preconcentration step is commonly necessary. In this paper, a simple analytical method for the quantification of eight triazines (three chlorotriazines, four methylthiotriazines and one methoxytriazine) in water samples by solid phase extraction-reversed phase liquid chromatography (LC) has been developed. LC shows good analytical performance for simultaneous multiple triazine analysis (repeatability <2%, reproducibility <3%), except for prometon (repeatability 5.52%, reproducibility 16%). The results, obtained by using carbograph and polymeric sorbents for solid phase extraction (SPE), have been compared. The limits of quantification achieved permit the application of the proposed SPE-LC method for the determination of eight triazines in water samples (0.0065-0.028 mug l(-1)). PMID:18968941
Dopico G, M S; Gonz�lez R, M V; Castro R, J M; Gonz�lez S, E; P�rez I, J; Rodr??guez T, M; Calleja, A; Vilari�o, J M L
Cucurbitacin B induced ATM-mediated DNA damage causes G2/M cell cycle arrest in a ROS-dependent manner.
Cucurbitacins are a class of triterpenoids widely distributed in plant kingdom with potent anti-cancer activities both in vitro and in vivo by inducing cycle arrest, autophagy, and apoptosis. Cucurbitacin B (Cuc B), could induce S or G2/M cell cycle arrest in cancer cells while the detailed mechanisms remain to be clear. This study was designed to precisely dissect the signaling pathway(s) responsible for Cuc B induced cell cycle arrest in human lung adenocarcinoma epithelial A549 cells. We demonstrated that low concentrations of Cuc B dramatically induced G2/M phase arrest in A549 cells. Cuc B treatment caused DNA double-strand breaks (DSBs) without affecting the signal transducer and activator of transcription 3 (STAT3), the potential molecular target for Cuc B. Cuc B triggers ATM-activated Chk1-Cdc25C-Cdk1, which could be reversed by both ATM siRNA and Chk1 siRNA. Cuc B also triggers ATM-activated p53-14-3-3-? pathways, which could be reversed by ATM siRNA. Cuc B treatment also led to increased intracellular reactive oxygen species (ROS) formation, which was inhibited by N-acetyl-l-cysteine (NAC) pretreatment. Furthermore, NAC pretreatment inhibited Cuc B induced DNA damage and G2/M phase arrest. Taken together, these results suggested that Cuc B induces DNA damage in A549 cells mediated by increasing intracellular ROS formation, which lead to G2/M cell phase arrest through ATM-activated Chk1-Cdc25C-Cdk1 and p53-14-3-3-? parallel branches. These observations provide novel mechanisms and potential targets for better understanding of the anti-cancer mechanisms of cucurbitacins. PMID:24505404
Guo, Jiajie; Wu, Guosheng; Bao, Jiaolin; Hao, Wenhui; Lu, Jinjian; Chen, Xiuping
Mathematical modeling of apoptosis
Apoptosis is a form of programmed cell death, which is fundamental to all multicellular organisms. Deregulation of apoptosis leads to a number of severe diseases including cancer. Apoptosis is initiated either by extrinsic signals via stimulation of receptors at the cellular surface or intrinsic signals, such as DNA damage or growth factor withdrawal. Apoptosis has been extensively studied using systems biology which substantially contributed to the understanding of this death signaling network. This review gives an overview of mathematical models of apoptosis and the potential of systems biology to contribute to the development of novel therapies for cancer or other apoptosis-related diseases.
Cannabisin B induces autophagic cell death by inhibiting the AKT/mTOR pathway and S phase cell cycle arrest in HepG2 cells.
This study investigates the anticancer properties of cannabisin B, purified from hempseed hull, in HepG2 human hepatoblastoma cells. The results indicate that cannabisin B significantly inhibited cell proliferation by inducing autophagic cell death rather than typical apoptosis. Cell viability transiently increased upon the addition of a low concentration of cannabisin B but decreased upon the addition of high concentrations. Cannabisin B-induced changes in cell viability were completely inhibited by pre-treatment with 3-methyladenine (3-MA), indicating that the induction of autophagy by cannabisin B caused cell death. Additionally, cannabisin B induced S phase cell cycle arrest in a dose-dependent manner. Moreover, cannabisin B was found to inhibit survival signaling by blocking the activation of AKT and down-stream targets of the mammalian target of rapamycin (mTOR). These findings suggest that cannabisin B possesses considerable antiproliferative activity and that it may be utilised as a promising chemopreventive agent against hepatoblastoma disease. PMID:23411211
Chen, Tianpeng; Hao, Jianxiong; He, Jinfeng; Zhang, Jianchun; Li, Yingcong; Liu, Rui; Li, Lite
spe-10 Encodes a DHHC-CRD Zinc-Finger Membrane Protein Required for Endoplasmic Reticulum\\/Golgi Membrane Morphogenesis During Caenorhabditis elegans Spermatogenesis
C. elegans spermatogenesis employs lysosome-related fibrous body-membranous organelles (FB-MOs) for transport of many cellular components. Previous work showed that spe-10 mutants contain FB-MOs that prematurely disassemble, resulting in defective transport of FB components into developing spermatids. Consequently, spe-10 spermatids are smaller than wild type and contain defective FB-MO derivatives. In this article, we show that spe-10 encodes a four-pass integral
Elizabeth J. Gleason; Wesley C. Lindsey; Tim L. Kroft; Andrew W. Singson; Steven W. L'Hernault
Cytochrome P450-catalysed reactive oxygen species production mediates the (-)schisandrin B-induced glutathione and heat shock responses in AML12 hepatocytes.
Sch B (schisandrin B), the most abundant dibenzocyclooctadiene lignan in Fructus schisandrae, can induce glutathione antioxidant and heat shock responses, as well as protect against oxidant-induced injury in various tissues, including the liver in rodents and AML12 (alpha mouse liver 12) hepatocytes. (-)Sch B is the most potent stereoisomer of Sch B in its cytoprotective action on AML12 hepatocytes. To define the role of ROS (reactive oxygen species) arising from CYP (cytochrome P450)-catalysed metabolism of (-)Sch B in triggering glutathione antioxidant and heat shock responses, the effects of a CYP inhibitor [ABT (aminobenzotriazole)] and antioxidants [DMTU (dimethylthiouracil) and TRX (trolox)] on (-)Sch B-induced ROS production and associated increases in cellular GSH level, as well as Hsp25/70 (heat-shock protein 25/70) production, were investigated in AML12 hepatocytes. The results indicated that (-)Sch B causes a dose dependent and sustained increase in ROS production over 6 h in AML12 hepatocytes, which was completely suppressed by pre-/co-treatment with ABT or DTMU/TRX. Incubation with (-)Sch B for 6 h caused optimal and dose-dependent increases in cellular GSH level and Hsp25/70 production at 16 h post-drug exposure in AML12 hepatocytes. These cellular responses were associated with protection against menadione-induced apoptosis. Pre-/co-treatment with ABT or antioxidants completely abrogated the (-)Sch B-induced glutathione antioxidant and heat shock responses, as well as protection against menadione-induced apoptosis. Experimental evidence obtained thus far supports the causal role of ROS arising from the CYP-catalysed metabolism of (-)Sch B in eliciting glutathione antioxidant and heat shock responses in AML12 hepatocytes. PMID:22070356
Leong, Pou Kuan; Chiu, Po Yee; Leung, Hoi Yan; Ko, Kam Ming
Analysis of pharmaceutical creams: a useful approach based on solid-phase extraction (SPE) and UV spectrophotometry.
Solid-phase extraction (SPE) using C-18, diol and ion-exchange sorbents followed by UV spectrophotometric (conventional and derivative mode) assay was applied to the analysis of basic, acidic and neutral drugs commercially available in creams. A representative set of drugs (promethazine, chlorhexidine, benzydamine, ketoprofen, ibuprofen, fentiazac, piroxicam, fluorouracil, crotamiton and hydrocortisone acetate) was selected, and for each drug the appropriate SPE conditions (adsorption, washing and elution) were investigated to obtain a practical and reliable sample clean-up. It was shown that the developed SPE procedures were capable of removing interfering cream components (excipients including preservatives) allowing accurate spectrophotometric analyses to be performed. In some applications, derivative spectrophotometry was advantageous over the conventional absorption mode with respect to higher selectivity and versatility. PMID:8634349
Bonazzi, D; Andrisano, V; Gatti, R; Cavrini, V
Fractionation and determination of aluminum and iron in soil water samples using SPE cartridges and ICP-AES.
The use of commercially available solid phase extraction (SPE) cartridges for the fractionation of Al and Fe in soil water is described. The quantitative determination was done by inductively coupled plasma atomic emission spectrometry (ICP-AES). Different types of SPE cartridges, based on cation exchange, anion exchange, and chelation were studied. To avoid pH changes, the SPE cartridge should be conditioned with a buffer that has a pH close to that of the sample. Both strong cation exchange (SCX) and chelation were found to work well, whereas low recovery was observed for Al when anion exchange was used. For Fe, the sum of the anionic and cationic fractions that passed through the cartridges was nearly 100%. The results obtained for Al for 23 soil water samples using a SPE/SCX cartridge and ICP-AES were compared with equilibrium calculations using the program ALCHEMI and also with a fractionation method that was based on separation on a manually prepared SCX column and detection by molecular spectrophotometry, after complexation with pyrocatechol violet (SCX-PCV method). The SPE/SCX-ICP-AES results for the labile Al fraction (Al bound to the SCX cartridge) showed an acceptable correlation with the results obtained by the equilibrium calculations, except for the samples with the highest DOC concentrations, whereas the values obtained for labile Al by the more traditional SCX-PCV method were much lower. We recommend that the SPE/SCX-ICP-AES procedure described in this work be selected for the fractionation of Al and Fe species in soil and freshwater samples. PMID:12521170
Tangen, Gaute; Wickstr�m, Torild; Lierhagen, Syverin; Vogt, Rolf; Lund, Walter
In leukemias and malignant tumors the balance between apoptosis and cell proliferation is dysregulated. This review deals with the apoptosis in acute leukemia. There are several publications about the molecular basis of decreased apoptosis in acute lymphoid leukemia (ALL) and AML. However, there have been contradictory results. Different results are published about the correlation of the spontaneous and induced apoptosis in leukemia with prognosis. The potential causes of these contradictions are discussed. PMID:15158085
Schuler, Dezso; Szende, B�la
Observing Silicate-Rich Asteroids in Technicolor: Detailed Compositional Constraints from SpeX.
NASA Astrophysics Data System (ADS)
We have recently begun a new survey of sili-cate-rich asteroids using SpeX, a low- to medium-resolution infrared spectrograph, at the Infrared Telescope Facility (IRTF) on Mauna Kea, Hawaii [1]. In its low-resolution mode (R approx.100), SpeX can produce spectra of faint asteroids from 0.8 to 2.5 microns with S/N comparable to data typically collected with visible wavelength CCDs. The SpeX data have been combined with visible CCD data measured during the SMASSII survey [2] to produce high S/N (often greater than100) spectra from 0.44 to 2.5 microns for several asteroids. This rich dataset includes subtle spectral signatures that we have analyzed with the Modified Gaussian absorption band model [3]. Among our results are clear unambiguous evidence for the presense of both high- and low-calcium pyroxene (HCP and LCP) as well as olivine and plagioclase. The quality of these data allow us to use the proportion of HCP relative to LCP, to constrain the petrologic history of these bodies. Very primitive bodies (e.g. primitive achondrites) have less than 10 percent HCP, while moderately evolved bodies (e.g. ordinary chondrites) have 15-20 percent HCP, and highly evolved bodies (e.g. basaltic achondrites) have more than 25 percent of their pyroxene as HCP. Our current dataset contains several asteroids, including members of the Merxia and Agnia families, with spectra that show little to no evidence for olivine, but instead are dominated by pyroxene absorptions and include significant (more than 40 percent) proportions of HCP. This HCP content implies a history of partial melting and silicate differentiation. As such we are actively examining smaller members of the Agnia and Merxia families to futher constrain the petrologic history of these bodies. References: [1] Rayner, J. T. et al. (1998) Proc. SPIE, 3354, 468-479. [2] Bus, S. J. et al. (2002) Icarus, 158, 106-145. [3] Sunshine, J. M. et al. (1990) JGR, 95, 6955-6966.
Sunshine, J. M.; Bus, S. J.; Burbine, T. H.; McCoy, T. J.; Binzel, R. P.
The Source Physics Experiments (SPE) at the Nevada National Security Site (NNSS): An Overview
NASA Astrophysics Data System (ADS)
Understanding the changes in seismic energy as it travels from the near field to the far field is the ultimate goal in monitoring for explosive events of interest. This requires a clear understanding of explosion phenomenology as it relates to seismic, infrasound, and acoustic signals. Although there has been much progress in modeling these phenomena, this has been primarily based in the empirical realm. As a result, the logical next step in advancing the seismic monitoring capability of the United States is to conduct field tests that can expand the predictive capability of the physics-based modeling currently under development. The Source Physics Experiment at the Nevada National Security Site (SPE) is the first step in this endeavor to link the empirically based with the physics-based modeling. This is a collaborative project between National Security Technologies (NSTec), Lawrence Livermore National Laboratory (LLNL), Los Alamos National Laboratory (LANL), Sandia National Laboratories (SNL), the Defense Threat Reduction Agency (DTRA), and the Air Force Technical Applications Center (AFTAC). The test series require both the simple and complex cases to fully characterize the problem, which is to understand the transition of seismic energy from the near field to the far field; to understand the development of S-waves in explosives sources; and how anisotropy controls seismic energy transmission and partitioning. The current series is being conducted in a granite body called the Climax Stock. This location was chosen for several reasons, including the fairly homogenous granite; the location of previous nuclear tests in the same rock body; and generally the geology has been well characterized. The simple geology series is planned for 7 shots using conventional explosives in the same shot hole surrounded by Continuous Reflectometry for Radius vs. Time Experiment (CORRTEX), Time of Arrival (TOA), Velocity of Detonation (VOD), down-hole accelerometers, surface accelerometers, infrasound, and a suite of seismic sensors of various frequency bands from the near field to the far field. This allows for the use of a single test bed in the simple geology case instead of multiple tests beds to obtain the same results. The shots are planned at various depths to obtain a Green's function, scaled-depth of burial data, nominal depth of burial data and damage zone data. SPE1 was conducted in May 2011 as a 220 lb (100 kg) TNT equivalent calibration shot at a depth of 180 ft (55 m). SPE2 was conducted in October 2011 as a 2200 lb (1000 kg) TNT equivalent calibration shot at a depth of 150 ft (46 m). SPE3 was conducted in July 2012 as a 2200 lb (1000 kg) TNT equivalent calibration shot at a depth of 150 ft (46 m) in the damaged zone. Over 400 data channels were recorded for each of these shots and data recovery was about 95% with high signal to noise ratio. Once the simple geology site data has been utilized, a new test bed will be developed in a complex geology site to test these physics based models. Ultimately, the results from this project will provide the next advances in the science of monitoring to enable a physics-based predicative capability. This work was done by National Security Technologies, LLC, under Contract No. DE-AC52-06NA25946 with the U.S. Department of Energy. DOE/NV/25946--1584
Snelson, C. M.; Chipman, V.; White, R. L.; Emmitt, R.; Townsend, M.; Barker, D.; Lee, P.
SPE-LC-FD determination of polycyclic aromatic hydrocarbon monohydroxy derivatives in cephalopods.
A new analytical methodology, based on liquid chromatography with fluorescence detection (LC-FD), after extraction, enzymatic hydrolysis, and solid-phase extraction (SPE) through Oasis HLB cartridges, was developed and validated for the simultaneous determination of three monohydroxy derivatives of polycyclic aromatic hydrocarbons (PAHs). The optimized analytical method is sensitive, accurate, and precise, with recoveries between 62 and 110% and limits of detection of 227, 9, and 45 ng/g for 1-hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene, respectively. Their levels were estimated in different cephalopod matrices (edible tissues and hemolymph). The methodology was applied to samples of the major cephalopod species consumed worldwide. Of the 18 samples analyzed, 39% were contaminated with 1-hydroxynaphthalene, which was the only PAH metabolite detected. Its concentration ranged from 786 to 1145 ng/g. This highly sensitive and specific method allows the identification and quantitation of PAH metabolites in forthcoming food safety and environmental monitoring programs. PMID:24588515
Louren�o, Diana; Silva, Liliana J G; Lino, Celeste M; Morais, Simone; Pena, Angelina
SpeB of Streptococcus pyogenes Differentially Modulates Antibacterial and Receptor Activating Properties of Human Chemokines
Background: CXC chemokines are induced by inflammatory stimuli in epithelial cells and some, like MIG\\/CXCL9, IP-10\\/ CXCL10 and I-TAC\\/CXCL11, are antibacterial for Streptococcus pyogenes. Methodology\\/Principal Findings: SpeB from S. pyogenes degrades a wide range of chemokines (i.e. IP10\\/CXCL10, I-TAC\\/ CXCL11, PF4\\/CXCL4, GROa\\/CXCL1, GROb\\/CXCL2, GROc\\/CXCL3, ENA78\\/CXCL5, GCP-2\\/CXCL6, NAP-2\\/CXCL7, SDF-1\\/CXCL12, BCA-1\\/CXCL13, BRAK\\/CXCL14, SRPSOX\\/CXCL16, MIP-3a\\/CCL20, Lymphotactin\\/XCL1, and Fractalkine\\/CX3CL1), has no activity on IL-8\\/CXCL8
Arne Egesten; Anders I. Olin; Helena M. Linge; Manisha Yadav; Matthias M�rgelin; Anna Karlsson; Mattias Collin
Uranus' cloud structure and scattering particle properties from IRTF SpeX observations
NASA Astrophysics Data System (ADS)
Observations of Uranus were made in August 2009 with the SpeX spectrograph at the NASA Infrared Telescope Facility (IRTF). Analysed spectra range from 0.8 to 1.8 ?m at a spatial resolution of 0.5" and a spectral resolution of R = 1,200. Spectra from 0.818 to 0.834 ?m, a region characterised by both strong hydrogen quadrupole and methane absorptions are considered to determine methane content. Evidence indicates that methane abundance varies with latitude. NEMESIS, an optimal estimation retrieval code with full-scattering capability, is employed to analyse the full range of data. Cloud and haze properties in the upper troposphere and stratosphere are characterised, and are consistent with other current literature. New information on single scattering albedos and particle size distributions are inferred.
Tice, D. S.; Irwin, P. G. J.; Fletcher, L. N.; Teanby, N. A.; Orton, G. S.; Davis, G. R.
Application of Nanofiber-packed SPE for Determination of Urinary 1-Hydroxypyrene Level Using HPLC
It is always desirable to achieve maximum sample clean-up, extraction, and pre-concentration with the minimum possible organic solvent. The miniaturization of sample preparation devices was successfully demonstrated by packing 10 mg of 11 electrospun polymer nanofibers into pipette tip micro column and mini disc cartridges for efficient pre-concentration of 1-hydroxypyrene in urine samples. 1-hydroxypyrene is an extensively studied biomarker of the largest class of chemical carcinogens. Excretory 1-hydroxypyrene was monitored with HPLC/fluorescence detector. Important parameters influencing the percentage recovery such as fiber diameter, fiber packing amount, eluent, fiber packing format, eluent volume, surface area, porosity, and breakthrough parameters were thoroughly studied and optimized. Under optimized condition, there was a near perfect linearity of response in the range of 1�1000 ?g/L with a coefficient of determination (r2) between 0.9992 and 0.9999 and precision (% RSD) ?7.64% (n = 6) for all the analysis (10, 25, and 50 ?g/L). The Limit of detection (LOD) was between 0.022 and 0.15 ?g/L. When compared to the batch studies, both disc packed nanofiber sorbents and pipette tip packed sorbents exhibited evident dominance based on their efficiencies. The experimental results showed comparable absolute recoveries for the mini disc packed fibers (84% for Nylon 6) and micro columns (80% for Nylon 6), although the disc displayed slightly higher recoveries possibly due to the exposure of the analyte to a larger reacting surface. The results also showed highly comparative extraction efficiencies between the nanofibers and conventional C-18 SPE sorbent. Nevertheless, miniaturized SPE devices simplified sample preparation, reducing back pressure, time of the analysis with acceptable reliability, selectivity, detection levels, and environmental friendliness, hence promoting green chemistry.
Ifegwu, Okechukwu Clinton; Anyakora, Chimezie; Chigome, Samuel; Torto, Nelson
Optimization of an SPE and GC/MS Method for Analyzing Endocrine Disrupting Compounds in Water
NASA Astrophysics Data System (ADS)
Endocrine disrupting compounds (EDCs) are compounds that interrupt hormonal functions in the body. The literature reports the presence of EDCs in all environmental matrices (air, water and soil) at concentrations of at least 1 nanogram/liter (ng/l), which may be high enough to induce adverse health effects. Therefore, reliable analytical methods for detecting trace amounts of EDCs in water is very important for investigating and controlling their concentrations in the environment. This study investigated a method for analyzing four known or suspected EDCs (chlorpyrifos, musk HHCB, diethyl phthalate, and butylated hydroxyanisole) in water samples. The analytical method was based on the USGS wastewater method developed by Zaugg et al. (2001), but modified, using solid phase extraction (SPE) followed by gas chromatography and mass spectrometry (GC/MS) analysis. The EDCs were extracted using 60mg Water Oasis hydrophilic-lipophillic balance (HLB) extraction cartridges. The SPE efficiency was investigated by using different initial extraction volumes and different EDC concentrations. The lowest concentration was 1ng/l and the lowest extraction volume was 100mL. Results of the study indicate that the initial oven temperature conditions and rate of temperature increases affects the peak signal to noise ratio and the sample run-time in the GC/MS. An increase in gas flow rate did not show any significant changes and hence was maintained at 1ml/min. Preliminary data suggests that the percent recovery of the compounds obtained using this method either met or exceeded those presented by Zaugg et al. (2001) as the USGS wastewater method.
Thomas, S. M.; Bodour, A.; Murray, K. E.
Pharmacologic Probing of Amphotericin B-Induced Renal Dysfunction in the Neonatal Rat. Gray, J.A., and Kavlock, R.J. (1988). Toxicol. Appl. Pharmacol. 93, 360-368. Acetazolamide, furosemide, chlorothiazide, and amiloride pharmacologic agents that act primarily in the proximal tub...
A Naturally Occurring Mutation in ropB Suppresses SpeB Expression and Reduces M1T1 Group A Streptococcal Systemic Virulence
Epidemiological studies of group A streptococcus (GAS) have noted an inverse relationship between SpeB expression and invasive disease. However, the role of SpeB in the course of infection is still unclear. In this study we utilize a SpeB-negative M1T1 clinical isolate, 5628, with a naturally occurring mutation in the gene encoding the regulator RopB, to elucidate the role of RopB and SpeB in systemic virulence. Allelic exchange mutagenesis was used to replace the mutated ropB allele in 5628 with the intact allele from the well characterized isolate 5448. The inverse allelic exchange was also performed to replace the intact ropB in 5448 with the mutated allele from 5628. An intact ropB was found to be essential for SpeB expression. While the ropB mutation was shown to have no effect on hemolysis of RBC's, extracellular DNase activity or survival in the presence of neutrophils, strains with the mutated ropB allele were less virulent in murine systemic models of infection. An isogenic SpeB knockout strain containing an intact RopB showed similarly reduced virulence. Microarray analysis found genes of the SpeB operon to be the primary target of RopB regulation. These data show that an intact RopB and efficient SpeB production are necessary for systemic infection with GAS.
Hollands, Andrew; Aziz, Ramy K.; Kansal, Rita; Kotb, Malak; Nizet, Victor; Walker, Mark J.
Yersinia enterocolitica YopT and Clostridium difficile Toxin B Induce Expression of GILZ in Epithelial Cells
Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype Yersinia enterocolitica which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different Yersinia mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, Clostridium difficile toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal GILZ promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the GILZ promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of GILZ promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.
Koberle, Martin; Goppel, David; Grandl, Tanja; Gaentzsch, Peer; Manncke, Birgit; Berchtold, Susanne; Muller, Steffen; Luscher, Bernhard; Asselin-Labat, Marie-Liesse; Pallardy, Marc; Sorg, Isabel; Langer, Simon; Barth, Holger; Zumbihl, Robert; Autenrieth, Ingo B.; Bohn, Erwin
Streptococcal SpeB Cleaved PAR-1 Suppresses ERK Phosphorylation and Blunts Thrombin-Induced Platelet Aggregation
Background The family of 4 related protease-activated receptors (PAR-1, 2, 3 & 4) expressed by mammalian cells allow to sense for and react to extracellular proteolytic activity. Since major human bacterial pathogens secret a wide array of protease(-s) we investigated whether they interfere with human PAR function. Methodology/Principal Findings Supernatants from cultures of major human bacterial pathogens were assayed for the presence of protease(-s) capable to cleave overexpressed human PAR-1, 2, 3 and 4 reporter constructs. Group A streptococcus (GAS) was found to secret a PAR-1-cleaving protease. Experiments involving genetical and pharmacological gain and loss of function identified streptococcal pyrogenic exotoxin B SpeB as the protease responsible. On the host�s side analysis of overexpressed PAR-1 carrying alanine substitutions and deletions showed the amino acid residue leucine44 on PAR-1�s extracellular N-terminus to be the only cleavage site. Complementary studies on endogenously expressed PAR-1 using PAR-1 blocking antibodies further supported our conclusion. Through PAR-1 cleavage SpeB efficiently blunted thrombin-induced induction of the ERK-pathway in endothelial cells and prevented platelets aggregation in response to thrombin. Conclusions/Significance Our results identify a novel function of the streptococcal virulence factor SpeB. By cleaving human PAR-1 at the N-terminal amino acid residue leucine44 SpeB rendered endothelial cells unresponsive to thrombin and prevented human platelets from thrombin-induced aggregation. These results suggest that by blunting PAR-1 signaling, SpeB modulates various innate host responses directed against invasive GAS potentially helping the invasive bacteria to escape. This may allow to tailor additional treatments in the future since upon invasion of the blood stream endothelial cells as well as platelets and mononuclear cells respond to PAR-1 agonists aiming to prevent further bacterial dissemination.
Ender, Miriam; Andreoni, Federica; Zinkernagel, Annelies Sophie; Schuepbach, Reto Andreas
Helicobacter pylori and apoptosis.
In an attempt to understand the diverse effects of infection with Helicobacter pylori on epithelial mucosal mass and consequent clinical outcome, the relationship between H. pylori infection and gastric epithelial cellular turnover has been investigated. Our results indicate that H. pylori increases epithelial cell proliferation and apoptosis in vivo, but that infection with bacteria of the cagA genotype leads to relatively more proliferation than apoptosis. This review explores the causes of the induction of apoptosis in gastric epithelial cells by H. pylori and the consequences of alterations in apoptosis to the maintenance of gastric mucosal homeostasis. Images Figure 1
Moss, S. F.
Molecularly imprinted microspheres as SPE sorbent for selective extraction of four Sudan dyes in catsup products.
A highly selective molecularly imprinted solid-phase extraction (MISPE) coupled with high performance liquid chromatography (HPLC) ultraviolet-visible detection was developed for the simultaneous isolation and determination of four Sudan dyes (I, II, III and IV) in catsup products. The novel molecularly imprinted microspheres (MIM) were synthesized by aqueous suspension polymerization using phenylamine and naphthol as template, which showed high affinity to Sudan dyes in aqueous solution. In order to develop a selective extraction protocol for simultaneous determination the four Sudan dyes from catsup products, the molecular recognition properties of MIM as a SPE sorbent were evaluated. Under the optimized condition, good linearity was obtained from 0.01 to 2.5 ?g g(-1) (r(2)? 0.9990) with the relative standard deviations of less than 3.4%. This proposed MISPE-HPLC procedure eliminated the effect of template leakage on quantitative analysis and could be applied to direct determination of four Sudan dyes in complicated food samples. PMID:21900053
Qiao, Fengxia; Geng, Yuru; He, Changqing; Wu, Yupei; Pan, Pengyu
Shear Wave Generation and Modeling Ground Motion From a Source Physics Experiment (SPE) Underground Explosion
NASA Astrophysics Data System (ADS)
We investigate the excitation and propagation of far-field (epicentral distance larger than 20 m) seismic waves by analyzing and modeling ground motion from an underground chemical explosion recorded during the Source Physics Experiment (SPE), Nevada. The far-field recorded ground motion is characterized by complex features, such as large azimuthal variations in P- and S-wave amplitudes, as well as substantial energy on the tangential component of motion. Shear wave energy is also observed on the tangential component of the near-field motion (epicentral distance smaller than 20 m) suggesting that shear waves were generated at or very near the source. These features become more pronounced as the waves propagate away from the source. We address the shear wave generation during the explosion by modeling ground motion waveforms recorded in the frequency range 0.01-20 Hz, at distances of up to 1 km. We used a physics based approach that combines hydrodynamic modeling of the source with anelastic modeling of wave propagation in order to separate the contributions from the source and near-source wave scattering on shear motion generation. We found that wave propagation scattering caused by the near-source geological environment, including surface topography, contributes to enhancement of shear waves generated from the explosion source. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-06NA25946/ NST11-NCNS-TM-EXP-PD15.
Pitarka, Arben; Mellors, Robert; Rodgers, Arthur; Vorobiev, Oleg; Ezzedine, Souheil; Matzel, Eric; Ford, Sean; Walter, Bill; Antoun, Tarabay; Wagoner, Jeffery; Pasyanos, Mike; Petersson, Anders; Sjogreen, Bjorn
ACME encoded speG abrogates the unique hypersensitivity of Staphylococcus aureus to exogenous polyamines
Polyamines, including spermine (Spm) and spermidine (Spd), are aliphatic cations that are reportedly synthesized by all living organisms. They exert pleiotropic effects on cells and are required for efficient nucleic acid and protein synthesis. Here, we report that the human pathogen Staphylococcus aureus lacks identifiable polyamine biosynthetic genes, and consequently produces no Spm/Spd or their precursor compounds putrescine and agmatine. Moreover, while supplementing defined medium with polyamines generally enhances bacterial growth, Spm and Spd exert bactericidal effects on S. aureus at physiologic concentrations. Small colony variants specifically lacking menaquinone biosynthesis arose after prolonged Spm exposure and exhibited reduced polyamine-sensitivity. However, other respiratory-defective mutants were no less susceptible to Spm implying menaquinone itself rather than general respiration is required for full Spm-toxicity. Polyamine hypersensitivity distinguishes S. aureus from other bacteria and is exhibited by all tested strains save those belonging to the USA-300 group of Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA). We identified one gene within the USA-300-specific Arginine Catabolic Mobile Element (ACME) encoding a Spm/Spd N-acetyltransferase that is necessary and sufficient for polyamine resistance. S. aureus encounters significant polyamine levels during infection, however the acquisition of ACME encoded speG allows USA-300 clones to circumvent polyamine-hypersensitivity, a peculiar trait of S. aureus.
Joshi, Gauri S.; Spontak, Jeffrey S.; Klapper, David G.; Richardson, Anthony R.
Development of methodology for determination of pesticides residue in water by SPE/HPLC/DAD.
To boost crop yield, sugarcane growers are using increasing amounts of pesticides to combat insects and weeds. But residues of these compounds can pollute water resources, such as lakes, rivers and aquifers. The present paper reports the results of a study of water samples from the Feij�o River, which is the source of drinking water for the city of S�o Carlos, S�o Paulo, Brazil. The samples were evaluated for the presence of four leading pesticides--ametryn, atrazine, diuron and fipronil--used on sugarcane, the dominant culture in the region. The samples were obtained from three points along the river: the headwaters, along the middle course of the river and just before the municipal water intake station. The pesticides were extracted from the water samples by solid-phase extraction (SPE) and then analyzed by liquid chromatography with diode array detection (LC-DAD). The analytical method was validated by traditional methods, obtaining recovery values between 90 and 95%, with precision deviations inferior to 2.56%, correlation coefficients above 0.99 and detection and quantification limits varying from 0.02 to 0.05 mg L(-1) and 0.07 to 0.17 mg L(-1), respectively. No presence of residues of the pesticides was detected in the samples, considering the detection limits of the method employed. PMID:23393971
Cappelini, Luciana Teresa Dias; Cordeiro, Daniela; Brondi, Silvia Helena Govoni; Prieto, K�tia Roberta; Vieira, Eny Maria
Apoptosis, autophagy, and more
Cell death has been subdivided into the categories apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). The boundary between Type I and II has never been completely clear and perhaps does not exist due to intrinsic factors among different cell types and the crosstalk among organelles within each type. Apoptosis can begin with autophagy, autophagy can
Richard A. Lockshin; Zahra Zakeri
Ultra sensitive measurement of endogenous epinephrine and norepinephrine in human plasma by semi-automated SPE-LC-MS/MS.
Measurement of endogenous epinephrine (E) and norepinephrine (NE) in human plasma is very challenging due to lower endogenous concentrations as compared with animal plasma. An LC-MS/MS in combination with alumina-based SPE and derivatization procedure was validated for the measurement of E and NE in human plasma with acceptable intra-day and inter-day accuracy and precision. Sample was extracted with semi-automated alumina 96-well solid phase extraction (SPE) cartridge. The resulting eluent was dried and derivatized using d4-acetaldehyde. The analytes were separated on a monolithic C(18) column. Extraction efficiencies were >66% for E and NE. The lower limit of quantitation (LLOQ) was 5.00 pg/mL for E and 20.0 pg/mL for NE. PMID:22483332
Zhang, Guodong; Zhang, Yizhong; Ji, Chengjie; McDonald, Thomas; Walton, Justin; Groeber, Elizabeth A; Steenwyk, Rick C; Lin, Zhaosheng
Development and validation of an SPE HG-AAS method for determination of inorganic arsenic in samples of marine origin.
The present paper describes a novel method for the quantitative determination of inorganic arsenic (iAs) in food and feed of marine origin. The samples were subjected to microwave-assisted extraction using diluted hydrochloric acid and hydrogen peroxide, which solubilised the analytes and oxidised arsenite (As(III)) to arsenate (As(V)). Subsequently, a pH buffering of the sample extract at pH 6 enabled selective elution of As(V) from a strong anion exchange solid-phase extraction (SPE) cartridge. Hydride generation atomic absorption spectrometry (HG-AAS) was applied to quantify the concentration of iAs (sum of As(III) and As(V)) as the total arsenic (As) in the SPE eluate. The results of the in-house validation showed that mean recoveries of 101-104% were achieved for samples spiked with iAs at 0.5, 1.0 and 1.5 mg�kg(-1), respectively. The limit of detection was 0.08 mg kg(-1), and the repeatability (RSD(r)) and intra-laboratory reproducibility (RSD(IR)) were less than 8% and 13%, respectively, for samples containing 0.2 to 1.5 mg kg(-1) iAs. The trueness of the SPE HG-AAS method was verified by confirming results obtained by parallel analysis using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry. It was demonstrated that the two sets of results were not significantly different (P < 0.05). The SPE HG-AAS method was applied to 20 marine food and feed samples, and concentrations of up to 0.14 mg kg(-1) of iAs were detected. PMID:22526669
Rasmussen, Rie R; Hedegaard, Rikke V; Larsen, Erik H; Sloth, Jens J
A New Piece of the Shigella Pathogenicity Puzzle: Spermidine Accumulationby Silencing of the speG Gene
The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a key determinant in the pathogenicity strategy adopted by this microrganism.
Barbagallo, Marialuisa; Di Martino, Maria Letizia; Marcocci, Lucia; Pietrangeli, Paola; De Carolis, Elena; Casalino, Mariassunta; Colonna, Bianca; Prosseda, Gianni
Uncertainty Estimation for the Determination of Ni, Pb and Al in Natural Water Samples by SPE-ICP-OES
In this paper we propose uncertainty estimation for the analytical results we obtained from determination of Ni, Pb and Al by solidphase extraction and inductively coupled plasma optical emission spectrometry (SPE-ICP-OES). The procedure is based on the retention of analytes in the form of 8-hydroxyquinoline (8-HQ) complexes on a mini column of XAD-4 resin and subsequent elution with nitric acid.
A. Ghorbani; M. Mahmoodi Farahani; M. Rabbani; F. Aflaki; Syed Waqifhosain
LC-MS-SPE-NMR for the Isolation and Characterization of neo-Clerodane Diterpenoids from Teucrium luteum subsp. flavovirens
neo-Clerodane diterpenes of plant origin are molecules difficult to monitor due to their nonspecific UV\\/vis absorption. The present work describes for the first time the application of the LC-MS-SPE-NMR technique for the isolation and characterization of three new neo-clerodane diterpenes, 3?-hydroxyteucroxylepin and teuluteumin A and teuluteumin B, from Teucrium luteum subsp. flavovirens, harvested from two different locations
A. Castro; S. I. A. Moco; J. Coll; J. J. M. Vervoort
Identification of the major constituents of Hypericum perforatum by LC\\/SPE\\/NMR and\\/or LC\\/MS
The newly established hyphenated instrumentation of LC\\/DAD\\/SPE\\/NMR and LC\\/UV\\/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one
Evangelos C. Tatsis; Sjef Boeren; Vassiliki Exarchou; Anastassios N. Troganis; Jacques Vervoort; Ioannis P. Gerothanassis
Colorimetric-Solid Phase Extraction Technology for Water Quality Monitoring: Evaluation of C-SPE and Debubbling Methods in Microgravity
NASA Technical Reports Server (NTRS)
Colorimetric-solid phase extraction (C-SPE) is being developed as a method for in-flight monitoring of spacecraft water quality. C-SPE is based on measuring the change in the diffuse reflectance spectrum of indicator disks following exposure to a water sample. Previous microgravity testing has shown that air bubbles suspended in water samples can cause uncertainty in the volume of liquid passed through the disks, leading to errors in the determination of water quality parameter concentrations. We report here the results of a recent series of C-9 microgravity experiments designed to evaluate manual manipulation as a means to collect bubble-free water samples of specified volumes from water sample bags containing up to 47% air. The effectiveness of manual manipulation was verified by comparing the results from C-SPE analyses of silver(I) and iodine performed in-flight using samples collected and debubbled in microgravity to those performed on-ground using bubble-free samples. The ground and flight results showed excellent agreement, demonstrating that manual manipulation is an effective means for collecting bubble-free water samples in microgravity.
Hazen-Bosveld, April; Lipert, Robert J.; Nordling, John; Shih, Chien-Ju; Siperko, Lorraine; Porter, Marc D.; Gazda, Daniel B.; Rutz, Jeff A.; Straub, John E.; Schultz, John R.; McCoy, J. Torin
Determination of abamectin in citrus fruits using SPE combined with dispersive liquid-liquid microextraction and HPLC-UV detection.
A new pretreatment method, SPE combined with dispersive liquid-liquid microextraction, was proposed for the determination of abamectin in citrus fruit samples for the first time. In this method, fruit samples were extracted by ultrasound-assisted extraction followed by SPE. Then, the SPE was used as a disperser solvent in the next dispersive liquid-liquid microextraction step for further purification and enrichment of abamectin. The effects of various parameters on the extraction efficiency of the proposed method were investigated and optimized. Good linearity of abamectin was obtained from 0.005 to 10.0 mg/kg for B1a and from 0.05 to 10.0 mg/kg for B1b with correlation coefficient (r(2)) of 0.998 for B1a and 0.991 for B1b, respectively. The LODs were 0.001 and 0.008 mg/kg (S/N = 3) for B1a and B1b, respectively. The relative recoveries at three spiked levels were ranged from 87 to 96% with the RSD less than 11% (n = 3). The method has been successfully applied to the determination of abamectin in citrus fruit samples. PMID:23913592
Rezaee, Mohammad; Mashayekhi, Hossein Ali; Saleh, Abolfazl; Abdollahzadeh, Yaser; Naeeni, Mohammad Hosein; Fattahi, Nazir
Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity ?SPE device.
We present a novel microfluidic solid-phase extraction (?SPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the ?SPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (?20 fmol) of membrane proteins could be isolated and recovered with ?89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the ?SPE device using computational simulations of different micropillar geometries to guide future device designs. PMID:24487280
Battle, Katrina N; Jackson, Joshua M; Witek, Ma?gorzata A; Hupert, Mateusz L; Hunsucker, Sally A; Armistead, Paul M; Soper, Steven A
Optimization of a pre-MEKC separation SPE procedure for steroid molecules in human urine samples.
Many steroid hormones can be considered as potential biomarkers and their determination in body fluids can create opportunities for the rapid diagnosis of many diseases and disorders of the human body. Most existing methods for the determination of steroids are usually time- and labor-consuming and quite costly. Therefore, the aim of analytical laboratories is to develop a new, relatively low-cost and rapid implementation methodology for their determination in biological samples. Due to the fact that there is little literature data on concentrations of steroid hormones in urine samples, we have made attempts at the electrophoretic determination of these compounds. For this purpose, an extraction procedure for the optimized separation and simultaneous determination of seven steroid hormones in urine samples has been investigated. The isolation of analytes from biological samples was performed by liquid-liquid extraction (LLE) with dichloromethane and compared to solid phase extraction (SPE) with C18 and hydrophilic-lipophilic balance (HLB) columns. To separate all the analytes a micellar electrokinetic capillary chromatography (MECK) technique was employed. For full separation of all the analytes a running buffer (pH 9.2), composed of 10 mM sodium tetraborate decahydrate (borax), 50 mM sodium dodecyl sulfate (SDS), and 10% methanol was selected. The methodology developed in this work for the determination of steroid hormones meets all the requirements of analytical methods. The applicability of the method has been confirmed for the analysis of urine samples collected from volunteers--both men and women (students, amateur bodybuilders, using and not applying steroid doping). The data obtained during this work can be successfully used for further research on the determination of steroid hormones in urine samples. PMID:24232737
Ol?dzka, Ilona; Kowalski, Piotr; Dziomba, Szymon; Szmudanowski, Piotr; B?czek, Tomasz
Geologic Assessment of the Damage Zone from the Second Test at Source Physics Experiment-Nevada (SPE-N)
The National Center for Nuclear Security (NCNS), established by the U.S. Department of Energy, National Nuclear Security Administration, is conducting a series of explosive tests at the Nevada National Security Site (NNSS; formerly the Nevada Test Site) that are designed to increase the understanding of certain basic physical phenomena associated with underground explosions. These tests will aid in developing technologies that might be used to detect underground nuclear explosions in support of verification activities for the Comprehensive Nuclear-Test-Ban Treaty (CTBT). The initial NCNS project is a series of explosive tests, known collectively as the Source Physics Experiment at the NNSS (SPE-N), being conducted in granitic rocks at the Climax stock in northern Yucca Flat. The SPE-N test series is designed to study the generation and propagation of seismic waves. The data will be used to improve the predictive capability of calculational models for detecting and characterizing underground explosions. The first SPE-N test (SPE-N-1) was a �calibration� shot conducted in May 2011, using 100 kilograms (kg) of explosives at the depth of 54.9 meters (m) (180 feet [ft]) in the U-15n source hole. SPE-N-2 was conducted in October 2011, using 1,000 kg of explosives at the depth of 45.7 m (150 ft) in the same source hole. Following the SPE-N-2 test, the core hole U-15n#10 was drilled at an angle from the surface to intercept the SPE-N-2 shot point location to obtain information necessary to characterize the damage zone. The desire was to determine the position of the damage zone near the shot point, at least on the northeast side, where the core hole penetrated it. The three-dimensional shape and symmetry of the damage zone are unknown at this time. Rather than spherical in shape, the dimensions of the damage zone could be influenced by the natural fracture sets in the vicinity. Geologic characterization of the borehole included geophysical logging, a directional survey, and geologic description of the core to document visual evidence of damage. Selected core samples were provided to Sandia National Laboratories (SNL) for laboratory tests (to be reported by SNL). A significant natural fault zone was encountered in the U-15n#10 angle core hole between the drilled depths of 149 and 155 ft (straight-line distance or range station [RS] from the shot point of 7.5 to 5.7 m). However, several of the fractures observed in the U-15n#10 hole are interpreted as having been caused by the explosion. These fractures are characterized by a �fresh,� mechanically broken look, with uncoated and very irregular surfaces. They tend to terminate against natural fractures and have orientations that differ from the previously defined natural fracture sets. The most distant fracture from the shot point that could be interpreted as having been caused by the explosion was seen at approximately RS 10.0 m. No other possibly explosion-induced fractures are apparent above the fault, but are common starting at RS 5.4 m, which is below the fault. It is unknown how the fault zone might have affected the propagation of seismic waves or how the materials in the fault zone (altered granite, breccia, gouge) were affected by the explosion. From RS 3.3 m to the end of the recovered core at RS 1.6 m, some of the core samples are softer and lighter in color, but do not appear to be weathered. It is thought this could be indicative of the presence of distributed microfracturing.
Development of a triple hyphenated HPLC�radical scavenging detection�DAD�SPE�NMR system for the rapid identification of antioxidants in complex plant extracts
A rapid method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining an HPLC with on-line radical scavenging using DPPH as a model radical and an HPLC�DAD�SPE�NMR system. Using this method a commercial rosemary extract was investigated. All major compounds present in the extract were collected on SPE cartridges after their separation.
Audrius Pukalskas; Teris A. van Beek; Pieter de Waard
Connective tissue growth factor mediates transforming growth factor b-induced collagen synthesis: down- regulation by cAMP
Connective tissue growth factor (CTGF) is a cysteine-rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF-b) that acts as a downstream mediator of TGF-b-induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF-b-induced fibroblast collagen synthesis. In vitro studies with normal rat kidney
Nerve Growth Factor, Neuropeptides, and Mast Cells in Ultraviolet-B-Induced Systemic Suppression of Contact Hypersensitivity Responses in Mice
The induction of systemic immunosuppression following ultraviolet B radiation exposure has been linked with the release of inflammatory and immunomodulatory mediators by cells of the epidermis and dermis. Nerve growth factor has not previously been linked with ultraviolet-B-induced immunosuppressive effects. Nerve growth factor antibodies abrogated ultraviolet-B-induced systemic suppression of contact hypersensitivity responses in BALB\\/C mice. Subcutaneous injection of nerve growth
Scott L. Townley; Michele A. Grimbaldeston; Ian Ferguson; Robert A. Rush; Shu-Hua Zhang; Xin-Fu Zhou; James M. Conner; John J. Finlay-Jones; Prue H. Hart
Translation control in apoptosis.
Regulation of protein synthesis, although known for many decades, has only recently begun to be recognized as a critical control mechanism for the maintenance of cellular homeostasis and cellular stress response. One of the key advantages of translational control is the ability of cells to rapidly reprogram the protein output in response to internal or external triggers. This is particularly important during cellular response to stress that may lead to apoptosis by providing cells with a fine tuning mechanism that tips the balance between cell survival or apoptosis. In the following review we highlight several distinct mechanisms of translation control and provide specific examples of translational control during apoptosis. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later". PMID:23070007
Liwak, U; Faye, M D; Holcik, M
\\u000a Apoptosis plays an important role in organ development and repair, however, it is becoming clear that it may also be involved\\u000a in a number of different diseases. In recent years, there has been growing evidence to implicate apoptosis in kidney development\\u000a [1, 2] and various renal disorders including immune-mediated renal disease, polycystic kidney disease and acute tubular necrosis.\\u000a Many of
Victoria Y. Wong; Shujath M. Ali; David P. Brooks
There is increasing evidence that some neuronal death after brain ischaemia is mediated by the action of cysteine-requiring aspartate-directed proteases (caspases), the proteases responsible for apoptosis in mammals, although this form of neuronal death is not always accompanied by the morphological changes that are typical of apoptosis in other tissues. Caspase-mediated neuronal death is more extensive after transient than permanent
Seth Love
Apoptosis and cancer chemotherapy.
Cytotoxic drugs currently remain as the basis for the chemotherapy of metastatic cancer. Why they fail to kill sufficient tumour cells in the major human solid cancers, such as the carcinomas, is suggested in this review to be due to the inherent inability of these cells to engage apoptosis after drug-induced damage. As a paradigm for drug resistant cancers, the resistance of bladder carcinoma cell lines to DNA damaging drugs is described here in terms of their response to the topoisomerase II poison etoposide. 60%-70% of bladder carcinomas have mutant p53; this can prevent the detection of and response to DNA damage. In vitro studies with a bladder carcinoma cell line containing a wild type p53 showed that it underwent a G1 checkpoint after etoposide, potentially allowing DNA damage repair, as well as apoptosis. In lines with mutant or non-functional p53 there is no checkpoint and no apoptosis. All lines showed constitutive expression of bcl-2 and bcl-XL (the suppressors of apoptosis) with low and non-inducible levels of bax (a promoter of apoptosis). Taken together, this menu of gene expression is more favourable to survival than apoptosis after the imposition of drug-induced DNA damage and may contribute to their inherent drug resistance. PMID:8950479
Chresta, C M; Arriola, E L; Hickman, J A
Ascorbic acid synergistically potentiates phloxine b-induced photocytotoxicity in human acute promyelocytic leukemia cells.
Ascorbic acid (AsA) is known as an antioxidant but concomitantly possesses a pro-oxidant property. Because the impact of AsA on photodynamic therapy response is unclear, we investigated the effect of AsA on photocytotoxicity induced by phloxine B in human acute promyelocytic leukemia HL-60 cells. AsA synergistically enhanced phloxine B-induced photocytotoxic effects, including inhibition of cell proliferation, DNA ladder formation, and caspase-3 activation, whereas AsA itself showed no photocytotoxicity. AsA also enhanced the consumption of the reduced glutathione level compared with the cells treated with phloxine B alone under the light condition. Combination of AsA with phloxine B under the light condition enhanced the phosphorylation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK). These effects were completely cancelled by catalase. These results suggest that AsA synergistically enhances phloxine B-induced photocytotoxicity, possibly through the extracellular oxidative stress-dependent MAPK pathway activation. PMID:24488945
Qi, Hang; Wu, Qian; Abe, Naomi; Saiki, Shunya; Zhu, Beiwei; Murata, Yoshiyuki; Nakamura, Yoshimasa
Cassia tora Linn Cream Inhibits Ultraviolet-B-Induced Psoriasis in Rats
The aim of present study was to determine the antipsoriatic activity of newly formulated O/W creams of methanolic extract of Cassia tora L. leaves by using ultraviolet-B-induced psoriasis in rat. The plant Cassia tora L. is traditionally claimed to be useful in the treatment of a number of skin diseases. However, there are no established scientific reports for its antipsoriatic activity. Methanolic Cassia tora L. leaves extract was used to prepare various concentrations of O/W creams and tested for acute dermal toxicity study. The different O/W creams showed good physical characteristics and passed the sensitivity, irritation, grittiness and bleeding test. The results of acute dermal toxicity showed that the creams were safe up to the dose of 2000?mg/kg. In case of psoriasis model, histopathological analysis revealed that there were absence of Munro's microabscess, elongation of rete ridges, and capillary loop dilation in the section in Test 2 (0.1%) and standard group. O/W creams and methanolic extract of Cassia tora L. leaves exhibited significant reduction in percentage of relative epidermal thickness and spleen index as compared to positive control. We concluded that topical O/W creams and crude extract containing methanolic extract of Cassia tora L. leaves have potent antipsoriatic activity in ultraviolet-B-induced psoriasis in rat.
Singhal, Manmohan; Kansara, Niraj
Apoptosis is a form of cell death that has been described as distinct from necrotic cell death. It is believed to be genetically programmed and occurs as a physiologic process in various organ systems of body. Although it has been tacitly believed that apoptosis does not occur in the terminally differentiated adult heart muscle cells, studies in endomyocardial biopsies from patients with dilated and ischemic cardiomyopathy and in explanted hearts from patients with end-stage heart failure undergoing cardiac transplantation have demonstrated histochemical evidence of apoptosis. It has been proposed that ventricular dilatation and neurohormonal activation during heart failure lead to upregulation of transcription factors, induce myocyte hypertrophy, and prepare the cell for entry into the cell cycle. However, terminally differentiated myocytes cannot divide, and failing to divide they undergo apoptosis. Initiation of apoptosis is associated with activation of upstream cascade, including the release of cytochrome c from mitochondria to cytoplasm and the processing of proteolytic caspases. The activation of caspases leads to fragmentation of various cytoplasmic proteins, including contractile proteins. However, the nuclear fragmentation and condensation is completed only rarely. It is hypothesized that the release of cytochrome c from mitochondria and cytoplasmic protein loss in a living heart muscle cell should lead to systolic dysfunction. PMID:10952426
Narula, J; Kolodgie, F D; Virmani, R
Clostridium difficile toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex.
Clostridium difficile infection (CDI) is a leading cause of health care-associated diarrhea and has increased in incidence and severity over the last decade. Pathogenesis is mediated by two toxins, TcdA and TcdB, which cause fluid secretion, inflammation, and necrosis of the colonic mucosa. TcdB is a potent cytotoxin capable of inducing enzyme-independent necrosis in both cells and tissue. In this study, we show that TcdB-induced cell death depends on assembly of the host epithelial cell NADPH oxidase (NOX) complex and the production of reactive oxygen species (ROS). Treating cells with siRNAs directed against key components of the NOX complex, chemical inhibitors of NOX function, or molecules that scavenge superoxide or ROS confers protection against toxin challenge. To test the hypothesis that chemical inhibition of TcdB-induced cytotoxicity can protect against TcdB-induced tissue damage, we treated colonic explants with diphenyleneiodonium (DPI), a flavoenzyme inhibitor, or N-acetylcysteine (NAC), an antioxidant. TcdB-induced ROS production in colonic tissue was inhibited with DPI, and both DPI and NAC conferred protection against TcdB-induced tissue damage. The efficacy of DPI and NAC provides proof of concept that chemical attenuation of ROS could serve as a viable strategy for protecting the colonic mucosa of patients with CDI. PMID:24167244
Farrow, Melissa A; Chumbler, Nicole M; Lapierre, Lynne A; Franklin, Jeffrey L; Rutherford, Stacey A; Goldenring, James R; Lacy, D Borden
Spaceflight Associated Apoptosis
NASA Technical Reports Server (NTRS)
Lymphoid tissues have been shown to atrophy in rats flown on Russian spaceflights. Histological examination indicated evidence for cell degradation. Lymphoid tissues from rats flown on Spacelab Life Sciences-2 mission were analyzed for apoptosis by evidence of fragmented lymphocytes, which could be engulfed by macrophages, or DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Apoptosis was not detected in the thymus and spleen collected inflight or from the synchronous ground rats but was detected in the thymus, spleen and inguinal lymph node of the flight animals on recovery. These results indicate that the apoptosis observed in the lymphatic tissues of the rats on recovery could have been induced by the gravitational stress of reentry, corroborating the findings from the early space-flight observations.
Ichiki, Albert T.; Gibson, Linda A.; Allebban, Zuhair
Pseudolaric Acid B Induced Cell Cycle Arrest, Autophagy and Senescence in Murine Fibrosarcoma L929 Cell
Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis. Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-?-galactosidase assay was used to detect senescence. Protein expression was examined by western blot. Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 ?mol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence. Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC.
hua Yu, Jing; yu Liu, Chun; bin Zheng, Gui; Zhang, Li Ying; hui Yan, Ming; yan Zhang, Wen; ying Meng, Xian; fang Yu, Xiao
Trace explosive detection in aqueous samples by solid-phase extraction ion mobility spectrometry (SPE-IMS).
Law enforcement agencies use ion mobility spectrometers for the detection of explosives, drugs of abuse, and chemical warfare agents. Ion mobility spectrometry (IMS) has the advantages of short analysis times, detections in the parts per billion concentrations, and high sensitivity. On-site environmental analysis of explosives or explosive residues in water is possible with ion mobility spectrometers. Unfortunately, the direct analysis of low levels of explosives in water is difficult. Extraction provides a method for pre-concentrating the analytes and removing interferents. Coupling solid-phase extraction (SPE) with IMS is useful for the identification of trace amounts of explosives in water. Commercially available SPE disks were used. After extraction, the sample disk is inserted into the ion mobility spectrometer, where the analytes are thermally desorbed from the disk. Concentrations as low as one part per trillion were detected with a Barringer Ionscan 350. An external computer and acquisition software (LabVIEW, National Instruments) were used to collect data. SIMPLISMA (SIMPLe-to-use-Interactive Self-modeling Mixture Analysis) was applied to the data to resolve features that vary with respect to time. PMID:14610961
Buxton, Tricia L; Harrington, Peter de B
Enrichment of steroid hormones in water with porous and hydrophobic polymer-based SPE followed by HPLC-UV determination.
There have been great concerns about the persistence of steroid hormones in surface water. Since the concentrations of these compounds in water samples are usually at a trace level, the efficient enrichment of steroid hormones is vital for further analysis. In this work, a porous and hydrophobic polymer was synthesized and characterized. The composition of solvent used as porogen in the synthetic process was shown to have an effect on the morphology of the polymer, which was successfully used as an SPE sorbent for simultaneously enriching steroid hormones in surface water samples. The recoveries of the steroid hormones on the custom-made polymer ranged from 93.4 to 106.2%, whereas those on commercialized ENVI-18, LC-18, and Oasis HLB ranged from 54.8 to 104.9, 66 to 93.6, and 77.2 to 106%, respectively. Five types of steroid hormones were simultaneously measured using HPLC-UV after they were enriched by the custom-made sorbent. Based on these findings, the SPE-HPLC method was developed. The LODs of this method for estriol, estradiol, estrone, androstenedione, progesterone were 0.07, 0.43, 0.61, 0.27, and 0.42 ?g/L, respectively, while precision and reproducibility RSDs were <6.40 and 7.49%, respectively. PMID:23956108
Hu, Yinfen; Zhang, Man; Tong, Changlun; Wu, Jianmin; Liu, Weiping
From retrospective assessment to prospective decisions in natural product isolation: HPLC-SPE-NMR analysis of Carthamus oxyacantha.
An extract of Carthamus oxyacantha (wild safflower) was investigated using two approaches: a traditional, nontarget fractionation by VLC and HPLC, and the hyphenated technique HPLC-PDA-HRMS-SPE-NMR followed by targeted isolation of selected constituents for inclusion in a screening library of pure natural products. While the nontarget fractionation involved considerable time spent on pursuing fractions containing well-known or undesired compounds, the hyphenated analysis was considerably faster and required less solvent and other consumables. The results were used to design and execute an optimized, HPLC-HRMS-guided, targeted isolation scheme aiming exclusively at a series of identified spiro compounds. Thus, HPLC-PDA-HRMS-SPE-NMR is a dereplication technique of choice, allowing economical acquisition of comprehensive data about compounds in crude extracts, which can be used for rational, prospective decisions about further isolation efforts. A total of 15 compounds were identified in the extract. Six spiro compounds, of which four have not previously been characterized, and tracheloside (a lignin glucoside) are presented with assigned 1H and 13C chemical shifts. PMID:22060189
Johansen, Kenneth T; Wubshet, Sileshi G; Nyberg, Nils T; Jaroszewski, Jerzy W
Assessment of constituents in Allium by multivariate data analysis, high-resolution ?-glucosidase inhibition assay and HPLC-SPE-NMR.
Bulbs and leaves of 35 Allium species and cultivars bought or collected in 2010-2012 were investigated with multivariate data analysis, high-resolution ?-glucosidase inhibition assays and HPLC-HRMS-SPE-NMR with the aim of exploring the potential of Allium as a future functional food for management of type 2 diabetes. It was found that 30 out of 106 crude extracts showed more than 80% inhibition of the ?-glucosidase enzyme at a concentration of 40mg/mL (dry sample) or 0.4g/mL (fresh sample). High-resolution ?-glucosidase biochromatograms of these extracts allowed fast identification of three analytes with ?-glucosidase inhibitory activity, and subsequent HPLC-HRMS-SPE-NMR experiments allowed identification of these as N-p-coumaroyloctopamine, N-p-coumaroyltyramine, and quercetin. The distribution of these three compounds was mapped for all samples by HPLC-ESI-HRMS. Unsupervised principal component analysis of samples from 2012 indicated that a major difference between fresh material and dried material is the increased amount of quercetin, a known ?-glucosidase inhibitor. PMID:24837940
Schmidt, Jeppe S; Nyberg, Nils T; Staerk, Dan
Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a Model for In Vivo Gene Expression�
For a pathogen such as Streptococcus pyogenes, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analyses of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we determined that the gene encoding the SpeB cysteine protease is up-regulated over the course of infection in a murine soft-tissue model. Conditions were identified, including growth phase, acidic pH, and an NaCl concentration of <0.1 M, that were required for expression of speB in vitro. Analysis of global expression profiles in response to these conditions in vitro identified a set of coregulated genes whose expression patterns showed a significant correlation with that of speB when examined during infection of murine soft tissues. This analysis revealed that a culture medium that promotes high levels of SpeB expression in vitro produced an expression profile that showed significant correlation to the profile observed in vivo. Taken together, these studies establish culture conditions that mimic in vivo expression patterns; that growth phase, pH, and NaCl may mimic relevant cues sensed by S. pyogenes during infection; and that identification of other environmental cues that alter expression of speB in vitro may provide insight into the signals that direct global patterns of gene expression in vivo.
Loughman, Jennifer A.; Caparon, Michael
Identification of major and minor constituents of Harpagophytum procumbens (Devil's claw) using HPLC-SPE-NMR and HPLC-ESIMS/APCIMS.
The HPLC-SPE-NMR technique, supported by HPLC-MS measurements, was used to determine structures of major as well as some minor constituents of ethanol and petroleum ether extracts of Harpagophytum procumbens (Devil's claw) roots. This method was also shown to be applicable for rapid and precise on-line identification of secondary metabolites present in commercial herbal products of H. procumbens. A total of 15 compounds (1-14 and 17) were identified from the ethanol and petroleum ether extracts, including a novel Diels-Alder dimer 14. Optimization of the HPLC-SPE-NMR experiments included quantitative (1)H NMR measurements, determination of trapping and elution efficiency, effect of multiple trapping of analytes, use of various deuterated solvents for SPE cartridge elution, and effect of post-column dilution ratio of eluent with water. Linear accumulation of apolar and relatively polar analytes was demonstrated for at least 8-10 repeated trappings, resulting in greatly improved signal-to-noise ratios in NMR spectra and reduced acquisition times. Thus, the HPLC-SPE-NMR technique provides an efficient means of identification of multiple components of crude extracts. By allowing on-line generation of high-quality 2D NMR data without traditional purification of extract components, the HPLC-SPE-NMR methodology represents a paradigm shift in natural products research with respect to structure elucidation. PMID:16989520
Clarkson, Cailean; Staerk, Dan; Hansen, Steen Honor�; Smith, Peter J; Jaroszewski, Jerzy W
Structural basis for the recognition of superantigen streptococcal pyrogenic exotoxin A (SpeA1) by MHC class II molecules and T-cell receptors.
Streptococcal pyrogenic exotoxin A (SpeA) is a superantigen produced by Streptococcus pyogenes and is associated with severe infections characterized by rash, hypotension, multiorgan failure and a high mortality rate. In this study, an allelic form of this toxin, SpeA1, was crystallized with four molecules in the crystallographic asymmetric unit and its crystal structure was determined at 2.6 A resolution. The crystallographic R-factor was 19.4% (33 497 reflections) for 7031 protein atoms and 88 water molecules. The overall structure of SpeA1 is considerably similar to that of other prototype microbial superantigens, either of staphylococcal or streptococcal origin, but has greatest similarity to staphylococcal enterotoxin C (SEC). Based on structural and mutagenesis data, we have mapped several important residues on the toxin molecule, which are involved in the recognition of major histocompatibility complex (MHC) class II molecules and T-cell receptors. Also, the toxin appears to possess a potential zinc-binding site which may have implications in binding to particular MHC class II molecules. Finally, we propose models for SpeA1-MHC class II and SpeA1-T-cell receptor association and the relevance of this phenomenon to the superantigenic action of this toxin is considered.
Papageorgiou, A C; Collins, C M; Gutman, D M; Kline, J B; O'Brien, S M; Tranter, H S; Acharya, K R
NF-?B inducing kinase: a key regulator in the immune system and in cancer.
NF-?B inducing kinase (NIK) is a kinase that activates the canonical and non-canonical NF-?B pathways to control transcriptional expression of certain proteins such as cytokines, chemokines and NF-?B signaling molecules. Many advances have been made in understanding the molecular mechanisms by which the stability of NIK is regulated to affect downstream signaling. Genetic mouse models suggest that NIK plays an essential role in the regulation of the immune system as well as in the bone microenvironment. Increasing evidence links NIK to the tumorigenesis of hematological cancers, such as multiple myeloma, and solid tumors, such as pancreatic carcinoma and melanoma. Understanding the mechanism by which NIK is de-regulated will potentially provide therapeutic options for certain diseases such as autoimmunity and cancer. PMID:20685151
Thu, Yee Mon; Richmond, Ann
Rhodamine B induces long nucleoplasmic bridges and other nuclear anomalies in Allium cepa root tip cells.
The cytogenetic toxicity of rhodamine B on root tip cells of Allium cepa was investigated. A. cepa were cultured in water (negative control), 10 ppm methyl methanesulfonate (positive control), and three concentrations of rhodamine B (200, 100, and 50 ppm) for 7 days. Rhodamine B inhibited mitotic activity; increased nuclear anomalies, including micronuclei, nuclear buds, and bridged nuclei; and induced oxidative stress in A. cepa root tissues. Furthermore, a substantial amount of long nucleoplasmic bridges were entangled together, and some nuclei were simultaneously linked to several other nuclei and to nuclear buds with nucleoplasmic bridges in rhodamine B-treated cells. In conclusion, rhodamine B induced cytogenetic effects in A. cepa root tip cells, which suggests that the A. cepa root is an ideal model system for detecting cellular interactions. PMID:24234815
Tan, Dehong; Bai, Bing; Jiang, Donghua; Shi, Lin; Cheng, Shunchang; Tao, Dongbing; Ji, Shujuan
Keratocyte Apoptosis after Corneal Surgery
PURPOSE. Programmed cell death (apoptosis) is the controlled death of cells that occurs with minimal collateral damage to surrounding cells or tissue during development, homeostasis, and wound healing. The authors hypothesize that keratocyte apoptosis is an initiating factor in the wound-healing response after refractive surgical procedures. To evaluate the effects of different corneal manipulations, keratocyte apoptosis was examined qualitatively and
Marco C Helena; Fiona Baerveldt; Woo-Jung Kim; Steven E. Wilson
Apoptosis: Genetically Programmed Cell Death
Extensively and successfully studied problems of programmed cell death are considered. Recent evidence on apoptosis genes is presented, including the bcl-2 family and other genes with similar functions. A scheme of pathways of the main apoptosis mechanism is constructed. Examples of associations of apoptosis and diseases are presented in a special section.
L. S. Popov; L. I. Korochkin
A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C6-Ceramide
Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [?-32P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C6-ceramide) as substrate. Its Km value (4??M) was comparable to that of N-hexanoyl-sphingenine (C6-ceramide). The produced fluorescent NBD-C6-ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126; IC50 4??M) and ceramide analogues (e.g., fenretinide, AMG-9810; IC50 1.1??M) affect CERK in vitro.
Van Overloop, Helena; Van der Hoeven, Gerd; Van Veldhoven, Paul P.
New SPE loading material for affinity-based separation of cyclodextrins from drug:CD complexes in order to overcome Beer's law deviations.
Molecular inclusion of guest molecules within CDs is known to alter guest molecule spectrophotometric absorptivity, making their determination, based on spectrophotometric data, inaccurate. Therefore specific analytical methods capable of quantifying the drugs as free molecules must be developed and validated. SPE was selected to simplify sample and avoid more time-consuming alternatives. A new solid phase was synthesized and characterized by infrared spectrometry, differential scanning calorimetry and elemental analysis. The competitive complexation of adamantane groups immobilized on the silica substrate facilitates drug:CD complex dissociation and elimination of CD from samples. The drug molecules, now free from CD, can be easily analysed by an already available HPLC method. This new SPE loading material was employed in the determination of ketoprofen in its CD complex as a representative example of the utility of this novel material. The calculated analytical errors were reduced from a maximum of 20.79% (without SPE) to a minimum of 3.99%. PMID:19777455
Rozou, Stavroula; Raftopoulos, Panayiotis; Hatziantoniou, Sophia; Antoniadou-Vyza, Ekaterini
Spermatogenesis-defective (spe) Mutants of the Nematode Caenorhabditis elegans Provide Clues to Solve the Puzzle of Male Germline Functions during Reproduction
In most species, each sex produces gametes, usually either sperm or oocytes, from its germline during gametogenesis. The sperm and oocyte subsequently fuse together during fertilization to create the next generation. This review focuses on spermatogenesis and the roles of sperm during fertilization in the nematode Caenorhabditis elegans, where suitable mutants are readily obtained. So far 186 mutants defective in the C. elegans male germline functions have been isolated, and many of these mutations are alleles for one of the ~60 spermatogenesis-defective (spe) genes. Many cloned spe genes are expressed specifically in the male germline, where they play roles during spermatogenesis (spermatid production), spermiogenesis (spermatid activation into spermatozoa), and/or fertilization. Moreover, several spe genes are orthologs of mammalian genes, suggesting that the reproductive processes of the C. elegans and the mammalian male germlines might share common pathways at the molecular level.
Nishimura, Hitoshi; L'Hernault, Steven W.
Apoptosis in neurodegenerative disorders
Neuronal death underlies the symptoms of many human neurological disorders, including Alzheimer's, Parkinson's and Huntington's diseases, stroke, and amyotrophic lateral sclerosis. The identification of specific genetic and environmental factors responsible for these diseases has bolstered evidence for a shared pathway of neuronal death � apoptosis � involving oxidative stress, perturbed calcium homeostasis, mitochondrial dysfunction and activation of cysteine proteases called
Mark P. Mattson
Enhanced knowledge of the significance of apoptosis in hepatic disease has justified much of the research reviewed in this chapter. Our improved knowledge of the process itself will lend itself to the development of new treatment approaches where therapeutic strategies can be aimed at more specific targets. New therapeutic routes are already being considered in hepatocyte culture and in hepatic
N. T. MUKWENA; Mohamed Al-Rubeai
National Technical Information Service (NTIS)
Reaper is a central regulator of apoptosis in the fly, Drosophila melanogaster. At the start of this proposal our laboratory identified what was believed to be a pro-apoptotic human homolog of Reaper. This was of extreme interest as no true Reaper homolog...
J. Perry
NGFI-B targets mitochondria and induces cardiomyocyte apoptosis in restraint-stressed rats by mediating energy metabolism disorder.
NGFI-B/Nur77/TR3, originally identified as an immediate-early gene rapidly induced by serum and growth factors, is a member of the steroid hormone nuclear receptor superfamily with no identified endogenous ligand. NGFI-B induces apoptosis in a number of cell lineages exposed to proapoptotic stimuli by directly targeting the mitochondria, inducing cytochrome c release. The present study was designed to determine the role of NGFI-B in cardiomyocytes of restraint-stressed rats. The NGFI-B content was increased in mitochondria and reduced in plasma as apoptosis increased. Analysis showed that NGFI-B induces cardiomyocyte apoptosis in restraint-stressed rats by mediating mitochondrial energy metabolism disorder. Several novel mitochondrial proteins, which correlate with NGFI-B, were reported in cardiomyocyte apoptosis of restraint-stressed rats. Five proteins associated with NGFI-B participate directly in mitochondrial energy metabolism. Studies of mitochondrial respiratory efficiency and ATP synthase activity strongly support the findings. These results provide significant information for comprehensively understanding the cellular mechanism of cardiovascular diseases. PMID:19412742
Wang, XinXing; Liu, XiaoHua; Kong, RuiRui; Zhan, Rui; Wang, XiaoMing; Leng, Xue; Gong, JingBo; Duan, Meng; Wang, LiQun; Wu, Lei; Qian, LingJia
Induction of T-cell apoptosis by human herpesvirus 6.
The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection.
Inoue, Y; Yasukawa, M; Fujita, S
Induction of T-cell apoptosis by human herpesvirus 6.
The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection. PMID:9094650
Inoue, Y; Yasukawa, M; Fujita, S
Inhibition of NF-kappa B activity induces apoptosis in murine hepatocytes.
Recently we have demonstrated that inhibition of the nuclear factor (NF)-kappa B/Rel family of transcription factors induces apoptosis of B cells. Interestingly, mice lacking the relA gene encoding the p65 subunit of NF-kappa B exhibit embryonic lethality at days 15 to 16 of gestation, accompanied by massive destruction of liver via apoptosis. To determine whether p65 protein plays a direct role in hepatocyte survival, we employed a nontransformed murine hepatocyte (NMH) cell line, which maintains to a high degree the differentiated hepatocyte phenotype. Exponentially growing NMH cells were found to possess a constitutive level of functional classical (p50/p65) NF-kappa B as assayed by electrophoretic mobility shift analysis, antibody supershift, and transient transfection assays. Treatment of NMH cells with the proteasome inhibitor lactacystin, which prevents degradation of the NF-kappa B inhibitor proteins I kappa B, induced apoptosis. Direct inhibition of the endogenous NF-kappa B activity by microinjection of NMH cells with purified specific inhibitor I kappa B-alpha-glutathione-S-transferase fusion protein or an antibody against p65 protein induced apoptosis. These findings suggest that expression of NF-kappa B/Rel activity in murine hepatocytes acts directly to promote survival of these cells and suggest that apoptosis observed in hepatocytes of mice lacking relA is a direct effect of p65 deficiency. Images Figure 1 Figure 2
Bellas, R. E.; FitzGerald, M. J.; Fausto, N.; Sonenshein, G. E.
Development of an HPLC method for the determination of ranitidine and cimetidine in human plasma following SPE.
A selective, sensitive and accurate high-performance liquid chromatographic method has been developed, validated and applied for the determination of ranitidine and cimetidine in plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of investigated drugs were investigated. Sample preparation was carried out by adding an internal standard, famotidine, and the clean-up procedure was accomplished using solid-phase extraction (SPE). This method uses ultraviolet detection, the separation used a Lichrocart Lichrospher 60 RP-select B column and the mobile phase consisted of 0.2% triethylamine (TEA), 0.04 mol l(-1) KH2PO4 at pH 6.8 and 14% acetonitrile. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma. The method has been implemented to monitor ranitidine levels in clinical samples. PMID:12972081
Zendelovska, Dragica; Stafilov, Trajce
Mortalin, apoptosis, and neurodegeneration.
Mortalin is a highly conserved heat-shock chaperone usually found in multiple subcellular locations. It has several binding partners and has been implicated in various functions ranging from stress response, control of cell proliferation, and inhibition/prevention of apoptosis. The activity of this protein involves different structural and functional mechanisms, and minor alterations in its expression level may lead to serious biological consequences, including neurodegeneration. In this article we review the most current data associated with mortalin's binding partners and how these protein-protein interactions may be implicated in apoptosis and neurodegeneration. A complete understanding of the molecular pathways in which mortalin is involved is important for the development of therapeutic strategies for cancer and neurodegenerative diseases. PMID:24970131
Londono, Carolina; Osorio, Cristina; Gama, Vivian; Alzate, Oscar
Mortalin, Apoptosis, and Neurodegeneration
Mortalin is a highly conserved heat-shock chaperone usually found in multiple subcellular locations. It has several binding partners and has been implicated in various functions ranging from stress response, control of cell proliferation, and inhibition/prevention of apoptosis. The activity of this protein involves different structural and functional mechanisms, and minor alterations in its expression level may lead to serious biological consequences, including neurodegeneration. In this article we review the most current data associated with mortalin�s binding partners and how these protein-protein interactions may be implicated in apoptosis and neurodegeneration. A complete understanding of the molecular pathways in which mortalin is involved is important for the development of therapeutic strategies for cancer and neurodegenerative diseases.
Londono, Carolina; Osorio, Cristina; Gama, Vivian; Alzate, Oscar
Mitochondrial alterations in apoptosis.
Besides their conventional role as energy suppliers for the cell, mitochondria in vertebrates are active regulators of apoptosis. They release apoptotic factors from the intermembrane space into the cytosol through a mechanism that involves the Bcl-2 protein family, mediating permeabilization of the outer mitochondrial membrane. Associated with this event, a number of additional changes affect mitochondria during apoptosis. They include loss of important mitochondrial functions, such as the ability to maintain calcium homeostasis and to generate ATP, as well as mitochondrial fragmentation and cristae remodeling. Moreover, the lipidic component of mitochondrial membranes undergoes important alterations in composition and distribution, which have turned out to be relevant regulatory events for the proteins involved in apoptotic mitochondrial damage. PMID:24732580
Cosentino, Katia; Garc�a-S�ez, Ana J
NSDL National Science Digital Library
A variety of key events in apoptosis focus on mitochondria, including the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins. The different signals that converge on mitochondria to trigger or inhibit these events and their downstream effects delineate several major pathways in physiological cell death.
Douglas Green (La Jolla Institute for Allergy and Immunology;); John Reed (Burnham Institute;)
BMP-7 represses albumin-induced chemokine synthesis in kidney tubular epithelial cells through destabilization of NF-?B-inducing kinase.
Protein overload activates proximal tubule epithelial cells (PTECs) to release chemokines. Bone morphogenetic protein-7 (BMP-7) reduces infiltrating cells and tissue damage in acute and chronic renal injuries. The present study examines the inhibitory effect and related molecular mechanism of BMP-7 on chemokine and adhesion molecule synthesis by PTECs activated with human serum albumin (HSA). The expression profiles of chemokines and adhesion molecules in cultured human PTECs were screened by PCR array. Expression of CXCL1, CXCL2 and vascular cell adhesion protein 1 (VCAM-1) by PTECs was significantly upregulated by HSA and reduced by BMP-7. HSA activated both the canonical and noncanonical nuclear factor (NF)-?B pathways in PTECs, as indicated by the increased nuclear translocation of NF-?B p50 and p52 subunits. The nuclear translocation of NF-?B p52 was completely abrogated by BMP-7, whereas NF-?B p50 activation was only partially repressed. BMP-7 increased the expression of cellular inhibitor of apoptosis 1 (cIAP1), tumor necrosis factor receptor-associated factor (TRAF)2 and TRAF3, but not of NF-?B-inducing kinase (NIK) that was significantly upregulated by HSA. Silencing NIK recapitulated the partial inhibitory effect on HSA-induced chemokine synthesis by BMP-7. Complete abolishment of the chemokine synthesis was only achieved by including additional blockade of the NF-?B p65 translocation on top of NIK silencing. Our data suggest that BMP-7 represses the NIK-dependent chemokine synthesis in PTECs activated with HSA through blocking the noncanonical NF-?B pathway and partially interfering with the canonical NF-?B pathway. PMID:24418819
Lim, Ai Ing; Chan, Loretta Y Y; Tang, Sydney C W; Yiu, Wai Han; Li, Ruixi; Lai, Kar Neng; Leung, Joseph C K
Brevenal Inhibits Pacific Ciguatoxin-1B-Induced Neurosecretion from Bovine Chromaffin Cells
Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and ?-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera.
Mattei, Cesar; Alvarez, Martha; Benoit, Evelyne; Bourdelais, Andrea J.; Lewis, Richard J.; Baden, Daniel G.; Molgo, Jordi; Meunier, Frederic A.
Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop
Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 ? resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC50 values for trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.
Gonzalez-Paez, Gonzalo E.; Wolan, Dennis W.
Effect of SPE-like proton or photon radiation on the kinetics of mouse peripheral blood cells and radiation biological effectiveness determinations.
Exploration missions outside low-Earth orbit are being planned; therefore, it is critical to understand the risk astronauts would be exposed to in the space environment, especially during extravehicular activities (EVAs). Reductions in white blood cell (WBC) numbers can occur as a result of exposure to solar particle event (SPE) radiation. The aim of the present study was to determine the duration of the effects on blood cell numbers from exposure to a single whole-body dose of SPE-like proton radiation or photon radiation as well as to determine the radiation biological effectiveness (RBE) values at those times when radiation exposure causes blood cell numbers to experience the most critical effects when using mice as a model. Our results indicate that both types of radiation cause significant reductions in the numbers of all blood cell types at different times post-irradiation. The RBE values were not significantly different from 1.0. These results indicate that the risk estimations for astronauts from exposure of mice to SPE-like proton radiation are comparable to those previously made for doses of standard reference radiations, suggesting that countermeasures should be developed for the decreases in blood cell counts observed following the exposure of mice to SPE radiation. PMID:23980767
Romero-Weaver, A L; Wan, X S; Diffenderfer, E S; Lin, L; Kennedy, A R
Effect of SPE-like Proton or Photon Radiation on the Kinetics of Mouse Peripheral Blood Cells and Radiation Biological Effectiveness Determinations
Abstract Exploration missions outside low-Earth orbit are being planned; therefore, it is critical to understand the risk astronauts would be exposed to in the space environment, especially during extravehicular activities (EVAs). Reductions in white blood cell (WBC) numbers can occur as a result of exposure to solar particle event (SPE) radiation. The aim of the present study was to determine the duration of the effects on blood cell numbers from exposure to a single whole-body dose of SPE-like proton radiation or photon radiation as well as to determine the radiation biological effectiveness (RBE) values at those times when radiation exposure causes blood cell numbers to experience the most critical effects when using mice as a model. Our results indicate that both types of radiation cause significant reductions in the numbers of all blood cell types at different times post-irradiation. The RBE values were not significantly different from 1.0. These results indicate that the risk estimations for astronauts from exposure of mice to SPE-like proton radiation are comparable to those previously made for doses of standard reference radiations, suggesting that countermeasures should be developed for the decreases in blood cell counts observed following the exposure of mice to SPE radiation. Key Words: Proton radiation�Gamma radiation�Blood cell counts�Solar particle event. Astrobiology 13, 570�577.
Romero-Weaver, A.L.; Wan, X.S.; Diffenderfer, E.S.; Lin, L.
Determination of crystal violet in seawater and seafood samples through off-line molecularly imprinted SPE followed by HPLC with diode-array detection.
A highly selective sample cleanup procedure combined with molecularly imprinted SPE was developed for the isolation of crystal violet from seawater and seafood samples. The molecularly imprinted polymer was prepared using crystal violet as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker. The crystal violet-imprinted polymer was used as the selective sorbent for the SPE of crystal violet. An off-line molecularly imprinted SPE method followed by HPLC with diode-array detection for the analysis of crystal violet was also established. Good linearity on the molecularly imprinted SPE columns was obtained from 0 to 200 ?g/L (R(2) > 0.99). The result demonstrated that the proposed method can be used for the direct determination of crystal violet in seawater and seafood samples. Finally, five samples were analyzed and the following crystal violet concentrations were obtained: 0.92 and 0.52 ?g/L in two seawater samples, as well as 0.36 and 0.27 ?g/kg in two seafood samples. There is no crystal violet detected in the third seawater sample. PMID:23390113
Lian, Ziru; Wang, Jiangtao
Modulation of ceramide metabolism in T-leukemia cell lines potentiates apoptosis induced by the cationic antimicrobial peptide bovine lactoferricin.
Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that selectively induces apoptosis in several different types of human cancer cells. However, the potential use of LfcinB as an anticancer agent is presently limited by the need for relatively high concentrations of the peptide to trigger apoptosis. Ceramide is a membrane sphingolipid that is believed to function as a second messenger during apoptosis. In this study, we investigated the role of ceramide in LfcinB-induced apoptosis in CCRF-CEM and Jurkat T-leukemia cell lines. Exposure to LfcinB caused nuclear condensation and fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in CCRF-CEM and Jurkat T-cell acute lymphoblastic leukemia cell lines. Treatment with C6 ceramide, a cell-permeable, short-chain ceramide analog, also induced apoptotic nuclear morphology, PARP cleavage, and DNA fragmentation in T-leukemia cells. Although LfcinB treatment did not cause ceramide to accumulate in CCRF-CEM or Jurkat cells, the addition of C6 ceramide to LfcinB-treated T-leukemia cells resulted in increased DNA fragmentation. Furthermore, modulation of cellular ceramide metabolism either by inhibiting ceramidases with D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol or N-oleoylethanolamine, or by blocking glucosylceramide synthase activity with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, enhanced the ability of LfcinB to trigger apoptosis in both Jurkat and CCRF-CEM cells. In addition, LfcinB-induced apoptosis of T-leukemia cells was enhanced in the presence of the antiestrogen tamoxifen, which has multiple effects on cancer cells, including inhibition of glucosylceramide synthase activity. We conclude that manipulation of cellular ceramide levels in combination with LfcinB therapy warrants further investigation as a novel strategy for the treatment of T cell-derived leukemias. PMID:18292930
Furlong, Suzanne J; Ridgway, Neale D; Hoskin, David W
Liquid Metering Centrifuge Sticks (LMCS): A Centrifugal Approach to Metering Known Sample Volumes for Colorimetric Solid Phase Extraction (C-SPE)
NASA Technical Reports Server (NTRS)
Phase separation is one of the most significant obstacles encountered during the development of analytical methods for water quality monitoring in spacecraft environments. Removing air bubbles from water samples prior to analysis is a routine task on earth; however, in the absence of gravity, this routine task becomes extremely difficult. This paper details the development and initial ground testing of liquid metering centrifuge sticks (LMCS), devices designed to collect and meter a known volume of bubble-free water in microgravity. The LMCS uses centrifugal force to eliminate entrapped air and reproducibly meter liquid sample volumes for analysis with Colorimetric Solid Phase Extraction (C-SPE). C-SPE is a sorption-spectrophotometric platform that is being developed as a potential spacecraft water quality monitoring system. C-SPE utilizes solid phase extraction membranes impregnated with analyte-specific colorimetric reagents to concentrate and complex target analytes in spacecraft water samples. The mass of analyte extracted from the water sample is determined using diffuse reflectance (DR) data collected from the membrane surface and an analyte-specific calibration curve. The analyte concentration can then be calculated from the mass of extracted analyte and the volume of the sample analyzed. Previous flight experiments conducted in microgravity conditions aboard the NASA KC-135 aircraft demonstrated that the inability to collect and meter a known volume of water using a syringe was a limiting factor in the accuracy of C-SPE measurements. Herein, results obtained from ground based C-SPE experiments using ionic silver as a test analyte and either the LMCS or syringes for sample metering are compared to evaluate the performance of the LMCS. These results indicate very good agreement between the two sample metering methods and clearly illustrate the potential of utilizing centrifugal forces to achieve phase separation and metering of water samples in microgravity.
Gazda, Daniel B.; Schultz, John R.; Clarke, Mark S.
Since the initial description of apoptosis, a number of different forms of cell death have been described. In this review we will focus on classic caspase-dependent apoptosis and its variations that contribute to diseases. Over fifty years of research have clarified molecular mechanisms involved in apoptotic signaling as well and shown that alterations of these pathways lead to human diseases. Indeed both reduced and increased apoptosis can result in pathology. More recently these findings have led to the development of therapeutic approaches based on regulation of apoptosis, some of which are in clinical trials or have entered medical practice.
Favaloro, B.; Allocati, N.; Graziano, V.; Di Ilio, C.; De Laurenzi, V.
Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin
The ubiquitin conjugating enzyme Rad6B is overexpressed in breast cancer and induces ?-catenin transcriptional activation and stabilization via K63-linked polyubiquitination. Here we identify ?-catenin and Rad6B interacting regions, identify potential Rad6B ubiquitination sites in ?-catenin, and characterize their breast cancer tissue expression. ?-catenin and Rad6B colocalize in breast carcinoma and coimmunoprecipitate from MDA-MB-231 cells. Pull-down assays using GST-?-catenin and His-Rad6B deletion mutants identified amino acids 131�181 and 50�116, respectively, as necessary for their interaction. Ubiquitination assays using ?-catenin deletion mutants mapped Rad6B-induced ubiquitination within ?-catenin 181�422 encompassing Armadillo repeats 2�7. Lysine to arginine mutations within repeats 5�7 identified K394 as the major Rad6B ubiquitination site in vitro and in vivo, and confirmed by Rad6B ubiquitination of a ?-catenin peptide encompassing K394. Ubiquitination of wild type- but not K394R-?-catenin was decreased by Rad6B silencing. Compared to wild type-, K312R-, K335R-, K345R-, or K354R-?-catenin, K394R mutation caused ~50% drop in TOP/Flash activity in Wnt-silent MCF-7 cells. Consistent with these data, expression of Rad6B, itself a ?-catenin/TCF transcriptional target, was also reduced in K394R-?-catenin transfected cells. Steady-state K394R-?-catenin levels are decreased compared to wild type-?-catenin. The decreased expression is not due to proteasomal degradation as treatment with MG132 failed to rescue its levels. Lymph node-positive breast carcinomas express higher levels of Rad6 protein and Rad6 activity, and K63-linked ubiquitinated ?-catenin than reduction mammoplasties. These data suggest that K394 is a novel site of ?-catenin ubiquitination that may be important for the stability and activity of ?-catenin in breast cancer.
Gerard, Brigitte; Sanders, Matthew A.; Visscher, Daniel W.; Tait, Larry; Shekhar, Malathy P.V.
Caffeic Acid Phenethyl Ester Protects against Amphotericin B Induced Nephrotoxicity in Rat Model
The present study was conducted to investigate whether caffeic acid phenethyl ester (CAPE), an active component of propolis extract, has a protective effect on amphotericin B induced nephrotoxicity in rat models. Male Wistar-Albino rats were randomly divided into four groups: (I) control group (n = 10), (II) CAPE group (n = 9) which received 10??mol/kg CAPE intraperitoneally (i.p.), (III) amphotericin B group (n = 7) which received one dose of 50?mg/kg amphotericin B, and (IV) amphotericin B plus CAPE group (n = 7) which received 10??mol/kg CAPE i.p. and one dose of 50?mg/kg amphotericin B. The left kidney was evaluated histopathologically for nephrotoxicity. Levels of malondialdehyde (MDA), nitric oxide (NO), enzyme activities including catalase (CAT), and superoxide dismutase (SOD) were measured in the right kidney. Histopathological damage was prominent in the amphotericin B group compared to controls, and the severity of damage was lowered by CAPE administration. The activity of SOD, MDA, and NO levels increased and catalase activity decreased in the amphotericin B group compared to the control group (P = 0.0001, P = 0.003, P = 0.0001, and P = 0.0001, resp.). Amphotericin B plus CAPE treatment caused a significant decrease in MDA, NO levels, and SOD activity (P = 0.04, P = 0.02, and P = 0.0001, resp.) and caused an increase in CAT activity compared with amphotericin B treatment alone (P = 0.005). CAPE treatment seems to be an effective adjuvant agent for the prevention of amphotericin B nephrotoxicity in rat models.
Altuntas, Atila; Y?lmaz, H. Ramazan; Altuntas, Aysegul; Uz, Efkan; Demir, Murat; Gokcimen, Alparslan; Aksu, Oguzhan; Bayram, Dilek Senol; Sezer, Mehmet Tugrul
Simultaneous online SPE-LC-MS/MS quantification of six widely used synthetic progestins in human plasma.
Co-administration of synthetic progestin containing hormonal contraceptives (HCs) and antiepileptic drugs (AEDs) is a common clinical situation which needs specific considerations due to drug interactions. Several studies have demonstrated that lamotrigine plasma levels are significantly decreased during co-medication with HCs, and that this interaction is associated with increased seizure frequency in most of the cases. Additionally, an increase in contraceptive failure and unintended pregnancy could be observed during co-medication. Hence, monitoring of progestin plasma levels in patients with AED co-medication is of interest. A rapid and reliable online solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (online SPE-LC-MS/MS) method using gradient elution in the LC domain was established and validated for the simultaneous quantitative determination of gestodene, dienogest, drospirenone, etonogestrel, cyproterone acetate, and levonorgestrel in human plasma. The online SPE-LC-MS/MS method covered a quantification concentration range of 5-100 ng/ml for dienogest, 1-100 ng/ml for etonogestrel and 2-100 ng/ml for all other analytes. Stable isotope-labeled internal standards were used for analyte quantification based on selected reaction monitoring experiments. Inter- and intra-assay precision and accuracy were determined from quality control (QC) samples at the lower limits of quantification and at low, medium, and high concentration levels within the calibration range. Inter-assay reproducibility at the QC levels was better than 10% (relative standard deviation, RSD), accuracy at these levels ranged from -3.7% to 11.3%. Total extraction efficiency, tested at three concentrations, ranged from 92.5% to 106.4%. Matrix interferences were excluded by post-column infusion experiments. To prove the applicability of the assay in clinical cohorts, a sample set (n = 298) stemming from study patients under AED/oral HC co-medication was screened for progestin plasma levels. This method has to be considered a research-use-only assay and must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the IVD directive (98/79/EG) of the European Community. PMID:22160205
Moser, Christina; Zoderer, Daniela; Luef, Gerhard; Rauchenzauner, Markus; Wildt, Ludwig; Griesmacher, Andrea; Seger, Christoph
Viral induced yeast apoptosis.
In an analogous system to mammals, induction of an apoptotic cell death programme (PCD) in yeast is not only restricted to various exogenous factors and stimuli, but can also be triggered by viral killer toxins and viral pathogens. In yeast, toxin secreting killer strains are frequently infected with double-stranded (ds)RNA viruses that are responsible for killer phenotype expression and toxin secretion in the infected host. In most cases, the viral toxins are either pore-forming proteins (such as K1, K2, and zygocin) that kill non-infected and sensitive yeast cells by disrupting cytoplasmic membrane function, or protein toxins (such as K28) that act in the nucleus by blocking DNA synthesis and subsequently causing a G1/S cell cycle arrest. Interestingly, while all these virus toxins cause necrotic cell death at high concentration, they trigger caspase- and ROS-mediated apoptosis at low-to-moderate concentration, indicating that even low toxin doses are deadly by triggering PCD in enemy cells. Remarkably, viral toxins are not solely responsible for cell death induction in vivo, as killer viruses themselves were shown to trigger apoptosis in non-infected yeast. Thus, as killer virus-infected and toxin secreting yeasts are effectively protected and immune to their own toxin, killer yeasts bear the intrinsic potential to dominate over time in their natural habitat. PMID:18291112
Schmitt, Manfred J; Reiter, Jochen
Apoptosis Resistance in Endometriosis
Introduction In a cytological analysis of endometriotic lesions neither granulocytes nor cytotoxic T-cells appear in an appreciable number. Based on this observation we aimed to know, whether programmed cell death plays an essential role in the destruction of dystopic endometrium. Disturbances of the physiological mechanisms of apoptosis, a persistence of endometrial tissue could explain the disease. Another aspect of this consideration is the proliferation competence of the dystopic mucous membrane. Methods Endometriotic lesions of 15 patients were examined through a combined measurement of apoptosis activity with the TUNEL technique (terminal deoxyribosyltransferase mediated dUTP Nick End Labeling) and the proliferation activity (with the help of the Ki-67-Antigens using the monoclonal antibody Ki-S5). Results Twelve out of 15 women studied showed a positive apoptotic activity of 3-47% with a proliferation activity of 2-25% of epithelial cells. Therefore we concluded that the persistence of dystopic endometrium requires proliferative epithelial cells from middle to lower endometrial layers. Conclusion A dystopia misalignment of the epithelia of the upper layers of the functionalism can be rapidly eliminated by apoptotic procedures.
Salmassi, Ali; Acar-Perk, Bengi; Schmutzler, Andreas G.; Koch, Kerstin; Pungel, Frank; Jonat, Walter; Mettler, Liselotte
[Neuronal development and apoptosis].
There are several distinct mechanisms of neuronal cell death that occurs during development. Nuclear type of cell death involves cell shrinkage and chromatin condensation both typical of apoptosis. Here in this short review we summarize some recent advances concerning the mechanism of nuclear type of neuronal death in vitro. Particular focus is placed on superior cervical ganglion (SCG) cells and neuronal PC12 cells both undergoing apoptosis when they are deprived of NGF or serum/NGF. With regard to presynaptic regulation of neuronal death/survival in SCG both cholinergic and non-cholinergic innervations have a pivotal role. The former may involve the modulation of intracellular free Ca2+ concentration, while the latter may exert its effect through a cAMP-dependent mechanism. Finally, neuronal death/survival mechanism of cerebellar granule cells is discussed in terms of cerebellum development and synaptogenesis. Physiological implications are also described about fyn knockout mouse in which naturally-occurring neuronal death may be greatly impaired. PMID:8025016
Koike, T; Tanaka, S; Ito, E
Apoptosis in the aging process
Although many hypotheses have been proposed to explain the aging process, the exact mechanisms are not well defined. Recent accumulating evidence indicates that dysregulation of the apoptotic process may be involved in some aging processes; however, it is still debatable how exactly apoptosis is expressed during aging in vivo. In this review, we discuss recent findings related to apoptosis of
Yoshikazu Higami; Isao Shimokawa
Real-Time Data Management, IP Telemetry, Data Integration, and Data Center Operations for the Source Physics Experiment (SPE), Nevada National Security Site
NASA Astrophysics Data System (ADS)
The Nevada Seismological Laboratory (NSL) manages time-series data and high-throughput IP telemetry for the National Center for Nuclear Security (NCNS) Source Physics Experiment (SPE), underway on the Nevada National Security Site (NNSS). During active-source experiments, SPE's heterogeneous systems record over 350 channels of a variety of data types including seismic, infrasound, acoustic, and electro-magnetic. During the interim periods, broadband and short period instruments record approximately 200 channels of continuous, high-sample-rate seismic data. Frequent changes in sensor and station configurations create a challenging meta-data environment. Meta-data account for complete operational histories, including sensor types, serial numbers, gains, sample rates, orientations, instrument responses, data-logger types etc. To date, these catalogue 217 stations, over 40 different sensor types, and over 1000 unique recording configurations (epochs). Facilities for processing, backup, and distribution of time-series data currently span four Linux servers, 60Tb of disk capacity, and two data centers. Bandwidth, physical security, and redundant power and cooling systems for acquisition, processing, and backup servers are provided by NSL's Reno data center. The Nevada System of Higher Education (NSHE) System Computer Services (SCS) in Las Vegas provides similar facilities for the distribution server. NSL staff handle setup, maintenance, and security of all data management systems. SPE PIs have remote access to meta-data, raw data, and CSS3.0 compilations, via SSL-based transfers such as rsync or secure-copy, as well as shell access for data browsing and limited processing. Meta-data are continuously updated and posted on the Las Vegas distribution server as station histories are better understood and errors are corrected. Raw time series and refined CSS3.0 data compilations with standardized formats are transferred to the Las Vegas data server as available. For better data availability and station monitoring, SPE is beginning to leverage NSL's wide-area digital IP network with nine SPE stations and six Rock Valley area stations that stream continuous recordings in real time to the NSL Reno data center. These stations, in addition to eight regional legacy stations supported by National Security Technologies (NSTec), are integrated with NSL's regional monitoring network and constrain a high-quality local earthquake catalog for NNSS. The telemetered stations provide critical capabilities for SPE, and infrastructure for earthquake response on NNSS as well as southern Nevada and the Las Vegas area.
Plank, G.; Slater, D.; Torrisi, J.; Presser, R.; Williams, M.; Smith, K. D.
Apoptosis and the Airway Epithelium
The airway epithelium functions as a barrier and front line of host defense in the lung. Apoptosis or programmed cell death can be elicited in the epithelium as a response to viral infection, exposure to allergen or to environmental toxins, or to drugs. While apoptosis can be induced via activation of death receptors on the cell surface or by disruption of mitochondrial polarity, epithelial cells compared to inflammatory cells are more resistant to apoptotic stimuli. This paper focuses on the response of airway epithelium to apoptosis in the normal state, apoptosis as a potential regulator of the number and types of epithelial cells in the airway, and the contribution of epithelial cell apoptosis in important airways diseases.
White, Steven R.
Detailed Comparison of Observed Dose-Time Profile of October 19-20, 1989 SPE on Mir with Model Calculations
NASA Technical Reports Server (NTRS)
The dose rate dynamics of the October 19-20,1989 solar energetic particle (SPE) event as observed by the Liulin instrument onboard the Mir orbital station was analyzed in light of new calculations of the geomagnetic cutoff and improved estimates of the less than 100 MeV energy spectra from the GOES satellite instrument. The new calculations were performed using the as-flown Mir orbital trajectory and includes time variations of the cutoff rigidity due to changes in the kappa (sub p) index. Although the agreement of total event integrated calculated dose to the measured dose is good, it results from some measured dose-time profile been higher and some lower than model calculations. They point to the need to include the diurnal variation of the geomagnetic cutoff and modifications of the cutoffs to variations in kappa (sub p) in model calculations. Understanding of such events in light of the upcoming construction of the International Space Station during the period of maximum solar activity needs to be vigorously pursued.
Badhwar, Gautam D.; Atwell, William
Selective enrichment of trace copper(II) from biological and natural water samples by SPE using ion-imprinted polymer.
A novel Cu(II)-imprinted polymer sorbent was prepared by an ion-imprinted polymer (IIP) technique using (2Z)-N,N'-bis(2-aminoethylic)but-2-enediamide as the functional monomer and pentaerythritol triacylate as a crosslinker. IR, XPS, and elemental analysis techniques were used to confirm the obtained product. Subsequently, when this polymer was used as sorbent in SPE, it exhibited excellent selectivity for template ion from an aqueous solution. Quantitative extraction of Cu(II) was achieved in the pH range of 4-7. The time needed to extract each sample was less than 30 min by the batch method. The distribution ratio (D) values of IIP for Cu(II) were greatly larger than that for other ions. At optimal pH value, the maximum extraction capacity of IIP and nonimprinted polymer (NIP) was found to be 29.8 and 7.0 mg/g, respectively. The adsorption behavior of Cu(II) on the sorbents could be described by Langmuir adsorption isotherm equation. The feasible flow rate of Cu(II)-containing solution for quantitative extraction onto the column packed with IIP was 1-4 mL/min, whereas for elution it was less than 1 mL/min. The developed method was successfully applied to the separation and enrichment of trace Cu(II) in biological and natural water samples with satisfactory results. PMID:18338364
Zhai, Yunhui; Yang, Dong; Chang, Xijun; Liu, Yongwen; He, Qun
Streptococcus pyogenes c-di-AMP Phosphodiesterase, GdpP, Influences SpeB Processing and Virulence
Small cyclic nucleotide derivatives are employed as second messengers by both prokaryotes and eukaryotes to regulate diverse cellular processes responding to various signals. In bacteria, c-di-AMP has been discovered most recently, and some Gram-positive pathogens including S. pyogenes use this cyclic nucleotide derivative as a second messenger instead of c-di-GMP, a well-studied important bacterial second messenger. GdpP, c-di-AMP phosphodiesterase, is responsible for degrading c-di-AMP inside cells, and the cellular role of GdpP in S. pyogenes has not been examined yet. To test the cellular role of GdpP, we created a strain with a nonpolar inframe deletion of the gdpP gene, and examined the properties of the strain including virulence. From this study, we demonstrated that GdpP influences the biogenesis of SpeB, the major secreted cysteine protease, at a post-translational level, susceptibility to the beta lactam antibiotic ampicillin, and is necessary for full virulence in a murine subcutaneous infection model.
Cho, Kyu Hong; Kang, Song Ok
Comparative Analysis of Apoptosis and Inflammation Genes of Mice and Humans
Apoptosis (programmed cell death) plays important roles in many facets of normal mammalian physiology. Host-pathogen interactions have provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses through NF?B induction. Proteins involved in apoptosis and NF?B induction commonly contain evolutionarily conserved domains that can serve as signatures for identification by bioinformatics methods. Using a combination of public (NCBI) and private (RIKEN) databases, we compared the repertoire of apoptosis and NF?B-inducing genes in humans and mice from cDNA/EST/genomic data, focusing on the following domain families: (1) Caspase proteases; (2) Caspase recruitment domains (CARD); (3) Death Domains (DD); (4) Death Effector Domains (DED); (5) BIR domains of Inhibitor of Apoptosis Proteins (IAPs); (6) Bcl-2 homology (BH) domains of Bcl-2 family proteins; (7) Tumor Necrosis Factor (TNF)-family ligands; (8) TNF receptors (TNFR); (9) TIR domains; (10) PAAD (PYRIN; PYD, DAPIN); (11) nucleotide-binding NACHT domains; (12) TRAFs; (13) Hsp70-binding BAG domains; (14) endonuclease-associated CIDE domains; and (15) miscellaneous additional proteins. After excluding redundancy due to alternative splice forms, sequencing errors, and other considerations, we identified cDNAs derived from a total of 227 human genes among these domain families. Orthologous murine genes were found for 219 (96%); in addition, several unique murine genes were found, which appear not to have human orthologs. This mismatch may be due to the still fragmentary information about the mouse genome or genuine differences between mouse and human repertoires of apoptotic genes. With this caveat, we discuss similarities and differences in human and murine genes from these domain families.
Reed, John C.; Doctor, Kutbuddin; Rojas, Ana; Zapata, Juan M.; Stehlik, Christian; Fiorentino, Loredana; Damiano, Jason; Roth, Wilfried; Matsuzawa, Shu-ichi; Newman, Ruchi; Takayama, Shinichi; Marusawa, Hiroyuki; Xu, Famming; Salvesen, Guy; Godzik, Adam
Use of diffusion-ordered NMR spectroscopy and HPLC-UV-SPE-NMR to identify undeclared synthetic drugs in medicines illegally sold as phytotherapies.
The informal (and/or illegal) e-commerce of pharmaceutical formulations causes problems that governmental health agencies find hard to control, one of which concerns formulas sold as natural products. The purpose of this work was to explore the advantages and limitations of DOSY and HPLC-UV-SPE-NMR. These techniques were used to identify the components of a formula illegally marketed in Brazil as an herbal medicine possessing anti-inflammatory and analgesic properties. DOSY was able to detect the major components present at higher concentrations. Complete characterization was achieved using HPLC-UV-SPE-NMR, and 1D and 2D NMR analyses enabled the identification of known synthetic drugs. These were ranitidine and a mixture of orphenadrine citrate, piroxicam, and dexamethasone, which are co-formulated in a remedy called Rheumazim that is used to relieve severe pain, but it is prohibited in Brazil because of a lack of sufficient pharmacokinetic and pharmacodynamic information. PMID:23818305
Silva, Lorena M A; Filho, Elenilson G A; Thomasi, S�rgio S; Silva, Bianca F; Ferreira, Antonio G; Ven�ncio, Tiago
Apoptosis in gingival overgrowth tissues.
Variations in the balance between cell proliferation and apoptosis could contribute to the etiology of gingival overgrowth. The aim of this study was to test the hypothesis that, in fibrotic gingival lesions, fibroblast proliferation is stimulated and apoptosis is decreased. Apoptotic index, caspase 3 expression, the proliferative index, FOXO1 expression, and histological inflammation were measured in situ. Analysis of data showed that apoptosis decreased in all forms of gingival overgrowth examined (p < 0.05), and inflammation caused a small but significant increase compared with non-inflamed tissues (p < 0.05). The greatest decrease of apoptosis occurred in the most fibrotic tissues. Cell proliferation was elevated in all forms of gingival overgrowth tested, independent of inflammation (p < 0.05). To identify potential mechanisms of transcriptional regulation of apoptosis, we assessed FOXO1 and caspase 3 expression levels and found them to correlate well with diminished apoptosis. Analysis of data suggests that increased fibroblast proliferation and a simultaneous decrease in apoptosis contribute to gingival overgrowth. PMID:17720861
Kantarci, A; Augustin, P; Firatli, E; Sheff, M C; Hasturk, H; Graves, D T; Trackman, P C
Apoptosis in Gingival Overgrowth Tissues
Variations in the balance between cell proliferation and apoptosis could contribute to the etiology of gingival overgrowth. The aim of this study was to test the hypothesis that, in fibrotic gingival lesions, fibroblast proliferation is stimulated and apoptosis is decreased. Apoptotic index, caspase 3 expression, the proliferative index, FOXO1 expression, and histological inflammation were measured in situ. Analysis of data showed that apoptosis decreased in all forms of gingival overgrowth examined (p < 0.05), and inflammation caused a small but significant increase compared with non-inflamed tissues (p < 0.05). The greatest decrease of apoptosis occurred in the most fibrotic tissues. Cell proliferation was elevated in all forms of gingival overgrowth tested, independent of inflammation (p < 0.05). To identify potential mechanisms of transcriptional regulation of apoptosis, we assessed FOXO1 and caspase 3 expression levels and found them to correlate well with diminished apoptosis. Analysis of data suggests that increased fibroblast proliferation and a simultaneous decrease in apoptosis contribute to gingival overgrowth.
Kantarci, A.; Augustin, P.; Firatli, E.; Sheff, M.C.; Hasturk, H.; Graves, D.T.; Trackman, P.C.
In vivo detection of apoptosis.
After several decades of debate, it is now widely acknowledged that apoptosis, also known as programmed cell death, is central to homoeostasis and normal development and physiology in all multicellular organisms, including humans. The dysregulation of apoptosis can lead to the destruction of normal tissues in a variety of disorders, including autoimmune and neurodegenerative diseases (too much apoptosis) or the growth of tumors (too little apoptosis). In addition, effective therapy of tumors requires the iatrogenic induction of programmed cell death by radiation, chemotherapy, or both. Given the central role of apoptosis, it would be desirable to have a noninvasive imaging method to serially detect and monitor this process in cancer patients undergoing conventional radiation and chemotherapy treatments as well as for the development and testing of new drugs. In this article, the latest modalities and contrast agents described in the literature for the imaging of apoptosis in vivo are reviewed. First, the most recent developments in the biochemical characterization of the many intracellular pathways involved in this complex process are discussed. Next, a variety of new radionuclide tracers, including radiolabeled annexin V and caspase inhibitors for PET and SPECT, are described. Finally, the use of MRI, MR spectroscopy, and ultrasound as possible alternative imaging modalities for the imaging of apoptosis is addressed. PMID:18523067
Blankenberg, Francis G
Simultaneous Analysis of Hexaconazole, Myclobutanil, and Tebuconazole Residues in Apples and Soil by SPE Clean-Up and GC with Nitrogen�Phosphorus Detection
A facile and sensitive method utilizing capillary gas chromatography with nitrogen phosphorus detection (GC�NPD) has been\\u000a developed and validated for simultaneous analysis of hexaconazole, myclobutanil, and tebuconazole, three broad-spectrum systemic\\u000a fungicides, in apples and soil. Two samples were fortified with the three pesticides and subjected to ultrasonic extraction,\\u000a followed by solid-phase extraction (SPE) to remove coextractives, before analysis by GC�NPD.
Zhubo Deng; Jiye Hu; Dongmei Qin; Hui Li
A novel staphylococcal cassette chromosomal element, SCCfusC, carrying fusC and speG in fusidic acid-resistant methicillin-resistant Staphylococcus aureus.
A high prevalence of fusC (16/46, 59%) was found in fusidic acid-resistant methicillin-resistant Staphylococcus aureus isolates collected from 2008 to 2010. Nucleotide sequencing of fusC and flanking regions revealed a novel staphylococcal cassette chromosome (SCC) structure, SCCfusC, which was integrated into rlmH and located upstream from SCCmec. The SCCfusC element contained speG, which may contribute to the polyamine resistance. PMID:24277045
Lin, Yu-Tzu; Tsai, Jui-Chang; Chen, Hsiao-Jan; Hung, Wei-Chun; Hsueh, Po-Ren; Teng, Lee-Jene
Impact of the SpeB protease on binding of the complement regulatory proteins factor H and factor H-like protein 1 by Streptococcus pyogenes.
Microbial pathogens often exploit human complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FH and FHL-1 binding protein expressed on the surface of the human pathogenic bacterium Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. Fba has been shown to contribute to phagocytosis resistance, intracellular invasion, and virulence in mice. Here, we look at the role of Fba in recruitment of FH and FHL-1 by five serotype M1 isolates of streptococci. Inactivation of fba greatly inhibited binding of FH and FHL-1 by all isolates, indicating that Fba is a major FH and FHL-1 binding factor of serotype M1 streptococci. For three isolates, FH binding was significantly reduced in stationary-phase cultures and correlated with high levels of protease activity and SpeB (an extracellular cysteine protease) protein in culture supernatants. Analysis of a speB mutant confirmed that SpeB accounts for the loss of Fba from the cell surface, suggesting that the protease may modulate FH and FHL-1 recruitment during infection. Comparisons of fba DNA sequences revealed that the FH and FHL-1 binding site in Fba is conserved among the M1 isolates. Although the ligand binding site is not strictly conserved in Fba from a serotype M49 isolate, the M49 Fba protein was found to bind both FH and FHL-1. Collectively, these data indicate that binding of FH and FHL-1 is a conserved function of Fba while modulation of Fba function by SpeB is variable. PMID:15784545
Wei, Lin; Pandiripally, Vinod; Gregory, Eugene; Clymer, Micaya; Cue, David
Distributions of H 2O and CO 2 ices on Ariel, Umbriel, Titania, and Oberon from IRTF\\/SpeX observations
We present 0.8�2.4 ?m spectral observations of uranian satellites, obtained at IRTF\\/SpeX on 17 nights during 2001�2005. The spectra reveal for the first time the presence of CO2 ice on the surfaces of Umbriel and Titania, by means of 3 narrow absorption bands near 2 ?m. Several additional, weaker CO2 ice absorptions have also been detected. No CO2 absorption is
W. M. Grundy; L. A. Young; J. R. Spencer; R. E. Johnson; E. F. Young; M. W. Buie
An SPE-assisted BODIPY fluorometric paper sensor for the highly selective and sensitive determination of Cd(2+) in complex sample: rice.
By using sensing technology, the individual component analysis at trace level in complex samples remains problematic simply because of various interfering species. For example, the determination of Cd(2+) in rice is difficult due to the co-existing interfering metal cations at thousands or even millions of times higher concentrations. In this study, a heavy-metal ion sensitive BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-based fluorometric paper sensor with assistance of solid phase extraction (SPE) was developed for the highly selective and sensitive determination of trace Cd(2+) in rice. SPE column packed with prepared sulfonated PS-DVB microspheres was employed to enrich trace Cd(2+) and meanwhile remove most interfering heavy-metal ions in simulated complex rice sample with oxalic acid as eluent, which was theoretically selected on the basis of f values. Mn(2+), as a major coexistent heavy-metal ion, could not be easily removed by SPE, but showed little fluorescent response for BODIPY fluorometric paper sensor even in excess amounts. Combining the separation and enrichment capability of SPE column with the selectivity of BODIPY-based fluorometric paper sensor, we were able to detect trace Cd(2+) in complex samples. The response of fluorometric paper sensor was linearly related with Cd(2+) concentrations in the range of 0.5-4 ?M, with a detection limit of 0.5 ?M. Twelve real rice samples spiked with Cd(2+) were analysed using this method and the results are in good agreement with ICP-MS measurements. PMID:24802241
Zhang, Yu; Li, Hui; Niu, Li-Ya; Yang, Qing-Zheng; Guan, Ya-Feng; Feng, Liang
A strategy for fast structural elucidation of metabolites in small volume plant extracts using automated MS-guided LC-MS-SPE-NMR
Fast and reliable metabolite identification based on automated MS-guided HPLC-MS-SPE-NMR metabolite extraction combined with an automated 1H NMR spectrum fitting was developed. Positional isomers as structure 1 and 2 were easily distinguished. In many metabolomics studies, metabolite identification by mass spectrometry (MS) often is hampered by the lack of good reference compounds, and hence, NMR information is essential for structural
Hooft van der J. J. J; V. V. Mihaleva; Vos de R. C. H; R. J. Bino; J. J. M. Vervoort
Characterisation of commercially available linear alkylbenzenesulfonates by LC-SPE-NMR\\/MS (liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy-mass spectroscopy)
Commercially available linear alkylbenzenesulfonates (LASs) are a mixture of various homologues and isomers, leading to 20 major species. In this work we investigated the commercial product by liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy-mass spectrometry (LC-SPE-NMR\\/MS). The commercial product was separated into 17 fractions by liquid chromatography (LC). After chromatographic separation, 5% of the flow was split to a mass
S. Schmidt; C. Piechotta; M. Godejohann; T. Win; I. Nehls; C. M�gge
Occurrence of polar organic contaminants in the dissolved water phase of the Danube River and its major tributaries using SPE-LC-MS 2 analysis
Polar water-soluble organic contaminants were analysed in the dissolved liquid water phase of river water samples from the Danube River and its major tributaries (within the Joint Danube Survey 2). Analyses were performed by solid-phase extraction (SPE) followed by triple-quadrupole liquid chromatography mass spectrometry (LC-MS2). In total, 34 different polar organic compounds were screened. Focus was given on pharmaceutical compounds
Robert Loos; Giovanni Locoro; Serafino Contini
[Apoptosis in the nervous system].
Apoptosis (programmed cell death) is a distinct form of controlled cell degeneration, different from necrosis. It serves multiple physiological functions, such as the control of cell numbers during development, the maintenance of tissue homeostasis and the deletion of abnormal cells. Apoptosis has unique morphological and biochemical features, especially at the nuclear level, in keeping with the idea of the active participation of the cell in its own demise. Gene regulation of apoptosis shows variability among different tissues, particularly regarding the signals that trigger cell death, but shares an effector phase highly conserved accross species. In the nervous system, genes have been identified which either i) promote apoptosis: Bax, Bcl-xS, c-fos, c-jun, p75NGFR and ICE-like proteases, or ii) block apoptosis: Bcl-2 and Bcl-xL. In addition, availability of trophic factors and expression of Trk membrane receptors allow for the fine adjustement of viable cells in each neuronal population. In some diseases, neuron loss takes place via apoptosis, whether exclusively or associated with necrosis, especially when cellular insults are of moderate intensity or death occurs in areas of the brain adjacent to necrotic foci. This has been shown in excitotoxicity, X-ray injury and hypoxia-ischemia. Activation of apoptosis occurs also in some neurodegenerative diseases. Infantile spinal muscular atrophy can be the first example of a pediatric hereditary disease where a deletion in the gene of a protein which inhibits neuron apoptosis has a pathogenic role. Last, some central nervous system infections produce abnormal activation of apoptosis. PMID:8974737
Macaya, A
Mitochondrial control of nuclear apoptosis
Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro- oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.
Application of Colorimetric Solid Phase Extraction (C-SPE) to Monitoring Nickel(II) and Lead(II) in Spacecraft Water Supplies
NASA Technical Reports Server (NTRS)
Archived water samples collected on the International Space Station (ISS) and returned to Earth for analysis have, in a few instances, contained trace levels of heavy metals. Building on our previous advances using Colorimetric Solid Phase Extraction (C-SPE) as a biocide monitoring technique, we are devising methods for the low level monitoring of nickel(II), lead(II) and other heavy metals. C-SPE is a sorption-spectrophotometric platform based on the extraction of analytes onto a membrane impregnated with a colorimetric reagent that are then quantified on the surface of the membrane using a diffuse reflectance spectrophotometer. Along these lines, we have determined nickel(II) via complexation with dimethylglyoxime (DMG) and begun to examine the analysis of lead(II) by its reaction with 2,5- dimercapto-1,3,4-thiadiazole (DMTD) and 4-(2- pyridylazo)-resorcinol (PAR). These developments are also extending a new variant of C-SPE in which immobilized reagents are being incorporated into this methodology in order to optimize sample reaction conditions and to introduce the colorimetric reagent. This paper describes the status of our development of these two new methods.
Diaz, Neil C.; Gazda, Daniel B.; Fritz, James S.; Porter, Marc D.; Rutz, Jeff; Mudgett, Paul; Schultz, John
Molecular mechanisms of hepatic apoptosis
Apoptosis is a prominent feature of liver diseases. Causative factors such as alcohol, viruses, toxic bile acids, fatty acids, drugs, and immune response, can induce apoptotic cell death via membrane receptors and intracellular stress. Apoptotic signaling network, including membrane death receptor-mediated cascade, reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, lysosomal permeabilization, and mitochondrial dysfunction, is intermixed each other, but one mechanism may dominate at a particular stage. Mechanisms of hepatic apoptosis are complicated by multiple signaling pathways. The progression of liver disease is affected by the balance between apoptotic and antiapoptotic capabilities. Therapeutic options of liver injury are impacted by the clear understanding toward mechanisms of hepatic apoptosis.
Wang, K
Advantages of online SPE coupled with UPLC/MS/MS for determining the fate of pesticides and pharmaceutical compounds.
Laboratory experimentation is essential for our understanding of the fate and behaviour of pollutants. Many analytical techniques exist, but they all have disadvantages either in terms of sensitivity or of selectivity. The number of samples that can be analysed, the low volume of samples available during the experiment and the need to identify different degradates are all obstacles that new techniques are able to overcome. The work presented here summarizes progress in the field of metrology as concerns online solid phase extraction technology coupled with liquid chromatography followed by tandem mass spectrometry detection. Recently developed analytical techniques were validated for both 18 pesticides and their degradates and 17 pharmaceuticals and their degradates. Limits of quantification from 20 to 70�ng�L(-1) for pharmaceuticals and from 15 to 25�ng�L(-1) for pesticides and metabolites have been obtained, with linearity range up to 1�?g�L(-1). The limits of quantification of a few nanograms per litre, the possibility of working on less than 1�mL of sample and the simultaneous quantification of the target products and their transformation products are all advantages that are demonstrated by two environmental applications. The first application concerns the evaluation of ecotoxicological effects of pesticides on aquatic organisms exposed in mesocosms. The second application aims to determine the adsorption constants of pharmaceutical molecules on soils and river sediments. For both applications, the robustness, range of linearity and limit of quantification of the developed analytical methods satisfy the requirements for laboratory experiments conducted under controlled conditions. Specific constraints generated by this type of experiment (adding CaCl2 for the adsorption study and filtration of the water coming from the mesocosms) were not found to limit the use of online SPE. These two preliminary studies show that new experimental fields are possible thanks to online solid phase extraction coupled with liquid chromatography. PMID:23907687
Togola, Anne; Baran, Nicole; Coureau, Charlotte
miR-125b induces cellular senescence in malignant melanoma
Background Micro RNAs (miRs) have emerged as key regulators during oncogenesis. They have been found to regulate cell proliferation, differentiation, and apoptosis. Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma. Our goal was therefore to further examine this theory. Methods We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis. Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and ?-galactosidase staining. The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization. Results In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm. A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53. Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours. Conclusions Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.
analyte retained SPE columns transferred 2nd dimension .... NRR API (colloidal oatmeal CO ) gave r2 prediction accuracy 0.971 ...... (without pre- post-column derivatization) pharmaceutical cream.
Did you mean: analyte retained SPE columns transferred 2nd dimension .... NRR API (colloidal oatmeal CO ) gave r2 prediction accuracy 0.971 ...... (without pre- post-column derivatization) pharmaceutical cream. ?
colloidal oatmeal analyte retained SPE columns transferred 2nd dimension .... NRR API (colloidal oatmeal CO ) gave r2 prediction accuracy 0.971 ...... (without pre- post-column derivatization) pharmaceutical cream.
Did you mean: colloidal oatmeal analyte retained SPE columns transferred 2nd dimension .... NRR API (colloidal oatmeal CO ) gave r2 prediction accuracy 0.971 ...... (without pre- post-column derivatization) pharmaceutical cream. ?
Apoptosis induced by parasitic diseases
Fatalities caused by parasitic infections often occur as a result of tissue injury that results from a form of host-cell death known as apoptosis. However, instead of being pathogenic, parasite-induced apoptosis may facilitate host survival. Consequently, it is of utmost importance to decipher and understand the process and the role of apoptosis induced or controlled by parasites in humans. Despite this, few studies provide definitive knowledge of parasite-induced host-cell apoptosis. Here, the focus is on a consideration of host-cell apoptosis as either a pathogenic feature or as a factor enabling parasite survival and development. Cell death by apoptotic-like mechanisms could be described as a ride to death with a return ticket, as initiation of the pathway may be reversed, with the potential that it could be manipulated for therapeutic purposes. The management of host-cell apoptosis could thus be an adjunctive factor for parasitic disease treatment. Evidence that the apoptotic process could be reversed by anti-apoptotic drugs has recently been obtained, leading to the possibility of host-cell rescue after injury. An important issue will be to predict the beneficial or deleterious effects of controlling human cell death by apoptotic-like mechanisms during parasitic diseases.
Peroxisome proliferator-activated receptors (PPARs) are ligand binding transcription factors which function in many physiological roles including lipid metabolism, cell growth, differentiation, and apoptosis. PPARs and their ligands have been shown to play a role in cancer. In particular, PPAR? ligands including endogenous prostaglandins and the synthetic thiazolidinediones (TZDs) can induce apoptosis of cancer cells with antitumor activity. Thus, PPAR? ligands have a potential in both chemoprevention and therapy of several types of cancer either as single agents or in combination with other antitumor agents. Accordingly, the involvement of PPAR? and its ligands in regulation of apoptosis of cancer cells have been extensively studied. Depending on cell types or ligands, induction of apoptosis in cancer cells by PPAR? ligands can be either PPAR?-dependent or -independent. Through increasing our understanding of the mechanisms of PPAR? ligand-induced apoptosis, we can develop better strategies which may include combining other antitumor agents for PPAR?-targeted cancer chemoprevention and therapy. This review will highlight recent research advances on PPAR? and apoptosis in cancer.
Elrod, Heath A.; Sun, Shi-Yong
Liver myofibroblasts up-regulate monocyte CD163 expression via PGE2 during hepatitis B induced liver failure
Background Although patients with liver failure exhibit a generalized inflammatory-imbalance status, substantial evidence indicates that this immunosuppressive or anti-inflammatory state may be deleterious. Increased expression of CD163 (known to be involved in several anti-inflammatory functions of the immune system) in patients with liver failure is significantly correlated with a fatal outcome. However, little is known of the regulatory mechanisms that influence the expression of CD163. Methods We assessed the expression of CD163 on monocytes from both circulating cells and the liver tissues of patients with hepatitis B induced liver failure using flow cytometry and isolated the myofibroblasts from diseased livers. The ability of human liver myofibroblasts to regulate CD163 expression on monocytes was studied in vitro. Results We showed that CD163+ monocytes were enriched primarily in diseased livers and that they were associated with liver myofibroblasts in the same area. Accordingly, liver myofibroblasts were significantly superior to normal skin fibroblasts in inducing the expression of CD163 on monocytes in vitro. Moreover, we found that liver myofibroblasts triggered the activation of monocytes by secreting PGE2. Inhibition of PGE2 production in liver myofibroblasts using NS-398 markedly reduced CD163 expression in vitro. Conclusion These results suggest that liver myofibroblasts play a direct role in regulating the expression of CD163 on monocytes in human liver tissues and thereby may regulate monocyte function during hepatitis B induced liver failure.
Inhibitory effects of resveratrol on melanin synthesis in ultraviolet B-induced pigmentation in Guinea pig skin.
Resveratrol is a polyphenolic compound found in various natural products such as grapes and berries and possesses anti-cancer, anti-hyperlipidemia, and anti-aging properties. Recently, it has been reported that resveratrol inhibits ?-melanocyte-stimulating hormone signaling, viability, and migration in melanoma cells. However, these effects have not been confirmed in vivo, specifically brownish guinea pigs. To evaluate the potential of resveratrol as a regulator of melanin for hyperpigmentation therapy, the influence of resveratrol on pigmentation was investigated by ultraviolet B-induced hyperpigmentation in brownish guinea pig skin. We found that resveratrol reduced the expression of melanogenesis-related proteins tyrosinase, tyrosinase-related proteins 1 and 2, and microphthalmia-associated transcription factor in melanoma cells. Furthermore, topical application of resveratrol was demonstrated to significantly decrease hyperpigmentation on ultraviolet B-stimulated guinea pig skin in vivo. Based on our histological data, resveratrol inhibits melanin synthesis via a reduction in tyrosinase-related protein 2 among the melanogenic enzymes. This study is the first to provide evidence supporting resveratrol as a depigmentation agent, along with further clinical investigation of resveratrol in ultraviolet B-induced skin disorders such as hyperpigmentation and skin photoaging. PMID:24596619
Lee, Taek Hwan; Seo, Jae Ok; Baek, So-Hyeon; Kim, Sun Yeou
Histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and alters HagB-induced chemokine responses
NASA Astrophysics Data System (ADS)
Histatins are human salivary gland peptides with anti-microbial and anti-inflammatory activities. In this study, we hypothesized that histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and attenuates HagB-induced chemokine responses in human myeloid dendritic cells. Histatin 5 bound to immobilized HagB in a surface plasmon resonance (SPR) spectroscopy-based biosensor system. SPR spectroscopy kinetic and equilibrium analyses, protein microarray studies, and I-TASSER structural modeling studies all demonstrated two histatin 5 binding sites on HagB. One site had a stronger affinity with a KD1 of 1.9 ?M and one site had a weaker affinity with a KD2 of 60.0 ?M. Binding has biological implications and predictive modeling studies and exposure of dendritic cells both demonstrated that 20.0 ?M histatin 5 attenuated (p < 0.05) 0.02 ?M HagB-induced CCL3/MIP-1?, CCL4/MIP-1?, and TNF? responses. Thus histatin 5 is capable of attenuating chemokine responses, which may help control oral inflammation.
Borgwardt, Derek S.; Martin, Aaron D.; van Hemert, Jonathan R.; Yang, Jianyi; Fischer, Carol L.; Recker, Erica N.; Nair, Prashant R.; Vidva, Robinson; Chandrashekaraiah, Shwetha; Progulske-Fox, Ann; Drake, David; Cavanaugh, Joseph E.; Vali, Shireen; Zhang, Yang; Brogden, Kim A.
Smac mimetic sensitizes glioblastoma cells to Temozolomide-induced apoptosis in a RIP1- and NF-?B-dependent manner.
Inhibitor of apoptosis (IAP) proteins are expressed at high levels in many cancers and therefore represent attractive targets for therapeutic intervention. Here, we report for the first time that the second mitochondria-derived activator of caspases (Smac) mimetic BV6 sensitizes glioblastoma cells toward Temozolomide (TMZ), the first-line chemotherapeutic agent in the treatment of glioblastoma. BV6 and TMZ synergistically reduce cell viability and trigger apoptosis in glioblastoma cells (combination index <0.4-0.8), which is accompanied by increased loss of mitochondrial-membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Analysis of the molecular mechanisms reveals that BV6 causes rapid degradation of cIAP1, leading to stabilization of NF-?B-inducing kinase and NF-?B activation. BV6-stimulated NF-?B activation is critically required for sensitization toward TMZ, as inhibition of NF-?B by overexpression of the mutant I?B? super-repressor profoundly reduces loss of mitochondrial membrane potential, cytochrome c release, caspase activation and apoptosis. Of note, BV6-mediated sensitization to TMZ is not associated with increased tumor necrosis factor alpha (TNF?) production. Also, TNF?, CD95 or TRAIL-blocking antibodies or knockdown of TNFR1 have no or little effect on combination treatment-induced apoptosis. Interestingly, BV6 and TMZ cooperate to trigger the formation of a RIP1 (receptor activating protein 1)/caspase-8/FADD complex. Knockdown of RIP1 by small interfering RNA significantly reduces BV6- and TMZ-induced caspase-8 activation and apoptosis, showing that RIP1 is necessary for apoptosis induction. By demonstrating that BV6 primes glioblastoma cells for TMZ in a NF-?B- and RIP1-dependent manner, these findings build the rationale for further (pre)clinical development of Smac mimetics in combination with TMZ. PMID:22469979
Wagner, L; Marschall, V; Karl, S; Cristofanon, S; Zobel, K; Deshayes, K; Vucic, D; Debatin, K-M; Fulda, S
Organopalladium compound 7b targets mitochondrial thiols and induces caspase-dependent apoptosis in human myeloid leukemia cells.
The advances in the treatment of chronic myeloid leukemia (CML) during the last years were also accompanied by the development of evading strategies by tumor cells, resulting in chemotherapy resistance in some patients. Patented organopalladium compounds derived from the reaction of N,N-dimethyl-1-phenethylamine (dmpa) with [1,2-ethanebis(diphenylphosphine)] (dppe) exhibited a potent antitumor activity in vivo and in vitro in melanoma cells. We showed here that the cyclopalladated derivative [Pd2(R(+))C(2), N-dmpa)2(?-dppe)Cl2], named compound 7b, was highly effective to promote cell death in the K562 human leukemia cells and its mechanisms of action were investigated. It was shown that compound 7b was able to promote exclusively apoptotic cell death in K562 cells associated to cytochrome c release and caspase 3 activation. This cytotoxic effect was not observed in normal peripheral mononuclear blood cells. The compound 7b-induced intrinsic apoptotic pathway was triggered by the protein thiol oxidation that resulted in the dissipation of the mitochondrial transmembrane potential. The preventive effect of the dithiothreitol on the compound 7b-induced cell death and all downstream events associated to apoptosis confirmed that death signal was elicited by the thiol oxidation. These findings contribute to the elucidation of the palladacycle 7b-induced cell death mechanism and present this compound as a promising drug in the CML antitumor chemotherapy. PMID:23744358
Moraes, V W R; Caires, A C F; Paredes-Gamero, E J; Rodrigues, T
Organopalladium compound 7b targets mitochondrial thiols and induces caspase-dependent apoptosis in human myeloid leukemia cells
The advances in the treatment of chronic myeloid leukemia (CML) during the last years were also accompanied by the development of evading strategies by tumor cells, resulting in chemotherapy resistance in some patients. Patented organopalladium compounds derived from the reaction of N,N-dimethyl-1-phenethylamine (dmpa) with [1,2-ethanebis(diphenylphosphine)] (dppe) exhibited a potent antitumor activity in vivo and in vitro in melanoma cells. We showed here that the cyclopalladated derivative [Pd2(R(+))C2, N-dmpa)2(?-dppe)Cl2], named compound 7b, was highly effective to promote cell death in the K562 human leukemia cells and its mechanisms of action were investigated. It was shown that compound 7b was able to promote exclusively apoptotic cell death in K562 cells associated to cytochrome c release and caspase 3 activation. This cytotoxic effect was not observed in normal peripheral mononuclear blood cells. The compound 7b-induced intrinsic apoptotic pathway was triggered by the protein thiol oxidation that resulted in the dissipation of the mitochondrial transmembrane potential. The preventive effect of the dithiothreitol on the compound 7b-induced cell death and all downstream events associated to apoptosis confirmed that death signal was elicited by the thiol oxidation. These findings contribute to the elucidation of the palladacycle 7b-induced cell death mechanism and present this compound as a promising drug in the CML antitumor chemotherapy.
Moraes, V W R; Caires, A C F; Paredes-Gamero, E J; Rodrigues, T
Spontaneous apoptosis in human thymocytes.
Apoptosis seems to be involved in different stages of immune cell development. In particular, experimental evidence suggests that it is a major form of cell death in the thymus. The present analysis of human thymocytes reveals that a fraction of these cells, cultured in vitro, undergoes spontaneous apoptosis. This observation is based both on molecular (DNA fragmentation) and morphological (electron microscopic) investigations of the cells. The apoptotic thymocytes are CD3- or CD3lo, CD4lo, and CD8lo and do not express Bcl-2 protein. Furthermore, thymocytes die by apoptosis when exposed to pharmacological stimuli, such as tumor necrosis factor-alpha, dexamethasone, ATP, or Ca++ ionophore. Thus the apoptotic machinery in thymocytes can be triggered by an imbalance in growth factors in the in vitro culture media and can be modulated by various biochemical signals. The process of spontaneous apoptosis is independent of mRNA or protein synthesis, as actinomycin D and cycloheximide fail to inhibit this phenomenon. Furthermore, apoptosis seems to require active oxidative phosphorylation, as it is prevented by incubation of the cells with inhibitors of the respiratory chain. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8
Tiso, M.; Gangemi, R.; Bargellesi Severi, A.; Pizzolitto, S.; Fabbi, M.; Risso, A.
RTF+SpeX Near-IR Images and Spectra of the Moon Pre-impact and at Impact of SMART-1
NASA Technical Reports Server (NTRS)
We present SpeX 0.8-2.5 micron spectra and Br-gamma or J H or K-band images of the Moon on 2006 July 10 UT, 2006 Sep 1 UT, and 2006 Sep 3 UT. The first two dates are data taken in preparation (near Full Moon and 1 st quarter Moon) for observing the impact of ESA's SMART-1 Lunar satellite on 2006 Sep 3 UT. We hope to be presenting images (about 1 arc minute by 1 arc minute), and low resolution (Prism mode) spectra of the SMART-1 impact to be taken with the 60 arc second long slit.
Wooden, Diane H.; Ennico, Kimberly A.; Colaprete, Anthony; Lucey, Paul G.; Tokunaga, Alan T.; Rayner, John T.; Meech, Karen J.; Foing, B.; Ehrenfreund, P.
A multiresidue method with mobile on-site sampling based on SPE for ultratrace analysis of plant protectants in environmental waters
A new multiclass\\/multiresidue method for monitoring plant protectant residues in raw- and drinking waters with on-site sampling\\u000a using a mobile, self-contained sampling unit based on SPE was developed and validated. 38 active compounds with widely varying\\u000a chemical and physicochemical properties (acid-base properties, polarities, vapor pressures, solubilities) are measured from\\u000a just one sample and work-up. 100 mL water, acidified with acetic
Klaus Pittertschatscher; Norbert Inreiter; Andrea Schatzl; Hans Malissa
Perfluorinated compounds (PFCs) in groundwater and aqueous soil extracts: using inline SPE-LC-MS\\/MS for screening and sorption characterisation of perfluorooctane sulphonate and related compounds
Perfluorinated compounds (PFCs) have been recognised as emerging pollutants of global relevance. A fully automated method\\u000a with inline solid-phase extraction coupled to electrospray ionisation liquid chromatography-tandem mass spectrometry (SPE-LC-MS\\/MS)\\u000a is presented and used for characterisation of soil adsorption and desorption for six PFCs: perfluoroheptanoic acid (PFHpA),\\u000a perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorobutane sulphonate (PFBS),\\u000a and perfluorooctane
Rasmus Enevoldsen; Ren� K. Juhler
Profiling of impurities in p-methoxymethamphetamine (PMMA) by means of SPE/TLC method. Examination of the influence of experimental conditions according to 2(4) factorial.
In the present paper profiling of impurities in p-methoxymethamphetamine (PMMA) by means of SPE/TLC is reported. PMMA was synthesised by Leuckart procedure. The influence of experimental conditions on the profile quality was investigated. The experiments were carried out according to a 2(4) factorial. The proposed characteristics of the profile quality (optimisation criterions) are based on a matrix presentation of TLC patterns. They take into account, simultaneously, the number of spots revealed, differences between R(f) values and intensity of fluorescence. PMID:12850419
Kochana, J; Wilamowski, J; Parczewski, A
Molecular mechanisms of hepatic apoptosis.
Apoptosis is a prominent feature of liver diseases. Causative factors such as alcohol, viruses, toxic bile acids, fatty acids, drugs, and immune response, can induce apoptotic cell death via membrane receptors and intracellular stress. Apoptotic signaling network, including membrane death receptor-mediated cascade, reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, lysosomal permeabilization, and mitochondrial dysfunction, is intermixed each other, but one mechanism may dominate at a particular stage. Mechanisms of hepatic apoptosis are complicated by multiple signaling pathways. The progression of liver disease is affected by the balance between apoptotic and antiapoptotic capabilities. Therapeutic options of liver injury are impacted by the clear understanding toward mechanisms of hepatic apoptosis. PMID:24434519
Wang, K
Method of Modulating Apoptosis and Compositions Thereof.
National Technical Information Service (NTIS)
Disclosed herein are methods for modulating apoptosis. Methods are provided for inhibiting or preventing apoptosis by promoting formation of a complex of BAK and Voltage-Dependent Anion Channel 2 (VDAC2). The invention also provides methods of promoting o...
S. Korsmeyer E. Cheng
Granzyme B-Induced Neurotoxicity Is Mediated via Activation of PAR-1 Receptor and Kv1.3 Channel
Increasing evidence supports a critical role of T cells in neurodegeneration associated with acute and subacute brain inflammatory disorders. Granzyme B (GrB), released by activated T cells, is a cytotoxic proteinase which may induce perforin-independent neurotoxicity. Here, we studied the mechanism of perforin-independent GrB toxicity by treating primary cultured human neuronal cells with recombinant GrB. GrBactivated the protease-activated receptor (PAR)-1 receptor on the neuronal cell surface leading to decreased intracellular cyclic AMP levels. This was followed by increased expression and translocation of the voltage gated potassium channel, Kv1.3 to the neuronal cell membrane. Similar expression of Kv1.3 was also seen in neurons of the cerebral cortex adjacent to active inflammatory lesions in patients with multiple sclerosis. Kv1.3 expression was followed by activation of Notch-1 resulting in neurotoxicity. Blocking PAR-1, Kv1.3 or Notch-1 activation using specific pharmacological inhibitors or siRNAs prevented GrB-induced neurotoxicity. Furthermore, clofazimine protected against GrB-induced neurotoxicity in rat hippocampus, in vivo. These observations indicate that GrB released from T cells induced neurotoxicity by interacting with the membrane bound Gi-coupled PAR-1 receptor and subsequently activated Kv1.3 and Notch-1. These pathways provide novel targets to treat T cell-mediated neuroinflammatory disorders. Kv1.3 is of particular interest since it is expressed on the cell surface, only under pathological circumstances, and early in the cascade of events making it an attractive therapeutic target.
Wang, Tongguang; Lee, Myoung-Hwa; Choi, Elliot; Pardo-Villamizar, Carlos A.; Lee, Sung Bin; Yang, In Hong; Calabresi, Peter A.; Nath, Avindra
Apoptosis in Established and Healing Psoriasis
Background: Previous studies have described apoptosis in the stratum granulosum and in the stratum corneum, but not in the germinative compartment in normal skin. In psoriasis, an increased epidermal apoptosis has been observed in the differentiated compartment, suggesting that apoptosis has a key role in the pathogenesis of psoriasis, as a counteracting factor to the overproduction of cells. Little is
M. Laporte; P. Galand; D. Fokan; C. de Graef; M. Heenen
HSV-Induced Apoptosis in Herpes Encephalitis
HSV triggers and blocks apoptosis in cell type-specific fashion. This review discusses present understanding of the role of apoptosis and signaling cascades in neuronal pathogenesis and survival and summarizes present findings relating to the modulation of these strictly balanced processes by HSV infection. Underscored are the findings that HSV-1, but not HSV-2, triggers apoptosis in CNS neurons and causes encephalitis
L. Aurelian
Methods for determining Myc-induced apoptosis.
Although many oncoproteins promote cell growth and proliferation, some also possess the potential to induce cell death by apoptosis. Deregulated expression of the myc oncogene promotes apoptosis in both cultured cells and in some tissues in vivo. Here we describe techniques to detect Myc-induced apoptosis in vitro using flow cytometry and microscopy and in vivo using immunohistochemical staining. PMID:24006060
Lu, Dan; Littlewood, Trevor D
MEKC combined with SPE and sample stacking for multiple analysis of pesticides in water samples at the ng/L level.
In this work, a new multiresidue analytical method based on MEKC with UV detection combined with SPE as off-line preconcentration strategy, and reversed-electrode polarity stacking mode (REPSM) as on-line stacking procedure, has been developed for the monitoring of 12 pesticides (carbendazim, pirimicarb, metalaxyl, pyrimethanil, procymidone, nuarimol, azoxystrobin, tebufenozide, fenarimol, benalaxyl, penconazole, and tetradifon) that are currently being used in the Canary Islands (Spain). The optimized MEKC buffer, consisting of 100 mM sodium tetraborate and 30 mM SDS at pH 8.5 with 6% v/v 1-propanol, provided baseline resolution of the 12 pesticides in less than 20 min. The developed method was applied to the analysis of mineral, stagnant, and tap water samples. The proposed SPE-REPSM-MEKC-UV method showed high extraction efficiencies with detection limits (LODs) at the low ng/L level providing LOD values down to 64 ng/L for these real samples. PMID:17476718
Ravelo-P�rez, Lidia M; Hern�ndez-Borges, Javier; Cifuentes, Alejandro; Rodr�guez-Delgado, Miguel A
Identification of three novel polyphenolic compounds, origanine A-C, with unique skeleton from Origanum vulgare L. using the hyphenated LC-DAD-SPE-NMR/MS methods.
Identification of new compounds especially those with new skeletons from plant kingdom has long been a vital aspect for understanding phytochemistry, plant metabolisms and discovering new bioactive compounds. In this study, we identified and isolated three novel polyphenolic compounds, origanine A-C, from a well-researched plant Origanum vulgare L. using the hyphenated LC-DAD-SPE-NMR/MS methods. Based on the combined information from UV-visible, accurate mass and 2D NMR spectra together with computational calculations, we found that these compounds all had a novel skeleton of cyclohexenetetracarboxylic acids attached with some well-known bioactive moieties including 3,4-dihydroxyphenyl, 4-(?-d-glucopyranosyloxy)benzyl alcohol (gastrodin), and 3-(3,4-dihydroxyphenyl)lactic acid (danshensu) residues. These findings provided crucial information to fill the gaps in our knowledge in terms of the plant secondary metabolism. This study also indicated the necessity for further research in plant secondary metabolism for even well-studied plants and demonstrated the powerfulness of the hyphenated LC-DAD-SPE-NMR/MS methods for comprehensive analysis of plant metabolites in particular for discovering new natural compounds. PMID:22142199
Liu, Hongbing; Zheng, Anmin; Liu, Huili; Yu, Haiyan; Wu, Xiangyu; Xiao, Chaoni; Dai, Hui; Hao, Fuhua; Zhang, Limin; Wang, Yulan; Tang, Huiru
Ultrasonic extraction of veterinary antibiotics from soils and pig slurry with SPE clean-up and LC-UV and fluorescence detection.
A simple and rapid analytical method is presented in which the three veterinary antibiotics oxytetracycline (OTC), sulfachloropyridazine (SCP) and tylosin (TYL) are simultaneously extracted and determined in four different soils. Extractions were carried out by a combination of ultrasonic agitation and vortex mixing using a mixture of methanol, EDTA and McIlvaine buffer at pH 7 as the extractant solution. The extracts were then cleaned-up by a tandem solid phase extraction (SPE) method using an Isolute SAX anion exchange cartridge to remove natural organic matter and an Oasis HLB polymeric cartridge to retain the study compounds. Analysis was by HPLC-UV with additional fluorescence detection for SCP. Recoveries were in the range 68-85% for SCP in all soil types, 58-75% for OTC in sandy soils, 27-51% for OTC in clay containing soils, 74-105% for TYL and 47-61% in a clay soil. OTC and SCP were also extracted from liquid pig manure using a mixture of EDTA and McIlvaine buffer at pH 7 with ultrasonic agitation and vortex mixing with SPE clean-up and HPLC-UV analysis. Recoveries were greater than 77% and 58% for OTC and SCP, respectively. Limits of detection were 18mugkg(-1) for OTC and SCP and 40mugkg(-1) for TYL in soils and 70mugL(-1) for OTC and 140mugL(-1) for SCP in pig slurry. PMID:18969712
Blackwell, Paul A; Holten L�tzh�ft, Hans-Christian; Ma, Hai-Ping; Halling-S�rensen, Bent; Boxall, Alistair B A; Kay, Paul
Radiation dose predictions for SPE events during solar cycle 23 from NASA's Nowcast of Atmospheric Ionizing Radiation for Aviation Safety (NAIRAS) model
NASA Astrophysics Data System (ADS)
NASA's High Charge and Energy Transport (HZETRN) code is a deterministic model for rapid and accurate calculations of the particle radiation fields in the space environment. HZETRN is used to calculate dosimetric quantities on the International Space Station (ISS) and assess astronaut risk to space radiations, including realistic spacecraft and human geometry for final exposure evaluation. HZETRN is used as an engineering design tool for materials research for radiation shielding protection. Moreover, it is used to calculate HZE propagation through the Earth and Martian atmospheres, and to evaluate radiation exposures for epidemiological studies. A new research project has begun that will use HZETRN as the transport engine for the development of a nowcast prediction of air-crew radiation exposure for both background galactic cosmic ray (GCR) exposure and radiation exposure during solar particle events (SPE) that may accompany solar storms. The new air-crew radiation exposure model is called the Nowcast of Atmospheric Ionizing Radiation for Aviation Safety (NAIRAS) model, which utilizes real-time observations from ground-based, atmospheric, and satellite measurements. In this paper, we compute the global distribution of atmospheric radiation dose for several SPE events during solar cycle 23, with particular emphasis on the high-latitude and polar region. We also characterize the suppression of the geomagnetic cutoff rigidity during these storm periods and their subsequent influence on atmospheric radiation exposure.
Mertens, Christopher; Blattnig, Steve; Slaba, Tony; Kress, Brian; Wiltberger, Michael; Solomon, Stan
Diclofenac induces apoptosis in hepatocytes
Hepatotoxicity is one of the side effects associated with the administration of diclofenac, a non-steroidal anti-inflammatory drug widely used clinically. The effect of diclofenac on the early events that trigger apoptosis cascade have been evaluated in rat hepatocytes. To do this, early and late apoptotic markers, associated with the pivotal steps of the execution phase, have been evaluated after incubation
Xavier Ponsoda; Enrique O'Connor; Teresa Donato; R Jover; Jos� V Castell
Methylselenium and Prostate Cancer Apoptosis.
National Technical Information Service (NTIS)
The purpose of this research is to gain a better understanding of the biochemical pathways and molecular targets for the selective induction of apoptosis signaling and execution of PCa cells by methyl selenium (Se)/selenol. We hypothesized that methyl sel...
J. Lu
Pancreatic carcinogenesis: apoptosis and angiogenesis.
Apoptosis and angiogenesis are critical biologic processes that are altered during carcinogenesis. Both apoptosis and angiogenesis may play an important role in pancreatic carcinogenesis. Despite numerous advances in the diagnosis and treatment of pancreatic cancer, its prognosis remains dismal and a new therapeutic approach is much needed. Recent research has revealed that apoptosis and angiogenesis are closely interrelated. Several reports show that a tumor suppresser gene that is expressed in pancreatic carcinoma and related to malignant potential can induce apoptosis and also inhibit angiogenesis. At present, it is generally accepted that tumor growth in cancers, including pancreatic cancer, depends on angiogenesis. We have identified 2 new angiogenesis inhibitors from a conditioned medium of human pancreatic carcinoma cell line (BxPC-3): antiangiogenic antithrombin III (aaAT-III) and vitamin D binding protein-macrophage activating factor (DBP-maf). These molecules were able to regress tumors in severe combined immunodeficiency disease (SCID) mice, demonstrating potent inhibition of endothelial cell proliferation. Moreover, the angiogenesis inhibitors induced tumor dormancy in the animal model. These results suggest that antiangiogenic therapy using angiogenesis inhibitors may become a new strategy for treatment of pancreatic cancer in the near future. PMID:15084979
Onizuka, Shinya; Kawakami, Shunsuke; Taniguchi, Ken; Fujioka, Hikaru; Miyashita, Kosei
Viral Control of Mitochondrial Apoptosis
Throughout the process of pathogen�host co-evolution, viruses have developed a battery of distinct strategies to overcome biochemical and immunological defenses of the host. Thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. Since the elimination of infected cells via programmed cell death is one of the most ancestral defense mechanisms against
Lorenzo Galluzzi; Catherine Brenner; Eugenia Morselli; Zahia Touat; Guido Kroemer
Apoptosis induced by anticancer drugs
Most of the cytotoxic anticancer drugs in current use have been shown to induce apoptosis in susceptible cells. The fact that disparate agents, which interact with different targets, induce cell death with some common features (endonucleolytic cleavage of DNA, changes in chromatin condensation) suggests that cytotoxicity is determined by the ability of the cell to engage this so-called �programmed� cell
John A. Hickman
Mitochondrial Control of Nuclear Apoptosis
Summary Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the exist- ence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplas- mic structures including mitochondria have been shown to participate in the control of apop- totic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they
Naoufal Zamzami; Santos A. Susin; Philippe Marchetti
Maximal adamantyl-substituted retinoid-related molecule-induced apoptosis requires NF-?B noncanonical and canonical pathway activation
NF-?B transcription factors have a critical role in regulating cell survival and apoptosis. We have previously shown that 4-(3-Cl-(1-adamantyl)-4-hydroxyphenyl)-3-chlorocinnamic acid (3-Cl-AHPC), an adamantyl-substituted retinoid molecule, induced apoptosis and required NF-?B activation in prostate and breast carcinoma cells. Here, we show that 3-Cl-AHPC activated both I?B kinase (IKK)? and IKK? with subsequent activation of the canonical and noncanonical NF-?B pathways in the human breast carcinoma and leukemia cell lines. 3-Cl-AHPC-mediated activation of the NF-?B canonical pathway occurred within 6?h, whereas maximal activation of the NF-?B noncanonical pathway required 48?h. Knockout of IKK? or IKK? expression in mouse embryonic fibroblast cells and knockdown of IKK? or IKK? in MDA-MB-468 cells resulted in the inhibition of 3-Cl-AHPC-mediated apoptosis, indicating that activation of canonical and noncanonical pathways are required for maximal 3-Cl-AHPC-mediated apoptosis. 3-Cl-AHPC activation of the noncanonical pathway was preceded by caspase-mediated decrease in the E3-ligase c-IAP1 with subsequent stabilization of NF-?B-inducing kinase (NIK) expression, increased binding of NIK by TRAF3, activation of IKK?, and the resultant increased levels of RelB and p52. Increased expression of c-IAP1 blocked 3-Cl-AHPC-mediated stabilization of NIK levels and 3-Cl-AHPC-mediated apoptosis. Cdc37 expression was required for activation of IKK? and IKK? by 3-Cl-AHPC. These findings suggest that NF-?B pathways have an important role in 3-Cl-AHPC-mediated apoptosis.
Farhana, L; Dawson, M I; Murshed, F; Fontana, J A
Intervention for apoptosis in cardiomyopathy
Apoptosis plays an important role in pathogenesis of primary and secondary cardiomyopathies. It is proposed that antiapoptotic\\u000a interventions may constitute an effective strategy for these diseases. Some of the antiapoptotic interventions are �old wine\\u000a in a new bottle� measures already included in the conventional pharmacotherapy. As specific antiapoptotic treatment, caspase\\u000a inhibitors and anti-TNF-? antibody are in early phases of clinical
Hiroyuki Yaoita; Yukio Maruyama
APOPTOSIS: Life and Death Decisions
NSDL National Science Digital Library
Access to the article is free, however registration and sign-in are required. Screening small molecules for their ability to perturb a cellular pathway, a strategy called forward chemical genetics, can yield unexpected information about other pathway components. This is well illustrated by new work (Jiang et al.), as Nicholson and Thornberry discuss in their Perspective. Discovery of a small-molecule activator of apoptosis implicated a tumor suppressor protein and an oncoprotein in the regulation of the mitochondrial cell death pathway.
Donald W. Nicholson (Merck Frosst Centre for Therapeutic Research, Merck Research Laboratories;); Nancy A. Thornberry (Merck Research Laboratories;)
Nitric Oxide Functions as a Signal in Ultraviolet-B-Induced Baicalin Accumulation in Scutellaria baicalensis Suspension Cultures
Stress induced by ultraviolet-B (UV-B) irradiation stimulates the accumulation of various secondary metabolites in plants. Nitric oxide (NO) serves as an important secondary messenger in UV-B stress-induced signal transduction pathways. NO can be synthesized in plants by either enzymatic catalysis or an inorganic nitrogen pathway. The effects of UV-B irradiation on the production of baicalin and the associated molecular pathways in plant cells are poorly understood. In this study, nitric oxide synthase (NOS) activity, NO release and the generation of baicalin were investigated in cell suspension cultures of Scutellaria baicalensis exposed to UV-B irradiation. UV-B irradiation significantly increased NOS activity, NO release and baicalin biosynthesis in S. baicalensis cells. Additionally, exogenous NO supplied by the NO donor, sodium nitroprusside (SNP), led to a similar increase in the baicalin content as the UV-B treatment. The NOS inhibitor, N?-nitro-l-arginine (LNNA), and NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) partially inhibited UV-B-induced NO release and baicalin accumulation. These results suggest that NO is generated by NOS or NOS-like enzymes and plays an important role in baicalin biosynthesis as part of the defense response of S. baicalensis cells to UV-B irradiation.
Zhang, Jin-Jie; Li, Xue-Qin; Sun, Jun-Wei; Jin, Song-Heng
Development, validation, and uncertainty measurement of multi-residue analysis of organochlorine and organophosphorus pesticides using pressurized liquid extraction and dispersive-SPE techniques.
Simple and efficient multi-residue analytical methods were developed and validated for the determination of 13 organochlorine and 17 organophosphorous pesticides from soil, spinach and eggplant. Techniques namely accelerated solvent extraction and dispersive SPE were used for sample preparations. The recovery studies were carried out by spiking the samples at three concentration levels (1 limit of quantification (LOQ), 5 LOQ, and 10 LOQ). The methods were subjected to a thorough validation procedure. The mean recovery for soil, spinach and eggplant were in the range of 70-120% with median CV (%) below 10%. The total uncertainty was evaluated taking four main independent sources viz., weighing, purity of the standard, GC calibration curve and repeatability under consideration. The expanded uncertainty was well below 10% for most of the pesticides and the rest fell in the range of 10-20%. PMID:21210211
Sanyal, Doyeli; Rani, Anita; Alam, Samsul; Gujral, Seema; Gupta, Ruchi
Determination of fluoroquinolones in bovine milk samples using a pipette-tip SPE step based on multiwalled carbon nanotubes prior to CE separation.
A simple CE-UV method was developed for the simultaneous determination of ciprofloxacin, norfloxacin, and ofloxacin in milk samples. The optimum separation was obtained using a 20 mM ammonium dihydrogenphosphate solution with 2 mM cetyltrimethylammonium bromide at pH 3.0 as the BGE. Satisfactory resolution for structurally very similar analytes, like norfloxacin and ciprofloxacin, was achieved without including any organic solvent. Milk samples were prepared using a simple/extraction procedure based on acidic protein precipitation followed by an SPE step using only 5 mg of multiwalled carbon nanotubes as the sorbent material. The LODs for the three compounds were between 7.5 and 11.6 ?g/L and the RSDs for the peak areas were between 2.6 and 4.9%. The complete method was applied to spiked real milk samples with satisfactory recoveries for all analytes (84-106%). PMID:24227292
Springer, Valeria; Jacks�n, Johan; Ek, Patrik; Lista, Adriana G; Emmer, Asa
Perfluorinated compounds (PFCs) in groundwater and aqueous soil extracts: using inline SPE-LC-MS/MS for screening and sorption characterisation of perfluorooctane sulphonate and related compounds.
Perfluorinated compounds (PFCs) have been recognised as emerging pollutants of global relevance. A fully automated method with inline solid-phase extraction coupled to electrospray ionisation liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) is presented and used for characterisation of soil adsorption and desorption for six PFCs: perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorobutane sulphonate (PFBS), and perfluorooctane sulphonate (PFOS). The method reduces sample turnaround time and solvent consumption and is suitable for low volume sampling. The only sample preparation necessary for water samples was sedimentation by centrifugation. The method has a total runtime of 21 min including inline sample cleanup (2 min for injection and SPE, 14 min for the chromatographic separation, 5 min for reconditioning). Negative AP-ESI with selective reaction monitoring (SRM) was used and the method was documented for quantification of the six environmentally important PFCs in subsoil matrix and related aqueous matrixes (groundwater and drainage water). Linearity was demonstrated in the range 5 to 2,500 ng/l and the LOD was between 2 and 8 ng/l in groundwater. Adsorption was characterised by linear Freundlich isotherms for all six compounds in two agricultural top soils (A horizon, sandy and clayey soil).Variability in sorption characteristics for soil types as well as compound properties were found, and correlation between the organic carbon normalised sorption coefficient (K (OC)) and PFC molecular weight was demonstrated. The K (d) values were in the range 0.1 to 33 (l/kg), and 0.3 to 65 (l/kg) for sorption and desorption respectively. PMID:20740279
Enevoldsen, Rasmus; Juhler, Ren� K
Butyric acid induces apoptosis in inflamed fibroblasts.
Butyric acid, an extracellular metabolite from periodontopathic bacteria, induces apoptosis in murine and human T- and B-cells, whereas intact gingival fibroblasts isolated from healthy humans are resistant to butyric-acid-induced apoptosis. We examined the susceptibility of inflamed gingival fibroblasts isolated from adult persons with periodontitis to butyric-acid-induced apoptosis. Butyric acid significantly suppressed the viability of inflamed gingival fibroblasts and induced apoptosis in a dose-dependent manner. The incubation of inflamed gingival fibroblasts with butyric acid induced DNA fragmentation and apoptotic changes such as chromatin condensation, hypodiploid nuclei, and mitochondrial injury. Furthermore, butyric-acid-induced apoptosis in inflamed gingival fibroblasts was reduced by caspase-3/7, -6, -8, and -9 inhibitors. Thus, inflamed gingival fibroblasts from adult persons with periodontitis appear to be highly susceptible to mitochondria- and caspase-dependent apoptosis induced by butyric acid, compared with healthy gingival fibroblasts. PMID:18096893
Kurita-Ochiai, T; Seto, S; Suzuki, N; Yamamoto, M; Otsuka, K; Abe, K; Ochiai, K
AMPK activator, AICAR, inhibits palmitate-induced apoptosis in osteoblast
Osteoblast apoptosis reduces bone mineral density. Apoptosis can be induced in a variety of cells by palmitate, which is one of the most common saturated fatty acids in dietary fat. The AMPK activator, AICAR, has been shown to inhibit palmitate-induced apoptosis. However, the role of palmitate in osteoblast apoptosis is currently unknown. This study examined whether palmitate could induce apoptosis
Ji-Eun Kim; Myun-Whan Ahn; Suk-Hwan Baek; In Kyu Lee; Yong-Woon Kim; Jong-Yeon Kim; Jin-Myoung Dan; So-Young Park
Apoptosis: focus on sea urchin development
It has been proposed that the apoptosis is an essential requirement for the evolution of all animals, in fact the apoptotic\\u000a program is highly conserved from nematodes to mammals. Throughout development, apoptosis is employed by multicellular organisms\\u000a to eliminate damaged or unnecessary cells. Here, we will discuss both developmental programmed cell death (PCD) under normal\\u000a conditions and stress induced apoptosis,
Maria Agnello; Maria Carmela Roccheri
Apoptosis in Lung Injury and Disease
Pulmonary cell death may contribute significantly to acute and chronic lung injuries caused by various adverse environmental\\u000a agents. Pulmonary cells may die by necrosis, apoptosis, and other forms of regulated cell death. Apoptosis exerts a homeostatic\\u000a function in lung defense and development, through the removal of dysfunctional cells and by regulating cellular proliferation.\\u000a Lung cell apoptosis can occur as a
Stefan W. Ryter; Hong Pyo Kim; Augustine M. K. Choi
Apoptosis and aging: increased resistance to apoptosis enhances the aging process
Apoptosis is a vital component in the evolutionarily conserved host defense system. Apoptosis is the guardian of tissue integrity\\u000a by removing unfit and injured cells without evoking inflammation. However, apoptosis seems to be a double-edged sword since\\u000a during low-level chronic stress, such as in aging, increased resistance to apoptosis can lead to the survival of functionally\\u000a deficient, post-mitotic cells with
Antero Salminen; Johanna Ojala; Kai Kaarniranta
Apoptosis in cancer: from pathogenesis to treatment
Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. It is also one of the most studied topics among cell biologists. An understanding of the underlying mechanism of apoptosis is important as it plays a pivotal role in the pathogenesis of many diseases. In some, the problem is due to too much apoptosis, such as in the case of degenerative diseases while in others, too little apoptosis is the culprit. Cancer is one of the scenarios where too little apoptosis occurs, resulting in malignant cells that will not die. The mechanism of apoptosis is complex and involves many pathways. Defects can occur at any point along these pathways, leading to malignant transformation of the affected cells, tumour metastasis and resistance to anticancer drugs. Despite being the cause of problem, apoptosis plays an important role in the treatment of cancer as it is a popular target of many treatment strategies. The abundance of literature suggests that targeting apoptosis in cancer is feasible. However, many troubling questions arise with the use of new drugs or treatment strategies that are designed to enhance apoptosis and critical tests must be passed before they can be used safely in human subjects.
Death-Defining Immune Responses After Apoptosis
Apoptosis is a programmed form of cell death whereby characteristic internal cellular dismantling is accompanied by the preservation of plasma membrane integrity. Maintaining this order during apoptosis prevents the release of cellular contents and ensures a noninflammatory death. Here, we consider examples of apoptosis in different contexts and discuss how the same form of cell death could have different immunological consequences. Multiple parameters such as cell death as a result of microbial infection, the nature of the inflammatory microenvironment, the type of responding phagocytic cells and the genetic background of the host organism all differentially influence the immunological consequences of apoptosis.
Campisi, L.; Cummings, R. J.; Blander, J. Magarian
Apoptosis and Molecular Targeting Therapy in Cancer
Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction.
Hassan, Mohamed; Watari, Hidemichi; AbuAlmaaty, Ali; Ohba, Yusuke; Sakuragi, Noriaki
The cellular decision between apoptosis and autophagy
Apoptosis and autophagy are important molecular processes that maintain organismal and cellular homeostasis, respectively. While apoptosis fulfills its role through dismantling damaged or unwanted cells, autophagy maintains cellular homeostasis through recycling selective intracellular organelles and molecules. Yet in some conditions, autophagy can lead to cell death. Apoptosis and autophagy can be stimulated by the same stresses. Emerging evidence indicates an interplay between the core proteins in both pathways, which underlies the molecular mechanism of the crosstalk between apoptosis and autophagy. This review summarizes recent literature on molecules that regulate both the apoptotic and autophagic processes.
Fan, Yong-Jun; Zong, Wei-Xing
Determination of analytes in medical herbs extracts by SPE coupled with two-dimensional planar chromatography in combination with diode array scanning densitometry and HPLC-diode array detector.
The purpose of this study is to demonstrate an application of 2-D high-performance planar chromatography-diode array detector (DAD) and HPLC-DAD after solid-phase extraction (SPE) for identification and quantitative analysis of pesticides (isoproturon, aziprotryne, hexazinone, flufenoxuron, methabenzthiazuron, procymidone, and ?-cypermethrin) in Melissa officinalis L. (Labiatae) samples. The procedure described for the determination of compounds is inexpensive and can be applied to routine analysis of analytes in medical herbs' samples after preliminary cleanup and concentration by SPE. Average recoveries on C18 SPE cartridges of pesticides eluted with 5 mL tetrahydrofuran by the proposed HPLC-DAD method, before and after 2-D-high-performance planar chromatography separation of analytes from M. officinalis L. samples spiked with pesticide at a concentration level of 10 ?g/g in plant material are presented. Method validation parameters for the quantification of pesticides by the proposed HPLC-DAD after SPE method are also presented. PMID:21171173
Tuzimski, Tomasz
Liquid chromatography/tandem mass spectrometric bioanalysis using normal-phase columns with aqueous/organic mobile phases - a novel approach of eliminating evaporation and reconstitution steps in 96-well SPE.
Bioanalytical methods using automated 96-well solid-phase extraction (SPE) and liquid chromatography with electrospray tandem mass spectrometry (LC/MS/MS) are widely used in the pharmaceutical industry. SPE methods typically require manual steps of drying of the eluates and reconstituting of the analytes with a suitable injection solvent possessing elution strength weaker than the mobile phase. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well SPE by using normal-phase LC/MS/MS methods with low aqueous/high organic mobile phases, which consisted of 70-95% organic solvent, 5-30% water, and small amount of volatile acid or buffer. While the commonly used SPE elution solvents (i.e. acetonitrile and methanol) have stronger elution strength than a mobile phase on reversed-phase chromatography, they are weaker elution solvents than a mobile phase for normal-phase LC/MS/MS and therefore can be injected directly. Analytical methods for a range of polar pharmaceutical compounds, namely, omeprazole, metoprolol, fexofenadine, pseudoephedrine as well as rifampin and its metabolite 25-desacetyl-rifampin, in biological fluids, were developed and optimized based on the foregoing principles. As a result of the time saving, a batch of 96 samples could be processed in one hour. These bioanalytical LC/MS/MS methods were validated according to "Guidance for Industry - Bioanalytical Method Validation" recommended by the Food and Drug Administration (FDA) of the United States. PMID:12362389
Naidong, Weng; Shou, Wilson Z; Addison, Thomas; Maleki, Saber; Jiang, Xiangyu
Melatonin decreases apoptosis and expression of apoptosis-associated proteins in acute puromycin aminonucleoside nephrosis
Background. The anti-apoptotic properties of melato- nin have been demonstrated previously in several in vivo and in vitro studies. Previous reports have shown increased apoptosis during puromycin aminonucleo- side nephrosis (PAN). The aim of this study was to determine if melatonin (MEL) can prevent apoptosis and modify oxidative stress, an apoptosis inducer, in this experimental model. Methods. Rats were injected
Adriana Pedreanez; Jaimar Rincon; Maritza Romero; Ninoska Viera; Jesus Mosquera
A cell-intrinsic requirement for NF-?B-inducing kinase in CD4 and CD8 T cell memory.
NF-?B-inducing kinase [(NIK), MAP3K14] is an essential kinase linking a subset of TNFR family members to the noncanonical NF-?B pathway. To assess the cell-intrinsic role of NIK in murine T cell function, we generated mixed bone marrow chimeras using bone marrow from NIK knockout (KO) and wild-type (WT) donor mice and infected the chimeras with lymphocytic choriomeningitis virus (LCMV). The chimeras possess an apparently normal immune system, including a mixture of NIK KO and WT T cells, and the virus was cleared normally. Comparison of the NIK KO and WT CD4 and CD8 T cell responses at 8 d post infection revealed modest but significant differences in the acute response. In both CD4 and CD8 compartments, relatively fewer activated (CD44(hi)) NIK KO T cells were present, but within the CD44(hi) population, a comparable percentage of the activated cells produced IFN-? in response to ex vivo stimulation with antigenic LCMV peptides, although IL-7R expression was reduced in the NIK KO CD8 T cells. Assessment of the LCMV-specific memory at 65 d post infection revealed many more LCMV-specific WT memory T cells than NIK KO memory T cells in both the CD4 and the CD8 compartments, although the small number of surviving NIK KO memory T cells responded to secondary challenge with virus. These results demonstrate a cell-intrinsic requirement for NIK in the generation and/or maintenance of memory T cells in response to acute viral infection. PMID:24006459
Rowe, Alexander M; Murray, Susan E; Rau�, Hans-Peter; Koguchi, Yoshinobu; Slifka, Mark K; Parker, David C
Diclofenac induces apoptosis in hepatocytes.
Hepatotoxicity is one of the side effects associated with the administration of diclofenac, a non-steroidal anti-inflammatory drug widely used clinically. The effect of diclofenac on the early events that trigger apoptosis cascade have been evaluated in rat hepatocytes. To do this, early and late apoptotic markers, associated with the pivotal steps of the execution phase, have been evaluated after incubation with the drug. The results show that the apoptotic effect of diclofenac occurs after exposure to sub-cytotoxic concentrations of the drug (maximal non toxic concentration, MNTC, after 24-h treatment was 450 microM), without overlapping with cell necrosis (LDH leakage evaluation). Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 3 activation (3-5-fold control) was maximal after 12 h of exposure to 350 microM of the drug. The involvement of the mitochondrial permeability transition (MPT) in diclofenac-induced apoptosis was investigated. Cyclosporine A and decylubiquinone, MPT specific inhibitor, prevented the activation of caspase 3, thus showing that diclofenac opened the MPT pore. Treatment of hepatocytes with antioxidants (alpha-tocopherol, N,N-dimethylthiourea, superoxide dismutase) were able to prevent caspase cascade activation by diclofenac, revealing that oxidative stress at the mitochondrial level is involved in MPT induction. Finally, the differential cytotoxic and apoptotic effect produced in hepatocytes and non-metabolizing hepatoma cells suggest that CYP-mediated metabolism of diclofenac apoptosis may be related to the apoptotic effect of the drug. PMID:14599462
G�mez-Lech�n, Maria-Jos�; Ponsoda, Xavier; O'Connor, Enrique; Donato, Teresa; Jover, R; Castell, Jos� V
APOPTOSIS: Death by Crowd Control
NSDL National Science Digital Library
Access to the article is free, however registration and sign-in are required. Cells often die by way of a controlled suicide called apoptosis. The proteins most responsible for the deed are caspases, specific proteases that are carefully regulated in the cell so that they only become activated when absolutely necessary. In his Perspective, Hengartner discusss results reported by Yang et al. in the same issue on how some of the central caspases responsible for cell death, such as CED-3, are activated by oligomerization, a process that is regulated by the anti-death protein CED-9, a member of the large Bcl-2 family.
Michael Hengartner (Cold Spring Harbor Laboratory;)
Apoptosis in oral lichen planus.
Apoptotic cell death may be a contributory cause of basal cell destruction in oral lichen planus (OLP). Therefore. the purpose of this study was to investigate the rate of apoptosis in OLP and the expression of two proteins (FasR and FasL) regulating this process. Biopsies from 18 patients with histologically diagnosed OLP were investigated, with comparison to normal oral mucosa of healthy persons. For visualisation of DNA fragmentation, the TUNEL method was used. In order to characterise the infiltrating cell population (CD3. CD4, CD8) and expression of FasR and FasL, we used an immunohistochemical technique. The results showed that T cells dominated in the subepithelial cell infiltrate. Within the epithelium the apoptotic cells were confined to the basal cell layer, and more apoptotic cells were seen in areas with basal cell degeneration and atrophic epithelium. There was a prominent expression of FasR/FasL in OLP. with a rather uniform distribution throughout the inflammatory cell infiltrate. In the epithelium, the FasR/FasL expression was more abundant in the basal cell area compared to the suprabasal cell layer. In conclusion, apoptosis within the epithelium is significantly increased in situ in OLP compared to normal oral mucosa, and seems to be related to the epithelial thickness. PMID:11695759
Neppelberg, E; Johannessen, A C; Jonsson, R
Abstract In multicellular organisms, cell proliferation and death must be regulated to maintain tissue homeostasis. Many observations suggest that this regulation may be achieved, in part, by coupling the process of cell cycle progression and programmed cell death by using and controlling a shared set of factors. An argument in favor of a link between the cell cycle and apoptosis arises from the accumulated evidence that manipulation of the cell cycle may either prevent or induce an apoptotic response. This linkage has been recognized for tumor suppressor genes such as p53 and RB, the dominant oncogene, c-Myc, and several cyclin-dependent kinases (Cdks) and their regulators. These proteins that function in proliferative pathways may also act to sensitize cells to apoptosis. Indeed, unregulated cell proliferation can result in pathologic conditions including neoplasias if it is not countered by the appropriate cell death. Translating the knowledge gained by studying the connection between cell death and cell proliferation may aid in identifying novel therapies to circumvent disease progression or improve clinical outcome.
Pucci, Bruna; Kasten, Margaret; Giordano, Antonio
HIV-1 Induced Bystander Apoptosis
Apoptosis of uninfected bystander cells is a key element of HIV pathogenesis and believed to be the driving force behind the selective depletion of CD4+ T cells leading to immunodeficiency. While several viral proteins have been implicated in this process the complex interaction between Env glycoprotein expressed on the surface of infected cells and the receptor and co-receptor expressing bystander cells has been proposed as a major mechanism. HIV-1 utilizes CD4 as the primary receptor for entry into cells; however, it is the viral co-receptor usage that greatly influences CD4 decline and progression to AIDS. This phenomenon is relatively simple for X4 viruses, which arise later during the course of the disease, are considered to be highly fusogenic, and cause a rapid CD4+ T cell decline. However, in contrast, R5 viruses in general have a greater transmissibility, are encountered early during the disease and have a lesser pathogenic potential than the former. The above generalization gets complicated in numerous situations where R5 viruses persist throughout the disease and are capable of causing a rigorous CD4+ T cell decline. This review will discuss the multiple factors that are reported to influence HIV induced bystander apoptosis and pathogenesis including Env glycoprotein phenotype, virus tropism, disease stage, co-receptor expression on CD4+ T cells, immune activation and therapies targeting the viral envelope.
Garg, Himanshu; Mohl, Jonathon; Joshi, Anjali
CELL BIOLOGY: Apoptosis--the Calcium Connection
NSDL National Science Digital Library
Access to the article is free, however registration and sign-in are required. Calcium ions are crucial to many cellular processes including apoptosis. In their Perspective, Demaurex and Distelhorst explain new work that shows how calcium ions flowing between the endoplasmic reticulum (ER) and mitochondria regulate programmed cell death, highlighting the ER as a new gateway to apoptosis (Scorrano et al.).
Nicolas Demaurex (University of Geneva Medical Center;Department of Physiology); Clark Distelhorst (Case Western Reserve University Medical School;Department of Medicine)
Regulation of Apoptosis in Prostate Cancer
Transformation and malignant progression of prostate cancer is regulated by the inability of prostatic epithelial cells to undergo apoptosis rather than by increased cell proliferation. The basic apoptotic machinery of most prostate cancer cells is intact and the inability to undergo apoptosis is due to molecular alterations that result in failure to initiate or execute apoptotic pathways. This review discusses
Sushma Gurumurthy; Krishna Murthi Vasudevan; Vivek M. Rangnekar
Serine\\/Threonine Protein Kinases and Apoptosis
Over the past decade, our understanding of apoptosis, or programmed cell death, has increased greatly, with the identification of some of the major components of the apoptotic programme and the processes regulating their activation. Although apoptosis is an intrinsic process present in all cells, it can be regulated by extrinsic factors, including hormones, growth factors, cell surface receptors, and cellular
Timothy G. Cross; Dagmar Scheel-Toellner; Nick V. Henriquez; Elizabeth Deacon; Mike Salmon; Janet M. Lord
Intracellular mediators of erucylphosphocholine-induced apoptosis.
Induction of apoptosis contributes to the cytotoxic action of the intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC). To define molecular requirements for ErPC-induced apoptosis, activation of caspases-8, -9 and -3 and cleavage of the caspase-3 substrates PARP and ICAD were tested in normal Jurkat T cells, Jurkat cells resistant to death receptor (CD95 or TNFalpha-related apoptosis inducing ligand (TRAIL)-induced apoptosis, Jurkat cells lacking caspase-8 or Fas-associated death domain (FADD) Jurkat cells expressing a dominant-negative caspase-9 or overexpressing Bcl-2 as well as BJAB B-lymphoma cells expressing a dominant-negative FADD (FADD-DN). ErPC induced a time- and dose-dependent apoptotic cell death in Jurkat and BJAB cells, which was characterized by breakdown of the phosphatidylserine asymmetry, depolarization of the mitochondrial membrane potential, release of cytochrome c, activation of caspases-9, -8 and -3, cleavage of PARP and ICAD, as well as chromatin condensation. ErPC-induced apoptosis was independent from CD95-receptor signaling and FADD since CD95- and TRAIL-resistant, caspase-8- and FADD-negative Jurkat cells, as well as BJAB cells expressing FADD-DN were sensitive to ErPC-induced apoptosis. In contrast, inhibition of caspase-9 and overexpression of Bcl-2 significantly reduced ErPC-induced caspase activation and apoptosis. Thus, ErPC triggers apoptosis via a Bcl-2-dependent mitochondrial but death receptor-independent pathway. PMID:12730676
Jendrossek, Verena; M�ller, Ilka; Eibl, Hansj�rg; Belka, Claus
Histochemical detection of apoptosis in Parkinson's disease
Apoptosis may be an important mechanism of cell death in some experimental disease models as well as in human neurodegenerative disorders. We report evidence to indicate apoptosis in the substantia nigra of Parkinson's disease patients. We studied the midbrains of 7 patients with late onset PD, 4 patients with young onset PD and 6 control subjects using the nick-end labeling
Hideki Mochizuki; Keigo Goto; Hideo Mori; Yoshikuni Mizuno
Morphine promotes apoptosis in Jurkat cells
Patients with intravenous heroin addic- tion are prone to recurrent infections and at times these infections are fatal. We evaluated the effect of morphine on the apoptosis of Jurkat cells and freshly isolated human T lymphocytes. Morphine promoted apoptosis of both the Jurkat cells and the freshly isolated T lymphocytes in a dose-dependent manner. DAGO, a specific � receptor agonist,
Pravin C. Singhal; Aditi A. Kapasi; Krishna Reddy; Nicholas Franki; Nora Gibbons; Guohua Ding
Enhancing antimelanoma immune responses through apoptosis
We examined the feasibility of using tumor apoptosis at accessible sites to enhance antimelanoma immune responses in a model of spontaneous canine melanoma. We show that priming peripheral blood mononuclear cells with apoptotic melanoma cells significantly enhanced autologous and allogeneic lymphokine-activated killing of tumor cells. Since various pathways required for intrinsic apoptosis are often inactivated in melanoma, we used Fas
Stacie R Bianco; Juan Sun; Susan P Fosmire; Kenneth Hance; Marcia L Padilla; Michelle G Ritt; David M Getzy; Richard C Duke; Stephen J Withrow; Susan Lana; David T Matthiesen; Steven W Dow; Donald Bellgrau; Gary R Cutter; Stuart C Helfand; Jaime F Modiano
The role of apoptosis in neurodegeneration in developing animals and in adults has become increasingly apparent in the past ten years. Normal apoptosis occurs in the CNS from the embryonic stage through senescence, with different cells in each region of the nervous system having ...
The Paracrine Role of Stem Cell Factor\\/c-kit Signaling in the Activation of Human Melanocytes in Ultraviolet-B-Induced Pigmentation
The interaction of stem cell factor with its receptor, c-kit, is well known to be critical to the survival of melanocytes. Little is known about the role(s) of the stem cell factor\\/c-kit interaction in epidermal pigmentation, however. To clarify whether the stem cell factor\\/c-kit signaling has a paracrine role in ultraviolet-B-induced pigmentation, we determined whether the exposure of human keratinocytes,
Akira Hachiya; Akemi Kobayashi; Atsushi Ohuchi; Yoshinori Takema; Genji Imokawa
Phospholipase C? has a crucial role in ultraviolet B-induced neutrophil-associated skin inflammation by regulating the expression of CXCL1\\/KC
Phospholipase C (PLC) ? is a phosphoinositide-specific PLC regulated by small GTPases including Ras and Rap. We previously demonstrated that PLC? has an important role in the development of phorbol ester-induced skin inflammation. In this study, we investigated the role of PLC? in ultraviolet (UV) B-induced acute inflammatory reactions in the skin. Wild-type (PLC?+\\/+) and PLC? gene knockout (PLC??\\/?) mice
Masahiro Oka; Hironori Edamatsu; Makoto Kunisada; Lizhi Hu; Nobuyuki Takenaka; Masanobu Sakaguchi; Tohru Kataoka; Chikako Nishigori
Role of nitric oxide in UV-B-induced activation of PAL and stimulation of flavonoid biosynthesis in Ginkgo�biloba callus
The role of nitric oxide (NO) in UV-B-induced secondary metabolite accumulation in Ginkgo�biloba callus was investigated. Overall, UV-B irradiation induced multiple biological responses in callus of G.�biloba, including increased both NO production and nitric oxide synthase (NOS) activity, and subsequent activation of phenylalanine\\u000a ammonium lyase (PAL) and synthesis of flavonoids. Application of NO via the donor sodium nitroprusside (SNP) enhanced
Gangping Hao; Xihua Du; Faxing Zhao; Renjiu Shi; Jianmei Wang
VASP Activation via the G?13/RhoA/PKA Pathway Mediates Cucurbitacin-B-Induced Actin Aggregation and Cofilin-Actin Rod Formation
Cucurbitacin B (CuB), a potent antineoplastic agent of cucurbitacin triterpenoids, induces rapid disruption of actin cytoskeleton and aberrant cell cycle inhibiting carcinogenesis. However, the underlying molecular mechanism of such anticancer effects remains incompletely understood. In this study, we showed that CuB treatment rapidly induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation (i.e. activation) at the Ser157 residue and generated VASP clumps which were co-localized with amorphous actin aggregates prior to the formation of highly-ordered cofilin-actin rods in melanoma cells. Knockdown of VASP or inhibition of VASP activation using PKA-specific inhibitor H89 suppressed CuB-induced VASP activation, actin aggregation and cofilin-actin rod formation. The VASP activation was mediated by cAMP-independent PKA activation as CuB decreased the levels of cAMP while MDL12330A, an inhibitor of adenylyl cyclase, had weak effect on VASP activation. Knockdown of either G?13 or RhoA not only suppressed VASP activation, but also ameliorated CuB-induced actin aggregation and abrogated cofilin-actin rod formation. Collectively, our studies highlighted that the CuB-induced actin aggregation and cofilin-actin rod formation was mediated via the G?13/RhoA/PKA/VASP pathway.
Zhang, Yan-Ting; Xu, Li-Hui; Lu, Qun; Liu, Kun-Peng; Liu, Pei-Yan; Ji, Fang; Liu, Xiao-Ming; Ouyang, Dong-Yun; He, Xian-Hui
Mitochondrial apoptosis is amplified through gap junctions
The death of one cell can precipitate the death of nearby cells in a process referred to as the bystander effect. We investigated whether mitochondrial apoptosis generated a bystander effect and, if so, by which pathway. Microinjection with cytochrome c mimicked function of the mitochondrial apoptosis-induced channel MAC and caused apoptosis of both target and nearby osteoblasts. This effect was suppressed by inhibiting gap junction intercellular communication. A bystander effect was also observed after exogenous expression of tBid, which facilitates MAC formation and cytochrome c release. Interestingly, in connexin-43 deficient osteoblasts, microinjection of cytochrome c induced apoptosis only in the target cell. These findings indicate that a death signal was generated downstream of MAC function and was transmitted through gap junctions to amplify apoptosis in neighboring cells. This concept may have implications in development of new therapeutic approaches.
Peixoto, Pablo M.; Ryu, Shin-Young; Pietkiewicz-Pruzansky, Dawn; Kuriakose, Maria; Gilmore, Andrew; Kinnally, Kathleen W.
Analysis of Apoptosis in Caenorhabditis elegans.
The nematode worm Caenorhabditis elegans has provided researchers with a wealth of information on the molecular mechanisms controlling programmed cell death (apoptosis). Its genetic tractability, optical clarity, and relatively short lifespan are key advantages for rapid assessment of apoptosis in vivo. The use of forward and reverse genetics methodology, coupled with in vivo imaging, has provided deep insights into how a multicellular organism orchestrates the self-destruction of specific cells during development and in response to exogenous stresses. Strains of C. elegans carrying mutations in the core elements of the apoptotic pathway, or in tissue-specific regulators of apoptosis, can be used for genetic analyses to reveal conserved mechanisms by which apoptosis is regulated in the somatic and reproductive (germline) tissue. Here we present an introduction to the study of apoptosis in C. elegans, including current techniques for visualization, analysis, and screening. PMID:24786497
Lant, Benjamin; Derry, W Brent
Apoptosis inducers in chronic lymphocytic leukemia
Chronic lymphocytic leukemia (CLL) is characterized by a typical defect in apoptosis and is still an incurable disease. Numerous apoptosis inducers have been described. These synthetic compounds and natural products (mainly derived from plants) display antileukemic properties in vitro and in vivo and some have even been tested in the clinic in CLL. They act through several different mechanisms. Most of them involve proteins of the Bcl-2 family, which are the key regulators in triggering the mitochondrial pathway of caspase-dependent apoptosis. Thus, the Mcl-1/Noxa axis appeared as a target. Here I overview natural and synthetic apoptosis inducers and their mechanisms of action in CLL cells. Opportunities for developing novel, apoptosis-based therapeutics are presented.
Billard, Christian
The tumor suppressor DAL-1/4.1B and protein methylation cooperate in inducing apoptosis in MCF-7 breast cancer cells
Background DAL-1 (Differentially Expressed in Adenocarcinoma of the Lung)/4.1B is a member of the protein 4.1 superfamily that has been shown to suppress growth in lung, breast and brain tumor cells. In the case of the caspase-3 deficient MCF-7 breast cancer cells, this growth suppression has been shown to be partially mediated by the induction of apoptosis. However the exact mechanism of action of DAL-1/4.1B is unknown. Recently, protein arginine N-methyltransferase 3 (PRMT3) was identified as a DAL-1/4.1B interacting protein. Protein arginine methyltransferases (PRMTs) posttranslationally methylate the arginine residues of proteins, a modification which has been implicated in the regulation of multiple cellular processes including nuclear-cytoplasmic transport, signal transduction, and transcription. Results To investigate the role of protein methylation in cell death induced by DAL-1/4.1B, DAL-1/4.1B-inducible MCF-7 cells were examined for apoptosis and caspase activation in the absence and presence of the protein methylation inhibitor adenosine dialdehyde (AdOX). Flow cytometry analysis revealed that apoptosis was primarily associated with the activation of caspase 8, and inhibition of this activation blocked the ability of DAL-1/4.1B to induce cell death. Conclusion These results suggest that protein methylation cooperates with DAL-1/4.1B-associated caspase 8-specific activation to induce apoptosis in breast cancer cells.
Jiang, Wei; Newsham, Irene F
Bcl-2 blocks p53-dependent apoptosis.
Adenovirus E1A expression recruits primary rodent cells into proliferation but fails to transform them because of the induction of programmed cell death (apoptosis). The adenovirus E1B 19,000-molecular-weight protein (19K protein), the E1B 55K protein, and the human Bcl-2 protein each cause high-frequency transformation when coexpressed with E1A by inhibiting apoptosis. Thus, transformation of primary rodent cells by E1A requires deregulation of cell growth to be coupled to suppression of apoptosis. The product of the p53 tumor suppressor gene induces apoptosis in transformed cells and is required for induction of apoptosis by E1A. The ability of Bcl-2 to suppress apoptosis induced by E1A suggested that Bcl-2 may function by inhibition of p53. Rodent cells transformed with E1A plus the p53(Val-135) temperature-sensitive mutant are transformed at the restrictive temperature and undergo rapid and complete apoptosis at the permissive temperature when p53 adopts the wild-type conformation. Human Bcl-2 expression completely prevented p53-mediated apoptosis at the permissive temperature and caused cells to remain in a predominantly growth-arrested state. Growth arrest was leaky, occurred at multiple points in the cell cycle, and was reversible. Bcl-2 did not affect the ability of p53 to localize to the nucleus, nor were the levels of the p53 protein altered. Thus, Bcl-2 diverts the activity of p53 from induction of apoptosis to induction of growth arrest, and it is thereby identified as a modifier of p53 function. The ability of Bcl-2 to bypass induction of apoptosis by p53 may contribute to its oncogenic and antiapoptotic activity. Images
Chiou, S K; Rao, L; White, E
Lymphocyte apoptosis in murine Pneumocystis pneumonia
Background Apoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due to Pneumocystis murina, an infection localized to the lungs. Methods Control mice and mice depleted of CD4+ lymphocytes were inoculated with Pneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins. Results In control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim. Conclusion Apoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response to Pneumocystis but is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins.
Shi, Xin; LeCapitaine, Nicole J; Rudner, Xiaowen L; Ruan, Sanbao; Shellito, Judd E
APOPTOSIS: Death of a Monopoly?
NSDL National Science Digital Library
Access to the article is free, however registration and sign-in are required. Hunot and Flavell discuss recent findings in Nature by Joza et al. that point to the existence of a new sort of cell death. Cell death is required during the development of an organism to remove portions of the body that will not be needed; this cell death usually requires destructive enzymes called caspases to perform the final demolition of the cell. But cavitation, an early developmental process in the mouse embryo that requires cell death, has now been suggested to occur through a caspase-independent pathway that instead uses a mitochondrial protein called AIF (apoptosis-inducing factor).
St�©phane Hunot (Yale University School of Medicine;Section of Immunobiology and the Howard Hughes Medical Institute); Richard A. Flavell (Yale University School of Medicine;Section of Immunobiology and the Howard Hughes Medical Institute)
Determination of urinary aromatic amines in smokers and nonsmokers using a MIPs-SPE coupled with LC-MS/MS method.
Urinary aromatic amines (AAs) could be used as biomarkers for human exposure to AAs in cigarette smoke. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of urinary AAs (i.e. 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), 3-aminobiphenyl (3-ABP) and 4-aminobiphenyl (4-ABP)) in smokers and nonsmokers. A molecularly imprinted polymers (MIPs) solid phase extraction (SPE) cartridge was applied to purify urine samples and no derivatization reaction was involved. Each analytes used respective stable isotope internal standards, which could well compensate matrix effect. Lower limit of detections (LODs) for four AAs were obtained and in the range of 1.5-5ngL(-1). Recovery ranged from 87.7�4.5% to 111.3�6.4% and precision were less than 9.9%. The method was applied to analyze urine samples of 40 smokers and 10 nonsmokers. The 24h urinary excretion amounts of total AAs were higher for smokers compared with nonsmokers. What's more, 1-NA, 3-ABP and 4-ABP excretion amounts showed significant differences (p<0.05) between smokers and nonsmokers. PMID:24735928
Yu, Jingjing; Wang, Sheng; Zhao, Ge; Wang, Bing; Ding, Li; Zhang, Xiaobing; Xie, Jianping; Xie, Fuwei
Preconcentration of organochlorine pesticides in aqueous samples by dispersive liquid-liquid microextraction based on solidification of floating organic drop after SPE with multiwalled carbon nanotubes.
SPE joined with dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) as a novel technique combined with GC with electron-capture detection has been developed as a preconcentration technique for the determination of organochlorine pesticides (OCPs) in water samples. Aqueous samples were loaded onto multiwalled carbon nanotubes as sorbent. After the elution of the desired compounds from the sorbent by using acetone, the DLLME-SFO technique was performed on the obtained solution. Variables affecting the performance of both steps such as sample solution flow rate, breakthrough volume, type and volume of the elution, type and volume of extraction solvent and salt addition were studied and optimized. The new method provided an ultra enrichment factor (8280-28221) for nine OCPs. The calibration curves were linear in the range of 0.5-1000 ng/L, and the LODs ranged from 0.1-0.39 ng/L. The RSD, for 0.01 ?g/L of OCPs, was in the range of 1.39-13.50% (n = 7). The recoveries of method in water samples were 70-113%. PMID:24288158
Mirzaei, Mohammad; Rakh, Mojgan
SpeX SPECTROSCOPY OF UNRESOLVED VERY LOW MASS BINARIES. I. IDENTIFICATION OF 17 CANDIDATE BINARIES STRADDLING THE L DWARF/T DWARF TRANSITION
We report the identification of 17 candidate brown dwarf binaries whose components straddle the L dwarf/T dwarf transition. These sources were culled from a large near-infrared spectral sample of L and T dwarfs observed with the Infrared Telescope Facility SpeX spectrograph. Candidates were selected on the basis of spectral ratios which segregate known (resolved) L dwarf/T dwarf pairs from presumably single sources. Composite templates, constructed by combining 13,581 pairs of absolute flux-calibrated spectra, are shown to provide statistically superior fits to the spectra of our 17 candidates as compared to single templates. Ten of these candidates appear to have secondary components that are significantly brighter than their primaries over the 1.0-1.3 {mu}m band, indicative of rapid condensate depletion at the L dwarf/T dwarf transition. Our results support prior indications of enhanced multiplicity amongst early-type T dwarfs; 53% +- 7% of the T0-T4 dwarfs in our spectral sample are found to be either resolved or unresolved (candidate) pairs, although this is consistent with an intrinsic (volume complete) brown dwarf binary fraction of only 15%. If verified, this sample of spectral binaries more than doubles the number of known L dwarf/T dwarf transition pairs, enabling a broader exploration of this poorly understood phase of brown dwarf atmospheric evolution.
Burgasser, Adam J. [Center for Astrophysics and Space Science, University of California San Diego, La Jolla, CA 92093 (United States); Cruz, Kelle L. [Astronomy Department, California Institute of Technology, Pasadena, CA 91125 (United States); Cushing, Michael; Looper, Dagny L. [Institute for Astronomy, University of Hawaii, 2680 Woodlawn Drive, Honolulu, HI 96822 (United States); Gelino, Christopher R.; Kirkpatrick, J. Davy [Infrared Processing and Analysis Center, California Institute of Technology, Pasadena, CA 91125 (United States); Faherty, Jacqueline K. [Department of Astrophysics, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10034 (United States); Reid, I. Neill, E-mail: email@example.com [Space Telescope Science Institute, 3700 San Marin Drive, Baltimore, MD 21218 (United States)
Occurrence of polar organic contaminants in the dissolved water phase of the Danube River and its major tributaries using SPE-LC-MS(2) analysis.
Polar water-soluble organic contaminants were analysed in the dissolved liquid water phase of river water samples from the Danube River and its major tributaries (within the Joint Danube Survey 2). Analyses were performed by solid-phase extraction (SPE) followed by triple-quadrupole liquid chromatography mass spectrometry (LC-MS(2)). In total, 34 different polar organic compounds were screened. Focus was given on pharmaceutical compounds (such as ibuprofen, diclofenac, sulfamethoxazole, carbamazepine), pesticides and their degradation products (e.g. bentazone, 2,4-D, mecoprop, atrazine, terbutylazine, desethylterbutylazine), perfluorinated acids (PFOS; PFOA), and endocrine disrupting compounds (nonylphenol, NPE(1)C, bisphenol A, estrone). The most relevant polar compounds identified in the Danube River basin in terms of frequency of detection, persistency, and concentration levels were 1H-benzotriazole (median concentration 185 ng/L), caffeine (87 ng/L), tolyltriazole (73 ng/L), nonylphenoxy acetic acid (49 ng/L), carbamazepine (33 ng/L), 4-nitrophenol (29 ng/L), 2,4-dinitrophenol (19 ng/L), PFOA (17 ng/L), sulfamethoxazole (16 ng/L), desethylatrazine (11 ng/L), and 2,4-D (10 ng/L). The highest contamination levels were found in the area around Budapest and in the tributary rivers Arges (Romania), Timok (Bulgaria), Rusenski Lom (Bulgaria), and Velika Morava (Serbia). PMID:20074769
Loos, Robert; Locoro, Giovanni; Contini, Serafino
Development of a targeted method for twenty-three metabolites related to polyphenol gut microbial metabolism in biological samples, using SPE and UHPLC-ESI-MS/MS.
An increasing number of studies have concerned the profiling of polyphenol microbial metabolites, especially in urine or plasma, but only a few have regarded their accurate quantification. This study reports on a new ultra-performance liquid chromatography tandem mass spectrometry method with electrospray ionisation (UHPLC-ESI-MS/MS) using a simple clean-up step with solid phase extraction (SPE) and validation on different biological matrices. The method was tested with spiked samples of liver, heart, kidneys, brain, blood and urine. The purification procedure, after the evaluation of three different cartridges, makes it possible to obtain cleaner samples and better quantification of putative trace metabolites, especially related to dietary studies, with concentrations below ng/g in tissue and for urine and blood, starting from ng/ml. Limits of detection and linear range were also assessed using mixed polyphenol metabolite standards. Short chromatographic separation was carried out for 23 target compounds related to the polyphenol microbial metabolism, coupled with a triple quadrupole mass spectrometer for their accurate quantification. By analysing different spiked biological samples we were able to test metabolite detection in the matrix and validate the overall recovery of the method, from purification to quantification. The method developed can be successfully applied and is suitable for high-throughput targeted metabolomics analysis related to nutritional intervention, or the study of the metabolic mechanism in response to a polyphenol-rich diet. PMID:25059152
Gasperotti, Mattia; Masuero, Domenico; Guella, Graziano; Mattivi, Fulvio; Vrhovsek, Urska
Use of SPE-GC/EIMS for residue analysis in wine elaborated from musts spiked with formulations of chlorpyriphos-methyl, methiocarb, dicofol, and cyproconazol.
The likely presence in wine of residues of the active ingredient and its degradation products, besides the byproducts and excipients of the commercial formulation, has been investigated for four pesticides. Formulations containing chlorpyriphos-methyl, methiocarb, dicofol, and cyproconazol were added to must, which was subjected to a usual vinification. The wines elaborated from must spiked with the formulation of chlorpyriphos-methyl contained two pyridinol compounds in addition to excipients such as alkylbenzenes, naphthalene, and methylnaphthalenes. Methiocarb was hydrolyzed to yield the corresponding phenol, and various unidentified compounds related to cyproconazol were observed in wine. The residues of the dicofol-containing formulation resulted to be dechlorination products; impurities from its commercial formulation were also detected in must and wine extracts. White wines contained higher amounts of residues than red wines. The residues were detected after an SPE followed by GC/EIMS in the scan mode. The concentrations of the active ingredients were determined by a matrix-matched calibration to avoid quantitative errors arising from the matrix. PMID:17444223
Jim�nez, Juan Jos�; Bernal, Jos� Luis; del Nozal, Mar�a J; Toribio, Laura; Bernal, Jos�
Stable isotopic internal standard correction for quantitative analysis of hydroxyeicosatetraenoic acids (HETEs) in serum by on-line SPE-LC-MS/MS in selected reaction monitoring mode.
The influence of the inclusion of a stable isotopic labeled internal standard (SIL-IS) on the quantitative analysis of hydroxyeicosatetranoic acids (HETEs) in human serum is evaluated in this research. A solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) platform, one of the preferred approaches for targeted analysis of biofluids through the selected reaction monitoring (SRM) operational mode, was used to determine HETEs. These compounds were chosen as targeted metabolites because of their involvement in cardiovascular disease, cancer and osteoporosis. 15HETE-d8 was chosen as internal standard to evaluate matrix effects. Thus, the physico-chemical properties of the SIL-IS were the basis to evaluate the analytical features of the method for each metabolite through four calibration models. Two of the models were built with standard solutions at different concentration levels, but one of the calibration sets was spiked with an internal standard (IS). The other two models were built with the serum pool from osteoporotic patients, which was spiked at different concentrations with the target analytes. In this case, one of the serum calibration sets was also spiked with the IS. The study shows that the IS allowed noticeable correction of matrix effects for some HETE isomers at certain concentration levels, while accuracy was decreased at low concentration (15ng/mL) of them. Therefore, characterization of the method has been properly completed at different concentration levels. PMID:24881549
Fern�ndez-Peralbo, M A; Ferreiro Vera, C; Priego-Capote, F; Luque de Castro, M D
Optimization and validation of a simple and fast method for the determination of fungicides in must and wine samples by SPE and GC/MS.
A rapid, simple, and low-cost method based on SPE was optimized and validated for simultaneous determination of eight fungicides belonging to different chemical classes in must and wine. The method involves extraction of 10 mL of must or wine samples with a C18 cartridge using 5 mL of dichloromethane as the elution solvent. Separation and final determination of the fungicides (vinclozolin, dichlofluanid, penconazol, captan, quinoxyfen, fluquinconazol, boscalid, and pyraclostrobin) was performed by GC coupled to single quadrupole MS. Recoveries at 10, 50, and 100 microg/L were between 71 and 106% in both matrixes for the fungicides evaluated. The calculated LOQ ranged from 1.5 to 3.4 microg/L in must and 1.1 to 3.8 microg/L in wine. Matrix effects observed for wine and must samples were overcome by using matrix-matched calibration. The developed method was linear at concentrations within the tested interval, with coefficients of determination higher than 0.999. The expanded uncertainties at 10 microg/L were <20% for all analytes. Intralaboratory precision in terms of the Horwitz ratio of the fungicides evaluated was below 0.5, suggesting the ruggedness of the method. The proposed method was applied to determine fungicide residues in must samples obtained from red grapes treated with two new commercial formulations, as well as in their corresponding final wines. PMID:23175987
Lagunas-Allu�, Laura; Sanz-Asensio, Jes�s; Mart�nez-Soria, Maria-Teresa
Novel magnetic SPE method based on carbon nanotubes filled with cobalt ferrite for the analysis of organochlorine pesticides in honey and tea.
A novel magnetic SPE method based on magnetic cobalt ferrite filled carbon nanotubes (MFCNTs) coupled with GC with electron capture detection was developed to determine organochlorine pesticides (OCPs) in tea and honey samples. The MFCNTs were prepared through the capillarity of carbon nanotubes for drawing mixed cobalt and iron nitrates solution into their inner cavity followed by heating to 550�C under Ar to form the cobalt ferrite nanoparticles. SEM images provided visible evidence of the filled cobalt ferrite nanoparticles in the multiwalled nanotubes. X-ray photoelectron spectroscopy indicated no adhesion of cobalt ferrite nanoparticles and metal salts on the outer surface of the MFCNTs. Eight OCPs were extracted with the MFCNTs. The enrichment factors were in the range of 52-68 for eight OCPs. The LODs for the eight OCPs were in the range of 1.3-3.6 ng/L. The recoveries of the OCPs for honey and tea samples were 83.2-128.7 and 72.6-111.0%, respectively. The RSDs for these samples were below 6.8%. The new method is particularly suited to extract nonpolar and weakly polar analytes from a complex matrix and could potentially be extended to other target analytes. PMID:23926126
Du, Zhuo; Liu, Miao; Li, Gongke
Determination of parent and substituted polycyclic aromatic hydrocarbons in high-fat salmon using a modified QuEChERS extraction, dispersive SPE and GC-MS
A fast and easy modified QuEChERS (quick, easy, cheap, rugged and safe) extraction method has been developed and validated for determination of 33 parent and substituted polycyclic aromatic hydrocarbons (PAHs) in high-fat smoked salmon that greatly enhances analyte recovery compared to traditional QuEChERS procedures. Sample processing includes extraction of PAHs into a solution of ethyl acetate, acetone and iso-octane followed by cleanup with dispersive SPE and analysis by GC-MS in SIM mode. Method performance was assessed in spike recovery experiments (500 ng/g wet weight) in three commercially available smoked salmon with 3 � 11% fat. Recoveries of some 2, 3 and 5-ring PAHs were improved 50 � 200% over traditional methods, while average recovery across all PAHs was improved 67%. Method precision was good with replicate extractions typically yielding relative standard deviations < 10% and detection limits were in the low ng/g range. With this method, a single analyst could extract and cleanup ? 60 samples for PAH analysis in an 8 hour work day.
Forsberg, Norman D.; Wilson, Glenn R.; Anderson, Kim A.
Determination of parent and substituted polycyclic aromatic hydrocarbons in high-fat salmon using a modified QuEChERS extraction, dispersive SPE and GC-MS.
A fast and easy modified QuEChERS (quick, easy, cheap, rugged and safe) extraction method has been developed and validated for determination of 33 parent and substituted polycyclic aromatic hydrocarbons (PAHs) in high-fat smoked salmon that greatly enhances analyte recovery compared to traditional QuEChERS procedures. Sample processing includes extraction of PAHs into a solution of ethyl acetate, acetone and isooctane followed by cleanup with dispersive SPE and analysis by GC-MS in SIM mode. Method performance was assessed in spike recovery experiments (500 ?g/g wet weight) in three commercially available smoked salmon with 3-11% fat. Recoveries of some 2-, 3- and 5-ring PAHs were improved 50-200% over traditional methods, while average recovery across all PAHs was improved 67%. Method precision was good with replicate extractions typically yielding relative standard deviations <10%, and detection limits were in the low ng/g range. With this method, a single analyst could extract and clean up ?60 samples for PAH analysis in an 8 h work day. PMID:21732651
Forsberg, Norman D; Wilson, Glenn R; Anderson, Kim A
An update for human blood plasma pretreatment for optimized recovery of low-molecular-mass peptides prior to CE�MS and SPE�CE�MS.
Protein precipitation and centrifugal filtration are well-established methods for concentrating and purifying peptides with a low relative molecular mass (Mr) from human blood plasma before proteomic and peptidomic studies using high-performance separation techniques, but there is little information on peptide recoveries. Here, we evaluate acetonitrile precipitation followed by a range of centrifugal filtration conditions for the analysis of low Mr peptides in human blood plasma before CE�MS and SPE coupled online to CE�MS. Three opioid peptides were used as model compounds, that is, dynorphin A 1�7, endomorphin 1, and methionine enkephalin and 3, 10, and 30 K Mr cut-off cellulose acetate filters (Amicon� Ultra-0.5) and 10 K Mr cut-off polyethersulfone filters (Vivaspin� 500) were studied. Unexpectedly, recoveries and repeatability were only optimum after passivating the 10 K Mr cut-off cellulose acetate filters with PEG to avoid peptide adsorption on the inner walls of the plastic sample reservoir. PMID:24151123
Pont, Laura; Benavente, Fernando; Barbosa, Jos�; Sanz-Nebot, Victoria
Determination of priority pesticides in water samples combining SPE and SPME coupled to GC-MS. A case study: Suqu�a River basin (Argentina).
This study reports a combined method using solid phase extraction (SPE), followed by solid phase microextraction (SPME) to concentrate different pesticides, including chlorinated, organophosphorus, triazines, pyretroids and chloroacetamides, present at trace levels in water samples. Identification and quantification was carried out by gas chromatography coupled to Mass Spectrometry (GC-MS). The optimized methodology showed LOQs at ng L(-1) levels (ranging 0.2-3.5 ng L(-1)) in addition to acceptable precision and robustness (recoveries ranged 63-104%, RSD from 4% to 23%), presenting a novel method to reach trace levels, similar to that obtainable using EC detector, with structural confirmation by MS during the analysis of a wide range of environmental pollutants. This method was applied to the study of temporal and spatial distribution of pesticides in the Suqu�a River basin (C�rdoba-Argentina). As expected, highest levels of agrochemicals were observed in areas with intensive agricultural practices, being atrazine (max.=433.9 ng L(-1)), alpha-cypermetrine (max.=121.7 ng L(-1)) and endosulfan sulfate (max.=106.7 ng L(-1)) predominant. In urban areas, the prevalent pesticide was alpha-cypermethrine. These results draw attention to the need of pesticide monitoring programs in rivers, considering both urban and rural sections. PMID:23177716
Bonansea, Roc�o In�s; Am�, Mar�a Valeria; Wunderlin, Daniel Alberto
Pharmacokinetic study of six flavones in rat plasma and tissues after oral administration of 'JiangYaBiFeng' using SPE-HPLC-DAD.
In this study, a high performance liquid chromatography (HPLC) coupled with diode array detection (DAD) for simultaneous determination of six flavones including baicalein, sophoricoside, rutin, baicalin, quercetin and genistein in rat plasma and tissues after oral administration of JiangYaBifeng (JYBF) tablets was developed. The investigated analytes in plasma and tissues were extracted and purified with liquid-liquid extraction and solid phase extraction (SPE). Chromatographic separation was accomplished on a DIONEX Acclaim C18 column (250mm�4.6mm, 5.0?m particle size) with a simple linear gradient elution. The calibration curves for all the flavones had good linearity in the measured range with R(2) higher than 0.9983. The relative errors (REs) of the intra- and inter-day accuracy at different flavones levels were all less than �10%. The proposed method enables unambiguous identification and quantification of investigated flavones in vivo. This is the first report on determination of the major flavones in rat plasma and tissues after oral administration of JYBF tablets. The results provided a meaningful basis for evaluating the clinical application of this medicine. PMID:21831556
Zeng, Hua-jin; Yang, Ran; Guo, Cheng; Wang, Qing-wen; Qu, Ling-bo; Li, Jian-jun
Placental Apoptosis in Health and Disease
Apoptosis, programmed cell death, is an essential feature of normal placental development but is exaggerated in association with placental disease. Placental development relies upon effective implantation and invasion of the maternal decidua by the placental trophoblast. In normal pregnancy, trophoblast apoptosis increases with placental growth and advancing gestation. However, apoptosis is notably exaggerated in the pregnancy complications, hydatidiform mole, pre-eclampsia, and intra-uterine growth restriction (IUGR). Placental apoptosis may be initiated by a variety of stimuli, including hypoxia and oxidative stress. In common with other cell-types, trophoblast apoptosis follows the extrinsic or intrinsic pathways culminating in the activation of caspases. In contrast, the formation of apoptotic bodies is less clearly identified, but postulated by some to involve the clustering of apoptotic nuclei and liberation of this material into the maternal circulation. In addition to promoting a favorable maternal immune response, the release of this placental-derived material is thought to provoke the endothelial dysfunction of pre-eclampsia. Widespread apoptosis of the syncytiotrophoblast may also impair trophoblast function leading to the reduction in nutrient transport seen in IUGR. A clearer understanding of placental apoptosis and its regulation may provide new insights into placental pathologies, potentially suggesting therapeutic targets.
Sharp, Andrew N.; Heazell, Alexander E.P.; Crocker, Ian P.; Mor, Gil
Manumycin induces apoptosis in prostate cancer cells
Background Manumycin exhibits an antitumor effect in a variety of cancer cell lines, including prostate cancer cell lines (DU145 and PC-3). Our previous studies demonstrated that manumycin induced the apoptosis of anaplastic thyroid cancer cells and leukemia cells via the intrinsic apoptosis pathway. In the current study, we further evaluated the effect of manumycin in two prostate cancer cell lines (LNCaP and 22Rv1), and here we elucidate some of the underlying mechanisms. Materials and methods The cell viability of prostate cancer cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after treatment with manumycin for 48 hours. Apoptosis was detected by flow cytometry using annexin V and propidium iodide. The expressions of B-cell lymphoma (Bcl)-2 family members and the activations of caspase-9 and caspase-3 were detected by Western blotting. Results Manumycin treatment resulted in significant decreases in the viabilities of the two prostate cancer cell lines in a dose-dependent manner through apoptosis, and this apoptosis involved caspase-9 activation. A specific inhibitor of caspase-9 protected cells from caspase-3 activation, apoptosis, and cytotoxicity induced by manumycin. We also found that manumycin downregulated Bcl-2 expression and upregulated Bax expression. Conclusion Our data suggest that manumycin induces apoptosis in prostate cancer cells through regulation of the Bcl-2 family involving caspase-9 activation. These results suggest that manumycin may be beneficial for the treatment of prostate cancer.
Li, Jing-Gao; She, Miao-Rong; Lu, Ci-Yong; Wei, Shan-Shan; Xia, Ping-Fang; Lu, Ze-Sheng; Peng, Qi
Honey and apoptosis in human gastric mucosa.
Background: Gastric cancer is the fourth most common malignancy in the world. Honey is a complex mixture of special biological active constituents. Honey possesses antioxidant and antitumor properties. Nutritional studies have indicated that consumption of honey modulates the risk of developing gastric cancer. On the other hand, apoptosis has been reported to play a decisive role in precancerous changes. Our chief study was conducted to assess the relationship between consumption of honey and apoptosis in human gastric mucosa. Method: This cross-sectional study was conducted on 98 subjects over 18 years old, referred to two hospitals in Tabriz, Iran. Subjects were undergone an upper gastrointestinal endoscopy, 62 subjects were finally enrolled. Honey consumption was assessed by a Food Frequency Questionnaire (FFQ) and apoptosis was detected by TUNEL technique. We tested polynomial curve to find the best fit between honey consumption and apoptosis. Results: A positive relation between honey consumption and apoptosis was found (P=0.024). Our results indicated that the final and the best fit curve was: apoptosis = 1.714+1.648(honey amount) - 0.533(honey amount)2 +1.833�10-5(honey amount)7. Conclusion: Honey consumption had positive effects on gastric cancer by inducing apoptosis in gastric mucosa. PMID:24688918
Ghaffari, Aida; Somi, Mohammad H; Safaiyan, Abdolrasoul; Modaresi, Jabiz; Ostadrahimi, Alireza
[Neuroprotection by neurotrophic factors in apoptosis].
During development, excess neurons are produced about half of which die. The time of cell death (apoptosis) is limited to the period of formation of synapses with the target cells, and the neurons which fail to obtain sufficient amounts of trophic factor(s) released from the target cells are eliminated. This selection system is considered to be a mechanism to ensure formation of a physiologically relevant neuronal network. Mature neurons which correctly execute their functions, however, undergo apoptosis in response to exogenous toxic stimuli. Such stimuli may be responsible for neurodegenerative diseases. The mechanism underlying cell death has been analyzed using in vitro model systems. In the present communication, we used cultured rat cerebellar granule neurons, in which low potassium concentration (LK+) in the medium induces apoptosis, and this apoptosis is prevented by high concentration of potassium (HK+), BDNF. One of the lipid-modifying kinases, phosphatidylinositol 3-kinase (PI3-K), is also activated by trophic factors including neurotrophins. BDNF and high K+ prevented low K(+)-induced apoptosis via PI3-K. BDNF also promotes the survival of basal forebrain cholinergic neurons cultured from postnatal 2-week-old (P2w) rats. The mechanism of neuronal apoptosis induced by oxidative stress using CNS neurons and PC12 cells was investigated, and we found that generation of reactive oxygen species (ROS) is highly associated with apoptosis. High oxygen induced neuronal apoptosis, which was blocked by protein or RNA synthesis inhibitors. Neurotrophic factors and Bcl-2 prevented this apoptotic cell death. Exposure to hydrogen peroxide, lipid hydroperoxide or serum deprivation triggered apoptosis associated with increased generation of ROS as determined using a ROS-specific fluorescent probe. In cultured cerebellar granule neurons from 15-day-old wild-type and p53-deficient mice, we examine the role of p53 in regulating the life and death of CNS neurons. When exposure of gamma-ray or bleomycin to neurons died in p53 dependent manner. These neuronal deaths were partially prevented by actinomycin D or cycloheximide. The pycnotic nuclei observed in these dying neurons indicated that cell death occurs via apoptosis. Although there are many evidences that p53 is involved in apoptosis in proliferating cells, it is interesting that p53 is also involved in apoptosis in postmitotic neurons as shown in this study. PMID:10190124
Hatanaka, H
A multi-residue method for the analysis of organophosphorus residues in cooked and polished rice using accelerated solvent extraction and dispersive-solid phase extraction (D-SPE) technique and uncertainty measurement
Quick, simple and efficient multi-residue analytical methods were developed and validated for the determination of organophosphorous insecticides from polished and cooked rice. Polished rice was extracted using a simple, automated technique namely accelerated solvent extraction (ASE) using dichloromethane as the extraction solvent. Cooked rice was extracted with acetone and cleaned up using dispersive-solid phase extraction (D-SPE) technique. The single step
Doyeli Sanyal; Anita Rani; Samsul Alam
Nuclear translocation of cytochrome c during apoptosis.
Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis. We showed that cytochrome c, released from mitochondria upon apoptosis induction, gradually accumulates in the nucleus as evidenced by both immunofluorescence and subcellular fractionation. Parallel to nuclear accumulation of cytochrome c, acetylated histone H2A, but not unmodified H2A, was released from the nucleus to the cytoplasm. Addition of purified cytochrome c to isolated nuclei recapitulated the preferential release of acetylated, but not deacetylated, histone H2A. Cytochrome c was also found to induce chromatin condensation. These results suggest that the nuclear accumulation of cytochrome c may be directly involved in the remodeling of chromatin. Our results provide evidence of a novel role for cytochrome c in inducing nuclear apoptosis. PMID:15073175
Nur-E-Kamal, Alam; Gross, Stephane R; Pan, Zui; Balklava, Zita; Ma, Jianjie; Liu, Leroy F
Coupling HPLC-SPE-NMR with a microplate-based high-resolution antioxidant assay for efficient analysis of antioxidants in food--validation and proof-of-concept study with caper buds.
This work describes the coupling of a microplate-based antioxidant assay with a hyphenated system consisting of high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-NMR/high-resolution antioxidant assay, for the analysis of complex food extracts. The applicability of the microplate-based antioxidant assay for high-resolution screening of common food phenolics as well as parameters related to their trapping efficiency, elution behavior, and recovery on/from SPE cartridges are described. It was found that the microplate-based high-resolution antioxidant assay is an attractive and easy implementable alternative to direct on-line screening methods. Furthermore, it was shown that Resin SH and Resin GP SPE material are superior to RP C18HD for trapping of phenolic compounds. Proof-of-concept study was performed with caper bud extract, revealing the most important antioxidants to be quercetin, kaempferol, rutin, kaempferol-3-O-?-rutinoside and N(1),N(5),N(10)-triphenylpropenoyl spermidine amides. Targeted isolation of the latter, and comprehensive NMR experiments showed them to be N(1),N(10)-di-(E)-caffeoyl-N(5)-p-(E)-coumaroyl spermidine, N(1)-(E)-caffeoyl-N(5),N(10)-di-p-(E)-coumaroyl spermidine, N(10)-(E)-caffeoyl-N(1),N(5)-di-p-(E)-coumaroyl spermidine, and N(1),N(5),N(10)-tri-p-(E)-coumaroyl spermidine amides. PMID:23993578
Wiese, Stefanie; Wubshet, Sileshi G; Nielsen, John; Staerk, Dan
Chemopreventive Effects of Sarcotriol on Ultraviolet B-induced Skin Tumor Development in SKH-1 Hairless Mice
Sarcotriol (ST) has been shown to be chemopreventive on 7,12-dimethyl-benz(a)anthracene (DMBA) initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumor development in CD-1 mice in recent studies from our laboratory. The objective of this study was to determine the chemopreventive effects of ST on ultraviolet B (UVB)-induced skin tumor development in female SKH-1 hairless mice, an experimental model relevant to human skin cancer development, and its possible mechanisms of action. Female SKH-1 mice were divided into two groups: Control and ST treated. Control was topically treated with 100 ?L acetone and ST treated group administered with 30 ?g ST in 100 ?L acetone one hour before UVB exposure. For UVB-induced tumorigenesis, carcinogenesis was initiated and promoted by UVB (180 mJ/cm2). Group weights and tumor counts were taken once every week. After 30 weeks, mice were sacrificed and dorsal skin samples were collected. The proteins from the skin sample were further used for SDS-PAGE and Western blotting using specific antibodies against caspase-3, caspase-8, caspase-9 and p53. Tumor multiplicity was found 19.6, 5.2 in the control and ST treated groups respectively. Caspase-3, -8, -9 and p53 were significantly (P < 0.05) upregulated in ST treated group compared to Control group. Together, this study for the first time identifies the chemopreventive effects of ST in UVB-induced carcinogenesis possibly by inducing apoptosis and upregulating p53.
Kundoor, Vipra; Zhang, Xiaoying; Bommareddy, Ajay; Khalifa, Sherief; Fahmy, Hesham; Dwivedi, Chandradhar
RSF1 is a positive regulator of NF-?B-induced gene expression required for ovarian cancer chemoresistance.
Overexpression or amplification of the RSF1 gene has been associated with poor prognosis in various human cancers, including ovarian cancer. In previous work, RSF1 was identified as an amplified gene that facilitated the development of paclitaxel-resistant ovarian cancer. In the present study, we further demonstrated that RSF1 expression inversely correlated with paclitaxel response in patients with ovarian cancer and the mouse xenograft model. In addition, RSF1-overexpressing paclitaxel-resistant ovarian cancer cell lines were found to express elevated levels of genes regulated by NF-?B, including some involved with the evasion of apoptosis (CFLAR, XIAP, BCL2, and BCL2L1) and inflammation (PTGS2). In addition, ectopic expression of RSF1 using Tet-off inducible SKOV3 cells significantly enhanced NF-?B-dependent gene expression and transcriptional activation of NF-?B. An RSF1 knockdown using short hairpin RNAs suppressed these same pathways. Moreover, pretreatment with NF-?B inhibitors or downregulation of NF-?B-regulated gene expression considerably enhanced paclitaxel sensitivity in RSF1-overexpressing OVCAR3 and/or RSF1-induced SKOV3 cells. A coimmunoprecipitation assay revealed that RSF1 interacts with NF-?B and CREB-binding protein, a ubiquitous coactivator for NF-?B. Recruitment of RSF1 to the NF-?B binding element in the PTGS2 and XIAP promoters was demonstrated by the chromatin immunoprecipitation assay. Furthermore, hSNF2H, a well-known binding partner of RSF1, was partially involved in the interaction between RSF1 and NF-?B. Taken together, these data suggest that RSF1 may function as a coactivator for NF-?B, consequently augmenting expression of genes necessary for the development of chemoresistance in ovarian cancer cells. PMID:24566868
Yang, Yeong-In; Ahn, Ji-Hye; Lee, Kyung-Tae; Shih, Ie-Ming; Choi, Jung-Hye
Molecular Mechanisms and Apoptosis in Pdt
NASA Astrophysics Data System (ADS)
Photodynamic Therapy (PDT) is a successful new therapy for malignant and non-malignant diseases. It is based on the activation of a photosensitizing dye by visible light in the target tissue, followed by production of cytotoxic substances. The article gives a short overview on the field of PDT with main focus on molecular mechanisms and apoptosis. It includes photodynamic principles, clinical application and procedures, biological effects, molecular mechanisms of damage processing and apoptosis.
Krammer, Barbara; Verwanger, Thomas
Regulation of apoptosis in prostate cancer
Transformation and malignant progression of prostate cancer is regulated by the inability of prostatic epithelial cells to\\u000a undergo apoptosis rather than by increased cell proliferation. The basic apoptotic machinery of most prostate cancer cells\\u000a is intact and the inability to undergo apoptosis is due to molecular alterations that result in failure to initiate or execute\\u000a apoptotic pathways. This review discusses
Sushma Gurumurthy; Krishna Murthi Vasudevan; Vivek M. Rangnekar
Apoptosis: A Way to Maintain Healthy Individuals
\\u000a Apoptosis, the best known form of programmed cell death, is tightly regulated by a number of sensors, signal transducers and\\u000a effectors. Apoptosis is mainly active during embryonic development, when deletion of redundant cellular material is required\\u000a for the correct morphogenesis of tissues and organs; moreover, it is essential for the maintenance of tissue homeostasis during\\u000a cell life. Cells also activate
Chiara Mondello; A. Ivana Scovassi
Apoptosis regulators as targets for cancer therapy
Apoptosis serves to remove excess or damaged cells and its dysregulation may lead to a number of pathological disorders including\\u000a cancer. Studies during the last 20 years have unravelled much of the molecular mechanisms that control apoptosis. Whether\\u000a a cell dies in response to diverse apoptotic stimuli, including DNA-damaging agents, is determined largely by interactions\\u000a between proteins of the Bcl-2
J. L. Fern�ndez-Luna
Abortive Apoptosis and Sperm Chromatin Damage
\\u000a The term apoptosis refers to a morphologically distinct form of cell death that plays a major role during the normal development and homeostasis\\u000a of multicellular organisms. This mode of cell death is a tightly regulated series of energy-dependent molecular and biochemical\\u000a events orchestrated by a genetic program. Apoptosis is either developmentally regulated (launched in response to specific\\u000a stimuli, such as
Hasan M. El-Fakahany; Denny Sakkas
Apoptosis in Cancer Biology and Cancer Therapeutics
Most anticancer therapies commonly used in the treatment of human cancer, e.g., chemotherapy, ?-irradiation, immunotherapy,\\u000a or suicide gene therapy, kill tumor cells by triggering cell death pathways including apoptosis in cancer cells. Hence, the\\u000a failure to activate such pathways may lead to the resistance of cancers to current treatment approaches. A better understanding\\u000a of the molecular events that regulate apoptosis
Simone Fulda
Inhibition of thymocyte apoptosis by berberine
To find anti-apoptotic substances in plant resources, a microassay method for estimating DNA fragmentation was established using fluorochrome 3,5-diaminobenzoic acid dihydrochloride. Examination was made of various herbal medicines for inhibitory effects on glucocorticoid-induced apoptosis in thymocytes. Several Kampo medicines, e.g. Oren-gedoku-to and San'o-shashin-to, were found to inhibit dexamethasone-induced apoptosis in murine thymocytes. Some of these medicines contain Coptidis rhizoma (CR)
Naoko Miura; Masahiro Yamamoto; Toshiyuki Ueki; Toshiyuki Kitani; Kazunori Fukcuda; Yasuhiro Komatsu
Activation of human herpesvirus replication by apoptosis.
A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance. PMID:23885073
Prasad, Alka; Remick, Jill; Zeichner, Steven L
Umbelliprenin Induces Apoptosis in CLL Cell Lines.
Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V-FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490
Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood
Anaplasma phagocytophilum Reduces Neutrophil Apoptosis In Vivo
Ovine neutrophils spontaneously underwent apoptosis during culture in vitro, as assessed by morphological changes and exposure of annexin V binding sites on their cell surfaces. The addition of conditioned medium from concanavalin A-treated ovine peripheral blood mononuclear cells (PBMC) could partially protect against this progression into apoptosis, but dexamethasone and sodium butyrate could not. Actinomycin D accelerated the rate at which ovine neutrophils underwent apoptosis. Neutrophils isolated from sheep experimentally infected with Anaplasma phagocytophilum showed significantly delayed apoptosis during culture ex vivo, and the addition of conditioned medium from PBMC to these cells could not delay apoptosis above the protective effects observed after in vivo infection. The ability of neutrophils from A. phagocytophilum-infected sheep to activate a respiratory burst was increased compared to the activity measured in neutrophils from uninfected sheep, but chemotaxis was decreased in neutrophils from infected sheep. These data are the first demonstration that in vivo infection with A. phagocytophilum results in changes in rates of apoptosis of infected immune cells. This may help explain how these bacteria replicate in these normally short-lived cells.
Scaife, Helena; Woldehiwet, Zerai; Hart, C. Anthony; Edwards, Steven W.
Umbelliprenin Induces Apoptosis in CLL Cell Lines
Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V�FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate.
Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood
Optimization and validation of a SPE-HPLC-PDA-fluorescence method for the simultaneous determination of drugs used in combined cardiovascular therapy in human plasma.
This paper reports the chemometrical optimization and the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-Fluo) method for the simultaneous analysis, in human plasma, of drugs usually combined in cardiovascular therapy. Separation of chlorthalidone (CLTD), valsartan (VAL), valsartan-M1 (VAL-M1), fluvastatin (FLUV) and the internal standard (IS) candesartan cilexetil was performed on a dC18 Atlantis column (100 mm x 3.9 mm, 3 microm) using a gradient with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% of formic acid and 10 mM of ammonium formate at pH 4.1. UV and fluorimetric (valsartan, its metabolite and fluvastatin) detectors were used. The sample preparation consisted of protein precipitation using acetonitrile suited to a solid-phase extraction (SPE) on a Strata-X cartridge for sample clean-up. Method validation was developed following the recommendations for bioanalytical method validation of International Conference on Harmonisation (ICH) and Food and Drug Administration (FDA) organizations. The method showed good linearity (31-3000 microg/l for chlorthalidone, 20-1000 microg/l for valsartan-M1, 10-5000 microg/l for valsartan and 14-1000 microg/l for fluvastatin), precision and accuracy. Recoveries were in the range of 78-91%. This method allowed the determination of these drugs in human plasma samples obtained from patients under cardiovascular treatment. PMID:19070983
Gonzalez, Oskar; Iriarte, Gorka; Ferreir�s, Nerea; Maguregui, Miren Itxaso; Alonso, Rosa Maria; Jim�nez, Rosa Maria
WINGS-SPE II: A catalog of stellar ages and star formation histories, stellar masses and dust extinction values for local clusters galaxies
NASA Astrophysics Data System (ADS)
Context. The WIde-field Nearby Galaxy clusters Survey (wings) is a project whose primary goal is to study the galaxy populations in clusters in the local universe (z < 0.07) and of the influence of environment on their stellar populations. This survey has provided the astronomical community with a high quality set of photometric and spectroscopic data for 77 and 48 nearby galaxy clusters, respectively. Aims: In this paper we present the catalog containing the properties of galaxies observed by the wings SPEctroscopic (wings-spe) survey, which were derived using stellar populations synthesis modelling approach. We also check the consistency of our results with other data in the literature. Methods: Using a spectrophotometric model that reproduces the main features of observed spectra by summing the theoretical spectra of simple stellar populations of different ages, we derive the stellar masses, star formation histories, average age and dust attenuation of galaxies in our sample. Results: ~ 5300 spectra were analyzed with spectrophotometric techniques, and this allowed us to derive the star formation history, stellar masses and ages, and extinction for the wings spectroscopic sample that we present in this paper. Conclusions: The comparison with the total mass values of the same galaxies derived by other authors based on sdss data, confirms the reliability of the adopted methods and data. Based on observations taken at the Anglo Australian Telescope (3.9 m- AAT), and at the William Herschel Telescope (4.2 m- WHT).Full Table 2 is available in electronic form both at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (126.96.36.199) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/526/A45, and by querying the wings database at http://web.oapd.inaf.it/wings/new/index.html
Fritz, J.; Poggianti, B. M.; Cava, A.; Valentinuzzi, T.; Moretti, A.; Bettoni, D.; Bressan, A.; Couch, W. J.; D'Onofrio, M.; Dressler, A.; Fasano, G.; Kj�rgaard, P.; Moles, M.; Omizzolo, A.; Varela, J.
1H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations
Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, 1H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that 1H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d6. Unexpected or unwanted extract constituents were also easily identified in the 1H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of 1H NMR spectral segments (buckets or bins of 0.02�ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that 1H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts. Electronic supplementary material The online version of this article (doi:10.1007/s11306-009-0195-x) contains supplementary material, which is available to authorized users.
Agnolet, Sara; Jaroszewski, Jerzy W.; Verpoorte, Robert
SPE-HPLC method for the determination of four flavonols in rat plasma and urine after oral administration of Abelmoschus manihot extract.
A SPE-HPLC method was developed and validated for the simultaneous determination of flavonols, isoquercitrin (1), hibifolin (2), myricetin (3), quercetin-3'-O-d-glucoside (4) and quercetin (5) in rat plasma and urine after oral administration of the total flavonoids from Abelmoschus manihot (TFA). The astragalin (6) and kaempferol (7) were used as internal standards (IS). Plasma and urine samples were pretreated by solid-phase extraction using Winchem C(18) reversed-phase cartridges. Analysis of the plasma and urinary extract was performed on YMC-Pack ODS-A C(18) and Thermo ODS-2HYEPRSIL C(18) reversed-phase column, respectively and a mobile phase of acetonitrile-0.1% phosphoric acid was employed. HPLC analysis was conducted with different elution gradients. The flow rate was 1.0 mL/min and the detection wavelength was set at 370 nm. Calibration ranges in plasma for flavonols 2-5 were at 0.011-2.220, 0.014-2.856, 0.022-4.320, and 0.028-5.600 microg/mL, respectively. In urine calibration ranges for flavonols 1, 2, 4 and 5 were at 2.00-16.00, 8.56-102.72, 2.70-21.60, and 3.00-24.00 microg/mL, respectively. The RSD of intra- and inter-day was less than 5.40% and 4.89% in plasma, and less than 3.96% and 6.85% in urine for all the analyses. A preliminary experiment to investigate the plasma concentration and urinary excretion of the flavonols after oral administration of TFA to rats demonstrated that the present method was suitable for determining the flavonols in rat plasma and urine. PMID:17258944
Lai, Xianyin; Zhao, Yuying; Liang, Hong; Bai, Yanjing; Wang, Bin; Guo, Dean
Solvent-assisted dispersive micro-SPE by using aminopropyl-functionalized magnetite nanoparticle followed by GC-PID for quantification of parabens in aqueous matrices.
In this research, solvent-assisted dispersive micro-SPE was introduced as a simple modified technique for the determination of parabens in water and cosmetic samples. Aminopropyl-functionalized magnetite nanoparticles (MNPs) were successfully synthesized and applied. GC with photoionization detector was used for the separation and detection of parabens. In this method, hexylacetate (15 ?L) as a solvent and aminopropyl-functionalized MNPs (5 ?g) as a sorbent were added to an aqueous sample (10 mL) and then the sample was sonicated. Dispersed magnetite was collected in the bottom of the conical tube by using a strong magnet and then ACN was added as a desorption solvent. Forty microliters of this solvent was transferred into a microvial and then acetic anhydride and pyridine were added, thus derivatization was performed by acetic anhydride. After evaporation, 1 ?L of derivatized sample was injected into a gas chromatograph for analysis. Several important parameters, such as kind of organic solvent, desorption solvent and volume, amount of aminopropyl-functionalized MNPs and effect of salt addition were investigated. Under optimum conditions, the limits of detection achieved were between 50 and 300 ng/L, with RSDs (n = 5) lower than 8%. Under the optimum conditions, the enrichment factors ranged from 217 to 1253 and the extraction recoveries ranged from 10 to 62%. The recoveries were obtained for the analytes in river water and mouthwash solution and hand cream in the range of 87-103%. The advantages of proposed method are simplicity of operation, rapidity, high extraction yields, and environmental friendly character. PMID:23197331
Abbasghorbani, Maryam; Attaran, Abdolmohammad; Payehghadr, Mahmood
Effects of ginsenoside Rg2 on the ultraviolet B-induced DNA damage responses in HaCaT cells.
Our previous study demonstrated the increase in the repair of UVB damage by mRg2, a mixture of ginsenosides containing 60% Rg2 in NIH3T3 cells. In the present study, the effects of purified Rg2 on the repair and apoptosis in ultraviolet B (UVB)-exposed HaCaT cells were investigated on gene expression levels. When cells were exposed to UVB and post-incubated in normal medium for 24 h, the cell viability decreased to about 50% of that in nontreated control. When Rg2 was post-incubated, however, the UVB-induced cytotoxicity was significantly prevented in an Rg2 concentration- and time-dependent manner. The apoptotic nuclear fragmentation resulting from UVB exposure was also significantly protected by the Rg2 post-incubation. Microarray analysis showed that the genes stimulated by the Rg2-alone treatment include those involved in p53 signaling pathway such as GADD45alpha, GADD45beta, and cell communication genes. RT-PCR analysis showed that the Rg2-alone treatment slightly upregulated the p53 and GADD45 transcript and protein levels by about 1.5-fold as compared with the nontreated control. The mRNA levels of p53 and GADD45 in cells exposed to UVB and post-incubated with Rg2 for 24 h decreased in an Rg2 concentration-dependent manner as compared with that post-incubated in normal medium. However, the mRNA level of the UVB-exposed cells post-incubated with 5 microM retinol was essentially the same as that post-incubated in normal medium. Time course experiment showed that the mRNA levels of p53 and GADD45 in UVB-exposed cells were upregulated by post-incubation with 50 microM Rg2 until 6 and 9 h, respectively, and then gradually decreased until 24 h. By Western blot analysis, it was also revealed that the Rg2 post-incubation decreases the expression of p53, phospho-p53, GADD45, and ATM in UVB-exposed cells. Time course analysis also indicated that these decreased expressions were due to the earlier upregulation of p53 and GADD45 proteins. When UVB-exposed cells were post-incubated with Rg2 for 24 h after UVB exposure, the level of remaining cyclobutane pyrimidine dimers decreased in both Rg2 concentration- and time-dependent manner. All these results suggest that Rg2 protects cells against UVB-induced genotoxicity by increasing DNA repair, in possible association with modulation of protein levels involved in p53 signaling pathway. PMID:20508917
Ha, Se Eun; Shin, Dae Hyun; Kim, Hyung Do; Shim, Sun Mi; Kim, Hack Soo; Kim, Bo Hyeon; Lee, Jung Sup; Park, Jong Kun
Induction of Apoptosis in Skeletal Tissues: Phosphate-Mediated Chick Chondrocyte Apoptosis is Calcium Dependent
In an earlier study, we have shown that Pi induced apoptosis of terminally differentiated hypertrophic chondrocytes. To ascertain whether Ca 2+ modulates Pi-induced cell death, we asked the following two questions: First, can we prevent Pi-induced apoptosis by removing Ca 2+ from the culture medium; alternatively, can we potentiate cell death by increasing the Ca 2+ concentration? Second, can we
K. Mansfield; B. Pucci; C. S. Adams; I. M. Shapiro
Initial apoptosis is followed by increased proliferation of apoptosis-resistant endothelial cells
We have demonstrated that VEGF receptor blockade in combination with chronic hypoxia causes in rats severe angioproliferative pulmonary hypertension (SAPH) associated with arterial occlusion by proliferating endothelial cells, and we postulate that the established, lumen- occluding lesions are the result of the emergence of apoptosis-resistant proliferating cells. To study the dependence of exuberant endothelial cell proliferation on initial apoptosis, we
Seiichiro Sakao; Laimute Taraseviciene-Stewart; Jong Deog Lee; Kathy Wood; Carlyne D. Cool; Norbert F. Voelkel
Regulation of membrane release in apoptosis.
Apoptosis is a fundamental process of cell regulation whereby cells execute one or more biochemical programs leading to cell death. Several mechanisms have been evaluated and suggested to play roles in the regulation of apoptosis, including the activation of phospholipase A2 (PLA2), usually measured as release of 3H-labelled arachidonic acid (AA) from prelabelled cells. The current study was aimed at examining the role of PLA2 in regulating apoptosis in response to several inducers (such as vincristine and etoposide) in lymphoid cell lines. Cells were labelled with [3H]fatty acids and the released radioactivity was characterized. These studies indicated that the AA release assay did not reflect release of non-esterified fatty acid via activation of the PLA2 pathway. Rather, studies using TLC and electron microscopy showed that AA release reflected a previously unsuspected shedding of a heterogeneous population of membrane vesicles and fragments, probably as components of apoptotic bodies. Further studies demonstrated that this process is an integral part of apoptosis. Overexpression of Bcl-2 or the addition of caspase peptide inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethane prevented the characteristic morphological changes of cell death, and completely inhibited the release of membrane vesicles and fragments. On the other hand, release of membrane vesicles and fragments was caused by various inducers of apoptosis, as measured by release of either 3H-labelled AA or palmitic acid. Thus the present study demonstrates that the release of membrane lipids during apoptosis defines a new assay for apoptosis and has allowed the investigation of the mechanisms regulating formation of apoptotic bodies.
Zhang, J; Driscoll, T A; Hannun, Y A; Obeid, L M
Dichloroacetate Induces Apoptosis in Endometrial Cancer Cells
Purpose A recent landmark study demonstrated that Dichloroacetate (DCA) treatment promoted apoptosis in lung, breast, and glioblastoma cancer cell lines by shifting metabolism from aerobic glycolysis to glucose oxidation coupled with NFAT-Kv1.5 axis remodeling. The objective of this study was to determine whether DCA induces apoptosis in endometrial cancer cells and to assess apoptotic mechanism. Methods A panel of endometrial cancer cell lines with varying degrees of differentiation was treated with DCA and analyzed for apoptosis via flow cytometry. Biological correlates such as gene expression, intracellular Ca2+, and mitochondrial membrane potential were examined to assess apoptotic mechanism. Results Initiation of apoptosis was observed in five low to moderately invasive cancer cell lines including Ishikawa, RL95-2, KLE, AN3CA, and SKUT1B while treatment had no effect on non-cancerous 293T cells. Two highly invasive endometrial adenocarcinoma cell lines, HEC1A and HEC1B, were found to be resistant to DCA induced apoptosis. Apoptotic responding cell lines had a significant increase in early and late apoptotis, a decrease in mitochondrial membrane potential, and decreased Survivin transcript abundance, which are consistent with a mitochondrial-regulated mechanism. DCA treatment decreased intracellular calcium levels in most apoptotic responding cell lines which suggests a contribution from the NFAT-Kv1.5-mediated pathway. DCA treatment increased p53 upregulated modulator of apoptosis (PUMA) transcripts in cell lines with an apoptotic response, suggesting involvement of a p53-PUMA-mediated mechanism. Conclusions Dichloroacetate effectively sensitizes most endometrial cancer cell lines to apoptosis via mitochondrial, NFAT-Kv1.5, and PUMA mediated mechanisms. Further investigation of the cancer therapeutic potential of DCA is warranted.
Wong, Jason Y.Y.; Huggins, Gordon S.; Debidda, Marcella; Munshi, Nikhil C.; De Vivo, Immaculata
Emergence of castration-resistant metastatic prostate cancer is due to activation of survival pathways, including apoptosis suppression and anoikis resistance, and increased neovascularization. Thus targeting of apoptotic players is of critical significance in prostate cancer therapy since loss of apoptosis and resistance to anoikis are critical in aberrant malignant growth, metastasis and conferring therapeutic failure. The majority of therapeutic agents act through intrinsic mitochondrial, extrinsic death receptor pathways or endoplasmic reticulum stress pathways to induce apoptosis. Current therapeutic strategies target restoring regulatory molecules that govern the pro-survival pathways such as PTEN which regulates AKT activity. Other strategies focus on reactivating the apoptotic pathways either by down-regulating anti-apoptotic players such as BCL-2 or by up-regulating pro-apoptotic protein families, most notably, the caspases. Caspases are a family of cystine proteases which serve critical roles in apoptotic and inflammatory signaling pathways. During tumorigenesis, significant loss or inactivation of lead members in the caspase family leads to impairing apoptosis induction, causing a dramatic imbalance in the growth dynamics, ultimately resulting in aberrant growth of human cancers. Recent exploitation of apoptosis pathways towards re-instating apoptosis induction via caspase re-activation has provided new molecular platforms for the development of therapeutic strategies effective against advanced prostate cancer as well as other solid tumors. This review will discuss the current cellular landscape featuring the caspase family in tumor cells and their activation via pharmacologic intervention towards optimized anti-cancer therapeutic modalities. This article is part of a Special Issue entitled �Apoptosis: Four Decades Later�.
Fiandalo, M.V.; Kyprianou, N.
Enhanced platelet apoptosis in chronic uremic patients.
Abstract Background: There is a paucity of research on platelet apoptosis and its contribution to platelet dysfunction in uremic patients. The present study sought to analyze platelets apoptosis in uremic patients who underwent different dialysis modalities. Methods: Sixteen chronic uremic patients (5 on hemodialysis, 6 on peritoneal dialysis and 5 on non-dialysis) and 16 controls were studied. Platelet-rich plasma was detected for apoptotic events including depolarization of mitochondrial inner membrane potential (??m), phosphatidylserine (PS) exposure, activation of caspases-3 and Bcl-2 family proteins variations by Flow Cytometry or by Western-Blot. Washed normal platelets were incubated with normal or uremic platelet poor plasma and then were detected apoptotic events. Platelets function was assessed by ristocetin induced aggregative function test. Results: Compared to controls, uremic platelets demonstrated greater apoptosis for the ??m depolarization (43.48?�?9.58 vs. 52.76?�?15.36, p?=?0.005) as well as PS exposure (1.36?�?0.51 vs. 0.99?�?0.27, p?0.001). There was no significant difference among different treatment groups (for the ??m depolarization f?=?0.16, p?=?0.85; for the PS exposure f?=?1.06, p?=?0.36). Western Blot analyses showed caspase-3 activation and pro-apoptotic Bcl-2 family proteins expression. Platelets exposed to uremic plasma exhibited distinct apoptosis phenomena. Ristocetin induced platelet aggregation was markedly diminished in uremic patients and treated platelets. Conclusions: These findings indicate that platelets are incurred apoptosis in uremia patients. Uremic plasma accelerates apoptosis of normal platelets, resulting in a dysfunctional pattern of platelets in uremia. Uremic platelets apoptosis has no relationship with dialysis modality. PMID:24655051
Li, Ming; Wang, Zhaoyue; Ma, Tongling; Lu, Guoyuan; Yan, Rong; Zhao, Lili; Deng, Kun; Dai, Kesheng
Cell cycle arrest, apoptosis and autophagy induced by iminosugars on K562 cells.
Iminosugars have gained a remarkable importance as new therapeutic agents since 1966. In this study, compounds A and B, two iminosugar analogs synthesized previously, showed an inhibition of the growth of K562 cells. They allowed cell cycle arrested at the G0/G1 phase, promoted apoptotic activities and also lowered the mitochondrial membrane potential. Further exploration of the apoptosis mechanism revealed that compound B significantly suppressed the expression of Hsp70, which is a major anti-apoptotic molecular chaperone. Significant decrease was also found in the expression of Akt, a serine/threonine-specific protein kinase with anti-apoptosis activities also known as protein kinase B (PKB). At mitochondria level in comparison with compound A, compound B brought a better promotion in the expression of pro-apoptotic protein Bad in Bcl-2 family. As a result of the promotion, the expression of anti-apoptotic protein Bcl-xL was down-regulated. Cytochrome c was released, activating the intrinsic signaling pathways of caspase and resulting in the occurrence of cascade reaction. In addition, compound B stimulated autophagy effectively by up-regulating Beclin 1, thus causing the conversion of LC3-I to LC3-II through Akt/mTOR signaling pathway. In summary, these results indicated that compounds A and B induced cell death through multiple pathways. The disclosed results not only provide an evidence of antitumor activity of iminosugars as a foundation for further studies, but also may find potential applications in chronic myeloid leukemia therapy as new heat shock protein inhibitors and autophagy inducer. PMID:24657462
Zhu, Jingjing; Zhou, Yifa; Wang, Guan-Nan; Tai, Guihua; Ye, Xin-Shan
BASP1 promotes apoptosis in diabetic nephropathy.
Apoptosis contributes to the development of diabetic nephropathy (DN), but the mechanisms that lead to diabetes-induced cell death are not fully understood. Here, we combined a functional genomics screen for cDNAs that induce apoptosis in vitro with transcriptional profiling of renal biopsies from patients with DN. Twelve of the 138 full-length cDNAs that induced cell death in human embryonic kidney cells matched upregulated mRNA transcripts in tissue from human DN. Confirmatory screens identified induction of BASP1 in tubular cross sections of human DN tissue. In vitro, apoptosis-inducing conditions such as serum deprivation, high concentrations of glucose, and proinflammatory cytokines increased BASP1 mRNA and protein in human tubular epithelial cells. In normal cells, BASP1 localized to the cytoplasm, but in apoptotic cells, it colocalized with actin in the periphery. Overexpression of BASP1 induced cell death with features of apoptosis; conversely, small interfering RNA (siRNA)-mediated knockdown of BASP1 protected tubular cells from apoptosis. Supporting possible involvement of BASP1 in renal disease other than DN, we also observed significant upregulation of renal BASP1 in spontaneously hypertensive rats and a trend toward increased tubulointerstitial BASP1 mRNA in human hypertensive nephropathy. In summary, a combined functional genomics approach identified BASP1 as a proapoptotic factor in DN and possibly also in hypertensive nephropathy. PMID:20110383
Sanchez-Ni�o, Maria Dolores; Sanz, Ana Belen; Lorz, Corina; Gnirke, Andrea; Rastaldi, Maria Pia; Nair, Viji; Egido, Jesus; Ruiz-Ortega, Marta; Kretzler, Matthias; Ortiz, Alberto
Baicalin induced dendritic cell apoptosis in vitro.
This study was aimed to investigate the effects of baicalin (BA), a major flavonoid constituent found in the herb Baikal skullcap, on dendritic cells (DCs). DCs were generated by culturing murine bone marrow (BM) cells for 6 days with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4, and lipopolysaccharide (LPS) was added on day 5 to stimulate DCs maturation. The expression levels of DC maturity markers (CD80/CD86) were assessed by flow cytometry using direct immunofluorescence method. IL-12 levels in the culture supernatants were assayed by ELISA. Apoptosis of DCs was analyzed by flow cytometry after annexin V/propidium iodide staining. The mitochondrial membrane potential (??(m)) changes were measured by using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Exposure of DCs to BA (2-50??M) during BM cell differentiation showed no effects on the up-regulation of CD80/CD86 expression on DCs in response to LPS stimulation, but reduced DCs recovery by inducing apoptosis, and significantly inhibited the release of IL-12 to culture supernatants. BA-induced DC apoptosis in a time- and dose-dependent way, and immature DCs were more sensitive for BA-induced apoptosis than mature DC. BA also induced ??(m) changes in DCs. These results demonstrate that BA induces selective apoptosis in immature DCs possibly through mitochondria-mediated pathway. PMID:21687510
Zhang, Huahua; Jiao, Qingqing; Gong, Qianfeng; Zhang, Yan; Zhang, Weidong; Hu, Zhenlin
Sodium nitroprusside induces apoptosis of rabbit chondrocytes
NASA Astrophysics Data System (ADS)
Osteoarthritis (OA) is characterized by a slowly progressing degradation of the matrix and destruction of articular cartilage. Apoptosis of chondrocyte is accounted for the mechanism of OA. Nitric oxide (NO), as a stimulus, has been shown to induce chondrocyte apoptosis by activating the matrix metalloproteinases (MMPs), increasing the expression of cyclooxygenase 2 (COX-2) and the level of prostaglandin E2 (PGE2), inhibiting the proteoglycan synthesis and type II collagen expression. In this study, sodium nitroprusside (SNP) was administered to be the NO donor to explore the mechanism of NO-induced apoptosis of rabbit chondrocytes obtained from six weeks old New Zealand rabbits. CCK-8 assay revealed the inhibitory effect of SNP on cell viability. We used flow cytometry (FCM) to assess the form of cell death by Annexin-V/propidium iodide (PI) double staining, and evaluate the change of mitochondrial membrane potential (??m). We found that the SNP induced chondrocyte apoptosis in a dose- and time-dependent manner and an observable reduction of ??m. In conclusion, our findings indicate that SNP induces apoptosis of rabbit chondrocytes via a mitochondria-mediated pathway.
Liang, Qian; Wang, Xiao-Ping; Chen, Tong-Sheng
Enhancing antimelanoma immune responses through apoptosis.
We examined the feasibility of using tumor apoptosis at accessible sites to enhance antimelanoma immune responses in a model of spontaneous canine melanoma. We show that priming peripheral blood mononuclear cells with apoptotic melanoma cells significantly enhanced autologous and allogeneic lymphokine-activated killing of tumor cells. Since various pathways required for intrinsic apoptosis are often inactivated in melanoma, we used Fas ligand (FasL) overexpression to promote extrinsic apoptosis. FasL induced apoptosis in five of six cell lines. Each of the susceptible lines, but not the resistant one, expressed Fas mRNA. In addition, direct intratumoral administration of FasL DNA to tumor-bearing dogs was safe, with no adverse events reported over 7 days of observation. A reduction of tumor burden was seen in three of five dogs treated. The reduction of tumor volume was correlated with Fas expression by the tumors, although one dog with a Fas-negative tumor survived for 82 weeks after treatment. Our data show that overexpression of FasL is suitable to promote apoptosis of Fas(+) melanomas, and support the notion that priming immune responder cells with apoptotic tumor cells may enhance antitumor responses. The results also suggest that intratumoral administration of FasL offers a safe route for therapeutic gene delivery. PMID:12944992
Bianco, Stacie R; Sun, Juan; Fosmire, Susan P; Hance, Kenneth; Padilla, Marcia L; Ritt, Michelle G; Getzy, David M; Duke, Richard C; Withrow, Stephen J; Lana, Susan; Matthiesen, David T; Dow, Steven W; Bellgrau, Donald; Cutter, Gary R; Helfand, Stuart C; Modiano, Jaime F
Biology and medicinal chemistry approaches towards various apoptosis inducers.
Apoptosis is a genetically in-built process whereby organisms remove unwanted cells. Apoptosis can serve as a regulatory and defense mechanism in the formation of the shape and size of the human body and also to eradicate surplus amount of cells. The regulation of apoptosis is relevant and differentiates between a normal cells of body and cancer cells by loss of control. Apoptosis being an intricate process regulated by much more than just a biological mechanism. The induction of the apoptosis manifests the control on the tumour size and number of tumour cells hence establishing the application of apoptotic inducers as vital components in the treatment of cancer. During apoptosis, cells die in a controlled and regulated fashion which makes apoptosis distinct from necrosis (uncontrolled cell death). Protein components and regulators for apoptosis signaling pathways can involve the mitochondria (intrinsic pathway) or signal through death receptors (extrinsic pathway). Many different drug and gene therapy approaches are being tested for initiating apoptosis. Resistance to apoptosis is considered a hallmark of cancer. Therapeutic approaches attempted to date include traditional small molecules, antisense oligonucleotides, monoclonal antibodies, recombinant proteins and several classes of chemical compounds discussed in this review. These compounds may serve as precursor molecules for more effective drugs, all aimed at developing clinically effective therapeutics, targeting key apoptosis regulatory mechanism. This review will discuss the current understanding of apoptosis induced by various chemical agents and highlighting the role of apoptosis inducing agents as emerging opportunities for cancer therapy. PMID:22721391
Vyas, Vivek K; Chintha, Chetan; Pandya, Mitul R
Noninvasive imaging of apoptosis in cardiovascular disease
Recent advances in molecular imaging have permitted the noninvasive imaging of apoptosis, a critical process underlying the pathogenesis of many diseases of the cardiovascular system including atherosclerotic vascular disease, myocardial ischemia and reperfusion injury, chronic heart failure, myocarditis, and cardiac allograft rejection. Multiple molecular targets including phosphatidylserine, phosphatidylinositol 3-kinase, and caspases have been targeted by a variety of imaging agents and modalities such as nuclear scintigraphy, PET, MRI, and fluorescent and bioluminescent imaging. Translationally, methods utilizing radiolabeled annexin V have proven promising in several clinical trials of ischemia-reperfusion injury and cardiac allograft rejection. New approaches using novel molecular imaging agents show great potential for the ability to image apoptosis in the research and clinical setting. Ultimately the ability to detect apoptosis noninvasively would help to identify patients for emerging anti-apoptotic therapies and guide clinical management with the aim of maximal myocardial preservation.
Korngold, Ethan Chauncey; Jaffer, Farouc Amin; Weissleder, Ralph
Apoptosis and aging: increased resistance to apoptosis enhances the aging process.
Apoptosis is a vital component in the evolutionarily conserved host defense system. Apoptosis is the guardian of tissue integrity by removing unfit and injured cells without evoking inflammation. However, apoptosis seems to be a double-edged sword since during low-level chronic stress, such as in aging, increased resistance to apoptosis can lead to the survival of functionally deficient, post-mitotic cells with damaged housekeeping functions. Senescent cells are remarkably resistant to apoptosis, and several studies indicate that host defense mechanisms can enhance anti-apoptotic signaling, which subsequently induces a senescent, pro-inflammatory phenotype during the aging process. At the molecular level, age-related resistance to apoptosis involves (1) functional deficiency in p53 network, (2) increased activity in the NF-?B-IAP/JNK axis, and (3) changes in molecular chaperones, microRNAs, and epigenetic regulation. We will discuss the molecular basis of age-related resistance to apoptosis and emphasize that increased resistance could enhance the aging process. PMID:21116678
Salminen, Antero; Ojala, Johanna; Kaarniranta, Kai
Role and Interrelationship of G? Protein, Hydrogen Peroxide, and Nitric Oxide in Ultraviolet B-Induced Stomatal Closure in Arabidopsis Leaves1[C][W
Heterotrimeric G proteins have been shown to transmit ultraviolet B (UV-B) signals in mammalian cells, but whether they also transmit UV-B signals in plant cells is not clear. In this paper, we report that 0.5 W m?2 UV-B induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by eliciting a cascade of intracellular signaling events including G? protein, hydrogen peroxide (H2O2), and nitric oxide (NO). UV-B triggered a significant increase in H2O2 or NO levels associated with stomatal closure in the wild type, but these effects were abolished in the single and double mutants of AtrbohD and AtrbohF or in the Nia1 mutants, respectively. Furthermore, we found that UV-B-mediated H2O2 and NO generation are regulated by GPA1, the G?-subunit of heterotrimeric G proteins. UV-B-dependent H2O2 and NO accumulation were nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cG?). In addition, exogenously applied H2O2 or NO rescued the defect in UV-B-mediated stomatal closure in gpa1 mutants, whereas cG? AtrbohD/AtrbohF and cG? nia1 constructs exhibited a similar response to AtrbohD/AtrbohF and Nia1, respectively. Finally, we demonstrated that G? activation of NO production depends on H2O2. The mutants of AtrbohD and AtrbohF had impaired NO generation in response to UV-B, but UV-B-induced H2O2 accumulation was not impaired in Nia1. Moreover, exogenously applied NO rescued the defect in UV-B-mediated stomatal closure in the mutants of AtrbohD and AtrbohF. These findings establish a signaling pathway leading to UV-B-induced stomatal closure that involves GPA1-dependent activation of H2O2 production and subsequent Nia1-dependent NO accumulation.
He, Jun-Min; Ma, Xian-Ge; Zhang, Ying; Sun, Tie-Feng; Xu, Fei-Fei; Chen, Yi-Ping; Liu, Xiao; Yue, Ming
Glucocorticoid-induced apoptosis in the thymus.
Destruction of thymus cells was one of the earliest observed properties of adrenal glucocorticoids. The cells affected are primarily immature, CD4/CD8 double-positive lymphocytes. This process has been clearly shown in vivo and in vitro to be apoptosis, as characterized by cell shrinkage, membrane alterations, nuclear collapse and chromatin fragmentation into oligonucleosomes. Glucocorticoid-induced thymocyte death requires new mRNA and protein synthesis. A beginning has been made in identifying the genes involved in thymocyte apoptosis. A case is made for the death of unselected thymocytes in vivo being regulated by endogenous glucocorticoids. PMID:1286164
Cohen, J J
APOPTOSIS: Mitochondria--the Death Signal Integrators
NSDL National Science Digital Library
Access to the article is free, however registration and sign-in are required. Many of the intricate pathways of apoptosis that instruct a cell to kill itself involve the convergence of key proteins on the membranes of mitochondria. Such proteins induce the permeabilization of mitochondrial membranes and the release of caspase enzymes and nuclease activators that set in motion the final stages of programmed cell death. Now, as Brenner and Kroemer discuss in their Perspective, a proapoptotic transcription factor called TR3 has been found to move from its normal location in the nucleus to the mitochondria and to promote release of cytochrome c, a key event in apoptosis (Li et al.)
Catherine Brenner (Institut Gustave Roussy; Universit�© de Technologie de Compi�¨gne ;Apoptosis, Cancer and Immunity Laboratory associated with the National League Against Cancer); Guido Kroemer (Institut Gustave Roussy; Universit�© de Technologie de Compi�¨gne ;Apoptosis, Cancer and Immunity Laboratory associated with the National League Against Cancer)
Apoptosis after reperfused myocardial infarction: Role of angiotensin II
Angiotensin II (Ang II) plays a significant role in apoptosis after myocardial infarction (MI) and reperfused MI. Cumulative evidence suggests that Ang II is a major contributor to cardiomyocyte (CM) apoptosis and left ventricular (LV) dysfunction after acute reperfused MI and that apoptosis mediates a major portion of early LV dysfunction. Importantly, blockade of the Ang II type 1 receptor (AT1R) limits CM apoptosis and LV dysfunction after acute reperfused MI. Ang II type 2 receptor activation during AT1R blockade contributes to these beneficial effects. The role of Ang II and apoptosis in chronic LV remodelling, healing and post-MI heart failure is more complex and involves effects on the CMs, fibroblasts and vascular cells. The long-term effects of agents targeting apoptosis after reperfused MI, including AT1R blockade, on apoptosis in different cell types, windows of enhanced apoptosis and the appropriate timing of therapy need to be considered.
Jugdutt, Bodh I
Emergence of the epidemic methicillin-resistant Staphylococcus aureus strain USA300 coincides with horizontal transfer of the arginine catabolic mobile element and speG-mediated adaptations for survival on skin.
The arginine catabolic mobile element (ACME) is the largest genomic region distinguishing epidemic USA300 strains of methicillin-resistant Staphylococcus aureus (MRSA) from other S. aureus strains. However, the functional relevance of ACME to infection and disease has remained unclear. Using phylogenetic analysis, we have shown that the modular segments of ACME were assembled into a single genetic locus in Staphylococcus epidermidis and then horizontally transferred to the common ancestor of USA300 strains in an extremely recent event. Acquisition of one ACME gene, speG, allowed USA300 strains to withstand levels of polyamines (e.g., spermidine) produced in skin that are toxic to other closely related S. aureus strains. speG-mediated polyamine tolerance also enhanced biofilm formation, adherence to fibrinogen/fibronectin, and resistance to antibiotic and keratinocyte-mediated killing. We suggest that these properties gave USA300 a major selective advantage during skin infection and colonization, contributing to the extraordinary evolutionary success of this clone. Importance: Over the past 15 years, methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem. It is likely that adaptations in specific MRSA lineages (e.g., USA300) drove the spread of MRSA across the United States and allowed it to replace other, less-virulent S. aureus strains. We suggest that one major factor in the evolutionary success of MRSA may have been the acquisition of a gene (speG) that allows S. aureus to evade the toxicity of polyamines (e.g., spermidine and spermine) that are produced in human skin. Polyamine tolerance likely gave MRSA multiple fitness advantages, including the formation of more-robust biofilms, increased adherence to host tissues, and resistance to antibiotics and killing by human skin cells. PMID:24345744
Planet, Paul J; LaRussa, Samuel J; Dana, Ali; Smith, Hannah; Xu, Amy; Ryan, Chanelle; Uhlemann, Anne-Catrin; Boundy, Sam; Goldberg, Julia; Narechania, Apurva; Kulkarni, Ritwij; Ratner, Adam J; Geoghegan, Joan A; Kolokotronis, Sergios-Orestis; Prince, Alice
Ultraviolet-B-Induced Stomatal Closure in Arabidopsis Is Regulated by the UV RESISTANCE LOCUS8 Photoreceptor in a Nitric Oxide-Dependent Mechanism.
UV RESISTANCE LOCUS8 (UVR8) signaling involves CONSTITUTIVELY PHOTOMORPHOGENIC1, the ELONGATED HYPOCOTYL5 (HY5) transcription factor, and the closely related HY5 HOMOLOG. Some UV-B responses mediated by UVR8 are also regulated by nitric oxide (NO), a bioactive molecule that orchestrates a wide range of processes in plants. In this study, we investigated the participation of the UVR8 pathway and its interaction with NO in UV-B-induced stomatal movements in Arabidopsis (Arabidopsis thaliana). Stomata in abaxial epidermal strips of Arabidopsis ecotype Landsberg erecta closed in response to increasing UV-B fluence rates, with maximal closure after 3-h exposure to 5.46 ?mol m(-2) s(-1) UV-B. Both hydrogen peroxide (H2O2) and NO increased in response to UV-B, and stomatal closure was maintained by NO up to 24 h after the beginning of exposure. Stomata of plants expressing bacterial NO dioxygenase, which prevents NO accumulation, did not close in response to UV-B, although H2O2 still increased. When the uvr8-1 null mutant was exposed to UV-B, stomata remained open, irrespective of the fluence rate. Neither NO nor H2O2 increased in stomata of the uvr8-1 mutant. However, the NO donor S-nitrosoglutathione induced closure of uvr8-1 stomata to the same extent as in the wild type. Experiments with mutants in UVR8 signaling components implicated CONSTITUTIVELY PHOTOMORPHOGENIC1, HY5, and HY5 HOMOLOG in UV-B-induced stomatal closure. This research provides evidence that the UVR8 pathway regulates stomatal closure by a mechanism involving both H2O2 and NO generation in response to UV-B exposure. PMID:24586043
Tossi, Vanesa; Lamattina, Lorenzo; Jenkins, Gareth I; Cassia, Ra�l O
Neuronal remodeling and apoptosis require VCP-dependent degradation of the apoptosis inhibitor DIAP1.
The regulated degeneration of axons or dendrites (pruning) and neuronal apoptosis are widely used during development to determine the specificity of neuronal connections. Pruning and apoptosis often share similar mechanisms; for example, developmental dendrite pruning of Drosophila class IV dendritic arborization (da) neurons is induced by local caspase activation triggered by ubiquitin-mediated degradation of the caspase inhibitor DIAP1. Here, we examined the function of Valosin-containing protein (VCP), a ubiquitin-selective AAA chaperone involved in endoplasmic reticulum-associated degradation, autophagy and neurodegenerative disease, in Drosophila da neurons. Strong VCP inhibition is cell lethal, but milder inhibition interferes with dendrite pruning and developmental apoptosis. These defects are associated with impaired caspase activation and high DIAP1 levels. In cultured cells, VCP binds to DIAP1 in a ubiquitin- and BIR domain-dependent manner and facilitates its degradation. Our results establish a new link between ubiquitin, dendrite pruning and the apoptosis machinery. PMID:21343367
Rumpf, Sebastian; Lee, Sung Bae; Jan, Lily Yeh; Jan, Yuh Nung
X-linked inhibitor of apoptosis (XIAP) confers human trophoblast cell resistance to Fas-mediated apoptosis
Apoptosis occurs in the placenta throughout gestation, with a greater frequency near term in comparison to the first trimester. The Fas\\/FasL system represents one of the main apoptotic pathways controlling placental apoptosis. Although first trimester trophoblast cells express both Fas and FasL, they are resistant to Fas-induced apoptosis. Therefore, trophoblast resistance to Fas-mediated apoptosis may be due to the inhibition
Shawn L. Straszewski-Chavez; Vikki M. Abrahams; Edmund F. Funai; Gil Mor
Calcium Signalling and the Regulation of Apoptosis
Apoptotic cell death is characterized by cell shrinkage, chromatin condensation and fragmentation, formation of apoptotic bodies and phagocytosis (Kerr et al., 1972). At the molecular level, activation of a family of cysteine proteases, caspases, related to interleukin-1?-converting enzyme is believed to be a crucial event in apoptosis. This is associated with the proteolysis of nuclear and cytoskeletal proteins, cell shrinkage,
M. I. P�rn-Ares; M. P. S. Ares; S. Orrenius
Cadmium-induced apoptosis in mouse liver.
Apoptosis is a process of active cell death, distinct from necrosis and characterized by specific morphological and biochemical features. Although the acute hepatotoxic effects of cadmium (Cd) are well described, little is known about the occurrence of apoptosis in Cd toxicity. Therefore, mice were injected with 5-60 mumol/kg i.p. of Cd and their livers were removed 1.5-48 h later and examined by light microscopy. Cd induced both a time- and dose-dependent increase in apoptotic index, severity of necrosis, and mitotic index. Apoptotic index peaked at 9-14 h after Cd administration and then decreased. The time course of apoptotic DNA fragmentation index, monitored by quantification of oligonucleosomal DNA fragments, correlated with the results obtained by histopathological analysis and a commercial in situ apoptotic DNA detection kit. Liver necrosis, as demonstrated by histology and serum alanine aminotransferase and sorbitol dehydrogenase assays, was most severe 14-48 h after Cd injection. Apoptosis was decreasing by 24 h while necrosis persisted. Replacement of liver tissue by blood lakes (peliosis hepatis) was observed after 14 h. The mitotic index increased gradually with time, indicating compensatory liver cell regeneration. There was a progressive increase in the severity of necrosis, apoptotic index, and mitotic index with increasing dose of Cd. These data demonstrate that apoptosis is a major mode of elimination of critically damaged cells in acute Cd hepatotoxicity in the mouse, and it precedes necrosis. PMID:9571989
Habeebu, S S; Liu, J; Klaassen, C D
Drosophila happyhour modulates JNK-dependent apoptosis.
Mitogen-activated protein kinase kinase kinase kinase-3 (MAP4K3) is a Ste20 kinase family member that modulates multiple signal transduction pathways. We recently identified MAP4K3 as proapoptotic kinase using an RNA interference screening approach. In mammalian cells, MAP4K3 enhances the mitochondrial apoptosis pathway through the post-transcriptional modulation of selected proapoptotic Bcl-2 homology domain 3-only proteins. Recent data suggest that MAP4K3 mutations contribute to pancreatic cancer, which highlights the importance of studying the in vivo function of this kinase. To determine whether the cell death function is conserved in vivo and which downstream signalling pathways are involved, we generated transgenic flies expressing happyhour (hppy), the Drosophila MAP4K3 orthologue. Here, we show that the overexpression of hppy promotes caspase-dependent apoptosis and that the hypothetical kinase domain is essential for inducing cell death. In addition, we show that hppy expression triggers the activation of both the c-Jun N-terminal kinase (JNK) and target of rapamycin (TOR) signalling pathways; however, only JNK signalling is required for apoptosis. Together, our results show that hppy has a JNK-dependent proapoptotic function in Drosophila, which reinforces the hypothesis that MAP4K3 might act as tumour suppressor by regulating apoptosis in higher eukaryotes. PMID:21364671
Lam, D; Shah, S; de Castro, I P; Loh, S H Y; Martins, L M
Spontaneous and inducible apoptosis in oesophageal adenocarcinoma
The use of neoadjuvant chemoradiotherapy prior to surgery in the treatment of oesophageal adenocarcinoma has increased in recent years, and up to 25% of patients will have a complete pathological response to the neoadjuvant therapy. Many patients will not respond, however, and the knowledge of molecular factors predicting response or resistance to chemoradiotherapy is required to enhance treatment results. An understanding of apoptosis and cell proliferation may be relevant and this study focused on apoptotic indices and cell-cycle related (Ki-67, p53 and bcl-2) protein expression in a cohort of 42 patients with primary oesophageal adenocarcinoma. We documented that apoptosis occurs among viable (proliferating) tumour cells in all adenocarcinoma cases examined in this study. Pre-operative chemoradiotherapy significantly increased apoptosis and significantly decreased cell proliferation (estimated by Ki-67 expression). Immunohistochemically detected p53 and bcl-2 gene products had no regulatory role in the apoptotic process. The cumulative expression of p53 protein is significantly associated with increasing proliferation activity. Evaluation of apoptosis in pre-treatment specimens may have potential utility in predicting the efficacy of treatment. Assessment of the tumours proliferation activity by Ki-67 expression might identify patients who are at risk of developing metastastic disease. � 2001 Cancer Research Campaign http://www.bjcancer.com
Raouf, A; Evoy, D; Carton, E; Mulligan, E; Griffin, M; Sweeney, E; Reynolds, J V
Apoptosis-based therapies and drug targets
The pathogenesis of many diseases is most closely connected with aberrantly regulated apoptotic cell death. The past 15 years have witnessed an explosion in the basic knowledge of mechanisms that regulate apoptosis and the mediators that either trigger or inhibit cell death. Consequently, great interest has emerged in devising therapeutic strategies for modulating the key molecules of life-and-death decisions. Numerous
U Fischer; K Schulze-Osthoff
Cytomegalovirus infection blocks apoptosis in cancer cells
Recent pathological findings reveal a higher frequency of human cytomegalovirus (HCMV) in tumor cells from different tumors compared with surrounding tissues. Experimental investigations suggest possible supportive effects of HCMV for tumor development and progression. One HCMV effect on tumor cells is the inhibition of apoptosis, leading to the promotion of tumor cell survival. Decreased sensitivity to treatment-induced tumor cell death
M. Michaelis; R. Kotchetkov; J.-U. Vogel; H. W. Doerr; J. Cinatl
Apoptosis in brain-specific autoimmune disease
Recent neuropathological studies of experimental autoimmune encephalomyelitis have focused attention on the high number of cells in the lesions that show typical morphological features of apoptosis. Surprisingly, it has turned out that the vast majority of apoptotic cells are T lymphocytes and that they actually represent the antigen-specific T-cell population responsible for the induction of the disease. Taken together, these
Jan Bauer; Hartmut Wekerle; Hans Lassmann
TRAIL Induced Apoptosis - A Prostate Cancer Therapy.
National Technical Information Service (NTIS)
Five of six prostate cancer cell lines undergo apoptosis when incubated with% TRAIL. Cell viability curves of six prostate cancer cell lines using varying amounts of TRAIL protein were completed. Prostate cancer cell lines Alva 31, PC-3 and DU 145 are hig...
X. Lu D. M. Rodman
Liposomal Sphingolipids to Target Breast Adenocarcinoma Apoptosis.
National Technical Information Service (NTIS)
The purpose of these studies was to develop key data for the approach to the proof-of-principle, efficacy studies in Aim 5 using orthotopic Her-2/neu over-expressing human breast adenocarcinoma models. Investigations pertinent to Aims 1 (apoptosis), 2 (fo...
J. Klostergaard
Liposomal Sphingolipids to Target Breast Adenocarcinoma Apoptosis.
National Technical Information Service (NTIS)
The purpose of these studies was: (1) to evaluate the interactions between Wortmannin-mediated inhibition of the Akt/PKB pathway and sphingolipid- induced apoptosis in a panel of human breast adenocarcinoma cell lines with basal or over-expression of HER-...
J. Klostergaard
The molecular legacy of apoptosis in transplantation.
Transplanted organs have to cope with diverse immunologic and metabolic stressors that augment the percentage of stressed and dying cells. Cell death, whether apoptotic or necrotic, is crucial in various transplantation-associated conditions. Necrosis, a proinflammatory type of cell death classically considered as accidental, is increasingly recognized as a highly controlled death program. Apoptosis, the classical programmed cell death mode program, is tightly orchestrated and culminates in the activation of caspases. Apoptosis was classically regarded as a silent form of cell death, but mounting evidence indicates that apoptotic cells "don't go silently" and leave a heritage to the local microenvironment. This apoptotic legacy, embedded within the effector phase of apoptosis, is aimed, at least in part, at controlling leukocyte trafficking and fostering tissue remodeling at sites of apoptotic cell deletion and can promote maladaptive remodeling pathways of importance for obliterative vascular remodeling. Moreover, apoptotic cells can transfer bioactive molecules by the release of apoptotic membrane vesicles that, in turn, shapes the phenotype and functions of immune cells. In this review, we summarize recent data highlighting the importance of apoptosis-associated intercellular communication networks in the regulation of allograft remodeling and immune responses in transplantation. PMID:22420581
Pallet, N; Dieud�, M; Cailhier, J; H�bert, M
Bin1, Apoptosis, and Prostate Cancer.
National Technical Information Service (NTIS)
In this research project, we had proposed to investigate the utility of the Bin1 gene as a diagnostic and/or prognostic marker in prostate malignancy and to perform proof-of-principle tests of its ability to induce apoptosis in androgen independent cancer...
F. J. Rauscher
Heat Shock Proteins Increase Resistance to Apoptosis
Heat shock treatment of cells increases their survival and resistance to apoptosis. The kinetics of development of this resistance correlates with the kinetics of synthesis of heat shock proteins (hsps). U937 and Wehi-s cells were cultured for 1 h at 42�C, conditions which induced the synthesis of heat shock proteins 27, 70, and 90. The cells were subsequently permitted to
Afshin Samali; Thomas G. Cotter
Death penalty for keratinocytes: apoptosis versus cornification
Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a
S Lippens; G Denecker; P Ovaere; P Vandenabeele; W Declercq
A novel method for detection of apoptosis
There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II*). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the $20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.
Zagariya, Alexander M., E-mail: firstname.lastname@example.com
Cleavage and inactivation of ATM during apoptosis.
The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance-the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling. PMID:10454555
Smith, G C; d'Adda di Fagagna, F; Lakin, N D; Jackson, S P
The modulation of apoptosis by oncogenic viruses
Transforming viruses can change a normal cell into a cancer cell during their normal life cycle. Persistent infections with these viruses have been recognized to cause some types of cancer. These viruses have been implicated in the modulation of various biological processes, such as proliferation, differentiation and apoptosis. The study of infections caused by oncogenic viruses had helped in our understanding of several mechanisms that regulate cell growth, as well as the molecular alterations leading to cancer. Therefore, transforming viruses provide models of study that have enabled the advances in cancer research. Viruses with transforming abilities, include different members of the Human Papillomavirus (HPV) family, Hepatitis C virus (HCV), Human T-cell Leukemia virus (HTLV-1), Epstein Barr virus (EBV) and Kaposi�s Sarcoma Herpesvirus (KSHV). Apoptosis, or programmed cell death, is a tightly regulated process that plays an important role in development and homeostasis. Additionally, it functions as an antiviral defense mechanism. The deregulation of apoptosis has been implicated in the etiology of diverse diseases, including cancer. Oncogenic viruses employ different mechanisms to inhibit the apoptotic process, allowing the propagation of infected and damaged cells. During this process, some viral proteins are able to evade the immune system, while others can directly interact with the caspases involved in apoptotic signaling. In some instances, viral proteins can also promote apoptosis, which may be necessary for an accurate regulation of the initial stages of infection.
Fluorescence spectroscopy to assess apoptosis in myocardium
NASA Astrophysics Data System (ADS)
Apoptosis induced mitochondrial destruction and dysfunction has been shown to play an important role in the pathogenesis of both acute cardiac ischemia-reperfusion injury and chronic myocardial infarction-induced ventricular remodeling. Unfortunately this understanding has not translated into effective therapeutic strategies for either condition-mostly due to an inability to assess mitochondrial dysfunction/apoptosis effectively in humans. All current measures of apoptosis are pseudo-quantitative and require invasive tissue biopsy. Our group has developed an optical, non-tissue destructive catheter based device that allows the quantitative regional assessment of this pathological process in vivo. This instrument has been designed to acquire fluorescence signals of intrinsic mitochondrial fluorophores, Nicotinamide Adenine Dinucleotide (NAD) and Flavoprotein (FP). The normalized ratio of these fluorophores (FP/FP+NADH) called the redox ratio, is an indicator of the in vivo mitochondrial dysfunction.1-3 We have demonstrated in a rabbit reperfusion model of apoptotic myocyte injury that this redox ratio is drastically increased which is consistent with profound apoptosis-induced "unhinging" of the mitochondrial respiratory function.
Ranji, Mahsa; Matsubara, Muneaki; Grosso, Michael A.; Jaggard, Dwight L.; Chance, Britton; Gorman, Robert C.; Gorman, Joseph H., III
Apoptosis-inducing protein derived from hepatocyte selectively induces apoptosis in lymphocytes
The liver is where lymphocytes undergo activation-induced cell death (AICD) at the resolution phase of an immune response, which is crucial for homeostasis of the immune system and prevention of autoimmunity. Exploring the machinery of AICD in the liver, we found that a primary culture supernatant of murine hepatocytes had an antiproliferative effect on antigen-stimulated T clone and T lymphoma cells. Biological study showed that the antiproliferation was due to induction of apoptosis in a caspase-dependent manner. The apoptosis-inducing potential was sensitive to trypsin, heat (> 70�) and acid (< pH 5) treatment but could not be neutralized by anti-tumour necrosis factor-?, anti-Fas ligand, or anti-transforming growth factor-? antibodies. Biochemical study of the isolated and purified apoptosis-inducing component from the supernatant showed that it was a protein with a molecular mass of about 68 000�70 000. It induced apoptotic change in murine T and B cells, and to a lesser degree, in human lymphoid cells, but not in macrophages. Biochemical and biological characteristics distinguish this protein from others that have been reported to induce apoptosis of lymphocytes. The identification of an apoptosis-inducing protein derived from murine hepatocytes, which selectively induces apoptosis in lymphocytes, suggests one possible mechanism for immune suppression in the liver.
Ikeda, Masanobu; Yoshikawa, Hideshi; Liu, Jie; Nakajima, Yasuo; Akahane, Yoshihiro; Tasaka, Kachio
Participation of chloroplasts in plant apoptosis.
Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN(-)-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O2 were shown to prevent the CN(-)-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN- effect. The light-dependent activation of CN(-)-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell. PMID:14570380
Samuilov, Vitaly D; Lagunova, Elena M; Kiselevsky, Dmitry B; Dzyubinskaya, Elena V; Makarova, Yana V; Gusev, Mikhail V
Caspase cleavage product lacking amino-terminus of IkappaBalpha sensitizes resistant cells to TNF-alpha and TRAIL-induced apoptosis.
In response to a diverse array of signals, IkappaBalpha is targeted for phosphorylation-dependent degradation by the proteasome, thereby activating NF-kappaB. Here we demonstrate a role of the cleavage product of IkappaBalpha in various death signals. During apoptosis of NIH3T3, Jurkat, Rat-1, and L929 cells exposed to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas, serum deprivation, or TNF-alpha, respectively, IkappaBalpha was cleaved in a caspase-dependent manner. In vitro and in vivo cleavage assays and site-directed mutagenesis showed that caspase-3 cleaved IkappaBalpha between Asp31 and Ser32. Expression of the cleavage product lacking amino-terminus (1-31), DeltaIkappaBalpha, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF-alpha or TRAIL, and HeLa tumor cells to TNF-alpha. DeltaIkappaBalpha was more pro-apoptotic compared to wild type or cleavage-resistant (D31E)IkappaBalpha mutant and the sensitization elicited by DeltaIkappaBalpha was as effective as that by the dominant negative mutant, (S32,36A)IkappaBalpha, in NIH3T3 cells. DeltaIkappaBalpha suppressed the transactivation of NF-kappaB induced by TNF-alpha or TRAIL, as reflected by luciferase-reporter activity. Conversely, expression of the p65 subunit of NF-kappaB suppressed TNF-alpha-, TRAIL-, and serum deprivation-induced cell death. On the contrary, DeltaIkappaBalpha was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that DeltaIkappaBalpha generated by various death signals sensitizes cells to apoptosis by suppressing NF-kappaB activity. PMID:11948689
Kim, Ki-Woo; Kim, Byung Ju; Chung, Chul-Woong; Jo, Dong-Gyu; Kim, In-Ki; Song, You-Hyun; Kwon, Yun-Kyung; Woo, Ha-Na; Jung, Yong-Keun
[The role of apoptosis in the development of virus hepatitis].
In present review hepatocyte apoptosis is presented as universal defensive reaction of liver, to the damages. Hepatocyte apoptosis may be caused by hepatotropic virus's direct affection, or by the immune reactions initiated by viruses. Apoptosis development caused by virus direct affection varies and contains at lest two mechanisms: production of specific proteins: B virus - X protein and C virus - core-protein; expression of the receptors leading the induction of this process on the hepatocyte membrane, for example, increasing of Fas-receptor and cell sensation to apoptosis stimulus. In apoptosis induced by immune reaction T-lymphocytes could trigger off apoptosis in two principal ways: by releasing perporines that produce holes through hepatocyte membrane and according to this process granzyms are permetted inside the cells. By destroying of caspases by proteases that initiate apoptosis cascade. In this article molecular mechanisms of the processes mentioned above are also discussed. PMID:21685531
Kipiani, N V; Topuridze, M L; Kipiani, V A; Pavliashvili, N S; Kipiani, N V
emm1/Sequence Type 28 Strains of Group A Streptococci That Express covR at Early Stationary Phase Are Associated with Increased Growth and Earlier SpeB Secretion?
Streptococcus pyogenes (group A streptococcus [GAS]) is a versatile human pathogen, and emm1/sequence type 28 (ST28) is the most frequently isolated type from GAS infections. The emm1/ST28 strain is associated with necrotizing fasciitis and streptococcal toxic shock syndrome. Growth-phase regulation is one of the important regulatory mechanisms in GAS, which controls gene expression at restricted phases of growth. CovRS, a two-component regulatory system, is considered the regulator of streptococcal pyrogenic exotoxin B (SpeB) and is thought to be activated in the exponential phase of growth. In the present study, Northern hybridization analysis showed that 52% of the analyzed GAS strains expressed covR at the exponential phase, but 48% of the strains expressed covR at the early stationary phase of growth. Strains transcribing covR at the early stationary phase showed better growth and earlier SpeB expression than the other group of strains. Multilocus sequence typing and pulsed-field gel electrophoresis analysis showed only emm1/ST28 strains (which comprise a clonal cluster) were expressing covR at the early stationary phase of growth, indicating that emm1/ST28 strains have special characteristics which may be related to their worldwide distribution.
Chiang-Ni, Chuan; Zheng, Po-Xing; Ho, Yueh-Ren; Wu, Hsiu-Mei; Chuang, Woei-Jer; Lin, Yee-Shin; Lin, Ming-T.; Liu, Ching-Chuan; Wu, Jiunn-Jong
Sustained adenosine exposure causes lung endothelial apoptosis: a possible contributor to cigarette smoke-induced endothelial apoptosis and lung injury
Pulmonary endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. Cigarette smoke (CS) causes lung EC apoptosis and emphysema. In this study, we show that CS exposure increased lung tissue adenosine levels in mice, an effect associated with increased lung EC apoptosis and the development of emphysema. Adenosine has a protective effect against apoptosis via adenosine receptor-mediated signaling. However, sustained elevated adenosine increases alveolar cell apoptosis in adenosine deaminase-deficient mice. We established an in vitro model of sustained adenosine exposure by incubating lung EC with adenosine in the presence of an adenosine deaminase inhibitor, deoxycoformicin. We demonstrated that sustained adenosine exposure caused lung EC apoptosis via nucleoside transporter-facilitated intracellular adenosine uptake, subsequent activation of p38 and JNK in mitochondria, and ultimately mitochondrial defects and activation of the mitochondria-mediated intrinsic pathway of apoptosis. Our results suggest that sustained elevated adenosine may contribute to CS-induced lung EC apoptosis and emphysema. Our data also reconcile the paradoxical effects of adenosine on apoptosis, demonstrating that prolonged exposure causes apoptosis via nucleoside transporter-mediated intracellular adenosine signaling, whereas acute exposure protects against apoptosis via activation of adenosine receptors. Inhibition of adenosine uptake may become a new therapeutic target in treatment of CS-induced lung diseases.
Sakhatskyy, Pavlo; Newton, Julie; Shamirian, Paul; Hsiao, Vivian; Curren, Sean; Gabino Miranda, Gustavo Andres; Pedroza, Mesias; Blackburn, Michael R.; Rounds, Sharon
Increased leucocyte apoptosis in transfused ?-thalassaemia patients.
This exploratory study assessed apoptosis in peripheral blood leucocytes (PBL) from ?-thalassaemia patients receiving chronic transfusions and chelation therapy (deferasirox or deferoxamine) at baseline, 1, 6, and 12�months. At baseline, thalassaemic PBLs presented 50% greater levels of Bax (BAX), 75% higher caspase-3/7, 48% higher caspase-8 and 88% higher caspase-9 activities and 428% more nucleosomal DNA fragmentation than control subjects. Only neutrophils correlated significantly with apoptotic markers. Previously, we showed that over the treatment year, hepatic iron declined; we now show that the ratio of Bax/Bcl-2 (BCL2), (-27�3%/year), and caspase-9 activity (-13�3%/year) declined in both treatment groups, suggesting that chelation decreases body iron and indicators of PBL apoptosis. PMID:23216540
Walter, Patrick B; Porter, John; Evans, Patricia; Kwiatkowski, Janet L; Neufeld, Ellis J; Coates, Thomas; Giardina, Patricia J; Grady, Robert W; Vichinsky, Elliott; Olivieri, Nancy; Trachtenberg, Felicia; Alberti, Daniele; Fung, Ellen; Ames, Bruce; Higa, Annie; Harmatz, Paul
Apoptosis, Stem Cells, and Tissue Regeneration
NSDL National Science Digital Library
Tissue regeneration after wounding or amputation generally requires cell proliferation. Work in several model organisms, including Drosophila, Hydra, Xenopus, and mouse, revealed a surprising function for caspases in cell proliferation after tissue damage, in addition to their known role in a form of cell death called apoptosis. In apoptotic cells, caspases can stimulate the production of secreted cytokines, such as Wnt, bone morphogenetic protein (BMP), transforming growth factor–β (TGF-β), Hedgehog family members, and prostaglandins, which in turn induce proliferation of neighboring cells, thus promoting tissue regeneration and homeostasis. These pathways can also contribute to tumorigenesis. This Review summarizes findings on this phenomenon, termed "apoptosis-induced compensatory proliferation," and contains three figures and 110 references.
Andreas Bergmann (MD Anderson Cancer Center;Department of Biochemistry and Molecular Biology REV); Hermann Steller (Rockefeller University;Howard Hughes Medical Institute REV)
Ubiquitin-mediated regulation of apoptosis.
Ubiquitin is a protein modifier that is conjugated to target proteins either as a single moiety or as polyubiquitin chains. Over the past several years, an increasing number of ubiquitin ligases and ubiquitin-deconjugating enzymes have been identified; these modulate cell survival by degradative and non-degradative means. Mutations that affect ubiquitin-mediated signalling are tightly linked to various human pathologies including tumorigenesis. Unravelling how the ubiquitin-signal is conjugated, edited and 'read' is crucial to understanding cellular processes such as endocytic trafficking, NF-kappaB signalling, gene expression, DNA repair and apoptosis. In this review, we summarize recent advances that start to elucidate how the ubiquitin message is used as a versatile tool to regulate apoptosis, for example in the conjugation of ubiquitin to caspases. This results in steric interference with substrate entry and allosteric conformational impairment of the catalytic pocket of the caspase. PMID:19217783
Broemer, Meike; Meier, Pascal
Clinical implications of apoptosis in ischemic myocardium.
Apoptosis, a genetically programmed form of cell death, contributes to myocyte cell loss in a variety of cardiac pathologies, including cardiac failure and those related to ischemia/reperfusion injury. The apoptotic program is complex, involving both pro- and anti-apoptotic proteins, and apoptosis occurs when the equilibrium between these opposing factors is perturbed. Some of these factors are intrinsic to the apoptotic pathway, such as the pro- and anti-apoptotic members of the Bcl2 family. Other, extrinsic, cellular factors can also modify the outcome of the response to an apoptotic stimulus. In this review, we have focused on some of these extrinsic factors, such as STAT-1 as a pro-apoptotic agent and the urocortins and Bag-1 as anti-apoptotic factors, since these may be potential therapeutic targets. In addition, we discuss the profound cytoprotective effects of the antibiotic, minocycline. PMID:16503249
Scarabelli, Tiziano M; Knight, Richard; Stephanou, Anastasis; Townsend, Paul; Chen-Scarabelli, Carol; Lawrence, Kevin; Gottlieb, Roberta; Latchman, David; Narula, Jagat
Alymphoplasia ( aly )-type Nuclear Factor k B-inducing Kinase (NIK) Causes Defects in Secondary Lymphoid Tissue Chemokine Receptor Signaling and Homing of Peritoneal Cells to the Gut-associated Lymphatic Tissue System
Alymphoplasia ( aly ) mice, which carry a point mutation in the nuclear factor k B-inducing ki- nase (NIK) gene, are characterized by the systemic absence of lymph nodes and Peyer's patches, disorganized splenic and thymic architectures, and immunodeficiency. Another unique feature of aly\\/aly mice is that their peritoneal cavity contains more B1 cells than normal and aly\\/ 1 mice.
Sidonia Fagarasan; Reiko Shinkura; Tadashi Kamata; Fumiaki Nogaki; Koichi Ikuta; Kei Tashiro; Tasuku Honjo
Extracellular NAD(+) inhibits human neutrophil apoptosis.
Regulation of neutrophil apoptosis plays a critical role in the inflammatory response. Inflammation has previously been shown to increase levels of extracellular ?-nicotinamide adenine dinucleotide (NAD(+)). The present study demonstrates that extracellular NAD(+) at concentrations found in the inflamed tissues profoundly delays spontaneous apoptosis of human neutrophils as was evidenced by inhibition of phosphatidylserine (PS) exposure, DNA fragmentation and caspase-3 activation. The effect was abrogated by NF157, an antagonist of P2Y11 receptor, and was pertussis toxin-insensitive. The NAD(+)-mediated delay of neutrophil apoptosis was reversed by 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, and Rp-8-Br-cAMPS, an inhibitor of type I cAMP-dependent protein kinase A (PKA). Blocking of NAD(+)-induced influx of extracellular Ca(2+) with EGTA did not abolish the pro-survival effect of NAD(+). Extracellular NAD(+) inhibited proteasome-dependent degradation of Mcl-1 upstream of caspase activation and, furthermore, suppressed Bax translocation to the mitochondria and attenuated both dissipation of mitochondrial transmembrane potential (??m) and cytochrome c release from the mitochondria into the cytosol. Finally, we found that extracellular NAD(+) inhibited spontaneous activation of caspase-9, but not caspase-8, and the pro-survival effect of extracellular NAD(+) was abrogated by the inhibitor of caspase-9, but not by the inhibitor of caspase-8. Together, these results demonstrate that extracellular NAD(+) inhibits neutrophil apoptosis via P2Y11 receptor and cAMP/PKA pathway by regulating Mcl-1 level, Bax targeting to the mitochondria and mitochondrial apoptotic pathway. Thus, extracellular NAD(+) acts as a neutrophil survival factor that can contribute to prolonged neutrophil lifespan in inflammatory response. PMID:24292505
Pliyev, Boris K; Ivanova, Anna V; Savchenko, Valery G
NMR exposure sensitizes tumor cells to apoptosis
NMR technology has dramatically contributed to the revolution of image diagnostic. NMR apparatuses use combinations of microwaves\\u000a over a homogeneous strong (1 Tesla) static magnetic field. We had previously shown that low intensity (0.3�66�mT) static magnetic\\u000a fields deeply affect apoptosis in a Ca2+ dependent fashion (Fanelli et�al., 1999 FASEBJ., 13;95�102). The rationale of the present study is to examine whether
L. Ghibelli; C. Cerella; S. Cordisco; G. Clavarino; S. Marazzi; M. De Nicola; S. Nuccitelli; M. D'Alessio; A. Magrini; A. Bergamaschi; V. Guerrisi; L. M. Porfiri
Regulation of apoptosis in prostate cancer.
Transformation and malignant progression of prostate cancer is regulated by the inability of prostatic epithelial cells to undergo apoptosis rather than by increased cell proliferation. The basic apoptotic machinery of most prostate cancer cells is intact and the inability to undergo apoptosis is due to molecular alterations that result in failure to initiate or execute apoptotic pathways. This review discusses the role of anti-apoptotic proteins such as Bcl-2/BclXL, NF-kappaB, IGF, caveolin, and Akt, and pro-apoptotic molecules such as PTEN, p53, Bin1, TGF-beta, and Par-4 that can regulate progression of prostate cancer. In addition to highlighting the salient features of these molecules and their relevance in apoptosis, this review provides an appraisal of their therapeutic potential in prostate cancer. Molecular targeting of these proteins and/or their innate pro- or anti-apoptotic pathways, either singly or in combination, may be explored in conjunction with conventional and currently available experimental strategies for the treatment of both hormone-sensitive and hormone-resistant prostate cancer. PMID:12085964
Gurumurthy, S; Vasudevan, K M; Rangnekar, V M
Bcl-3 regulates UVB-induced apoptosis.
B cell leukemia-3 (Bcl-3) has been defined as an anti-apoptotic gene; however, the exact mechanisms through which Bcl-3 influences apoptosis have been elusive. To determine the specific role of Bcl-3 in apoptosis, we evaluated the effect of its silencing on the expression of proteins involved in either the extrinsic or intrinsic apoptotic pathways induced by ultraviolet light B-mediated DNA damage. We found that, in Bcl-3-silenced cells, caspase-3, caspase-8 and caspase-9 activation is accelerated and tBid mitochondrial content is increased. It is important to note that, although mitochondrial Smac levels were reduced after UV exposure, the rate of reduction was slightly higher in Bcl-3 silenced cells than in control cells. Additionally, p53 levels diminished in Bcl-3 silenced cells compared to control cells, as did those of DNA-PK, a DNA repair protein. Altogether, our data indicate that Bcl-3 protects cells from apoptosis by regulating both apoptotic pathways. PMID:23494744
Garc�a, Ingrid; Cos�o, Gabriela; Liz�rraga, Floria; Mart�nez-Ruiz, Gustavo; Mel�ndez-Zajgla, Jorge; Ceballos, Gisela; Espinosa, Magali; Pacheco, Rosario; Maldonado, Vilma
Lithium protects ethanol-induced neuronal apoptosis
Lithium is widely used for the treatment of bipolar disorder. Recent studies have demonstrated its neuroprotective effect. Ethanol is a potent neurotoxin that is particularly harmful to the developing nervous system. In this study, we evaluated lithium's neuroprotection against ethanol-induced apoptosis. Transient exposure of infant mice to ethanol caused apoptotic cell death in brain, which was prevented significantly by administering a low dose of lithium 15 min later. In cultured cerebellar granule neurons, ethanol-induced apoptosis and activation of caspase-3/9, both of which were prevented by lithium. However, lithium's protection is not mediated by its commonly known inhibition of glycogen synthase3{beta}, because neither ethanol nor lithium has significant effects on the phosphorylation of Akt (ser473) or GSK3{beta} (ser9). In addition, the selective GSK-3{beta} inhibitor SB-415286 was unable to prevent ethanol-induced apoptosis. These data suggest lithium may be used as a potential preventive measure for ethanol-induced neurological deficits.
Zhong Jin [Departments of Pediatrics, Anatomy and Cell Biology, Riley Hospital for Children, 702 Barnhill Drive, Room 2641, Indiana University School of Medicine, Indianapolis, IN 46202 (United States)]. E-mail: firstname.lastname@example.com; Yang Xianlin [Departments of Pediatrics, Anatomy and Cell Biology, Riley Hospital for Children, 702 Barnhill Drive, Room 2641, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Yao Weiguo [Departments of Pediatrics, Anatomy and Cell Biology, Riley Hospital for Children, 702 Barnhill Drive, Room 2641, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Lee Weihua [Departments of Pediatrics, Anatomy and Cell Biology, Riley Hospital for Children, 702 Barnhill Drive, Room 2641, Indiana University School of Medicine, Indianapolis, IN 46202 (United States)
Pharmacological regulation of neutrophil activity and apoptosis
Novel strategies of antiinflammatory therapy are based upon pharmacological agents capable to enhance the resolution � i.e. the termination of the beneficial inflammation before it may turn into an adverse chronic stage. In contrast to the current therapy, which antagonises the formation of proinflammatory mediators, the �proresolving� therapy promotes natural antiinflammatory processes. It is likely that several drugs and phytochemicals would act in this way, but this point has not been investigated and thus might be totally overlooked. In this paper, effects of curcumin (diferuloylmethane) were analysed, considering the ability of this natural compound to affect resolution of inflammation through modulation of its important inputs � activity and apoptosis of neutrophils. The presented data indicate that, besides its well-known ability to suppress mechanisms engaged at the onset and progression of inflammation, curcumin could support resolution of inflammation through decreased activity and enhanced apoptosis of neutrophils. This substance decreased the formation of oxidants in neutrophils, both under in vitro conditions and after oral administration to arthritic rats. Moreover, curcumin accelerated spontaneous apoptosis of neutrophils, as indicated by increased externalisation of phosphatidylserine, by intercalation of propidium iodide and by enhanced activity of the executioner caspase-3.
Jancinova, Viera; Perecko, Tomas; Nosal, Radomir; Mihalova, Danica; Bauerova, Katarina; Drabikova, Katarina
Resistance of actin to cleavage during apoptosis.
A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1beta-converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation. PMID:8990178
Song, Q; Wei, T; Lees-Miller, S; Alnemri, E; Watters, D; Lavin, M F
Cell-Intrinsic Role for NF-kappa B-Inducing Kinase in Peripheral Maintenance but Not Thymic Development of Foxp3+ Regulatory T Cells in Mice
NF-?B inducing kinase (NIK, MAP3K14) is a key signaling molecule in non-canonical NF-?B activation, and NIK deficient mice have been instrumental in deciphering the immunologic role of this pathway. Global ablation of NIK prevents lymph node development, impairs thymic stromal development, and drastically reduces B cells. Despite altered thymic selection, T cell numbers are near normal in NIK deficient mice. The exception is CD4+ regulatory T cells (Tregs), which are reduced in the thymus and periphery. Defects in thymic stroma are known to contribute to impaired Treg generation, but whether NIK also plays a cell intrinsic role in Tregs is unknown. Here, we compared intact mice with single and mixed BM chimeric mice to assess the intrinsic role of NIK in Treg generation and maintenance. We found that while NIK expression in stromal cells suffices for normal thymic Treg development, NIK is required cell-intrinsically to maintain peripheral Tregs. In addition, we unexpectedly discovered a cell-intrinsic role for NIK in memory phenotype conventional T cells that is masked in intact mice, but revealed in BM chimeras. These results demonstrate a novel role for NIK in peripheral regulatory and memory phenotype T cell homeostasis.
Murray, Susan E.
Activation of NF-?B by the intracellular expression of NF-?B-inducing kinase acts as a powerful vaccine adjuvant
There is a pressing need for adjuvants that will enhance the effectiveness of genetic vaccines. This is particularly important in cancer and infectious disease such as HIV and malaria for which successful vaccines are desperately needed. Here, we describe an approach to enhance immunogenicity that involves the activation of NF-?B by the transgenic expression of an intracellular signaling molecule, NF-?B-inducing kinase (NIK). In vitro, NIK increases dendritic cell antigen presentation in allogeneic and antigen-specific T cell proliferation assays by potently activating NF-?B and consequently up-regulating the expression of cytokines (TNF-?, IL-6, IL-12, IL-15, and IL-18), chemokines [IL-8, RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein-1?, monocyte chemoattractant protein-1, and monocyte chemoattractant protein-3], MHC antigen-presenting molecules (class I and II), and costimulatory molecules (CD80 and CD86). In vivo, NIK enhances immune responses against a vector-encoded antigen and shifts them toward a T helper 1 immune response with increased IgG2a levels, T cell proliferation, IFN-? production, and cytotoxic T lymphocyte responses more potently than complete Freund's adjuvant, a very efficacious T helper 1-inducing adjuvant. These findings define NIK, and possibly other inducers of NF-?B activation, as a potent adjuvant strategy that offers great potential for genetic vaccine development.
Andreakos, E.; Williams, R. O.; Wales, J.; Foxwell, B. M.; Feldmann, M.
Apigenin, a bioactive flavonoid from Lycopodium clavatum, stimulates nucleotide excision repair genes to protect skin keratinocytes from ultraviolet B-induced reactive oxygen species and DNA damage.
In this study, we examined the antioxidative and the DNA protective potentials of apigenin, a flavonoid polyphenol isolated from Lycopodium clavatum, in both in-vitro (HaCaT skin keratinocytes) and in-vivo (mice) models against UV-B radiation. We used DAPI staining in UV-B-irradiated HaCaT skin keratinocytes pre-treated with and without apigenin to assess DNA damage. We also used a flow-cytometric analysis in mice exposed to UV-B radiation with or without topical application of apigenin to assess, through a comet assay, chromosomal aberrations and quanta from reactive oxygen species (ROS) generation. Data from the stability curves for the Gibb's free energy determined from a melting-temperature profile study indicated that apigenin increased the stability of calf thymus DNA. Immunofluorescence studies revealed that apigenin caused a reduction in the number of cyclobutane pyrimidine dimers (CPDs) after 24 h, the time at which the nucleotide excision repair (NER) genes were activated. Thus, apigenin accelerated reversal of UV-B-induced CPDs through up-regulation of NER genes, removal of cyclobutane rings, inhibition of ROS generation, and down-regulation of NF-?B and MAPK, thereby revealing the precise mechanism of DNA repair. PMID:24139463
Das, Sreemanti; Das, Jayeeta; Paul, Avijit; Samadder, Asmita; Khuda-Bukhsh, Anisur Rahman
NF-?B inducing kinase, a central signaling component of the non-canonical pathway of NF-?B, contributes to ovarian cancer progression.
Ovarian cancer is one of the leading causes of female death and the development of novel therapeutic approaches is urgently required. Nuclear factor-?B (NF-?B) is constitutively activated in several types of cancer including ovarian cancer and is known to support the survival of cancer cells. However, molecular mechanisms of persistent activation of NF-?B in ovarian cancer remain largely unknown. We report here that, in addition to the previously reported canonical activation, NF-?B is activated through the noncanonical pathway in ovarian cancer cells. RNA interference-mediated silencing of NF-?B inducing kinase (NIK), a central regulator of the noncanonical pathway, reduced the NF-?B2/p52 DNA binding activity and NF-?B-dependent reporter gene expression as well as NF-?B target gene expression. Notably, anchorage-dependent and -independent cell growth was impaired in NIK-depleted cells. Depletion of NIK also suppressed tumor formation in the nude mouse xenograft assay. These results indicate that NIK plays a key role in constitutive NF-?B activation and the progression of ovarian cancer cells and suggest that NIK represents an attractive therapeutic target for ovarian cancer. PMID:24533079
Uno, Masaya; Saitoh, Yasunori; Mochida, Kanako; Tsuruyama, Eri; Kiyono, Tohru; Imoto, Issei; Inazawa, Johji; Yuasa, Yasuhito; Kubota, Toshiro; Yamaoka, Shoji
NF-?B Inducing Kinase, a Central Signaling Component of the Non-Canonical Pathway of NF-?B, Contributes to Ovarian Cancer Progression
Ovarian cancer is one of the leading causes of female death and the development of novel therapeutic approaches is urgently required. Nuclear factor-?B (NF-?B) is constitutively activated in several types of cancer including ovarian cancer and is known to support the survival of cancer cells. However, molecular mechanisms of persistent activation of NF-?B in ovarian cancer remain largely unknown. We report here that, in addition to the previously reported canonical activation, NF-?B is activated through the noncanonical pathway in ovarian cancer cells. RNA interference-mediated silencing of NF-?B inducing kinase (NIK), a central regulator of the noncanonical pathway, reduced the NF-?B2/p52 DNA binding activity and NF-?B-dependent reporter gene expression as well as NF-?B target gene expression. Notably, anchorage-dependent and -independent cell growth was impaired in NIK-depleted cells. Depletion of NIK also suppressed tumor formation in the nude mouse xenograft assay. These results indicate that NIK plays a key role in constitutive NF-?B activation and the progression of ovarian cancer cells and suggest that NIK represents an attractive therapeutic target for ovarian cancer.
Uno, Masaya; Saitoh, Yasunori; Mochida, Kanako; Tsuruyama, Eri; Kiyono, Tohru; Imoto, Issei; Inazawa, Johji; Yuasa, Yasuhito; Kubota, Toshiro; Yamaoka, Shoji
Inhibitor of apoptosis proteins (IAPs) as regulatory factors of hepatic apoptosis.
IAPs are a group of regulatory proteins that are structurally related. Their conserved homologues have been identified in various organisms. In human, eight IAP members have been recognized based on baculoviral IAP repeat (BIR) domains. IAPs are key regulators of apoptosis, cytokinesis and signal transduction. The antiapoptotic property of IAPs depends on their professional role for caspases. IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. IAPs impede apoptotic process via membrane receptor-dependent (extrinsic) cascade and mitochondrial dependent (intrinsic) pathway. IAP-mediated apoptosis affects the progression of liver diseases. Therapeutic options of liver diseases may depend on the understanding toward mechanisms of the IAP-mediated apoptosis. PMID:23770286
Wang, Kewei; Lin, Bingliang
Aspartame-induced apoptosis in PC12 cells.
Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR. Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity. PMID:24355796
Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki
Artesunate induces AIF-dependent apoptosis in A549 cells
NASA Astrophysics Data System (ADS)
Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.
Zhou, Chen-Juan; Chen, Tong-Sheng
Crk is required for apoptosis in Xenopus egg extracts.
Apoptosis is essential for the development and homeostasis of multicellular organisms. Recently, a cell-free extract prepared from Xenopus eggs was shown to recapitulate intracellular apoptotic pathways in vitro. While many stimuli have been shown to trigger apoptosis in a variety of cell types, the intracellular signaling pathways involved in apoptosis remain largely unknown. Here we show that addition of a recombinant protein containing the phosphotyrosine binding (SH2) domain from the adaptor protein crk, but not those derived from a panel of other signaling proteins, can prevent apoptosis in the Xenopus egg extract system. Furthermore, immunodepletion of endogenous crk protein from the egg extracts, or addition of anti-crk antisera to these extracts, prevents apoptosis. The ability to undergo apoptosis can be restored to these extracts by addition of recombinant crk protein. These results directly demonstrate that crk participates in apoptotic signaling.
Evans, E K; Lu, W; Strum, S L; Mayer, B J; Kornbluth, S
Cucurbitacin B inhibits growth and induces apoptosis through the JAK2/STAT3 and MAPK pathways in SH?SY5Y human neuroblastoma cells.
Cucurbitacin�B (CuB) is a tetracyclic triterpene that is contained in extracts from cucurbitaceous plants and has been demonstrated to have anticancer and anti?in?ammatory activities. The purpose of the present study was to determine whether CuB exhibits anticancer effects on SH?SY5Y human neuroblastoma cells and to analyze the underlying molecular mechanism. The results demonstrated that CuB not only induced cell cycle arrest at the G2/M phase, but also induced apoptosis as characterized by positive Annexin�V staining, downregulation of phospho?Janus kinase�2 (p?JAK2), phospho?signal transducer and activator of transcription�3 (p?STAT3), phospho?extracellular signal?regulated kinases and the activation of c?Jun N?terminal kinase and p38 mitogen activated protein kinase (MAPK). CuB also altered the expression of gene products that mediated cell proliferation (Cyclin B1 and cyclin?dependent kinase�1), cell survival (B?cell lymphoma�2, Bcl2?associated�X protein) and increased the expression of p53 and p21. These results provide the evidence that JAK2/STAT3 and MAPKs have crucial roles in CuB?induced growth inhibition and apoptosis in SH?SY5Y human neuroblastoma cells. PMID:24789581
Zheng, Qian; Liu, Yunyi; Liu, Weiwei; Ma, Fengyun; Zhou, Yi; Chen, Mingjie; Chang, Junli; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan
Germ cell apoptosis in the testes of normal stallions
Apoptosis in testicular germ cells has been demonstrated in many mammalian species. However, little is known about the stallion (Equus caballus) and rates of apoptosis during spermatogenesis. Morphological and biochemical features of apoptosis reported in other species were used to confirm that the TdT-mediated dUTP Nick end labeling (TUNEL) assay is an acceptable method for identification and quantification of apoptotic
Noah L Heninger; Christophe Staub; Terry L Blanchard; Larry Johnson; Dickson D Varner; David W Forrest
Apoptosis Mediated by a Novel Leucine Zipper Protein Par4
The prostate apoptosis response-4 (par-4) gene was isolated in a differential screen for immediate-early genes that are up-regulated during apoptosis of prostate cancer cells. Unlike most other immediate-early genes, par-4 is exclusively induced during apoptosis. The expression or induction of par-4 is not restricted to prostatic cells. The par-4 gene is widely expressed in diverse normal tissues and cell types
V. M. Rangnekar
Apoptosis by Par4 in cancer and neurodegenerative diseases
Prostate apoptosis response-4 (par-4) is a pro-apoptotic gene identified in prostate cancer cells undergoing apoptosis. Par-4 protein, which contains a leucine zipper domain at the carboxy-terminus, functions as a transcriptional repressor in the nucleus. Par-4 selectively induces apoptosis in androgen-independent prostate cancer cells and Ras-transformed cells but not in androgen-dependent prostate cancer cells or normal cells. Cells that are resistant
Nadia El-Guendy; Vivek M Rangnekar
Par-4 inducible apoptosis in prostate cancer cells.
Prostate cancer is associated with the inability of prostatic epithelial cells to undergo apoptosis rather than with increased cell proliferation. Prostate apoptosis response-4 (Par-4) is a unique pro-apoptotic molecule that is capable of selectively inducing apoptosis in cancer cells when over-expressed, sensitizing the cells to diverse apoptotic stimuli and causing regression of tumors in animal models. This review discusses the salient functions of Par-4 that can be harnessed to prostate cancer therapy. PMID:14755681
Gurumurthy, Sushma; Rangnekar, Vivek M
Apoptosis versus oncotic necrosis in hepatic ischemia\\/reperfusion injury
Warm and cold hepatic ischemia followed by reperfusion leads to necrotic cell death (oncosis), which often occurs within minutes of reperfusion. Recent studies also suggest a large component of apoptosis after ischemia\\/reperfusion. Here, we review the mechanisms underlying adenosine triphosphate depletion�dependent oncotic necrosis and caspase-dependent apoptosis, with emphasis on shared features and pathways. Although apoptosis causes internucleosomal DNA degradation that
Hartmut Jaeschke; John J Lemasters
Antinuclear antibodies recognize cellular autoantigens driven by apoptosis
Objectives. � Present study addresses the issue whether cellular antigens recognised by antinuclear autoantibodies are driven by apoptosis.Materials and methods. � HEp-2 cells were committed to apoptosis by camptothecin; DNA fragmentation and FasL and Bax expression monitored apoptosis. Autoantigens were probed by indirect immunofluorescence and Western blot with autoantibodies or monoclonals against: DNA, Ro60, La, U1-RNP, CENP-B, DNA Topoisomerase I,
Roxana Ram�rez-Sandoval; Sergio H. S�nchez-Rodr�guez; David Herrera-van Oostdam; Esperanza Avalos-D�az; Rafael Herrera-Esparza
Antinuclear antibodies recognize cellular autoantigens driven by apoptosis
Objectives. - Present study addresses the issue whether cellular antigens recognised by antinuclear autoantibodies are driven by apoptosis. Materials and methods. - HEp-2 cells were committed to apoptosis by camptothecin; DNA fragmentation and FasL and Bax expression monitored apoptosis. Autoantigens were probed by indirect immunofluorescence and Western blot with autoantibodies or monoclonals against: DNA, Ro60, La, U1-RNP, CENP-B, DNA Topoisomerase
Roxana Ram�rez-Sandoval; Sergio H. S�nchez-Rodr�guez; David Herrera-van Oostdam; Esperanza Avalos-D�az; Rafael Herrera-Esparza
Fate of the Nuclear Lamina during Caenorhabditis elegans Apoptosis
Invertebrates and in Drosophila, lamins and lamin-associated proteins are primary targets for cleavage by caspases. Eliminating mammalian lamins causes apoptosis, whereas expressing mutant lamins that cannot be cleaved by caspase-6 delay apoptosis. Caenorhabditis elegans has a single lamin protein, Ce-lamin, and a caspase, CED-3, that is responsible for most if not all somatic apoptosis. In this study we show that
Yonatan B. Tzur; Bradley M. Hersh; H. Robert Horvitz; Yosef Gruenbaum
Induction of gastric epithelial apoptosis by Helicobacter pylori
BACKGROUND--Helicobacter pylori may promote gastric carcinogenesis through increasing gastric epithelial cell proliferation. How H pylori does so is unknown. Programmed, non-necrotic, cell death (apoptosis) occurs throughout the gut and is linked to proliferation. It was hypothesised that H pylori may induce hyper-proliferation through increasing apoptosis. AIM--To measure the effect of H pylori infection on gastric epithelial apoptosis in situ. PATIENTS--Patients
S F Moss; J Calam; B Agarwal; S Wang; P R Holt
Apoptosis and rheumatoid arthritis: Past, present, and future directions
The current studies of apoptosis in rheumatoid arthritis (RA) suggest that molecules (Fas-related or TNF-related), pathways\\u000a (activation of pro-apoptosis or anti-apoptosis pathway), cell types (lymphocytes or synovial fibroblast), and the mechanism\\u000a that triggers apoptosis (tolerance induction-related, down-modulation of inflammationrelated, or DNA damage-related) all play\\u000a a fundamental role to determine the induction or prevention of RA. These series of defects at
John D. Mountz; Hui-Chen Hsu; Yasunori Matsuki; Huang-Ge Zhang
[Apoptosis, a protective mechanism for pathogens and their hosts].
In this review we summarize the great amount of recent information on the apoptosis in aspects of the host-parasite interaction. Although apoptosis is a form of programmed cell death which plays a pivotal role in normal tissue development a plethora of pathogens including parasitic protista and helminths are able to modulate host apoptosis pathways to their own advantage. Here in we present and discuss new research data and results describing the phenomenon as a process have been controlled by gene expression, biochemical reactions and receptor-ligand interactions at the cell membrane surface. Section 1 describes apoptosis as ongoing process in normal tissue development. Section 2 analyzes the role of apoptosis in outcome of infection and pathogenesis of several disorders evoked by viruses and bacteria. The cellular mechanisms of cell death during infection with unicellular parasites such as Leishmania sp. and Plasmodium sp. are described in Section 3. In the next paragraph the potency of parasitic protista and helmiths for modulation host apoptosis pathways to their own advantage is discussed. The involvement of apoptosis in immunoregulation of the host immune function was proposed as a one of possible mechanism in creation of the host-parasite relationship. The molecular and cellular mechanisms of parasite-induced immune response via apoptosis pathways are discussed. We conclude that novel strategies for the management of the host-parasite relationships need to be explained into the mechanisms by which parasites induced apoptosis in contribution to the activity of immune system of the host. PMID:16913499
Donskow-Schmelter, Katarzyna; Doligalska, Maria
Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation
The protease granzyme�B (GrB) plays a key role in the cytocidal activity during cytotoxic T lymphocyte (CTL)-mediated programmed cell death. Multiple caspases have been identified as direct substrates for GrB, suggesting that the activation of caspases constitutes an important event during CTL-induced cell death. However, recent studies have provided evidence for caspase-independent pathway(s) during CTL-mediated apoptosis. In this study, we demonstrate caspase-independent and direct cleavage of the 45 kDa unit of DNA fragmentation factor (DFF45) by GrB both in vitro and in vivo. Using a novel and selective caspase-3 inhibitor, we show the ability of GrB to process DFF45 directly and mediate DNA fragmentation in the absence of caspase-3 activity. Furthermore, studies with DFF45 mutants reveal that both caspase-3 and GrB share a common cleavage site, which is necessary and sufficient to induce DNA fragmentation in target cells during apoptosis. Together, our data suggest that CTLs possess alternative mechanism(s) for inducing DNA fragmentation without the requirement for caspases.
Sharif-Askari, Ehsan; Alam, Antoine; Rheaume, Eric; Beresford, Paul J.; Scotto, Christian; Sharma, Kamal; Lee, Dennis; DeWolf, Walter E.; Nuttall, Mark E.; Lieberman, Judy; Sekaly, Rafick-Pierre
The Endoplasmic Reticulum Stress-Mediated Apoptosis Signal Pathway Is Involved in Sepsis-Induced Abnormal Lymphocyte Apoptosis
Background\\/Aim: The mechanisms of abnormal lymphocyte apoptosis in sepsis are only partially defined. The present study was designed to investigate whether the endoplasmic reticulum (ER) is implicated in the extensive apoptosis of lymphocytes in sepsis. Methods: C57BL\\/6 mice were randomized into cecal ligation and puncture (CLP) and sham operation groups. Apoptosis was detected by the TUNEL method and flow cytometry.
T. Ma; L. Han; Y. Gao; L. Li; X. Shang; W. Hu; C. Xue
Cadherin engagement protects human ?-cells from apoptosis.
The aim of this study was to assess the expression of different types of cadherins in human islets and their role in human ?-cell apoptosis. Expression of E-, N-, and P-cadherins was studied by immunofluorescence on pancreas sections and islet cells, and by Western blotting on protein extracts of isolated islets and islet cells. The effects of specific cadherins on cell adhesion and apoptosis were studied using chimeric proteins containing functional E-, N-, or P-cadherin ectodomains fused to Fc fragment of Ig (E-cad/Fc, N-cad/Fc, and P-cad/Fc) and immobilized on glass substrate. ?-Cells were identified by immunofluorescence for insulin and apoptotic cells by terminal deoxynucleotide transferase-mediated 2'-deoxyuridine, 5'-triphosphate nick-end labeling. By immunofluorescence, we showed that E- and N-, and not P-, cadherins were expressed at the surface of islet cells. By triple staining, we showed that E-cadherin was expressed at similar extent in ?- and ?-cells, whereas N-cadherin was preferentially expressed in ?-cells. These results were confirmed by Western blot analysis using protein extracts from fluorescence-activated cell sorting-sorted ?- and non-?-cells. Adhesion tests showed that the affinity of islet cells for E-cad/Fc and N-cad/Fc and not for P-cad/Fc was increased compared with control. By terminal deoxynucleotide transferase-mediated 2'-deoxyuridine, 5'-triphosphate nick-end labeling, we showed that the percentage of apoptotic cells was lower in aggregated ?-cells compared with single ?-cells and that attachment to E-cad/Fc and N-cad/Fc and not to P-cad/Fc decreased apoptosis of single ?-cells compared with control. Our results show that at least E- and N-cadherins are expressed at the surface of human ?-cells and that these adhesion molecules are involved in the maintenance of ?-cell viability. PMID:21990317
Parnaud, G�raldine; Gonelle-Gispert, Carmen; Morel, Philippe; Giovannoni, Laurianne; Muller, Yannick D; Meier, Raphael; Borot, Sophie; Berney, Thierry; Bosco, Domenico
Apoptosis and immune responses to self.
The mystery that surrounds autoimmunity revolves around how the immune system of patients who have systemic autoimmune diseases becomes primed to recognize intracellular antigens, how the autoantibodies thus produced contribute to the pathogenesis of the disease, and how those autoantibodies access their target proteins. By examining the mechanisms that are involved in the normal cellular process of apoptosis, we are beginning to unravel this mystery. The intracellular autoantigen targets of many systemic autoimmune diseases become altered during apoptosis in ways that may change how they are perceived by the immune system. High concentrations of self-antigens, or in the case of viral infection, complexes of foreign and self-antigens, are packaged during generation of apoptotic cells. The packages also may contain altered fragments of self-antigens that have not been encountered previously by the immune system. Under normal circumstances, apoptotic cells are cleared rapidly by macrophages and DCs. The normal consequence of that clearance is that the apoptosis-altered self-antigens are either ignored by the immune system or tolerance to those antigens is maintained. Clearance is achieved through complex mechanisms that enable macrophages and DCs to recognize apoptotic cells as nonthreatening "self" particles. Defects in this process that cause a delay in clearance could change the appearance of apoptotic cells and cause them to be recognized as "foreign invaders," thereby stimulating an inflammatory response that, in turn, activates an immune response to self-antigens. By studying the mechanisms that are involved in recognition and clearance of apoptotic cells, we are uncovering clues to the defects that may underlie the development of systemic autoimmunity. PMID:15061575
Navratil, Jeannine S; Sabatine, Janice M; Ahearn, Joseph M
Cardiac Apoptosis in Severe Relapsing Fever Borreliosis
Previous studies revealed that the heart suffers significant injury during experimental Lyme and relapsing fever borreliosis when the immune response is impaired (D. Cadavid, Y. Bai, E. Hodzic, K. Narayan, S. W. Barthold, and A. R. Pachner, Lab. Investig. 84:1439-1450, 2004; D. Cadavid, T. O'Neill, H. Schaefer, and A. R. Pachner, Lab. Investig. 80:1043-1054, 2000; and D. Cadavid, D. D. Thomas, R. Crawley, and A. G. Barbour, J. Exp. Med. 179:631-642, 1994). To investigate cardiac injury in borrelia carditis, we used antibody-deficient mice persistently infected with isogenic serotypes of the relapsing fever agent Borrelia turicatae. We studied infection in hearts 1 to 2 months after inoculation by TaqMan reverse transcription-PCR and immunohistochemistry (IHC) and inflammation by hematoxylin and eosin and trichrome staining, IHC, and in situ hybridization (ISH). We studied apoptosis by terminal transferase-mediated DNA nick end labeling assay and measured expression of apoptotic molecules by RNase protection assay, immunofluorescence, and immunoblot. All antibody-deficient mice, but none of the immunocompetent controls, developed persistent infection of the heart. Antibody-deficient mice infected with serotype 2 had more severe cardiac infection and injury than serotype 1-infected mice. The injury was more severe around the base of the heart and pericardium, corresponding to sites of marked infiltration by activated macrophages and upregulation of interleukin-6 (IL-6). Infected hearts showed evidence of apoptosis of macrophages and cardiomyocytes as well as significant upregulation of caspases, most notably caspase-1. We conclude that persistent infection with relapsing fever borrelias causes significant loss of cardiomyocytes associated with prominent infiltration by activated macrophages, upregulation of IL-6, induction of caspase-1, and apoptosis.
Londono, Diana; Bai, Yunhong; Zuckert, Wolfram R.; Gelderblom, Harald; Cadavid, Diego
Canine coronavirus induces apoptosis in cultured cells.
Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups. In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown. We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in order to characterize the apoptotic effect in homologous cell system. Following CCoV infection A-72 cell line, which is permissive to CCoV, showed reduced growth rate, as detected by MTT assay, a standard colorimetric assay for measuring cellular proliferation, and underwent to apoptotic death. Starting from 24h after CCoV infection, cells morphology appeared dramatically changed, with cells rounding and detachment from culture surface. Morphologic and biochemical features of apoptosis, such as blebbing of the plasma membrane, translocation of phosphatidilserine to cell surface and annexin V positive staining, nuclear fragmentation, apoptotic bodies formation and DNA laddering, were detected in CCoV-infected cells. Propidium iodide staining of infected culture indicated the appearance of hypodiploid DNA peak corresponding to apoptotic cell population. Commonly to other animal coronavirus infection caspase-3 is likely to contribute to the execution phase of apoptosis induced by CCoV in A-72 cells since we found activation of enzymatic activity as well as procaspase-3 activating cleavage. Apoptotic death of infected cells is detrimental as it causes cell and tissue destruction as well as inflammatory responses. Therefore in the case of CCoV associated gastroenteritis, apoptosis of epithelial mucosa cells may be responsible for pathology induced by CCoV infection. PMID:17254720
Ruggieri, A; Di Trani, L; Gatto, I; Franco, M; Vignolo, E; Bedini, B; Elia, G; Buonavoglia, C
An increase of granulosa cell apoptosis mediates aqueous neem (Azadirachta indica) leaf extract-induced oocyte apoptosis in rat
Objective: Neem plant (Azadirachta indica) has been extensively used in Ayurvedic system of medicine for female fertility regulation for a long time, but its mechanism of action remains poorly understood. Hence, the present study was aimed to determine whether an increase of granulosa cell apoptosis is associated with aqueous neem leaf extract (NLE)-induced oocyte apoptosis. Materials and Methods: Sexually immature female rats of 20 days old were fed NLE (50 mg/day) for 10 days and then subjected to superovulation induction protocol. The morphological changes in cumulus oocyte complexes (COCs), rate of oocyte apoptosis, hydrogen peroxide (H2O2), total nitrite, and cytochrome c concentrations, inducible nitric oxide synthase (iNOS), cytochrome c, p53, Bcl2 and Bax expressions, deoxyribonucleic acid (DNA) fragmentation, and estradiol 17? level in granulosa cells collected from preovulatory COCs were analyzed. Results: Aqueous NLE increased H2O2 concentration and decreased catalase activity, increased iNOS expression and total nitrite concentration, increased p53, Bax, and p53 expressions but decreased Bcl2 expression, increased cytochrome c concentration and induced DNA fragmentation in granulosa cells. An increased granulosa cell apoptosis resulted in reduced estradiol 17? concentration and induced apoptosis in ovulated oocytes. Conclusion: We conclude that aqueous NLE-induced granulosa cell apoptosis through the mitochondria-mediated pathway, reduced estradiol 17? concentration and induced apoptosis in ovulated oocytes. Thus, granulosa cell apoptosis mediates NLE-induced oocyte apoptosis during female fertility regulation in rat.
Tripathi, Anima; Shrivastav, Tulsidas G; Chaube, Shail K
Detecting apoptosis in Drosophila tissues and cells.
In this chapter we discuss methods that can be used to study apoptotic cell death in the Drosophila embryo, ovary, as well as in cultured cell lines. These methods assay various aspects of the cell death process, from mitochondrial changes to caspase activation and DNA cleavage. The assays are useful for examining apoptosis in normal development and in response to developmental perturbations and external stresses. These techniques include Acridine Orange staining, TUNEL, cleaved caspase staining, caspase activity assays and assays for mitochondrial fission and permeabilization. PMID:24613678
Sarkissian, Tatevik; Timmons, Allison; Arya, Richa; Abdelwahid, Eltyeb; White, Kristin
Cis-urocanic acid suppresses UV-B-induced interleukin-6 and -8 secretion and cytotoxicity in human corneal and conjunctival epithelial cells in vitro
Purpose Urocanic acid (UCA) is a major ultraviolet (UV)-absorbing endogenous chromophore in the epidermis and is also an efficacious immunosuppressant. The anti-inflammatory and cytoprotective effects of cis-UCA were studied in ocular surface cell cultures exposed to UV-B irradiation. Methods Human corneal epithelial cells (HCE-2) and human conjunctival epithelial cells (HCECs) were incubated with 10, 100, 1,000, and 5,000 ?g/ml cis-UCA with and without a single UV-B irradiation dose. The concentrations of IL-1?, IL-6, IL-8, and TNF-? in the culture medium and caspase-3 activity in the cell extract sampled were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was measured by the colorimetric MTT (3-(4,5-dimethyldiazol- 2-yl)-2,5-diphenyltetrazolium bromide) assay. Results UV-B irradiation multiplied interleukin IL-6 and IL-8 secretion levels in HCE-2 cells and HCECs as analyzed with ELISA. Cell viability as measured by the MTT assay declined by 30%�50% in HCE-2 cells and by 20%�40% in HCECs after UV-B irradiation. Moreover, UV-B increased caspase-3 activity in both cell types as analyzed with ELISA. Treatment with 100 ?g/ml cis-UCA completely suppressed IL-6 and IL-8 secretion, decreased caspase-3 activity, and improved cell viability against UV-B irradiation. No significant effects on IL-6 or IL-8 secretion, caspase-3 activity, or viability of the non-irradiated cells were observed with 100 ?g/ml cis-UCA in both cell types. The 5,000 ?g/ml concentration was toxic. Conclusions These findings indicate that cis-UCA may represent a promising anti-inflammatory and cytoprotective treatment option to suppress UV-B-induced inflammation and cellular damage in human corneal and conjunctival epithelial cells.
Viiri, J.; Jauhonen, H.M.; Kauppinen, A.; Ryhanen, T.; Paimela, T.; Hyttinen, J.; Sorri, I.; Laihia, J.K.; Leino, L.
A novel NF-kappa B-inducing kinase-MAPK signaling pathway up-regulates NF-kappa B activity in melanoma cells.
Constitutive activation of NF-kappa B is an emerging hallmark of various types of tumors including breast, colon, pancreatic, ovarian, and melanoma. In melanoma cells, the basal expression of the CXC chemokine, CXCL1, is constitutively up-regulated. This up-regulation can be attributed in part to constitutive activation of NF-kappa B. Previous studies have shown an elevated basal I kappa B kinase (IKK) activity in Hs294T melanoma cells, which leads to an increased rate of I kappa B phosphorylation and degradation. This increase in I kappa B-alpha phosphorylation and degradation leads to an approximately 19-fold higher nuclear localization of NF-kappa B. However, the upstream IKK kinase activity is up-regulated by only about 2-fold and cannot account for the observed increase in NF-kappa B activity. We now demonstrate that NF-kappa B-inducing kinase (NIK) is highly expressed in melanoma cells, and IKK-associated NIK activity is enhanced in these cells compared with the normal cells. Kinase-dead NIK blocked constitutive NF-kappa B or CXCL1 promoter activity in Hs294T melanoma cells, but not in control normal human epidermal melanocytes. Transient overexpression of wild type NIK results in increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which is inhibited in a concentration-dependent manner by PD98059, an inhibitor of p42/44 MAPK. Moreover, the NF-kappa B promoter activity decreased with overexpression of dominant negative ERK expression constructs, and EMSA analyses further support the hypothesis that ERK acts upstream of NF-kappa B and regulates the NF-kappa B DNA binding activity. Taken together, our data implicate involvement of I kappa B kinase and MAPK signaling cascades in NIK-induced constitutive activation of NF-kappa B. PMID:11773061
Dhawan, Punita; Richmond, Ann
A Novel NF-?B-inducing Kinase-MAPK Signaling Pathway Up-regulates NF-?B Activity in Melanoma Cells*
Constitutive activation of NF-?B is an emerging hallmark of various types of tumors including breast, colon, pancreatic, ovarian, and melanoma. In melanoma cells, the basal expression of the CXC chemokine, CXCL1, is constitutively up-regulated. This up-regulation can be attributed in part to constitutive activation of NF-?B. Previous studies have shown an elevated basal I?B kinase (IKK) activity in Hs294T melanoma cells, which leads to an increased rate of I?B phosphorylation and degradation. This increase in I?B-? phosphorylation and degradation leads to an ~19-fold higher nuclear localization of NF-?B. However, the upstream IKK kinase activity is up-regulated by only about 2-fold and cannot account for the observed increase in NF-?B activity. We now demonstrate that NF-?B-inducing kinase (NIK) is highly expressed in melanoma cells, and IKK-associated NIK activity is enhanced in these cells compared with the normal cells. Kinase-dead NIK blocked constitutive NF-?B or CXCL1 promoter activity in Hs294T melanoma cells, but not in control normal human epidermal melanocytes. Transient overexpression of wild type NIK results in increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which is inhibited in a concentration-dependent manner by PD98059, an inhibitor of p42/44 MAPK. Moreover, the NF-?B promoter activity decreased with overexpression of dominant negative ERK expression constructs, and EMSA analyses further support the hypothesis that ERK acts upstream of NF-?B and regulates the NF-?B DNA binding activity. Taken together, our data implicate involvement of I?B kinase and MAPK signaling cascades in NIK-induced constitutive activation of NF-?B.
Dhawan, Punita; Richmond, Ann
Odoroside A and ouabain inhibit Na+/K+-ATPase and prevent NF-kappaB-inducible protein expression by blocking Na+-dependent amino acid transport.
Inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin-1 (IL-1), trigger the activation of transcription factor NF-kappaB that induces the expression of a variety of genes, including intercellular adhesion molecule (ICAM)-1. Odoroside A [3beta-O-(beta-D-diginosyl)-14-hydroxy-5beta,14beta-card-20(22)-enolide] was found to inhibit the cell-surface expression of ICAM-1 induced by TNF-alpha and IL-1 at comparable concentrations in human lung carcinoma A549 cells. In this study, the molecular mechanism underlying the inhibition of TNF-alpha-induced cell-surface ICAM-1 expression by odoroside A together with the specific Na(+)/K(+)-ATPase inhibitor ouabain was further investigated. Odoroside A and ouabain neither prevented IkappaBalpha degradation nor NF-kappaB translocation to the nucleus upon TNF-alpha stimulation. While odoroside A and ouabain had no inhibitory effect on the induction of ICAM-1 mRNA, they inhibited the TNF-alpha-induced ICAM-1 expression at the protein level. Consistent with these results, odoroside A and ouabain potently reduced de novo protein synthesis, largely due to its ability to block Na(+)-dependent transport of amino acids across the plasma membrane, but not to interfering with the translation machinery. As a direct molecular target, odoroside A was found to inhibit the ATP-hydrolyzing activity of Na(+)/K(+)-ATPase as potently as ouabain. These results clearly demonstrate that odoroside A and ouabain prevent NF-kappaB-inducible protein expression by blocking the Na(+)-dependent amino acid transport. PMID:19559678
Takada, Yohei; Matsuo, Kentaro; Ogura, Hirotsugu; Bai, Liming; Toki, Asami; Wang, Liyan; Ando, Masayoshi; Kataoka, Takao
miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells.
Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NF?B activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells. PMID:24091729
Amodio, Nicola; Bellizzi, Dina; Leotta, Marzia; Raimondi, Lavinia; Biamonte, Lavinia; D'Aquila, Patrizia; Di Martino, Maria Teresa; Calimeri, Teresa; Rossi, Marco; Lionetti, Marta; Leone, Emanuela; Passarino, Giuseppe; Neri, Antonino; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco
NF-?B-inducing kinase (NIK) prevents the development of hypereosinophilic syndrome (HES)-like disease in mice independent of IKK? activation
Immune cell-mediated tissue injury is the common feature of different inflammatory diseases, yet the pathogenetic mechanisms and cell types involved vary significantly. Hypereosinophilic Syndrome (HES) represents a group of inflammatory diseases that are characterized by increased numbers of pathogenic eosinophilic granulocytes in the peripheral blood and diverse organs. Based on clinical and laboratory findings, various forms of HES have been defined, yet the molecular mechanism and potential signaling pathways that drive eosinophil expansion remain largely unknown. Here we show that mice deficient of the serine/ threonine-specific protein kinase NF-?B inducing kinase (NIK) develop a HES-like disease, reflected by progressive blood and tissue eosinophilia, tissue injury and premature death at around 25�30 weeks of age. Similar to the lymphocytic form of HES, CD4+ T-cells from NIK-deficient mice express increased levels of T-helper 2 (Th2)-associated cytokines, and eosinophilia and survival of NIK deficient mice could completely be prevented by genetic ablation of CD4+ T-cells. Experiments based on bone marrow chimeric mice, however, demonstrated that inflammation in NIK-deficient mice depended on radiation-resistant tissues, implicating that NIK-deficient immune cells mediate inflammation in a non-autonomous manner. Surprisingly, disease development was independent of NIKs known function as IkappaB kinase (IKK)-? kinase, as mice carrying a mutation in the activation loop of IKK?, which is phosphorylated by NIK, did not develop inflammatory disease. Our data show that NIK activity in non-hematopoietic cells controls Th2-cell development and prevents eosinophil-driven inflammatory disease, most likely using a signaling pathway that operates independent of the known NIK substrate IKK?.
Hacker, Hans; Chi, Liying; Rehg, Jerold E.; Redecke, Vanessa
Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant Abstract HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...
Inhibitor of apoptosis protein hILP undergoes caspase-mediated cleavage during T lymphocyte apoptosis.
Several endogenous or viral inhibitors of apoptosis, including Bcl-2, Bcl-xL, FLIP, p35, and CrmA, have been shown to be cleaved by caspases during apoptosis. In this study, we demonstrate that the endogenous inhibitor of apoptosis, hILP/XIAP, is also cleaved in apoptotic T lymphocytes, generating at least one prominent fragment of 29 kDa. This p29 cleaved fragment was detected in Jurkat cells induced to apoptose by anti-Fas antibody, staurosporin, or VP-16. The cleavage of hILP appears to be caspase mediated because the production of the p29 protein was inhibited by the pan-caspase peptide inhibitor, Z-VAD.FMK. In Jurkat cells engineered to overexpress CrmA, cleavage of hILP in response to anti-Fas antibody or staurosporin was inhibited, whereas overexpression of Bcl-2 abrogated the cleavage in response to VP-16. Cleavage of hILP was also observed in cell-free reactions using in vitro translated hILP and recombinant caspase-3 or -7. Moreover, we found that the p29 hILP fragment retained the ability to bind caspase-3 and -7, as shown previously for full-length or BIR-2 hILP. The p29 cleavage product was also detected during T-cell receptor-mediated apoptosis in peripheral blood lymphocytes from normal donors. Furthermore, tumor-associated T lymphocytes purified from ascites of patients with ovarian cancer expressed fragmented hILP, which was not detected in control T cells purified from peripheral blood of normal donors. Our results suggest that the cleavage of hILP represents an important event in apoptosis of T lymphocytes in both normal and pathological in vivo settings. PMID:10766165
Johnson, D E; Gastman, B R; Wieckowski, E; Wang, G Q; Amoscato, A; Delach, S M; Rabinowich, H
microRNA-29b contributes to pre-eclampsia through its effects on apoptosis, invasion and angiogenesis of trophoblast cells.
PE (pre-eclampsia), a pregnancy-specific disorder, is characterized by increased trophoblast cell death and deficient trophoblast invasion and reduced trophoblast-mediated remodelling of spiral arteries. The present study was performed to determine the function of miR-29b (microRNA-29b) in trophoblast cells and its underlying role in the pathogenesis of PE. The prediction of miR-29b target genes was performed using computer-based programs, including Targetscan, Pictar and miRBase. The function of these target genes was analysed further by gene ontology (GO). The effects of miR-29b on apoptosis, and invasion and angiogenesis of trophoblast cell lines (HTR-8/SVneo, BeWo and JAR) were examined by flow cytometry and Matrigel assay respectively. We found that miR-29b induced apoptosis and inhibited invasion and angiogenesis of trophoblast cells. Further studies confirmed that miR-29b regulated the expression of MCL1 (myeloid cell leukaemia sequence 1), MMP2 (encoding matrix metallproteinase 2), VEGFA (vascular endothelial growth factor A) and ITGB1 (integrin ?1) genes by directly binding to their 3'-UTRs (untranslated regions). Moreover, we identified that there was an inverse correlation between miR-29b and its target genes in subjects with PE. Taken together, these findings support a novel role for miR-29b in invasion, apoptosis and angiogenesis of trophoblast cells, and miR-29b may become a new potential therapeutic target for PE. PMID:22716646
Li, Pengfei; Guo, Wei; Du, Leilei; Zhao, Junli; Wang, Yaping; Liu, Liu; Hu, Yali; Hou, Yayi