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Fluid Shear Stress Increases the Production of Granulocyte-Macrophage Colony-Stimulating Factor by Endothelial Cells via mRNA Stabilization
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Abstract—To investigate whether the production of colony-stimulating factors (CSFs) by vascular endothelial cells is regulated by hemodynamic force, we exposed cultured human umbilical vein endothelial cells (HUVECs) to controlled levels of shear stress in a flow-loading apparatus and examined changes in the production of CSFs at both the protein and mRNA level. Exposure of HUVECs to a shear stress of 15 and 25 dyne/cm2 markedly increased the release of granulocyte-macrophage CSF (GM-CSF) detected by ELISA to 5.0 and 9.5 times, respectively, the amount released by the static controls at 24 hours, but it had no significant influence on the release of granulocyte CSF or macrophage CSF. The results of reverse transcriptase–polymerase chain reaction demonstrated that GM-CSF mRNA began to increase as early as 2 hours after initiation of 15 dyne/cm2 shear stress and continued to increase with time, reaching a peak of about four times the control levels at 24 hours. This increase in GM-CSF mRNA levels in response to shear stress depended on protein synthesis, because it was blocked by cycloheximide. Neither nuclear run-on assay or luciferase assay using a reporter gene containing GM-CSF gene promoter showed any significant change in transcription of the GM-CSF gene even after 24-hour exposure to a shear stress of 15 dyne/cm2. Actinomycin D chase experiments using a competitive polymerase chain reaction showed that shear stress extended the half-life of GM-CSF mRNA from ≈23 to 42 minutes in HUVECs. These findings suggest that fluid shear stress increases the production of GM-CSF in HUVECs via mRNA stabilization.
Granulocyte-macrophage CSF, a member of the hematopoietic growth factor family, is produced and released by monocytes, macrophages, fibroblasts, and ECs and stimulates the proliferation and differentiation of granulocyte/macrophage progenitor cells.1 Recently, it has been learned that in addition to its effect on hematopoietic cells, GM-CSF can act on nonhematopoietic cells.2 Specific GM-CSF receptors have been detected on the surface of ECs, fibroblasts, trophoblasts, and keratinocytes,3 with ECs appearing to be particularly strongly influenced by GM-CSF. This cytokine induces the proliferation and migration of ECs in vitro4 and causes new capillary formation in vivo.5 It also upregulates the synthesis of prostacyclin6 and the expression of class I myosin heavy chain antigens7 and affects the adhesion of leukocytes to ECs by modulating adhesion molecule expression on the surface of ECs.8 In addition, GM-CSF activates the Na+-H+ exchanger9 and the transcription of the oncogene c-fos in ECs.4 Thus, GM-CSF produced by ECs may act on ECs themselves in an autocrine or paracrine manner and may play an important role in the regulation of blood vessel functions.
A variety of EC functions were thought to be controlled mainly by chemical stimuli, such as hormones, cytokines, and neurotransmitters, but recently, they have been found to also be regulated by mechanical stimuli generated by blood flow or blood pressure.10 A number of studies have indicated that the fluid shear stress created by blood flow modulates EC morphology and functions and, at the same time, alters the expression of the related EC genes.11 For instance, shear stress upregulates or downregulates the mRNA levels of genes coding tissue plasminogen activator,12 PDGF,13 14 15 endothelin,16 17 18 MCP-1,19 transforming growth factor-β,20 superoxide dismutase,21 22 angiotensin-converting enzyme,23 nitric oxide synthase,24 C-type natriuretic peptide,25 adrenomedulin,26 thrombomodulin,27 28 heparin-binding epidermal growth factor,29 tissue factor,30 intercellular cell adhesion molecule-1,31 and VCAM-1.32 More recently, cis-acting elements and transcriptional factors involved in the molecular mechanism of shear stress–mediated regulation of EC genes have been identified; these are SSRE and NF-κB, which have been implicated in the upregulation of the PDGF-B gene by shear stress,33 and TRE and AP-1, which are involved in the MCP-1 and VCAM-1 gene responses to shear stress.34 35 A great deal of attention has been focused on such EC gene responses to shear stress in relation to the mechanisms of blood flow–dependent phenomena, such as angiogenesis, vascular remodeling, and atherosclerosis, but a great deal still remains to be elucidated.
ECs constitutively produce small amounts of GM-CSF, but they markedly increase production in response to chemical stimuli, such as TPA, IL-1, TNF, lipopolysaccharide, and acetylated LDL. However, it was uncertain whether production of GM-CSF by ECs is affected by blood flow or shear stress, a critical physiological stimulus. To answer this question, we applied controlled levels of shear stress to cultured HUVECs in a flow-loading apparatus and examined changes in GM-CSF production at both the protein and mRNA levels. We also investigated the effect of shear stress on GM-CSF gene transcription and mRNA stability.
Materials and Methods
Cell Culture
Primary cultures of HUVECs were obtained from human umbilical cord veins by collagenase treatment and grown in a 1% gelatin-coated flask in medium 199 containing 15% FBS (Hyclone Laboratories), 2 mmol/L l-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin, 50 μg/mL heparin, and 30 μg/mL EC growth factor (Becton and Dickinson). Cells were routinely passaged by trypsinization in a 0.05% trypsin/2 mmol/L EDTA solution. At every passage, cell number was counted with a Coulter Counter (type ZM, Coulter Electronics Limited), and cumulative population doubling was determined. Cells with 8 to 14 cumulative population doubling (passages 4 to 7) were grown to confluence on gelatin-coated glass plates (55 mm×70 mm) for use in the experiments. The purity of the cultures was demonstrated by immunofluorescent staining for the presence of factor VIII–related antigen and by the uptake of fluorescence-labeled acetylated LDL.
Bovine ECs were isolated from the descending thoracic aorta of a bovine fetus by brief collagenase digestion and cultured in medium 199 containing 15% FBS, 2 mmol/L l-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin. Cells at passages 4 to 7 were used for transfection experiments.
Flow-Loading Apparatus
ECs were exposed to laminar flow in a parallel-plate type of flow chamber, as previously described.32 Briefly, one side of the flow chamber consisted of a glass plate on which the cultured ECs rested, and the other side was a polycarbonate plate. These two flat surfaces were held 200 μm apart by a polytetrafluoroethylene gasket. The intensity of shear stress (τ, dyne/cm2) acting on the EC layer was calculated by the following formula: τ=6μQ/a2b, where μ is the viscosity of the perfusate (poise), Q is flow volume (mL/s), and a and b are cross-sectional dimensions of the flow path (cm). A closed circuit was arranged with a silicone tube, and a depulsator was placed between the pump and flow chamber to eliminate ripples generated by the pump. Medium was constantly circulated with a roller/tube pump (Atto Co) at 37°C in an atmosphere of 95% room air and 5% CO2.
In preliminary experiments, the characteristics of flow through the chamber containing the coverslip were visually examined at various perfusion rates by circulating medium with suspended polystyrene flakes. Flow patterns, as analyzed with a high-speed video camera (MHS200, NAC), showed no visible turbulence. Since the maximum Reynolds number corresponding to the highest flow rate used in the present study was ≈40, we assumed the flow to be laminar.
In some experiments, 5% dextran (molecular weight, 162 000; Sigma Chemical Co) was added to the perfusate to raise the viscosity as much as four times that of the control medium. All conditioned culture media and perfusates obtained after flow-loading experiments were confirmed to be free of endotoxin by the limulus gelatin test.
The amount of CSFs released by the ECs was assayed by ELISA using commercially available kits (Ohtsuka Assay Co and R&D Systems). Briefly, 100 μL of perfusate was incubated in a microplate coated with anti-human monoclonal antibodies against each CSF for 2 hours. After a washing with the specified detergent, each CSF conjugate was added and incubated for 2 hours. After the washing, the color reagent was added, and incubation was performed for 20 minutes. Absorbance at a specified wavelength was measured with a microplate reader (model 3550, Bio-Rad), and the concentration of CSF in each sample was determined from the standard curve.
RT-PCR Analysis
RT-PCR was performed to quantify the mRNA levels of each CSF, as previously described.32 Briefly, total RNA was isolated from the cells by the acid guanidine thiocyanate–phenol–chloroform extraction method. Reverse transcription of RNA was carried out in 20 μL of a reaction mixture containing 1.0 μg total RNA, 200 U Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL), 0.5 μg oligo d(T) 12–18 (Perkin Elmer-Cetus), 40 U ribonuclease inhibitor (Perkin Elmer-Cetus), 2.5 mmol of each dNTP mixture, and 10 mmol dithiothreitol in a first-strand buffer of 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, and 3 mmol/L MgCl2. The mixture was incubated at 37°C for 1 hour, heated at 99°C for 5 minutes, and chilled at 4°C for 5 minutes. The cDNA samples were then coamplified by PCR with primer pairs for each CSF and GAPDH (Table⇓). Eighty microliters of a solution containing 0.25 U of ExTaq DNA polymerase (Takara), 370 kBq of [α-32P]dCTP (≈111 TBq/mmol) (Amersham), and 0.1 pmol of each primer in an ExTaq buffer of 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 4 mmol/L MgCl2, and 0.001% gelatin was added to each sample. Each temperature cycle consisted of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. Ten microliters of amplified product was sampled every other cycle and electrophoresed on a 5% polyacrylamide gel (Sigma).
For quantification of PCR products, the radioactivity of each band was measured with a GS363 Molecular Imager System (Bio-Rad) and plotted against the number of PCR cycles on a semilogarithmic scale, forming a sigmoid curve. From the curve, the cycle in which the operating range of the PCR was linear was selected, and the ratio of radioactivity between CSF and GAPDH in the cycle was calculated as a parameter of relative CSF mRNA levels.
To confirm that CSF mRNA and GAPDH mRNA were correctly amplified by those primers, the PCR product was cloned into the pCR II vector using the TA cloning system (Invitrogen) and sequenced by the cycle-sequencing methods using the ABIPRISSM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer-Cetus). The sequences showed complete homology to the parts of each gene of interest.
Nuclear Run-on Assay
HUVECs, incubated under static conditions or exposed to shear stress for 6 hours, were washed with ice-cold PBS, scraped, and pelleted by centrifugation at 1500 rpm for 5 minutes. The cell pellet was resuspended in 1 mL NP-40 lysis buffer (10 mmol/L Tris-HCl [pH 7.4], 10 mmol/L NaCl, 3 mmol/L MgCl2, and 0.5% [vol/vol] NP-40), incubated for 5 minutes on ice, and centrifuged at 3000 rpm for 5 minutes. The nuclear pellet was washed once with 1 mL NP-40 lysis buffer and centrifuged again at 3000 rpm for 5 minutes. Nuclei were resuspended in 100 μL of 50 mmol/L Tris-HCl (pH 8.3), 5 mmol/L MgCl2, 0.1 mmol/L EDTA, and 40% (vol/vol) glycerol and frozen in liquid N2. The nuclei were thawed and reacted in 100 μL reaction buffer consisting of 10 mmol/L Tris-HCl (pH 8.0), 5 mmol/L MgCl2, 300 mmol/L KCl, 0.5 mmol/L ATP, 0.5 mmol/L CTP, 0.5 mmol/L GTP, and 3.7 MBq [α-32P]UTP (≈111 TBq/mmol) for 30 minutes at 30°C. The 32P-labeled RNA was precipitated by trichloroacetic acid and purified with phenol/chloroform extraction. Plasmids containing human GM-CSF or human GAPDH fragment (Clontech Laboratories, Inc) were linearized by restriction enzyme digestion and denatured at 95°C. The DNA was spotted onto nylon membranes and fixed with 0.4N NaOH. Radiolabeled RNA was adjusted to 2.5×106 cpm/mL in a hybridization solution (5× SSPE, 50% formamide, 0.1% Denhardt's solution, and 0.1% SDS) and hybridized to DNA immobilized on nylon membranes for 48 hours at 42°C. Blots were washed twice in 2× SSPE and 0.1% SDS for 15 minutes at 42°C, once in 1× SSPE and 0.1% SDS for 30 minutes at 42°C, and twice in 0.1× SSPE and 0.1% SDS for 15 minutes at room temperature. Autoradiograms of the membranes were obtained using a GS363 Molecular Imager System (Bio-Rad).
Luciferase Assay
Two fragments of the GM-CSF gene, −2.5 to +1.2 kb or −4.3 to +0.2 kb, were cloned from a human genomic DNA library and inserted into pGL-3 enhancer luciferase vector plasmid (Promega). The reporter genes, named pGL-GMCSF (−2.5 kb) and pGL-GMCSF (−4.3 kb), were transfected into cultured bovine ECs (passages 4 to 7) with Transfectam (Biosepra). To evaluate the efficiency of transfection, pRL-SV40 vector was cotransfected using a Dual-Luciferase Reporter Assay System (Promega). The cells were then either incubated under static conditions or exposed to flow with a shear stress of 15 dyne/cm2 for 24 hours, and their cytoplasmic proteins were harvested for luciferase assay. Luciferase activity derived from the reporter genes was measured with a Berthold Lumat luminometer (model LB9501) and normalized with that from cotransfected pRL-SV40 vector.
mRNA Stability Assay by Competitive PCR
HUVECs that had either been incubated under static conditions or exposed to flow with a shear stress of 15 dyne/cm2 for 24 hours were treated with actinomycin D (5 μg/mL, Wako Chemical) for 30, 60, or 90 minutes. Total RNA was obtained from these cells and treated with RNase-free DNAse I (Message Clean Kit, Gene Hunter).
Competitive PCR was performed to quantify mRNA concentrations. After the total RNA (1 μg) obtained from the sample was reverse-transcribed into cDNA, the products were mixed with serial dilutions of the competitor DNA (pGL-GMCSF, −2.5 kb) spanning a range of concentrations from 0.01 to 10 pg. These mixtures were amplified in the presence of [α-32P]dCTP for 36 cycles (94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 60 seconds) and subjected to electrophoresis on 5% polyacrylamide gel. The sense (5′-TGGCTGCAGAGCCTGCTGCTCTTGGGCACT-3′) and anti-sense (5′-CTGGAGGTCAAACATTTCTGAGATGACTTCT-3′) primers locate exons 1 and 2 of the GM-CSF gene, respectively, and an intron of 97 bp is present between the two exons. Accordingly, the size of the amplification product from the competitor DNA consists of 290 bp, and that from the cDNA consists of 193 bp. mRNA concentrations in the test samples were estimated by comparing the intensity of bands derived from amplification of the competitor cDNA (290 bp) with those derived from the RNA sample being tested (193 bp). The concentration of competitive cDNA that gives rise to a band with a radioactivity of 1.47 (290/193) times the test sample corresponds to the concentration of mRNA in the test sample.
Gel Shift Assay
Cytoplasmic extracts of static and shear-stressed HUVECs (15 dyne/cm2, 24 hours) were prepared by freeze-thaw lysis in 25 mmol/L Tris-HCl (pH 7.9), 0.5 mmol/L EDTA, and 0.1 mmol/L phenylmethylsulfonyl fluoride, followed by centrifugation at 15 000 rpm for 15 minutes. The DNA was synthesized using complementary oligonucleotide containing 54 nucleotides from the AT-rich 3′ UTR of human GM-CSF (positions 641 to 694) and including seven repeats of the ATTTA sequence (GATCAGTAATATTATATATTTATATTTTTAAAATATTTATTTATTTATTTATTTAAGGATC). This "7×AT DNA" segment was cloned into EcoRI-digested pBluescript II (Stratagene), and sequencing was performed to verify sequence fidelity. Transcription of pBluescript DNA linearized with XbaI was performed in vitro using T3 RNA polymerase in the presence of [α-32P]UTP. Labeled RNA had a specific activity of ≈1×107 cpm/μg.
The cytoplasmic extracts (10 μg of protein, 7×104 cells) were incubated at 30°C for 10 minutes with 32P-labeled "7×AU" RNA (1×105 cpm) in 10% glycerol, 12 mmol/L HEPES (pH 7.9), 15 mmol/L KCl, 0.25 mmol/L EDTA, 0.25 mmol/L dithiothreitol, and 5 mmol/L MgCl2. For competition experiments, these extracts were incubated with unlabeled probe before incubation with 32P-labeled RNA. Nonspecific binding was reduced by adding 2 μg of yeast tRNA. The reaction mixture was cross-linked with 254-nm UV radiation using a UV chamber (GS Gene Linker, Bio-Rad). RNase A was added to a final concentration of 1 μg/μL, and the mixture was incubated at 37°C for 30 minutes. An equal volume of 2× sampling buffer containing 2-mercaptoethanol was added to each sample and boiled at 100°C for 3 minutes. The samples were then electrophoresed in polyacrylamide gels containing 0.1% SDS, and the 32P-labeled protein was analyzed by a image analyzer.
Statistical Analysis
Differences in CSF production between the static control and flow-loaded samples were evaluated by ANOVA followed by Bonferroni's multiple comparison test by using SPSS (version 6.07J for Windows, SPSS Inc). Significance was assumed at P<.05. To assess the shear rate or shear stress dependence of GM-CSF mRNA levels, the ratio test of the composite hypothesis of Neyman and Pearson was used, as previously reported by Ando et al.40
Laminar Flow Increases GM-CSF Production in HUVECs
Confluent monolayers of HUVECs were subjected to laminar flow with a shear stress of 15 dyne/cm2, and the amounts of GM-CSF, G-CSF, and M-CSF released into the perfusates were measured by ELISA. HUVECs released a small amount of GM-CSF under static conditions but markedly increased the production in response to laminar flow (Figure 1⇓). The cumulative production of GM-CSF was increased to >5-fold the level of production in the static controls at 8, 24, and 48 hours after the initiation of flow. The rate of production tended to diminish after 24 hours. The increase in GM-CSF production became more prominent as shear stress increased from 15 to 25 dyne/cm2. On the other hand, neither G-CSF nor M-CSF production changed significantly, even under flow conditions.
The stimulatory effect of shear stress on GM-CSF production was compared with that of PMA (Sigma). The amount of GM-CSF released after 24-hour exposure to a shear stress of 25 dyne/cm2 corresponded roughly to one-third that after 24-hour stimulation with a maximally effective concentration of PMA (100 ng/mL) (161.0±6.0 versus 482.2±6.4 pg/106 cells; mean±SD, n=6).
Laminar Flow Increases GM-CSF mRNA Levels in HUVECs
HUVECs were exposed to laminar flow with a shear stress of 15 dyne/cm2 for 24 hours, and the levels of CSF mRNA were determined by RT-PCR. The GM-CSF band on the gel was much thicker for shear-stressed cells than for static control cells (Figure 2A⇓), indicating that shear stress increased GM-CSF mRNA levels in the HUVECs. The G-CSF band became slightly thinner after exposure to shear stress, whereas the M-CSF band became slightly thicker (Figure 2A⇓). Quantitative analysis of these CSF bands by densitometry revealed that the ratio of the levels of GM-CSF, G-CSF, and M-CSF mRNA in shear-stressed cells to their levels in static control cells was 3.70±0.22 (P<.01), 0.71±0.10 (P=NS), and 1.61±0.13 (P=NS) (mean±SD, n=6), respectively. GM-CSF mRNA levels began to increase as early as 2 hours after the initiation of laminar flow and continued to increase over time, reaching a peak of about four times the static control levels at 24 hours, and then decreased but were still much higher than the control level at 48 hours (Figure 2B⇓).
To determine whether upregulation of GM-CSF mRNA by shear stress depends on new protein synthesis, we treated the HUVECs with the protein synthesis inhibitor cycloheximide before exposing them to shear stress. The 1.5-hour pretreatment did not change the basal level of GM-CSF mRNA. Cycloheximide blocked the upregulation of GM-CSF mRNA by shear stress (Figure 3⇓), indicating that the upregulation process depends on the synthesis of new protein.
Flow-Induced Elevations of GM-CSF mRNA Levels Are Shear Stress Dependent
To determine whether the increase in GM-CSF mRNA levels is shear stress or shear rate dependent, HUVECs were subjected to the flow of two perfusates with different viscosities for 6 hours. The mRNA levels increased as shear rate increased, but the levels increased to an even greater extent when viscosity or shear stress was higher at the same shear rate (Figure 4⇓, top). On the other hand, mRNA levels plotted against shear stress formed a straight line (Figure 4⇓, bottom). These findings indicate that flow-induced elevations in GM-CSF mRNA levels are shear stress rather than shear rate dependent.
Transcription of the GM-CSF Gene Is Unaffected by Shear Stress
We performed a nuclear run-on assay to determine whether shear stress directly affects GM-CSF gene transcription. Nuclei were prepared from static control or shear-stressed HUVECs (15 dyne/cm2, for 24 hours), and transcription was allowed to proceed in the presence of [32P]UTP. Purified radiolabeled RNA was hybridized to cDNA immobilized on nylon membranes. Transcription of the GM-CSF gene, which could be detected distinctly in static control cells, was unchanged even after exposure to shear stress (Figure 5A⇓).
We also performed a luciferase assay to evaluate the effect of shear stress on GM-CSF gene transcription in vitro. HUVECs transfected with the reporter gene, pGL-GMCSF (−2.5 kb) or pGL-GMCSF (−4.3 kb), were exposed to a shear stress of 15 dyne/cm2 for 24 hours, and luciferase activity was measured. pGL-GMCSF (−2.5 kb) contains the sequence SSRE (GAGACC) at +610 to +615 in an intron between exons 2 and 3, and pGL-GMCSF (−4.3 kb) has the TRE sequence (TGACTCA) at ≈−3.0 kb. Both SSRE and TRE have been found to function as a cis element for shear responsiveness. Luciferase activity, however, did not change significantly even after exposure to shear stress (Figure 5B⇑). These findings indicate that shear stress does not affect transcription of the GM-CSF gene in HUVECs and that neither SSRE nor TRE is involved in the upregulation of GM-CSF gene expression by shear stress.
Shear Stress Increases GM-CSF mRNA Stability
We performed actinomycin D chase experiments to determine whether GM-CSF mRNA is regulated by shear stress at posttranscriptional levels. HUVECs were treated with actinomycin D after either being incubated under static conditions or being exposed to a shear stress of 15 dyne/cm2 for 24 hours, and the changes in concentration of GM-CSF mRNA were measured by competitive PCR. GM-CSF mRNA decreased as exposure time to actinomycin D increased, but the rate of decrease was lower in the shear-stressed cells than in the static control cells. The estimated half-life of GM-CSF mRNA in the shear-stressed cells was 42.3±1.0 minutes (mean±SD, n=3), as opposed to 23.1±1.3 minutes in the static control cells (Figure 6⇓). This indicates that shear stress regulates GM-CSF gene expression in HUVECs posttranscriptionally via mRNA stabilization.
Effects of Shear Stress on the Binding Activity of AU-Binding Factors in HUVECs
To examine whether any AU-binding factors are involved in the modulation of GM-CSF mRNA stability, we analyzed HUVECs for the AU-binding factors that specifically bind the AU-rich region in the 3′ UTR of GM-CSF mRNA. Gel shift assay showed that ≈40-kD proteins in the cytoplasmic extracts specifically cross-linked to the 7×AU motif (Figure 7⇓). The binding activity of the AU-binding protein was unaltered by exposure of the cells to shear stress. Treatment of the cells with actinomycin D produced a small increase in the binding activity of the protein, but protein binding was unaffected by shear stress.
The present study demonstrated that laminar flow increases the production of GM-CSF in HUVECs at both the protein and mRNA level. It was already known that GM-CSF production by ECs is greatly regulated by chemical stimuli, such as TPA, IL-1, TNF, lipopolysaccharide, and modified LDL, but the present study demonstrated for the first time that GM-CSF production by ECs is regulated by a mechanical stimulus, shear stress.
There are two aspects of the effect of flow. One aspect is shear stress as a mechanical stimulus that deforms ECs, and the other aspect is change in mass transport. If a substance that stimulates ECs is present in the perfusate, the amount of that substance that reaches the cell surface will increase as flow rate or shear rate increases, leading to more potent stimulation. To differentiate between the two aspects, flow-loading experiments were performed with two perfusates having different viscosities, enabling us to apply different levels of shear stress to ECs at the same shear rate. The results showed that GM-CSF mRNA levels increased as shear rate increased but that at any given shear rate, the increases were always larger in the flow with higher viscosity or higher shear stress. This indicates that the stimulatory effect of laminar flow on GM-CSF mRNA levels is shear stress rather than shear rate dependent.
To date, indications that GM-CSF gene expression can be regulated transcriptionally or posttranscriptionally have been found in several cell lines.41 Many transcription factors (NF-κB, NF-GMa, NF-GMb, NF-GM2, Elf-1, and NF-ATp/AP-1) and cis elements implicated in the cytokine-mediated regulation of GM-CSF gene expression have been identified.42 For instance, the sequence CATTA/T that is present −37 to −48 upstream from the transcription start site has been shown to be a critical positive regulatory element in cell line MLA144 stimulated with PMA,43 and the AP-1 consensus element (TRE) at ≈−3 kb in the 5′ upstream region has been identified as an enhancer in PMA-treated T cells.44 In the present study, however, our run-on assay revealed that shear stress did not influence GM-CSF transcription in HUVECs. Furthermore, luciferase assay using the reporter gene containing −2.5 to +1.2 kb or −4.3 to +0.2 kb of the GM-CSF gene showed no significant change in transcriptional activity in response to shear stress. Since the GM-CSF fragments contain SSRE or TRE in their sequences, both of which have already been identified as a cis-acting element required for shear stress responsiveness in the PDGF-B or MCP-1 gene, the results suggest that these elements are not involved in the GM-CSF gene response to shear stress. The results of a gel shift assay also showed that none of the nuclear proteins derived from shear-stressed HUVECs were able to form complexes with oligonucleotides synthesized on the basis of sequences of the above-mentioned known enhancers of GM-CSF gene transcription (data not shown). All of this evidence taken together makes it seem unlikely that shear stress regulates GM-CSF gene expression at transcriptional levels.
We performed actinomycin D chase experiments to determine whether shear stress alters the stability of GM-CSF mRNA, and we used a competitive PCR to compare the half-life of GM-CSF mRNA in static control cells and shear-stressed cells. Because of its high sensitivity, this method is suitable for measuring small amounts of mRNAs, such as GM-CSF mRNA, and the fact that cDNA and its competitor are coamplified in the same tube using a common set of primers allowed quantitative evaluation of GM-CSF mRNA levels in the samples. The results showed that exposure to shear stress increased mRNA half-life to almost twice its level in static cells (23.1 versus 42.3 minutes). Thus, GM-CSF gene expression in HUVECs seems to be regulated by shear stress posttranscriptionally via mRNA stabilization. Similar posttranscriptional regulation of GM-CSF gene expression has been observed in fibroblasts stimulated with TNF or TPA45 and in mouse B cells treated with IL-1.46
How GM-CSF mRNA is stabilized by shear stress, however, remains unclear at the present time. This shear stress–induced stabilization seems to occur through a process that requires new protein synthesis, since cycloheximide blocked the effect of shear stress on GM-CSF mRNA levels. The GM-CSF gene contains eight AU-rich motifs (AUUUA) in the 3′ UTR that play an important role in rapid degradation of mRNA.47 48 When the AU-rich region of unstable GM-CSF mRNA was placed in the 3′ UTR of a stable mRNA, such as that of β-globin, it resulted in rapid degradation of the chimeric transcripts.49 Five distinct proteins, 70, 45, 40, 38, and 32.5 kD, which specifically bind to the AU-rich region of human GM-CSF 3′ UTR, were found in human embryonic lung fibroblasts.50 To clarify the role of AUBPs in the modulation of GM-CSF mRNA stability by shear stress in the present study, we analyzed the interactions between RNA containing the 7×AU motif and proteins from cytoplasmic extracts. An AUBP of ≈40 kD was identified in HUVECs, but their binding activity was unaffected by shear stress. Thus, the AU-rich element may be inadequate to function as the SSRE. There are other types of mRNA-binding proteins besides AUBPs that are thought to influence mRNA stability: poly(A)-binding protein, a protein that binds to specific 3′ UTR sites other than AU-rich elements, and a protein that binds to the coding region determinant of mRNA.51 Thus, regulation of GM-CSF mRNA stability appears to be rather complicated, not simple, and SSREs may be more widely distributed in GM-CSF mRNA. Further study will be required to identify the cis elements involved and the proteins targeting the elements.
Selected Abbreviations and Acronyms
|activator protein-1
|AU-rich element–binding protein
|colony-stimulating factor
|endothelial cell
|granulocyte CSF
|granulocyte-macrophage CSF
|human umbilical vein EC
|macrophage CSF
|monocyte chemotactic protein-1
|nuclear factor
|polymerase chain reaction
|platelet-derived growth factor
|phorbol 12-myristate-13-acetate
|reverse transcriptase–PCR
|shear stress–responsive element
|tumor necrosis factor
|12-O-tetradecanoylphorbol 13-acetate
|tumor-promoting agent response element
|untranslated region
|vascular cell adhesion molecule-1
This study was partly supported by grants-in-aid for scientific research and for scientific research on priority areas from the Japanese Ministry of Education, Science, and Culture; by a research grant for cardiovascular diseases from the Japanese Ministry of Health and Welfare; and by research funds from the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion, and Product Review of Japan.
Reprint requests to Joji Ando, Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, 7–3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
- Received December 1, 1997.
- Accepted February 5, 1998.
- © 1998 American Heart Association, Inc.
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Sieff CA. Hematopoietic growth factors. J Clin Invest. 1987;79:1549–1557.
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Dedhar S, Gaboury L, Galloway P, Eaves C. Human granulocyte-macrophage colony-stimulating factor is a growth factor active on a variety of cell types of nonhemopoietic origin. Proc Natl Acad Sci U S A.. 1988;85:9253–9257.
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Mitsumata M, Fishel RS, Nerem RM, Alexander RW, Berk BC. Fluid shear stress stimulates platelet-derived growth factor expression in endothelial cells. Am J Physiol. 1993;265:H3–H8.
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Malek AM, Greene AL, Izumo S. Regulation of endothelin 1 gene by fluid shear stress is transcriptionally mediated and independent of protein kinase C and cAMP. Proc Natl Acad Sci U S A.. 1993;90:5999–6003.
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Shyy YJ, Hsieh HJ, Usami S, Chien S. Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium. Proc Natl Acad Sci U S A.. 1994;91:4678–4682.
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Ohno M, Cooke, JP, Dzau, VJ, Gibbons, GH. Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production. J Clin Invest. 1995;95:1363–1369.
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Inoue N, Ramasamy S, Fukai T, Nerem RM, Harrison DG. Shear stress modulates expression of Cu/Zn superoxide dismutase in human aortic endothelial cells. Circ Res. 1996;79:32–37.
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Topper JN, Cai J, Falb D, Gimbrone MA Jr. Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-1, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress. Proc Natl Acad Sci U S A. 1996;93: 10417–10422.
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Nishida K, Harrison DG, Navas JP, Fisher AA, Dockery SP, Uematsu M, Nerem RM, Alexander RW, Murphy TJ. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest. 1992;90:2092–2096.
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Chun T, Itoh H, Ogawa Y, Tamura N, Takaya K, Igaki T, Yamashita J, Doi K, Inoue M, Masatusgu K, Korenaga R, Ando J, Nakao K. Shear stress augments expression of C-type natriuretic peptide and adrenomedulin. Hypertension. 1997;29:1296–1302.
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Malek AM, Jackman R, Rosenberg RD, Izumo S. Endothelial expression of thrombomodulin is reversibly regulated by fluid shear stress. Circ Res. 1994;74:852–860.
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Ando J, Tsuboi H, Korenaga R, Takada Y, Toyama-Sorimachi N, Miyasaka M, Kamiya A. Shear stress inhibits adhesion of cultured mouse endothelial cells to lymphocytes by downregulating VCAM-1 expression. Am J Physiol. 1994;267:C679–C687.
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Shyy JYJ, Lin MC, Han J, Lu Y, Petrime M, Chien S. The cis-acting phorbol ester '12-O-tetradecanoylphorbol 13-acetate'-responsive element is involved in shear stress-induced monocyte chemotactic protein 1 gene expression. Proc Natl Acad Sci U S A.. 1995;92:8069–8073.
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- Fluid Shear Stress Increases the Production of Granulocyte-Macrophage Colony-Stimulating Factor by Endothelial Cells via mRNA StabilizationKeisuke Kosaki, Joji Ando, Risa Korenaga, Takahide Kurokawa and Akira KamiyaCirculation Research. 1998;82:794-802, originally published April 20, 1998http://dx.doi.org/10.1161/01.RES.82.7.794
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- Fluid Shear Stress Increases the Production of Granulocyte-Macrophage Colony-Stimulating Factor by Endothelial Cells via mRNA StabilizationKeisuke Kosaki, Joji Ando, Risa Korenaga, Takahide Kurokawa and Akira KamiyaCirculation Research. 1998;82:794-802, originally published April 20, 1998http://dx.doi.org/10.1161/01.RES.82.7.794
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text: Blue Valentine: A Totally Wonderful BummerEric Limer1/05/13 9:00pmFiled to: movie nightBlue Valentine47EditPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Sometimes you just want to curl up on a Saturday night and watch a feel-good flick that'll make you feel warm inside so you can smile to yourself and ponder how all is right with the world. Blue Valentine is the perfect choice if you are feeling the exact opposite. Grab a drink. Advertisement Blue Valentine is the story of a relationship, but handles itself more like it's the story of two. First you have the story of the callous, moderately alcoholic jerkass who provides for his family but really walks the line when it comes to being outright emotionally abusive to his exhausted, and increasingly depressed wife. Then you also have the story of the dogged nice guy, chasing down the girl of his dreams after love at first sight; it's almost too good to be true. Oh and it is, because it's the same couple, and the scenes are interspersed just the right way to make your soul hurt.To go any further into the details robs the film of some of its juxtaposition power. Suffice it to say that it's a great film, well made, and is a joy to watch from that angle, but it's also such a total bummer that aims to show you how everything you ever though could be wonderful will actually turn to total shit if given enough amount of time. A meta-juxtaposition, if you will. Now that's my kind of movie! It just came to Netflix Instant, so go check it out. But get ready for a letdown, a really good one. [Netflix]Gear from Kinja DealsCasper's New Box Spring Is a Miracle of SimplicityEOS: A Better Lip Balm With a Smart Form FactorSave $13 On the Bluetooth-Enabled Xbox One S ControllerReply47 repliesLeave a reply


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Friendly mixed ability Pilates class. Course of 6 lessons starting on 24th March 2015. Classes are kept small so spaces are limited and booking is essential. Easter break April 7th, no class April 21st
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The ACGC Drama Department will present "Into the Woods, Jr." on Saturday, October 29th at 2pm and 7pm and on Sunday, October 30th at 2pm in the ACGC Jr.-Sr. High School Theater. Tickets are $6 for adults and $2 for students and senior citizens.
ACGC volleyball will begin Section 5A play offs at KMS on Thursday at 7:00 pm. The winner will play at BBE on Friday at 7:00 pm.
ACGC will begin Section 5AA football on Tuesday at 7:00 pm in Sauk Centre.
The winner of this game will play EVW on Saturday at 2:00 pm in Eden Valley.
ACGC High School and Schwan's Cares is partnering together to help raise money for our upcoming trip to Boston, NY City & Washington D.C. in March. The money raised will help reduce the student cost for this trip..
We are recruiting new members in grades 8-12 for this academic school activity which "empowers youth to partner with their community to create economic and environmental vitality through hands-on learning and team-based projects." (www.youthenergysummit.org) Current projects include the tree trench project at the ACGC Elementary School in Atwater, LED to Solar Parking Lot Lights, School Garden, and ongoing recycling efforts. Contact Ms. Lokken
At ACGC, we pay close attention to creating a safe and welcoming environment where students become successful
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CHARLOTTESVILLE, Va. - Virginia head coach Mark Bernardino has announced Anna Miller as an assistant coach on the Cavalier swimming and diving staff.
Miller will work primarily with the middle distance freestylers, stroke and individual medley swimmers at UVa in addition with assisting in recruiting efforts.
"We are really pleased to announce the appointment of Anna Miller to the University of Virginia swimming and diving staff," Bernardino said. "Anna brings a tremendous and unique skill set to our program. She is both an accomplished swimmer and student of the sport. Anna brings us a fresh perspective on the latest technical and mechanical aspects of our sport. Her skills in working with Olympians and national team members in respects to stroke techniques and mechanics are something we feel will really benefit our student-athletes here at UVa."
Miller, a native of Johnson City, Tenn., comes to Virginia after spending the summer as the national team intern with USA Swimming. She assisted with all duties performed by the High Performance Team including filming, race analysis and coach/athlete support.
Miller also accompanied the High Performance team to national and Grand Prix competitions.
Miller spent the 2010-11 school year in two different roles at her alma mater, the University of Georgia. She spent the year as a graduate assistant with Georgia's CHAMPS/Life Skills program, which focuses on the development of student-athletes within the university environment. In that role, Miller monitored study hall and assisted in organizing community service events. She also served as an administrative assistant with the Bulldog swimming and diving program during the spring semester, helping with home swim meets, financial reporting and camper registration for Georgia's summer camps.
Miller swam for four years (2005-08) on the Georgia women's swimming team and finished her career as a seven-time All-American. She was a member of both the 2005 NCAA championship squad and the 2006 SEC championship team. Miller, a team captain as a senior in 2008, concluded her career as a four-time SEC Academic All-American. She also qualified for the 2004 and 2008 Olympic Trials.
Miller earned her bachelor's degree in risk management and real estate from Georgia in 2009. She completed her master's degree in sports management and policy from UGA in 2011.
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Cavalier Football Notebook: UNC WeekFootball10/21/16At 3 p.m. Saturday, UVA (2-4, 1-1) hosts Coastal Division rival North Carolina (5-2, 3-1) in an ACC football game at Scott Stadium.Lucas Playing Key Role in TurnaroundField Hockey10/20/16Carrera Lucas' play at goalkeeper has helped No.9 Virginia, which hosts No. 7 Louisville on Friday night, win seven of its past eight games.Media Day NotebookMen's Basketball10/18/16Virginia, which advanced to the NCAA tournament's Elite Eight in 2015-16, opens the season Nov. 11 against UNC Greensboro at the Greensboro Coliseum.
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: Deputy commissioner, Mokokchung would soon convene a meeting to prepare contingency plan to respond effectively in the event of occurrence of landslides in major roads in the district which might obstruct vehicular movement. According to an official bulletin, even though there was no report of interruption in the vehicular movement in any part of the district, the district administration is keeping a close watch on occurrences of major landslides triggered by incessant rains during monsoon.DC Mokokchung, Lithrongla G. Chishi said the department; roads and bridges would be informed to mobilize manpower as well as machineries to deal with occurrence of landslides in any part of the district and the district administration would gear up community participation to deal with situation.
MEANING OF LAO-ONG MO: The Literary meaning of the word "Lao-Ong" can be explained as "Wiping off the activity of the annual harvest completely". It signifies the winding up of all


text:
French soldiers shoot car attacker at mosque
AFP · 2 Jan 2016, 08:29
Published: 02 Jan 2016 08:29 GMT+01:00
- Defiant French throng Paris to bid adieu to 2015 (01 Jan 16)
- Paris attacks 'directed by one man in Belgium' (30 Dec 15)
Police said the driver deliberately sped his vehicle at the troops, injuring one of them, as they were stationed in a car park outside a large mosque in a suburb of Valence.
The driver bore down on the team a first time, prompting them to shout a warning, and when he returned for a second pass, they opened fire, they said.
The impact of the car, a Peugeot 307 station wagon, left the soldier with injuries to his knee and shin, police said.
The driver was shot in the arm and the leg and was taken to hospital, the prefecture of the Drome department (county) said.
The defence and interior ministries in Paris said in a joint statement the driver's injuries were "serious but currently not life-threatening."
A stray bullet also hit a passerby, a worshipper at the mosque, in the calf.
Police said the driver's motive remained unclear.
Local mayor Nicolas Daragon praised the soldiers for what he said was "remarkable" self-control, and Prime Minister Manuel Valls, Interior Minister Bernard Cazeneuve and Defence Minister Jean-Yves Le Drian were swift to add their backing.
"My support for the soldiers attacked in Valence. Fully appreciate our forces, mobilised for the security of France," Valls said in a tweet.
Le Drian and Cazeneuve's statement said the soldiers "responded with defensive fire" against the assailant.
The incident occurred in mid-afternoon, between two prayer sessions, when there was a large crowd outside, the rector of the mosque, Abadallah Dliouah, said.
The prefecture said the mosque was usually very calm, "and worship there takes place peacefully."
The soldier is among 10,000 security personnel protecting sensitive sites around the nation after the deadly jihadist assaults in Paris in 2015.
Attacks in Paris on satirical newspaper Charlie Hebdo and a Jewish supermarket killed 17 people in January 2015, with a series of coordinated shootings and bombings in the city in November leaving 130 people dead and 350 wounded.
The traditional New Year's Eve fireworks display in Paris was cancelled over fears of a terrorist attack.
Instead, the large crowds on the Champs-Elysees avenue were treated to a giant video projection on the Arc de Triomphe featuring the French flag, people smiling and images of famous Parisian landmarks, intended as a show of national unity and solidarity.
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FRANCE: Designer Mercier To Leave Fashion House Balmain
Swiss designer Laurent Mercier is leaving French fashion house Balmain after one year at the helm it was announced today.
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Hanabishi By KyushuAsian, Japanese, Sushi
15355 Sherman Way
(818) 786-6005
Hibeebz M.
It was a ramen kind of day, so I took my sons (one had a cold, so it seemed a perfect match). It was pretty crowded for after lunch (1:40 p.m.) Nice...read more
Taylor H.
First of all, i like that they're open late. Second, they've got a decent happy hour which is always a plus. But best of all is Miguel the sushi chef....read more
Bryant O.
Needed something late night but nothing heavy (I.e. Fast food) and it was already midnight. So their 1am closing time was perfect. Read the reviews and...read more
Mediocre and tired: Thumbs way down for this spot. I've been a Kyushu patron since I was in high school (under the original owner). I'm now grown up and travel extensively, but every time I come home, I try to get to Kyushu. Unfortunately, the place is in…read more
Brenda H
Good Japanese Ramen: I loved eating at Kyushu Ramen. I had lunch with family and the food was quite delicious. I tried Yakisoba, a fried noodle,vegetable and chicken dish that was very tasty. The Roasted Pork soup noodle is excellent too. The prices are…read more
good but not great: I've had better...my last review was deleted because I mentioned another location. But I am a die-hard foodie and personally I'd like to see recommendations, so again I will try to leave one: Ramen Nippon in Reseda. The service was good…read more
Amazing food Amazing price: I have been going to this restaurant for a long time now and the food , service , pricing, and atmosphere have always been wonderful. This place is a real gem! The only thing that makes it tricky sometimes is that on the weekends it get's…read more
Kim T
Delicious ramen: One word...Delicious! I really enjoy ramen and especially did the other night after swim practice. I go to swim practice in Van Nuys, and we usually go eat afterwards at a local restaurant. I got the ramen with some veggies and beef in a…read more
Best place for ramen IN THE VALLEY.: I've been going to this ramen place for a few years now, and I'll say what I've been saying for years: its a good place to go if you're in the Valley and you need a ramen fix. By no means is the food terrible; its actually very good. …read more
One of the BEST kept secrets for Japanese food!: This place is FANTASTIC. I have never had a bad dish at this awesome neighboorhood noodle house. The food is deliciously prepared and the prices are unbelievably low. It gets pretty packed on the weekend nights, so I would recommend…read more
Yuho Q
Kyushu Ramen serves ramen and Izakaya style food: Kyushu Ramen is divided into 2 parts: One side serves ramen and the other side serves Japanese Izakaya type food. They are known for Kyushu style ramen, but both ramen and izakaya dishes are wonderful.read more
yet to be disappointed!: I have been going here for a while now and I must say that I like the service, the prices, the specials, and the FOOD! have yet to be disappointed.read more
Ramen in the SF Valley: I believe that I have eaten in every ramen shop in the valley. I always order the chasu ramen everywhere I...read more
mmm.. ramen!: I've been coming to this place for years. Tasty broth and super succulent ramen. The ownership has changed but its still my go-to place when I'm craving ramen. :)read more
Keiko N
Kyushu Ramen: Ramen which is Japanese hot soup noodles is good here. I usually get the plain shoyu or shio ramen. This restaurant is very small and a casual place but if you feel like Ramen and your in this area this a good place to visit.read more
Japanesie S
In sad decline: Thumbs way down for this spot. I've been a Kyushu patron since I was in high school (under the original owner). I'm now grown up and travel extensively, but every time I come home, I try to get to Kyushu. Unfortunately, the place is in…read more
: I've got 4 words for you: pork cutlet curry ramen. This is THE dish to order here, and it is savory magic.read more
I miss Kyushu: I have been coming here since I was 5 so it has been 24 years since I have established a relationship here. I do miss the old owner dearly and the atmosphere isn't the same, and the prices have gone up but this is my only complaint. There…read more
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How tall is Stellan Skarsgård?
Stellan Skarsgård Height: 6' 3" (190.5 cm)
Standing at a height of 6 feet, 3 inches tall Stellan Skarsgård is taller than 97.9% of all men, as reflected by the figure's fill height %.
Conversely, at this height Stellan Skarsgård is not as tall as 2.1% of all men.


text:
Image Copyright © S&G Barratts/EMPICS Archive
Media ID 1725881 from PA Photos
Watermarking and Website Address do not appear on finished products
Creation Date: 14th May 1957
belt, fashionbook, fashionemma, fashionfinal, fifties, full, g2325-17, heart, ladieswear1, length, printstoreok, showcaseammonite, smile, uigclarehhome » Britain in Pictures » 100 Years of Fashion
Thank you to the seller it's quality
reviewed by Ruzbi Nayel
7th June 2016
Slightly smaller than thought but otherwise just right.
reviewed by Penny Gilbert
11th July 2016
I needed this mug to arrive before the 2 July for a birthday gift so contacted the seller. They were most helpful and arranged for this to happen and the package arrived on the 2 July, in plenty of time. Brilliant!!!
reviewed by Mrs H
10th July 2016
Photo I hadn't seen before of Starlight Express. Happy to have in my collection. Good price and really fast delivery. Very satisfied.
reviewed by Amazon Customer
15th July 2016
Print arrived well within Estimated Delivery Date, very well packaged, excellent condition and as described, thanks.
reviewed by MJYP
23rd July 2016
Very well packed. Arrived earlier than quoted. Excellent service" Lin M, 26 July 201
reviewed by Lin M
26th July 2016


text:
Did you know that Pilates can be a wonderful way to complement your next winter getaway?
Branch Out! 5 Reasons to Try Pilates This Summer
Ask the Expert: 6 Steps to Sexy Summer Arms
Man Up: Pilates Isn't Just for the Ladies Anymore
Pilates 101: 5 Articles to Round Up Your Practice
What's the Value of Fusion-Style Pilates?
Expert Q&A: The Proper Pilates Breathing Method
Your Best Night of Sleep Ever
Flavor of the Week: Do You Yamuna?
Zeel Loved The Nalini Method. Now Vogue Does Too!
Flying South: Look For Zeel At SXSW!
Ask the Expert: Pilates for Muscular Re-Patterning
Ask the Expert: Pilates for Injury Prevention
Ask the Expert: Pilates for a Healthy Pregnancy
Zeel's Healthy Holiday Gift Guide (with 9 Giveaways!)
Aching Back? Yes, You Can Still Work Your Core!
West Side Story: 3 Reasons to Try the Nalini Method
Zeel Week at Athleta: Free Classes & Fab Instructors
Hollywood's 40-Somethings Dare to Bare
Hilary Swank's Workout: It's Not Just Good Genes
No Gym? No Problem! Desk Job Fitness
Right-Sizing Your Workout Gear
Mom Was Right: Stand Up Straight
Copyright 2016, Zeel Networks, Inc., 33 West 17th Street, 2nd Floor, New York, NY 10011. ZEEL®, ZEEL SPA℠, ZEEL CONCIERGE℠, BLOCK PROJECT℠, MASSAGE ZEELOT®, MASSAGE ON DEMAND®, WE "HEART" MASSAGE®, THE MASSAGE EXPERTS®, RELIEVING THE SIDE EFFECTS OF LIFE℠, and WE'VE GOT YOUR BACK™ are trademarks of Zeel Networks, Inc. Zeelously Made in NYC. The contents of the Zeel web site are for informational purposes only. None of the information on the site should be construed or used as professional medical advice or consultation. Please read our terms of use for more information.
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text:
This download looks like an unpacked addon. You can either convert the files to an addon or merge the folders in the zip with the folders in your Garry's Mod folder.
You should end up with a directory structure similar to below:
This option makes it difficult to remove the content should you no longer want it. Merge any folders from the zip with the equivalent folders in your Garry's Mod folder.
Don't know where the Garry's Mod folder is or how to extract zip files? See more useful installation tips.
absza15, itsmakeshift14, Кешик, Mazané másličko, DuchyHesse, Lisa the fabulous plant, dittotrainer123, aveno12, SuperDuckBoy, Connor Kenway, soggywheeners, joelsdecastro, therayninja, therayninja, Dhacker8667, ristomarcus3, aaron.hevera, Vault 87, gadkareem06, kuralsizcocuk13, <MN>Rara1120, kuza0310, gabimagloviti, BlooodshotBiz, sazeomin, giangnguyencanh69, ThuMmPeR~, lopezkevin115.bo, ninouvirolois950, realitymalek59, schelumielepstein, slug56, cobcarr1, Jeffrey661, eletronicmusicbr, Malurhan, Manu_13_DA, Lrlax, Singlebow, Freezing-Point, saberamar482, jake_1526, ashtontorres40, 게못하는 게임백수, DarkShadows (ANTI-G.E.W.P), dylan.lf.22, ☆♬Green Hat♬☆ಠ一ಠ, vincenzinorubino, ChalkSauce, [L&G]Lancer +167 others


text:
gets a new look !
By continuing to use this website, you agree to the use of cookies in order to offer you content and services that are tailored to your interests.
A Short film by Leslie Villiaume
Production year : 2014
Within the framework of cultural exchanges and of initiation into international solidarity, ten young UNESCO ambassador's of a French college go to Benin in October, 2014.


text:
Medley Farms Llc
108 Jamestown Blvd Dothan, Alabama 36301(334) 671-3973
Print | Save | Directions
AboutMedley Farms Llc is located at the address 108 Jamestown Blvd in Dothan, Alabama 36301. They can be contacted via phone at (334) 671-3973 for pricing, hours and directions.
Medley Farms Llc has an annual sales volume of 501K - 999,999. .
For maps and directions to Medley Farms Llc view the map to the right. For reviews of Medley Farms Llc see below.
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text:
The typical asking rate per listing is in the $30's per square foot per year. The Douglas region is seeing a decrease in rates. In the last 3 months, the median rate decreased $8 per sqft per year. That's a 17% decline in rent.
Compared to the rest of the Gold Coast market, the Douglas submarket has greater rates, and you'll need to pay a premium to be in this hot submarket.
In the last 90 days, 2 listings have been posted online, which means new availabilities most likley are leasing quickly. You'll want to be prepared to make an offer immediately. There aren't many listings below 2,000 square feet posted, so finding a space for a small business might be tough.
Even though the median size is 5,660 sqft, there are still 0 new availabilities below 2,000 sqft. Most buildings in this area are close to 13 stories. Most buildings in this market were built in 1973 and are Class A.
Not much of the real estate is used for commercial space, so it might be hard to find office space. Public administration is the dominant industry in this market. Commute by bike is the most popular way to get to work amongst Douglas occupants. Commuting between 30 and 45 minutes a day is the norm for those who opt to reside in this part of town.


text:
32nd annual Wings Over Houston air show kicks off in Houston - 17 mins ago
Egypt cabinet to cut spending and diplomat missions abroad - 29 mins ago
UK's Transport Minister Chris Grayling says free trade with EU is in 'interests of both' - 30 mins ago
Halloween parade held in Chicago - 36 mins ago
Kurdish forces capture the town of Bashiqa near Mosul - 47 mins ago
Thais sing royal anthem in tribute to late Thai king - 48 mins ago
Expanding voter base in MCA main agenda for upcoming AGM, says Liow - 57 mins ago
Battle underwater: Robotic fish take part in sports games at 2016 World Robot Conference - about 1 hour ago
unauthorized check dam construction at Idukki - about 1 hour ago
DPM : Awareness level on early evacuation is moderate - 2 hours ago
Jiangsu tornado: In the aftermath of the storm
The rescue teams have been working for the past 14 hours. Now they have moved to one of the worst hit areas and are trying to remove a collapsed electricity pole blocking the main road in the village. CCTV's Shi Wenjing reports from a village in the worst-hit area.
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text:
Explore Stephanie Yewchuk, Paintings 3 and more!
Google Image Result for http://2.bp.blogspot.com/-TJyPQl2jW_8/TsZ-hmwmIUI/AAAAAAAAAak/QRe8V-RF7Po/s1600/new-york-snow-scene.jpg
The use of sharp lines and the colors on the image do a great job of highlighting the LOGO of VOGUE.
i want to make this!....its perfect blend of rustic yet classy. great color and design coffeegoeshere: Vintage turquoise coffee mug
Esse salto eu usaria!
Stephanie Ackerman using new Stampers Anonymous stamp sets. http://homegrownhospitality.typepad.com/homegrown_hospitality/2012/07/creative-best.html
ms. stephanie brown


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Rate Supervisor jobs
Neuvoo South Africa offers 134 rate supervisor jobs in South Africa. Submit your application now and land the best job available.
Rate Supervisor vacancies in South Africa
The rate supervisor is a profession in the Management category. In total on Neuvoo South Africa, more than 134 job vacancies are indexed from a multitude of sources such as company career sites, job boards, placement agencies and newspapers. Similar vacancies in this category like Quality Control Supervisor, Range Manager, Record Press Tender, Regional Director, Regional Manager or Relay Technician are also popular.
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Toad.IT Recruitment Services, Intelligent Placement, Communicate Cape Town Finance, What Next Recruitment, Execuplace Appointments or Selhurst Recruitment Consulting are among the top firms hiring profiles like this.
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Each month on average, 67 new job vacancies are offered on Neuvoo South Africa. Provinces where these profiles are most in demand are . Cities with the largest number of corresponding job vacancies available are Johannesburg, Cape Town, Port Elizabeth, Durban, Pretoria or Sandton .
Neuvoo South Africa: the largest job site in South Africa
Neuvoo South Africa offers over 174528 job opportunities in South Africa, 51,690 of which are in management and 134 of which are rate supervisor vacancies.Show more...
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For our growing Customer Service team, we are looking to hire a Call Centre Supervisor The Call Centre... Keep agents aware of inbound calls, calls waiting, abandonment rate, etc. review the past week's events...
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Ensure zero outstanding loads on web pages. Complete and submit rate request forms to commercial and... Rate review to be completed on the previous day plan, identify possible anomalies. Ensure special...
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Cape Town, Western Cape
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My client is looking for a Wayleave Supervisor. The ideal candidate will have connections in the Cape... Competitive Daily...
Frogg Recruitment SA |
(Unspecified city), Western Cape
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Well established Company is looking for a Loan Supervisor that will be responsible for the Lending... Checking that the loans on the system are correctly loaded. o Correct interest rate,. o payment,. o PV...
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Reliable and dynamic individual to fill the role of an Maintenance Supervisor. Qualified Artisan with... ..lines and equipment and achievement of set equipment Availability Rate and Operating speeds...
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Kempton Park, Gauteng
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..lines and equipment and achievement of set equipment availability rate and operations speeds... Must have proven management. supervisory experience. Intermediate level of MS Office. If you have not...
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For our growing Customer Service team, we are looking to hire a Call Centre Supervisor The Call Centre... Keep agents aware of inbound calls, calls waiting, abandonment rate, etc. review the past week's events...
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Johannesburg, Gauteng
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OSupport the monthly financial review and closing process. oCheck multipliers billing rate schedules in... oPerform any ad hoc duties as required by immediate supervisor or management. oAssist with queries...
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Cape Town, Western Cape
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OCall deposits. oCall withdrawals. oCall rate renegotiations. oFutures. oCreations and Liquidations... Escalation of all queries to supervisor. Assist Supervisor when required. Load manual Local and Foreign...
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text:
David Hill
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- Lexington Yellow Jackets face off against North Forsyth Vikingswschronicle.com
- SMount Kilimanjaro climb for Sussex charitiesshorehamherald.co.uk
- EExmouth's David happy with Paralympic resultexmouthjournal.co.uk
- New executive director prepares for upcoming festivalArkansas OnlineLow Key
- Trump tells gas industry officials they will love him as presidenttriblive.comUnited States
- TFederal grant looks to expand student involvement in ongoing drone researchtruomega.ca
- M$1.1-million for drone-ranching research projectmerrittherald.com
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- RFormer mayor of Rye dies at 87ryeandbattleobserver.co.ukAfrica
- One and Done: Why The Huddle, Won't be Around this Football Seasonboiseweekly.com
- Latin America's largest Ramsar Site facing 586 km transmission lineThe GuardianLima
- 'You're the first one I've seen': What Tory local politician said to black fireman on official visit to fire stationDaily Mail
- B&O American Brasserie to expand location with $250K renovationthedailyrecord.com
- In defence of the BBCThe Independent
- EExmouth's David heading for Paralympicsexmouthjournal.co.uk
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text:
Pocket Gophers and the Invasion and Restoration of Native Bunchgrass Communities
S. M. Watts, email email@example.com
More than 7 million hectares of California native plant communities are now dominated by exotic annual species, a biological invasion that has made native bunchgrass ecosystems in this region one of the most endangered ecosystems in North America. Many land use and environmental factors have contributed to the conversion of bunchgrass areas to annual grassland, but the role of gopher disturbance remains understudied. Here I report observational evidence that suggests gopher foraging is nonrandomly concentrated in the open spaces between clumps of bunchgrass and as a result may inhibit the recruitment of bunchgrass into these areas. Understanding patterns of direct gopher impact and coincident soil disturbance in bunchgrass versus annual grassland is important for successful restoration of bunchgrass habitats. While the prospect of excluding gophers is daunting, even short-term reductions in gopher populations would help to promote a restored community structure with a mature native bunchgrass and annual forb association.


text:
An asinine adventure through the world of high fantasy.4.47 / 5.00 2,323 Views
Nothing To Fear
Super villainy at its worst ...4.17 / 5.00 3,403 Views
The Ascent of Alfred
Make Alfred Great Again4.10 / 5.00 11,316 Views
I love this video cant wait to see the next killgore thumbs up big time
Totally deserves 5 stars. (Loads better then a lot of stuff on here.)
Some pointers though:
1. Minions looked way too cloned. Change the hair color or add facial hair to them. The different weapons they held helped though!
2. Lol. the 3 guys piked on Killgores horns was awesome but the horns stretching that long was a little silly. It works though.
3. One liners were okay, but the you being repeated twice was kind of pointless, but hey, it would really happen in a fight.
Have to say outstanding puppet work, lighting, and textures, and over all animated quality.
Keep this kind of work flow up, and have a nice one!
Glorious. All of it.
Great work - it's all super polished and smooth.
Thanks dude!
What a bad ass. Someone already stated this, but the action & one-liners were spot on. I'd love to see more.
Its not easy being a badass :P Thanks brooooooo!
If this was made into a game, everybody would play it.


text:
In benzene 7900, acetone 6500, chlorform 6300, carbon disulfide 5900, diethyl ether 5100, xylene 5000, iso-octanol 790, methanol 450 (all in g/kg, 25oC). Rate of hydrolysis increases with pH, and in the presence of copper and possibly of other metals that can form chelates. Packing: 200kg/iron drum
Sales Terms & Specifications:
|Place Of Origin
|Price Terms
|FOB / CIF / EXW(Factory Price)
|Terms Of Payment
|Delivery Lead Time
|Two weeks upon receiving the order
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PyridabenBiochemistry.: Inhibitor of mitochondrial electron transport at complex i Mode of action.: Non-systemic insecticide and acaricid
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