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Separation of the Stern and diffuse layer in coarse-grained models: The cases of phosphatidyl serine, phosphatidic acid, and PIP2 monolayers
NASA Astrophysics Data System (ADS)
We present here a method to separate the Stern and diffuse layer in general systems into two regions that can be analyzed separately. The Stern layer can be described in terms of Bjerrum pairing and the diffuse layer in terms of Poisson-Boltzmann theory (monovalent) or strong coupling theory plus a slowly decaying tail (divalent). We consider three anionic phospholipids: phosphatidyl serine, phosphatidic acid, and phosphatidylinositol(4,5)bisphosphate (PIP2), which we describe within a minimal coarse-grained model as a function of ionic concentration. The case of mixed lipid systems is also considered, which shows a high level of binding cooperativity as a function of PIP2 localization. Implications for existing experimental systems of lipid heterogeneities are also discussed.
Vangaveti, S.; Travesset, A.
Separation of the Stern and diffuse layer in coarse-grained models: the cases of phosphatidyl serine, phosphatidic acid, and PIP2 monolayers.
We present here a method to separate the Stern and diffuse layer in general systems into two regions that can be analyzed separately. The Stern layer can be described in terms of Bjerrum pairing and the diffuse layer in terms of Poisson-Boltzmann theory (monovalent) or strong coupling theory plus a slowly decaying tail (divalent). We consider three anionic phospholipids: phosphatidyl serine, phosphatidic acid, and phosphatidylinositol(4,5)bisphosphate (PIP2), which we describe within a minimal coarse-grained model as a function of ionic concentration. The case of mixed lipid systems is also considered, which shows a high level of binding cooperativity as a function of PIP2 localization. Implications for existing experimental systems of lipid heterogeneities are also discussed. PMID:25554186
Vangaveti, S; Travesset, A
Bermúdez, José Luis
Facile Synthesis of Phosphatidyl Saccharides for Preparation of Anionic Nanoliposomes with Enhanced Stability
Physical stability during storage and against processing such as dehyration/rehydration are the cornerstone in designing delivery vehicles. In this work, mono-, di- and tri-saccharides were enzymatically conjugated to phosphatidyl group through a facile approach namely phospholipase D (PLD) mediated transphosphatidylation in a biphasic reaction system. The purified products were structurally identified and the connectivities of carbohydrate to phosphatidyl moiety precisely mapped by 1H, 31P, 13C NMR pulse sequences and LC-ESI-FTMS. The synthetic phosphatidyl saccharides were employed as the sole biomimetic component for preparation of nanoliposomes. It was found that the critical micelle concentration (CMC) of phosphatidyl saccharides increases as more bulky sugar moiety (mono- to tri-) is introduced. Phosphatidyl di-saccharide had the largest membrane curvature. In comparison to the zwitterionic phosphatidylcholine liposome, all phosphatidyl saccharides liposomes are anionic and demonstrated significantly enhanced stability during storage. According to the confocal laser scan microscopy (CLSM) and atom force microscopy (AFM) analyses, the nanoliposomes formed by the synthetic phosphatidyl saccharides also show excellent stability against dehydration/rehydration process in which most of the liposomal structures remained intact. The abundance hydroxyl groups in the saccharide moieties might provide sufficient H-bondings for stabilization. This work demonstrated the synthesized phosphatidyl saccharides are capable of functioning as enzymatically liable materials which can form stable nanoliposomes without addition of stabilizing excipients. PMID:24069243
Song, Shuang; Cheong, Ling-Zhi; Falkeborg, Mia; Liu, Lei; Dong, Mingdong; Jensen, Henrik Max; Bertelsen, Kresten; Thorsen, Michael; Tan, Tianwei; Xu, Xuebing; Guo, Zheng
Docosahexaenoic acid-decient phosphatidyl serine and high K-tocopherol in a fetal mouse brain over-expressing
the polyunsaturated fatty acid (PUFA) profile in discrete phospholipid species, the K-tocopherol levels, a marker oxidative status. The PUFA profile in choline- and ethanolamine-phosphoglycerides was similar in tg polyunsaturated fatty acid (PUFA) component of brain membrane phospholipids (PLs) and is believed to participate
Groner, Yoram
The biosynthesis of glycine and l-serine is closely connected,\\u000a and both amino acids are produced in industry. However, whereas glycine is made chemically, l-serine\\u000a production relies largely on microbial processes. These include conversions of added glycine by C-1 utilizing\\u000a microorganisms. But such precursor conversions usually suffer from low yields, as did previous attempts\\u000a to produce l-serine from glucose. As more
Lothar Eggeling
Biased binding of class IA phosphatidyl inositol 3-kinase subunits to inducible costimulator (CD278)
To better understand T lymphocyte costimulation by inducible costimulator (ICOS; H4; CD278), we analyzed proteins binding\\u000a to ICOS peptides phosphorylated at the Y191MFM motif. Phosphorylated ICOS binds class IA phosphatidyl inositol 3-kinase (PI3-K) p85?, p50-55? and p85? regulatory subunits\\u000a and p110?, p110? and p110? catalytic subunits. Intriguingly, T cells expressed high levels of both p110? or p110? catalytic\\u000a subunits, yet
Yenny Y. Acosta; Maria Paz Zafra; Gloria Ojeda; Ilaria Seren Bernardone; Umberto Dianzani; Pilar Portol�s; Jose M. Rojo
NSDL National Science Digital Library
Serine is required for the metabolism of fat, tissue growth, and a healthy immune system. It assists in the production of immunoglobulins and antibodies. Some of its derivatives such as ethanolamine are important components of the phospholipids found in biological membranes. Serine is found in meat and dairy products, wheat gluten, peanuts, and soy products. It is not clear how toxic this substance can be, although it is known that high levels of serine may cause immune suppression and psychological symptoms such as cerebral allergies.
The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo
The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs. PMID:25049093
Boldrin, Francesca; Ventura, Marcello; Degiacomi, Giulia; Ravishankar, Sudha; Sala, Claudia; Svetlikova, Zuzana; Ambady, Anisha; Dhar, Neeraj; Kordulakova, Jana; Zhang, Ming; Serafini, Agnese; Vishwas, V. G.; Kolly, Ga�lle S.; Kumar, Naveen; Pal�, Giorgio; Guerin, Marcelo E.; Mikusova, Katarina; Cole, Stewart T.
Lysophosphatidylserine analogues differentially activate three LysoPS receptors.
Lysophosphatidylserine (1-oleoyl-2 R-lysophosphatidylserine, LysoPS) has been shown to have lipid mediator-like actions such as stimulation of mast cell degranulation and suppression of T lymphocyte proliferation, although the mechanisms of LysoPS actions have been elusive. Recently, three G protein-coupled receptors (LPS1/GPR34, LPS2/P2Y10 and LPS3/GPR174) were found to react specifically with LysoPS, raising the possibility that LysoPS serves as a lipid mediator that exerts its role through these receptors. Previously, we chemically synthesized a number of LysoPS analogues and evaluated them as agonists for mast-cell degranulation. Here, we used a transforming growth factor-? (TGF?) shedding assay to see if these LysoPS analogues activated the three LysoPS receptors. Modification of the serine moiety significantly reduced the ability of the analogues to activate the three LysoPS receptors, whereas modification of other parts resulted in loss of activity in receptor-specific manner. We found that introduction of methyl group to serine moiety (1-oleoyl-lysophosphatidylallothreonine) and removal of sn-2 hydroxyl group (1-oleoyl-2-deoxy-LysoPS) resulted in reduction of reactivity with LPS1 and LPS3, respectively. Accordingly, we synthesized a LysoPS analogue with the two modifications (1-oleoyl-2-deoxy-lysophosphatidylallothreonine) and found it to be an LPS2-selective agonist. These pharmacological tools will definitely help to identify the biological roles of these LysoPS receptors. PMID:25320102
Uwamizu, Akiharu; Inoue, Asuka; Suzuki, Kensuke; Okudaira, Michiyo; Shuto, Akira; Shinjo, Yuji; Ishiguro, Jun; Makide, Kumiko; Ikubo, Masaya; Nakamura, Sho; Jung, Sejin; Sayama, Misa; Otani, Yuko; Ohwada, Tomohiko; Aoki, Junken
A Fully Synthetic and Biochemically Validated Phosphatidyl Inositol-3-Phosphate Hapten via Asymmetric Synthesis and Native Chemical Ligation
We report the synthesis and biochemical validation of a phosphatidyl inositol-3 phosphate (PI3P) immunogen. The inositol stereochemistry was secured through peptide-catalyzed asymmetric phosphorylation catalysis, and the subsequent incorporation of a cysteine residue was achieved by native chemical ligation (NCL). Conjugation of the PI3P hapten to maleimide activated keyhole limpet hemocyanin (KLH) provided a PI3P immunogen, which was successfully used to generate selective PI3P antibodies. The incorporation of a sulfhydryl nucleophile into a phosphoinositide hapten demonstrates a general strategy to reliably access phosphoinositide immunogens. PMID:24344932
Chandler, Brent D.; Burkhardt, Anne L.; Foley, Klaudia; Cullis, Courtney; Driscoll, Denise; D�Amore, Natalie Roy; Miller, Scott J.
Serine phosphorylation of focal adhesion kinase in interphase and mitosis: a possible role in modulating binding to p130(Cas).
Focal adhesion kinase (FAK) is an important regulator of integrin signaling in adherent cells and accordingly its activity is significantly modulated during mitosis when cells detach from the extracellular matrix. During mitosis, FAK becomes heavily phosphorylated on serine residues concomitant with its inactivation and dephosphorylation on tyrosine. Little is known about the regulation of FAK activity by serine phosphorylation. In this report, we characterize two novel sites of serine phosphorylation within the C-terminal domain of FAK. Phosphorylation-specific antibodies directed to these sites and against two previously characterized sites of serine phosphorylation were used to study the regulated phosphorylation of FAK in unsynchronized and mitotic cells. Among the four major phosphorylation sites, designated pS1-pS4, phosphorylation of pS1 (Ser722) is unchanged in unsynchronized and mitotic cells. In contrast, pS3 and pS4 (Ser843 and Ser910) exhibit increased phosphorylation during mitosis. In vitro peptide binding experiments provide evidence that phosphorylation of pS1 (Ser722) may play a role in modulating FAK binding to the SH3 domain of the adapter protein p130(Cas). PMID:11160818
Ma, A; Richardson, A; Schaefer, E M; Parsons, J T
Serine Proteases of Parasitic Helminths
Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703
Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vall�e, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng
Wetting properties of dioleoyl-phosphatidyl-choline bilayers in the presence of trehalose: an X-ray diffraction study.
The combined effect of trehalose and temperature on the wetting properties of L-alpha-dioleoyl-phosphatidyl-choline (DOPC) model membranes in excess aqueous solutions has been analyzed by X-ray diffraction and extended electron density map reconstruction. At room temperature, DOPC in excess water forms a fluid lamellar L(alpha) phase. In the presence of trehalose, no phase transitions occur, but repeat and intermembrane distances increase considerably. Electron density maps show that trehalose in solution promotes a straightening of the hydrocarbon chain packing and a reduction of the molecular average area at the polar-apolar interface. Accordingly, the increased intermembrane distance is interpreted as a clear indication of a sugar screening effect of the van der Waals attractive contribution in the lamellar stacks, which overcomes the eventual decreasing of the repulsive fluctuations due to the hardening of the bilayer. By contrast to the thermally induced membrane swelling observed in excess water, in the presence of trehalose the DOPC bilayer thickness and repeat and intermembrane distances decrease continuously with temperature. While the thermal dependence of bilayer thickness is a consequence of the chain conformational disorder promoted by temperature, the changes in the intermembrane distance can be explained only assuming a trehalose-induced re-setting of the long-range force balance. The final results confirm the complex mechanism by which trehalose stabilize lipid bilayers. PMID:20435025
Di Gregorio, Giordano M; Ferraris, Paolo; Mariani, Paolo
G/sub o/ protein of fat cells: role in hormonal regulation of agonist-stimulated phosphatidyl inositol breakdown
Incubating rat fat cell membranes in the presence of (/sup 32/P)NAD/sup +/ and pertussis toxin (PT) results in the ADP-ribosylation of two peptides (M/sub r/ = 41,000 and 40,000). The 41,000-M/sub r/ peptide is the inhibitory G-protein of adenylate cyclase (G/sub i/). The 40,000-M/sub r/ peptide radiolabeled in the presence of (/sup 32/P)NAD/sup +/ and PT has been purified from rabbit heart and bovine brain, but has not been identified uniformly in membranes of fat cells. Two rabbit polyclonal antisera raised against the alpha-subunit of bovine brain G/sub o/ were used to probe the nature of the 40,000-M/sub r/ peptide in rat fat cell membranes that had been separated by gel electrophoresis in the presence of sodium dodecyl sulfate and transferred electrophoretically to nitrocellulose. Both antisera specific for the alpha-subunit of G/sub o/ recognized the M/sub r/ = 40,000 peptide of fat cells that is ADP-ribosylated in the presence of PT. PT treatment of rat fat cells blocks epinephrine-stimulated inositol 1,4,5 trisphosphate (IP/sub 3/) generation. The inhibition of IP/sub 3/ generation by PT suggests a role for either G/sub i/ or G/sub o/ in receptor-mediated phosphatidyl inositol breakdown in the rat fat cell.
Rapiejko, P.J.; Northup, J.K.; Malbon, C.C.
[Serine proteinase with lytic properties].
A thermophilic Bacillus licheniformis strain can synthesize extracellular serine proteinase, which is similar to subtilisin of the Carlsberg type in its amino acid composition, the specificity of its action, the character of its inhibition by certain compounds and immunochemical properties, but differs from the latter in its greater thermostability and in the ability to cause lysis of Gram-negative bacteria and yeast living cells. PMID:3141748
Pavlova, I N; Zholner, L G; Zakharova, I Ia; Tin'ianova, N Z; Chestukhina, G G
Studies on dodecyl betainate in combination with its degradation products or with phosphatidyl choline-phase behavior and hemolytic activity.
Surface active betaine esters contain a hydrolysable bond and give naturally occurring products (fatty alcohol and the amino acid betaine) on degradation. They are therefore interesting candidates for use as cationic surfactants in pharmaceutical applications. In this work the phase behavior of two systems of relevance for the utilization of dodecyl betainate as a pharmaceutical excipient is studied, namely dodecyl betainate/dodecanol/betaine hydrochloride/D2O and dodecyl betainate/phosphatidyl choline (PC)/ethanol/D2O. The techniques used for phase characterisation were 2H NMR measured on the solvent, small angle X-ray spectroscopy and optical microscopy. Dilute dodecyl betainate/PC dispersions were characterized using laser diffraction. It is shown that introduction of relatively small amounts of the hydrolysis products of dodecyl betainate, i.e., dodecanol and betaine (used in the form of betaine hydrochloride), has a strong effect on the phase behavior of the binary dodecyl betainate/D2O system. The degradation products change the average curvature of the surfactant film so that, instead of a hexagonal phase at concentrations above the micellar phase, a probably defective, lamellar phase seems to form. The dodecyl betainate/PC/ethanol/D2O system shows a large region of a highly swelling lamellar phase. Dispersions of dodecyl betainate/PC/ethanol in water can be prepared with low energy input; i.e., the preconcentrate can be regarded as a self-dispersing solution. Introduction of dodecyl betainate and its degradation products does not impair the ability of PC to form vesicles. Experiments for evaluating the toxicity of surface active betaine esters to erythrocytes were also performed. There are indications that the hemolytic activity of dodecyl betainate is lower than that of the stable surfactant tetradecyltrimethylammonium chloride, which has similar critical micelle concentration. A combination of dodecyl betainate and PC gives very low hemolytic activity. PMID:15450470
Lundberg, D; Ljusberg-Wahren, H; Norlin, A; Holmberg, K
Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves
In pulse-chase experiments with barley wilted leaves, label from (/sup 14/C)-ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When (/sup 14/C)monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine. The following experiments, in which tracer amounts of (/sup 14/C)ethanolamine or (/sup 14/C)formate were supplied to wilted barley leaves, implicated phosphoryl and phosphatidyl bases as intermediates in the methylation steps between ethanolamine and phosphatidylcholine. Label from both (/sup 14/C)ethanolamine and (/sup 14/C)formate entered phosphorylmonomethylethanolamine and phosphorylcholine very rapidly; these phosphoryl bases were the most heavily labeled products at 15 to 30 minutes after label addition and lost label rapidly as the fed /sup 14/C-labeled precursor was depleted. Phosphatidylmonomethylethanolamine and phosphatidylcholine were also significantly labeled from (/sup 14/C)ethanolamine and (/sup 14/)formate at early times; the corresponding free bases and nucleotide bases were not. Addition of a trapping pool of phosphorylcholine reduced (/sup 14/C)ethanolamine conversion to both phosphatidylcholine and betaine, and resulted in accumulation of labe in the trap. A computer model of the synthesis of betaine via phosphatidylcholine was developed from /sup 14/C kinetic data. The model indicates that about 20% of the total leaf phosphatidylcholine behaves as an intermediate in betaine biosynthesis and that a marked decrease (greater than or equal to2-fold) in the half-life of this metabolically active phosphatidylcholine fraction accompanies wilting.
Hitz, W.D.; Rhodes, D.; Hanson, A.
Enzymatic assay of d-serine using d-serine dehydratase from Saccharomyces cerevisiae
d-Serine is localized in the mammalian forebrain and modulates brain functions as a coagonist of an N-methyl-d-aspartate receptor. d-Serine is also found in human urine, although its physiological meaning is unclear. A method for rapid and simple assay of d-serine is probably useful for studying its physiological role and clinical relevance. Currently, d-serine is assayed by high-performance liquid chromatography after
Tomokazu Ito; Kei Takahashi; Tomoko Naka; Hisashi Hemmi; Tohru Yoshimura
Occurrence of D-serine in rice and characterization of rice serine racemase
Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D\\/(D+L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic
Yoshitaka Gogami; Katsuyoshi Ito; Yuji Kamitani; Yuki Matsushima; Tadao Oikawa
1 PS-1, Revision 6 Policy Statement Number: PS-01
1 PS-1, Revision 6 Policy Statement Number: PS-01 Title/Topic: Equal Opportunity Policy Effective Date: 2/5/2013 Revision Number: PS0001.R06 I. PURPOSE The purpose of this policy statement is to assert expression, religion, sex, national origin, age, mental or physical disability, or veteran's status, as well
Harms, Kyle E.
Sorafenib/Regorafenib and Phosphatidyl Inositol 3 Kinase/Thymoma Viral Proto-Oncogene Inhibition Interact to Kill Tumor Cells
The present studies were undertaken to determine whether the multikinase inhibitors sorafenib/regorafenib cooperated with clinically relevant , phosphatidyl inositol 3 kinase (PI3K)-thymoma viral proto-oncogene (AKT) inhibitors to kill tumor cells. In liver, colorectal, lung, breast, kidney, and brain cancer cells, at clinically achievable doses, sorafenib/regorafenib and the PI3K inhibitor acetic acid (1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-11-yl ester (PX-866) cooperated in a greater than additive fashion to kill tumor cells. Cells lacking phosphatase and tensin homolog were as sensitive to the drug combination as cells expressing the protein. Similar data were obtained using the AKT inhibitors perifosine and 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f] [1,6]naphthyridin-3(2H)-one hydrochloride (MK2206). PX-866 treatment abolished AKT/glycogen synthase kinase 3 (GSK3) phosphorylation, and cell killing correlated with reduced activity of AKT and mammalian target of rapamycin (mTOR). Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma�extra large or dominant negative caspase 9, but not cellular FLICE (FADD-like IL-1b�converting enzyme)-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, lysosome-associated membrane protein 2 (LAMP2), and microtubule-associated protein light chain (LC) 3 and LC3II that correlated with a large increase in LC3�green fluorescent protein (GFP) vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II, and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or autophagy-related 5 suppressed drug toxicity by ?40%. In vivo, sorafenib and PX-866 or regorafenib and MK2206 cooperated to suppress the growth of established HuH7 and HCT116 tumors, respectively. Collectively our data demonstrate that the combination of sorafenib family kinase inhibitors with inhibitors of the PI3K/AKT pathway kills tumor cells in vitro and in vivo. PMID:23877009
Sajithlal, Gangadharan B.; Hamed, Hossein A.; Cruickshanks, Nichola; Booth, Laurence; Tavallai, Seyedmehrad; Syed, Jahangir; Grant, Steven; Poklepovic, Andrew
Allosteric regulation of mouse brain serine racemase.
Serine racemase, purified from mouse brain, consisted of two isoforms. They had similar enzymatic properties and had molecular weights of about 55 kDa based on size exclusion chromatography. This is about twice that reported from its electrophoretic mobility on SDS gels or from the amino acid sequence of the recombinant enzyme. In addition to the previously reported requirements for pyridoxal phosphate and reducing agents, we found that both forms of the enzyme required Mg2+ and were strongly stimulated by yeast extract. The yeast extract could be replaced by ATP, GTP, or ADP and, to a lesser extent, by other nucleotides. In the presence of 1 mM ATP, the Km for L-serine decreased from 13 mM to 1.8 mM with little change in Vmax, indicating an allosteric mechanism for nucleotide activation. In addition to acting as a serine racemase, the enzyme has been reported to act on L-serine O-sulfate as a dehydratase. When measured by HPLC, after derivatization with 2,4 dinitrophenylhydrazine, we found, as expected, a very rapid formation of pyruvate from this substrate. L-serine was also converted to pyruvate at about twice the racemization rate. L-serine O-sulfate dehydration was inhibited by ATP, while L-serine dehydration, like racemization, was activated by nucleotides, indicating that, for L-serine, dehydration and racemization take place at the same site. PMID:12515328
Neidle, Amos; Dunlop, David S
Serine and glycine metabolism in cancer?
Serine and glycine are biosynthetically linked, and together provide the essential precursors for the synthesis of proteins, nucleic acids, and lipids that are crucial to cancer cell growth. Moreover, serine/glycine biosynthesis also affects cellular antioxidative capacity, thus supporting tumour homeostasis. A crucial contribution of serine/glycine to cellular metabolism is through the glycine cleavage system, which refuels one-carbon metabolism; a complex cyclic metabolic network based on chemical reactions of folate compounds. The importance of serine/glycine metabolism is further highlighted by genetic and functional evidence indicating that hyperactivation of the serine/glycine biosynthetic pathway drives oncogenesis. Recent developments in our understanding of these pathways provide novel translational opportunities for drug development, dietary intervention, and biomarker identification of human cancers. PMID:24657017
Amelio, Ivano; Cutruzzol�, Francesca; Antonov, Alexey; Agostini, Massimiliano; Melino, Gerry
Type II transmembrane serine proteases.
Analysis of genome and expressed sequence tag data bases at the turn of the millennium unveiled a new protease family named the type II transmembrane serine proteases (TTSPs) in a Journal of Biological Chemistry minireview (Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857-860). Since then, the number of known TTSPs has more than doubled, and more importantly, our understanding of the physiological functions of individual TTSPs and their contribution to human disease has greatly increased. Progress has also been made in identifying molecular substrates and endogenous inhibitors. This minireview summarizes the current knowledge of the rapidly advancing TTSP field. PMID:19487698
Bugge, Thomas H; Antalis, Toni M; Wu, Qingyu
Type II Transmembrane Serine Proteases*
Analysis of genome and expressed sequence tag data bases at the turn of the millennium unveiled a new protease family named the type II transmembrane serine proteases (TTSPs) in a Journal of Biological Chemistry minireview (Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857�860). Since then, the number of known TTSPs has more than doubled, and more importantly, our understanding of the physiological functions of individual TTSPs and their contribution to human disease has greatly increased. Progress has also been made in identifying molecular substrates and endogenous inhibitors. This minireview summarizes the current knowledge of the rapidly advancing TTSP field. PMID:19487698
Bugge, Thomas H.; Antalis, Toni M.; Wu, Qingyu
Abnormal serine phosphorylation of insulin receptor substrate 1 is associated with tau pathology in Alzheimer's disease and tauopathies
Neuronal insulin signaling abnormalities have been associated with Alzheimer's disease (AD). However, the specificity of this association and its underlying mechanisms have been unclear. This study investigated the expression of abnormal serine phosphorylation of insulin receptor substrate 1 (IRS1) in 157 human brain autopsy cases that included AD, tauopathies, ?-synucleinopathies, TDP-43 proteinopathies, and normal aging. IRS1-pS616, IRS1-pS312 and downstream target Akt-pS473 measures were most elevated in AD but were also significantly increased in the tauopathies: Pick's disease, corticobasal degeneration and progressive supranuclear palsy. Double immunofluorescence labeling showed frequent co-expression of IRS1-pS616 with pathologic tau in neurons and dystrophic neurites. To further investigate an association between tau and abnormal serine phosphorylation of IRS1, we examined the presence of abnormal IRS1-pS616 expression in pathological tau-expressing transgenic mice and demonstrated that abnormal IRS1-pS616 frequently co-localizes in tangle-bearing neurons. Conversely, we observed increased levels of hyperphosphorylated tau in the high-fat diet-fed mouse, a model of insulin resistance. These results provide confirmation and specificity that abnormal phosphorylation of IRS1 is a pathological feature of AD and other tauopathies, and provide support for an association between insulin resistance and abnormal tau as well as amyloid-?. PMID:25107476
Yarchoan, Mark; Toledo, Jon B.; Lee, Edward B.; Arvanitakis, Zoe; Kazi, Hala; Han, Li-Ying; Louneva, Natalia; Lee, Virginia M.-Y.; Kim, Sangwon F.; Trojanowski, John Q.; Arnold, Steven E.
1988, 2009 PHOTOCOUPLER PS2506-1,PS2506L-1 HIGH ISOLATION VOLTAGE AC INPUT, DARLINGTON TRANSISTOR-1 is lead bending type (Gull-wing) for surface mount. FEATURES · AC input response · High isolation voltage (BV = 5 000 Vr.m.s.) · High current transfer ratio (CTR = 2 000% TYP.) · High-speed switching (tr
Ravikumar, B.
Induction of serine racemase expression and D-serine release from microglia by amyloid ?-peptide
BACKGROUND: Roles for excitotoxicity and inflammation in Alzheimer's disease have been hypothesized. Proinflammatory stimuli, including amyloid ?-peptide (A?), elicit a release of glutamate from microglia. We tested the possibility that a coagonist at the NMDA class of glutamate receptors, D-serine, could respond similarly. METHODS: Cultured microglial cells were exposed to A?. The culture medium was assayed for levels of D-serine
Sheng-Zhou Wu; Angela M Bodles; Mandy M Porter; W Sue T Griffin; Anthony S Basile; Steven W Barger
Reactions of serine palmitoyltransferase with serine and molecular mechanisms of the actions of serine derivatives as inhibitors.
Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A to 3-ketodihydrosphingosine. We have succeeded in the overproduction of a water-soluble homodimeric SPT from Sphingomonas paucimobilis EY2395(T) in Escherichia coli. The recombinant SPT showed the characteristic absorption and circular dichroism spectra derived from its coenzyme pyridoxal 5'-phosphate. On the basis of the spectral changes of SPT, we have analyzed the reactions of SPT with compounds related to L-serine and product, and showed the following new aspects: First, we analyzed the binding of L-serine and 3-hydroxypropionate and found that the spectral change in SPT by the substrate is caused by the formation of an external aldimine intermediate and not by the formation of the Michaelis complex. Second, various serine analogues were also examined; the data indicated that the alpha-carboxyl group of L-serine was quite important for substrate recognition by SPT. Third, we focused on a series of SPT inhibitors, which have been used as convenient tools to study the cell responses caused by sphingolipid depletion. The interaction of SPT with myriocin suggested that such product-related compounds would strongly and competitively inhibit enzyme activity by forming an external aldimine in the active site of the enzyme. Beta-chloro-L-alanine and L-cycloserine were found to generate characteristic PLP-adducts that produced inactivation of SPT in an irreversible manner. The detailed mechanisms for the SPT inactivation were discussed. This is the first analysis of the inhibition mechanisms of SPT by these compounds, which will provide an enzymological basis for the interpretation of the results from cell biological experiments. PMID:14744154
Ikushiro, Hiroko; Hayashi, Hideyuki; Kagamiyama, Hiroyuki
Significance of the D-serine-deaminase and D-serine metabolism of Staphylococcus saprophyticus for virulence.
Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ?dsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ?dsdA mutant was attenuated in virulence murine model of urinary tract infection. PMID:24082071
Korte-Berwanger, Miriam; Sakinc, T�rkan; Kline, Kimberly; Nielsen, Hailyn V; Hultgren, Scott; Gatermann, S�ren G
Allosteric Regulation of Mouse Brain Serine Racemase
Serine racemase, purified from mouse brain, consisted of two isoforms. They had similar enzymatic properties and had molecular weights of about 55 kDa based on size exclusion chromatography. This is about twice that reported from its electrophoretic mobility on SDS gels or from the amino acid sequence of the recombinant enzyme. In addition to the previously reported requirements for pyridoxal
Amos Neidle; David S. Dunlop
Structure-Based Design of an Organoruthenium Phosphatidyl-inositol-3-Kinase Inhibitor Reveals a Switch Governing Lipid Kinase Potency and Selectivity
Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3K?/E5E2 complex reveals the molecular features that give rise to this potency and selectivity toward lipid kinases with implications for the design of a subsequent generation of PI3K-isoform-specific organometallic inhibitors.
Xie,P.; Williams, D.; Atilla-Gokcumen, G.; Milk, L.; Xiao, M.; Smalley, K.; Herlyn, M.; Meggers, E.; Marmorstein, R.
The effect of bacteriorhodopsin, detergent and hydration on the cubic-to-lamellar phase transition in the monoolein-distearoyl phosphatidyl glycerol-water system.
The cubic phase of monoolein (MO) has successfully been used for crystallization of membrane proteins. It is likely that the transition to a lamellar phase upon dehydration is important for the crystallization process, and that the internal dimensions of the lipid phases (i.e., water pore diameter) are crucial for the inclusion and the diffusion of membrane proteins. In the present study, we investigated the cubic-to-lamellar phase transitions in the MO-water and the MO-distearoyl phosphatidyl glycerol (DSPG) systems. The MO-water system was investigated by means of isothermal sorption and desorption microcalorimetry. We show that the transition from cubic to lamellar phase induced by desorption is driven by entropy. At 25 degrees C, this occurs at a water activity of 0.98 with a transition enthalpy of 860 J/mol (MO). The phase behavior was also investigated in the presence of a small amount of the transmembrane protein bacteriorhodopsin (bR), and a detergent, octyl glucoside (OG), and it was shown that both bR and OG stabilize the lamellar phase. Analogous results were obtained for the MO-DSPG-water system. The latter system resembles the MO-water system in that a cubic-to-lamellar phase transition is induced by dehydration, although the structural properties of these phases are slightly different. Finally, we demonstrate that bR can be crystallized from a cubic phase of MO-DSPG-buffer. PMID:15471581
Sparr, Emma; Wadsten, Pia; Kocherbitov, Vitaly; Engstr�m, Sven
Modulating the function of human serine racemase and human serine dehydratase by protein engineering.
D-Serine is a co-agonist of N-methyl D-aspartate, a glutamate receptor, which is a major excitatory neurotransmitter receptor in the brain. Human serine racemase (hSR) and serine dehydratase (hSDH) are two important pyridoxal-5'-phosphate-dependent enzymes that synthesize and degrade D-serine, respectively. hSR and hSDH have significant sequence homology (28% identity) and are similar in their structural folds (root-mean-square deviation, 1.12 �). Sequence alignment and structural comparison between hSR and hSDH reveal that S84 in hSR and A65 in hSDH play important roles in their respective enzyme activities. We surmise that exchange of these two amino acids by introducing S84A hSR and A65S hSDH mutants may result in switching their protein functions. To understand the modulating mechanism of the key residues, mutants S84A in hSR and A65S in hSDH were constructed to monitor the change of activities. The structure of A65S hSDH mutant was determined at 1.3 � resolution (PDB 4H27), elucidating the role of this critical amino acid. Our study demonstrated S84A hSR mutant behaved like hSDH, whereas A65S hSDH mutant acquired an additional function of using D-serine as a substrate. PMID:23112234
Wang, Cyong-Yi; Ku, Shan Chi; Lee, Cheng-Chung; Wang, Andrew H-J
NASA Astrophysics Data System (ADS)
ASASSN-15ez (ATel #7228) is an already discovered supernova < a href=http://star.pst.qub.ac.uk/ps1threepi/psdb/candidate/1085940131151112400/ > PS15mb < /a > . We apologize for any confusion we might have caused.
Stanek, K. Z.; ASAS-SN Team
OBJECTIVE: We review the experimental evaluations of several widely marketed nonprescription com- pounds claimed to be memory enhancers and treatments for age-related memory decline. We generally limit our review to double-blind placebo-controlled studies. The compounds examined are phosphatidyl- serine (PS), phosphatidylcholine (PC), citicoline, piracetam, vinpocetine, acetyl-L-carnitine (ALC), and antioxidants (particularly vitamin E). RESULTS: In animals, PS has been shown to
Mark A. McDaniel; Steven F. Maier; Gilles O. Einstein
Metal ion dependency of serine racemase from Dictyostelium discoideum.
D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2 ?M and 2.2 mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site. PMID:22311068
Ito, Tomokazu; Murase, Hirotaka; Maekawa, Motoki; Goto, Masaru; Hayashi, Shuhei; Saito, Hajime; Maki, Masatoshi; Hemmi, Hisashi; Yoshimura, Tohru
Hydrolysis of polyesters by serine proteases
The substrate specificity of a-chymotrypsin and other serine proteases, trypsin, elastase, proteinase K and subtilisin, towards hydrolysis of various polyesters was examined using poly(L-lactide) (PLA), poly(�-hydroxybutyrate) (PHB), poly(ethylene succinate) (PES), poly(ethylene adipate) (PEA), poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBS\\/A), poly[oligo(tetramethylene succinate)-co-(tetramethylane carbonate)] (PBS\\/C), and poly(?-caprolactone) (PCL). a-Chymotrypsin could degrade PLA and PEA with a lower activity on PBS\\/A. Proteinase K
Hyun-A Lim; Takao Raku; Yutaka Tokiwa
Serine Racemase Deletion Protects Against Cerebral Ischemia And Excitotoxicity
D-serine, formed from L-serine by serine racemase (SR), is a physiologic co-agonist at NMDA receptors. Using mice with targeted deletion of SR, we demonstrate a role for D-serine in NMDA receptor mediated neurotoxicity and stroke. Brain cultures of SR deleted mice display markedly diminished nitric oxide (NO) formation and neurotoxicity. In intact SR knockout mice NO formation and nitrosylation of NO targets are substantially reduced. Infarct volume following middle cerebral artery occlusion is dramatically diminished in several regions of the brains of SR mutant mice despite evidence of increased NMDA receptor number and sensitivity. PMID:20107067
Mustafa, Asif K.; Ahmad, Abdullah S.; Zeynalov, Emil; Gazi, Sadia K.; Sikka, Gautam; Ehmsen, Jeffrey T.; Barrow, Roxanne K.; Coyle, Joseph T.; Snyder, Solomon H.; Dor�, Sylvain
A bumblebee (Bombus ignitus) venom serine protease inhibitor that acts as a microbial serine protease inhibitor.
Serine protease inhibitors from bumblebee venom have been shown to block plasmin activity. In this study, we identified the protein BiVSPI from the venom of Bombus ignitus to be a serine protease inhibitor and an antimicrobial factor. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI is expressed in the venom gland and also found in the venom as an 8-kDa peptide. Recombinant BiVSPI that was expressed in baculovirus-infected insect cells exhibited inhibitory activity against chymotrypsin but not trypsin. BiVSPI also inhibited microbial serine proteases, such as subtilisin A (Ki=6.57nM) and proteinase K (Ki=7.11nM). In addition, BiVSPI was shown to bind directly to Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide evidence for a novel serine protease inhibitor in bumblebee venom that has antimicrobial functions. PMID:24158004
Wan, Hu; Kim, Bo Yeon; Lee, Kwang Sik; Yoon, Hyung Joo; Lee, Kyung Yong; Jin, Byung Rae
Sep 7, 2014 ... The first line lists the singularities, the second the smaller exponents, and .... standard Young tableau (SSYT) is a filling of such a diagram with positive ...... a quadrilateral depends on one parameter, the modulus of the�...
Oct 15, 2011 ... to an entire function, and the behavior at ? shows that this entire function. is a polynomial of degree ..... Now we describe the necessary modifications of this proof for the case that ... If c0 = ?, we may assume without loss ..... polynomial of least deviation from zero on [0,1] with respect to the weight. function�...
Mar 21, 2002 ... braic geometry, combinatorics and control theory as we describe below. ..... where a > 0 is a small parameter, whose range may depend on q. Thus ..... Elimination of u and y from (42), (43) gives the closed loop system.
symmetric case (that is when ak = (ibk)k with real bk), all eigenvalues but. finitely many are ..... From Step 2 applied with ? = 2?(m ? 1)/d, there is a Stokes line Um ..... [11] J. Jenkins and D. Spencer, Hyperelliptic trajectories, Ann. Math., 51.
Jul 27, 2006 ... K such that for every holomorphic map f from the unit disc to the complement ..... m(0) = m, and because fm wraps the disc around the sphere so as to cover ...... J. A. Jenkins, On explicit bounds in Landau's theorem II, Canad.
Synthesis of alkali metal indium thiophosphates containing the discrete anions [In(PS4)(PS5)2]6- and [In(PS4)2(PS5)]6-.
The first discrete anionic indium thiophosphate complexes are reported. The structures of K(6)[In(PS(4))(1.5)(PS(5))(1.5)] (1), Rb(6)[In(PS(4))(PS(5))(2)] (2), and Cs(6)[In(PS(4))(1.5)(PS(5))(1.5)] (3) all contain an anionic moiety consisting of octahedrally coordinated indium surrounded by the thiophosphate anions [PS(4)](3-) and the new [PS(5)](3-) ion. The conformation and bonding of the unsymmetric chelate ligand [PS(5)](3-) to indium give rise to different anions of the general formula [In(PS(4))(1+x)(PS(5))(2-x)](6-) (x = 0, 0.5). The anionic moiety in K(6)[In(PS(4))(1.5)(PS(5))(1.5)] (1) consists of cocrystallizing lambda-[In(PS(4))(2)(PS(5))](6-) and Lambdalambdadelta-[In(PS(4))(PS(5))(2)](6-) anions in a ratio of 1:1 (and their enantiomers). In Rb(6)[In(PS(4))(PS(5))(2)] (2), no cocrystallizatzion of anions was observed, and only the Lambdalambdalambda-[In(PS(4))(PS(5))(2)](6-) anion (and its enantiomer) is present. Cs(6)[In(PS(4))(1.5)(PS(5))(1.5)] (3) shows the same disorder between [PS(4)](3-) and [PS(5)](3-) ions as in 1. In 3, however, the octahedrally coordinated indium atom and thiophosphate ligands form a Lambdadeltadelta-[In(PS(4))(PS(5))(2)](6-) anion cocrystallizing with delta-[In(PS(4))(2)(PS(5))](6-). Additionally, ordered Rb(6)[In(PS(4))(PS(5))(2)] (2) was characterized by (31)P magic angle spinning NMR, Raman spectroscopy, UV-vis solid-state absorption spectroscopy, thermogravimetric analysis, differential thermal analysis, and energy dispersive X-ray analysis. PMID:20443630
Rothenberger, Alexander; Morris, Collin; Kanatzidis, Mercouri G
The pharmacological landscape and therapeutic potential of serine hydrolases
Serine hydrolases perform crucial roles in many biological processes, and several of these enzymes are targets of approved drugs for indications such as type 2 diabetes, Alzheimer's disease and infectious diseases. Despite this, most of the human serine hydrolases (of which there are more than 200) remain poorly characterized with respect to their physiological substrates and functions, and the vast
Daniel A. Bachovchin; Benjamin F. Cravatt
Cross genome comparisons of serine proteases in Arabidopsis and rice
BACKGROUND: Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa) genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. RESULTS: Despite the differences in
Lokesh P Tripathi; R Sowdhamini
Some biochemical and histochemical properties of human liver serine dehydratase
In rat, serine dehydratase (SDH) is abundant in the liver and known to be a gluconeogenic enzyme, while there is little information about the biochemical property of human liver serine dehydratase because of its low content and difficulty in obtaining fresh materials. To circumvent these problems, we purified recombinant enzyme from Escherichia coli, and compared some properties between human and
Tatsuhiko Kashii; Tomoharu Gomi; Takeshi Oya; Yoko Ishii; Hirofumi Oda; Muneharu Maruyama; Masashi Kobayashi; Tohru Masuda; Mitsuaki Yamazaki; Takuya Nagata; Kazuhiro Tsukada; Akinori Nakajima; Kazuhito Tatsu; Hisashi Mori; Fusao Takusagawa; Hirofumi Ogawa; Henry C. Pitot
D-Serine Regulation of NMDA Receptor Activity
NSDL National Science Digital Library
The N-Methyl-D-aspartate�type glutamate receptor (NMDAR) plays a key role in several important processes involving the nervous system, including brain development, synaptic plasticity, and learning. Unlike other neurotransmitter receptors, which are activated by individual neurotransmitters, activation of NMDARs requires the binding of a coagonist (D-serine or glycine) in addition to glutamate. Although previously considered an "unnatural" amino acid, D-serine is a key regulator of NMDAR activity and may be the main physiological ligand at the coagonist site. D-Serine is synthesized in the mammalian brain and is enriched in astrocytes, a class of glial cells that ensheath synapses in the brain. Astrocytes physiologically affect NMDAR neurotransmission by releasing D-serine, suggesting that D-serine acts as a gliotransmitter. However, recent findings indicate that D-serine signaling does not depend solely on glia, because D-serine and its biosynthetic enzyme are also present in substantial amounts in neurons. Here, we discuss these new findings, which begin to shed light on the relative roles of glia and neurons in D-serine signaling.
Herman Wolosker (Technion-Israel Institute of Technology; Department of Biochemistry REV)
Technology Transfer Automated Retrieval System (TEKTRAN)
Because of the importance of enzymes of the serine class as targets for novel controls of human diseases, the current study investigates the occurrence and function of serine proteases in Emeria tenella developmental stages. Using gel electrophoresis with casein imbedded gels (zymograms), bands of ...
Technology Transfer Automated Retrieval System (TEKTRAN)
Barley malt contains endoproteinases belonging to all four of the commonly occurring classes, including serine proteinases. It also contains low-molecular-weight proteins that inhibit the activities of many of these endoproteinases, but it had never been shown that any barley or malt serine protein...
3-Phosphoglycerate dehydrogenase deficiency: an inborn error of serine biosynthesis.
Serine concentrations were markedly decreased in the cerebrospinal fluid of two brothers with congenital microcephaly, profound psychomotor retardation, hypertonia, epilepsy, growth retardation, and hypogonadism. The youngest boy also had congenital bilateral cataract. Magnetic resonance imaging of the brain showed evidence of dysmyelination. Plasma serine as well as plasma and cerebrospinal fluid glycine concentrations were also decreased but to a lesser extent. Treatment with oral serine in the youngest patient significantly increased cerebrospinal fluid serine and abolished the convulsions. In fibroblasts of both patients, a decreased activity was demonstrated of 3-phosphoglycerate dehydrogenase, the first step of serine biosynthesis (22% and 13% of the mean control value). This is an unusual disorder as the great majority of aminoacidopathies are catabolic defects. It is a severe but potentially treatable inborn error of metabolism that has not been previously reported in man. PMID:8758134
Jaeken, J; Detheux, M; Van Maldergem, L; Foulon, M; Carchon, H; Van Schaftingen, E
Production of L-serine by Sarcina albida.
Conditions for the production of microbial L-serine hydroxymethyltransferase and for the conversion of glycine to L-serine were studied. A number of microorganisms were screened for their abilities to form and accululate L-serine from glycine, and Sarcina albida was selected as the best organism. Enzyme activity in this organism as high as 0.12 U/ml could be produced in shaken cultures at 30 degrees C in a medium containing glucose, ammonium sulfate, glycine, yeast extract, and inorganic salts. L-Serine was produced most efficiently by shaking cells at 30 degrees C in a reaction mixture containing 20% glycine, 5 X 10(-3) M formaldehyde, and 3 X 10(-4) M pyridoxal phosphate in yields of 22 mg of broth in 5 days. L-Serine was easily isolated in 84% yields by ion-exchange resin. PMID:39497
Ema, M; Kakimoto, T; Chibata, I
Catabolism of serine by Pediococcus acidilactici and Pediococcus pentosaceus.
The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from L- and D-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and ?-aminobutyrate. PMID:23241976
Irmler, Stefan; Bavan, Tharmatha; Oberli, Andrea; Roetschi, Alexandra; Badertscher, Ren�; Guggenb�hl, Barbara; Berthoud, H�l�ne
Immunohistochemical localization of D-serine dehydratase in chicken tissues.
Chicken D-serine dehydratase (DSD) degrades d-serine to pyruvate and ammonia. The enzyme requires both pyridoxal 5'-phosphate and Zn(2+) for its activity. d-Serine is a physiological coagonist that regulates the activity of the N-methyl-d-aspartate receptor (NMDAR) for l-glutamate. We have recently found in chickens that d-serine is degraded only by DSD in the brain, whereas it is also degraded to 3-hydroxypyruvate by d-amino acid oxidase (DAO) in the kidney and liver. In mammalian brains, d-serine is degraded only by DAO. It has not been clarified why chickens selectively use DSD for the control of d-serine concentrations in the brain. In the present study, we measured DSD activity in chicken tissues, and examined the cellular localization of DSD using a specific anti-chicken DSD antibody. The highest activity was found in kidney. Skeletal muscles and heart showed no activity. In chicken brain, cerebellum showed about 6-fold-higher activity (1.1 � 0.3 U/g protein) than cerebrum (0.19 � 0.03 U/g protein). At the cellular level DSD was demonstrated in proximal tubule cells of the kidney, in hepatocytes, in Bergmann-glia cells of the cerebellum and in astrocytes. The finding of DSD in glial cells seems to be important because d-serine is involved in NMDAR-dependent brain functions. PMID:24529545
Nishimura, Yoshihiro; Tanaka, Hiroyuki; Ishida, Tetsuo; Imai, Shinji; Matsusue, Yoshitaka; Agata, Yasutoshi; Horiike, Kihachiro
Catabolism of Serine by Pediococcus acidilactici and Pediococcus pentosaceus
The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from l- and d-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and ?-aminobutyrate. PMID:23241976
Bavan, Tharmatha; Oberli, Andrea; Roetschi, Alexandra; Badertscher, Ren�; Guggenb�hl, Barbara; Berthoud, H�l�ne
Fibrin(ogen)olytic activity of bumblebee venom serine protease
Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: > Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. > Bt-VSP activates prothrombin. > Bt-VSP directly degrades fibrinogen into fibrin degradation products. > Bt-VSP is a hemostatically active protein that is a potent clinical agent.
Qiu Yuling [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Joint Laboratory between Dong-A University and Shenyang Pharmaceutical University, Shenyang Pharmaceutical University, Shenyang (China); Choo, Young Moo [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Yoon, Hyung Joo [Department of Agricultural Biology, National Academy of Agricultural Science, Suwon (Korea, Republic of); Jia Jingming; Cui Zheng; Wang Dong [Joint Laboratory between Dong-A University and Shenyang Pharmaceutical University, Shenyang Pharmaceutical University, Shenyang (China); Kim, Doh Hoon [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Joint Laboratory between Dong-A University and Shenyang Pharmaceutical University, Shenyang Pharmaceutical University, Shenyang (China); Sohn, Hung Dae [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Jin, Byung Rae, E-mail: firstname.lastname@example.com [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Joint Laboratory between Dong-A University and Shenyang Pharmaceutical University, Shenyang Pharmaceutical University, Shenyang (China)
D-Serine metabolism in C6 glioma cells: Involvement of alanine-serine-cysteine transporter (ASCT2) and serine racemase (SRR) but not D-amino acid oxidase (DAO)
D-serine is an endogenous N-methyl-D-aspartate (NMDA) receptor coagonist. It is synthesized from L-serine by serine racemase (SRR), but many aspects of its metabolism remain unclear, especially in the forebrain, which lacks active D-amino acid oxidase (DAO), the major D-serine degradative enzyme. Candidate mechanisms include SRR operating in ?,?-eliminase mode (converting D-serine to pyruvate) and regulation by serine transport, in which the alanine-serine-cysteine transporter ASCT2 is implicated. Here we report studies in C6 glioma cells, which �simulate� the forebrain, in that the cells express SRR and ASCT2 but lack DAO activity. We measured D-serine, ASCT2, SRR, and DAO expression and DAO activity in two situations: after incubation of cells for 48 hr with serine isomers and after increased or decreased SRR expression by transfection and RNA interference, respectively. Incubation with serine enantiomers decreased [3H]D-serine uptake and ASCT2 mRNA and increased SRR immunoreactivity but did not alter DAO immunoreactivity, and DAO activity remained undetectable. SRR overexpression increased D-serine and pyruvate and decreased [3H]D-serine uptake and ASCT2 mRNA but did not affect DAO. SRR knockdown did not alter any of the parameters. Our data suggest that D-serine transport mediated by ASCT2 contributes prominently to D-serine homeostasis when DAO activity is absent. The factors regulating D-serine are important for understanding normal NMDA receptor function and because D-serine, along with DAO and SRR, is implicated in the pathogenesis and treatment of schizophrenia. � 2010 Wiley-Liss, Inc. PMID:20091774
Sikka, Pilleriin; Walker, Rosie; Cockayne, Rebecca; Wood, Matthew JA; Harrison, Paul J; Burnet, Philip WJ
- Standard - PS 3.18-2008 Digital Imaging and Communications in Medicine (DICOM) Part 18: Web and Pan American Copyright Conventions. #12;PS 3.18-2008 Page 2 - Standard - NOTICE AND DISCLAIMER that there is unanimous agreement among every person participating in the development of this document. NEMA standards
Rumolo, Giovanni
Console Hacking 2010 PS3 Epic Fail
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Touretzky, David S.
Mycobacterium tuberculosis Serine/Threonine Protein Kinases
The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354
D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...
Characterization of aL-serine dehydratase activity from Streptococcus faecalis
Characterization of aL-serine dehydratase activity from Streptococcus faecalis Le Lait, 191111 Streptococcus faecalis sp. produces pyruvate and ammonia from L-serine via a specifie L-serine dehydratase. The enzymatic activity is stimulated by Fe2 + ions. Key words: Streptococcus faecalis - L-serine dehydratase
Paris-Sud XI, Université de
Inhibition of serine proteases by peptidyl fluoromethyl ketones
Peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases ..cap alpha..-chymotrypsin and porcine pancreatic elastase were synthesized. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the ..gamma..-OH group of the active site serine to form a stable hemiacetal. ¹⁹F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF�¹ and Ac-ambo-Phe-CF� clearly
Barbara Imperiali; Robert H. Abeles
Amino Acids in Schizophrenia � Glycine, Serine and Arginine
\\u000a In recent years, there has been increased interest in the possible role of amino acids in the etiology and pharmacotherapy\\u000a of schizophrenia. Much of this research has focused on glutamate and ?-aminobutyric acid (GABA), and these are the subjects\\u000a of other chapters in this book. However, there have also been interesting findings reported with glycine, serine (particularly\\u000a D-serine) and arginine,
Glen B. Baker; Jaime E. C. Hallak; Alexandria F. Dilullo; Lisa Burback; Serdar M. Dursun
Complement-related serine proteases in tunicates and vertebrates
Serum mannose-binding lectin binds to pathogens in association with a serine protease termed MASP, and in this form, plays a crucial role in innate immunity by activating complement in a manner similar to activation via the classical pathway. MASP, C1r and C1s belong to the same family of serine proteases. In addition to its presence in advanced species, MASP also
Misao Matsushita; Yuichi Endo; Masaru Nonaka; Teizo Fujita
Astrocytes release D-serine by a large vesicle.
Long-term potentiation (LTP) of synaptic transmission in the CA1 region of the hippocampus depends on the activation of N-methyl-D-aspartate receptors (NMDARs), which can be regulated by Ca�?-dependent release of D-serine from astrocytes. The detailed mechanism underlying astrocytic d-serine release is still unknown. In hippocampal slices prepared from Sprague-Dawley rats, we found that clamping astrocytic [Ca�?] at 100-150 nM or puffing artificial cerebrospinal fluid (ACSF) into the extracellular space (weak mechanical stimulation) enhanced the synaptic activation of NMDARs. The enhancement was blocked by the NMDAR glycine site antagonist 5,7-dichlorokynurenic acid, glycine saturation, and infusion of astrocytes with D-amino acid oxidase and the serine racemase inhibitor L-erythro-3-hydroxyaspartate, suggesting the involvement of astrocytic D-serine release. Intracellular 100-150 nM [Ca�?] or puffing ACSF stimulated astrocytes to generate D-serine-containing large vesicles (1-3 ?m), exocytotic fusion of which released D-serine. The formation of astrocytic large vesicles involved the intracellular fusion of small vesicles and/or other organelles. Spontaneous fusion of large vesicles occurred occasionally in astrocytes at rest, contributing to baseline D-serine levels, which increased the rising slope of NMDAR post-burst potentiation (PBP) without altering the PBP peak amplitude. Thus, under physiological conditions, astrocytic D-serine release by large vesicles facilitated weak theta-burst (TBS consisting of five bursts), but not strong TBS (TBS consisting of 10 bursts) stimulation-induced LTP. PMID:23485803
Kang, N; Peng, H; Yu, Y; Stanton, P K; Guilarte, T R; Kang, J
Sequence and phylogenetic analysis of viper venom serine proteases
Snakebites are a major neglected tropical disease responsible for as many as 95000 deaths every year worldwide. Viper venom serine proteases disrupt haemostasis of prey and victims by affecting various stages of the blood coagulation system. A better understanding of their sequence, structure, function and phylogenetic relationships will improve the knowledge on the pathological conditions and aid in the development of novel therapeutics for treating snakebites. A large dataset for all available viper venom serine proteases was developed and analysed to study various features of these enzymes. Despite the large number of venom serine protease sequences available, only a small proportion of these have been functionally characterised. Although, they share some of the common features such as a C-terminal extension, GWG motif and disulphide linkages, they vary widely between each other in features such as isoelectric points, potential N-glycosylation sites and functional characteristics. Some of the serine proteases contain substitutions for one or more of the critical residues in catalytic triad or primary specificity pockets. Phylogenetic analysis clustered all the sequences in three major groups. The sequences with substitutions in catalytic triad or specificity pocket clustered together in separate groups. Our study provides the most complete information on viper venom serine proteases to date and improves the current knowledge on the sequence, structure, function and phylogenetic relationships of these enzymes. This collective analysis of venom serine proteases will help in understanding the complexity of envenomation and potential therapeutic avenues. PMID:23055627
Vaiyapuri, Sakthivel; Thiyagarajan, Nethaji; Hutchinson, E Gail; Gibbins, Jonathan M
A Serine Protease Isolated from the Bristles of the Amazonic Caterpillar, Premolis semirufa, Is a Potent Complement System Activator
Background The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called �Pararamose�, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa�s bristles extract could interfere with the human complement system. Results The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. Conclusion These data show that a serine protease present in the Premolis semirufa�s bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation. PMID:25760458
Villas Boas, Isadora Maria; Pidde-Queiroz, Giselle; Magnoli, Fabio Carlos; Gon�alves-de-Andrade, Rute M.; van den Berg, Carmen W.; Tambourgi, Denise V.
Serine and Threonine Catabolism in Saccharomyces cerevisiae: The CHAl Polypeptide Is Homologous With Other Serine and Threonine Dehydratases
The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHAl gene by complementation of a mutant, chal, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHAl region encompassing an open reading frame of
Claus Bornaes; Jens G. L. Petersen; Steen Holmberg
Processing of metals with ps-laser pulses in the range between 10ps and 100ps
NASA Astrophysics Data System (ADS)
The potential of pulsed laser system in the range of 10ps to 100ps pulse duration for material processing has been further investigated. In detail the dependency of the volume ablation rate, penetration depth and threshold fluence on the pulse duration and number of pulses applied to the material will be discussed. The experimental results show that in the case of copper and steel, better results in quality and efficiency of the ablated material are achieved with shorter pulse durations.
Schmid, Marc; Neuenschwander, Beat; Romano, Valerio; Jaeggi, Beat; Hunziker, Urs W.
D-Serine Production, Degradation, and Transport in ALS: Critical Role of Methodology
In mammalian systems, D-serine is perhaps the most biologically active D-amino acid described to date. D-serine is a coagonist at the NMDA-receptor, and receptor activation is dependent on D-serine binding. Because D-serine binding dramatically increases receptor affinity for glutamate, it can produce excitotoxicity without any change in glutamate per se. D-serine is twofold higher in the spinal cords of mSOD1 (G93A) ALS mice, and the deletion of serine racemase (SR), the enzyme that produces D-serine, results in an earlier onset of symptoms, but with a much slower rate of disease progression. Localization studies within the brain suggest that mSOD1 and subsequent glial activation could contribute to the alterations in SR and D-serine seen in ALS. By also degrading both D-serine and L-serine, SR appears to be a prime bidirectional regulator of free serine levels in vivo. Therefore, accurate and reproducible measurements of D-serine are critical to understanding its regulation by SR. Several methods for measuring D-serine have been employed, and significant issues related to validation and standardization remain unresolved. Further insights into the intracellular transport and tissue-specific compartmentalization of D-serine within the CNS will aid in the understanding of the role of D-serine in the pathogenesis of ALS. PMID:23029613
Crow, John P.; Marecki, John C.; Thompson, Misty
Endothelin-1 stimulates catalase activity through the PKC?-mediated phosphorylation of serine 167.
Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H2O2) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide ?V1.1, this phosphorylation was shown to be protein kinase C? (PKC?) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H2O2 compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKC? was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity. PMID:24211614
Rafikov, Ruslan; Kumar, Sanjiv; Aggarwal, Saurabh; Hou, Yali; Kangath, Archana; Pardo, Daniel; Fineman, Jeffrey R; Black, Stephen M
UPS study of NiPS 3 and FePS 3 crystals using synchrotron radiation
NASA Astrophysics Data System (ADS)
Ultraviolet photoemission spectroscopy (UPS) has been applied to the investigation of the valence band structures of nickel and iron trisulfo-phosphates (NiPS 3 and FePS 3) using synchrotron radiation. The careful observation of UPS spectra gives the accurate binding energy associated with each spectral features. The UPS spectral structures of NiPS 3 and FePS 3 change their intensities strongly with varying incident photon energy. The constant initial state spectroscopy, in conjunction with the photon energy dependence of the UPS spectra, provides the assignment of every UPS spectral feature. These results reveal that the electronic structures of NiPS 3 and FePS 3 differ from each other, suggesting an explanation for the difference in the electric properties of these compounds.
Miyazaki, T.; Fujimoto, H.; Ichimura, K.; Lee, K. P.; Hasegawa, S.; Inokuchi, H.
Phosphorylation alters backbone conformational preferences of serine and threonine peptides.
Despite the notion that a control of protein function by phosphorylation works mainly by inducing its conformational changes, the phosphorylation effects on even small peptide conformation have not been fully understood yet. To study its possible effects on serine and threonine peptide conformations, we recently carried out pH- and temperature-dependent circular dichroism (CD) as well as (1)H NMR studies of the phosphorylated serine and threonine peptides and compared them with their unphosphorylated analogs. In the present article, by performing the self-consistent singular value decomposition analysis of the temperature-dependent CD spectra and by analyzing the (3)J(H(N),H(?)) coupling constants extracted from the NMR spectra, the populations of the polyproline II (PPII) and ?-strand conformers of the phosphorylated Ser and Thr peptides are determined. As temperature is increased, the ?-strand populations of both phosphorylated serine and threonine peptides increase. However, the dependences of PPII/?-strand population ratio on pH are different for these two cases. The phosphorylation of the serine peptide enhances the PPII propensity, whereas that of the threonine peptide has the opposite effect. This suggests that the serine and threonine phosphorylations can alter the backbone conformational propensity via direct but selective intramolecular hydrogen-bonding interactions with the peptide N--H groups. This clearly indicates that the phosphoryl group actively participates in modulating the peptide backbone conformations. PMID:21989936
Kim, Su-Yeon; Jung, Youngae; Hwang, Geum-Sook; Han, Hogyu; Cho, Minhaeng
Microstructure and nanomechanical properties of enamel remineralized with asparagine-serine-serine peptide.
A highly biocompatible peptide, triplet repeats of asparagine-serine-serine (3 NSS) was designed to regulate mineral deposition from aqueous ions in saliva for the reconstruction of enamel lesions. Healthy human enamel was sectioned and acid demineralized to create lesions, then exposed to the 3 NSS peptide solution, and finally immersed in artificial saliva for 24h. The surface morphology and roughness were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. X-ray diffraction (XRD) was used to identify the phases and crystallinity of the deposited minerals observed on the enamel surface. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to quantitatively analyze the mineral variation by calculating the relative integrated-area of characteristic bands. Nanohardness and elastic modulus measured by nanoindentation at various treatment stages were utilized to evaluate the degree of recovery. Biomimetic effects were accessed according to the degree of nanohardness recovery and the amount of hydroxyapatite deposition. The charged segments in the 3 NSS peptide greatly attracted aqueous ions from artificial saliva to form hydroxyapatite crystals to fill enamel caries, in particular the interrod areas, resulting in a slight reduction in overall surface roughness. Additionally, the deposited hydroxyapatites were of a small crystalline size in the presence of the 3 NSS peptide, which effectively restrained the plastic deformations and thus resulted in greater improvements in nanohardness and elastic modulus. The degree of nanohardness recovery was 5 times greater for remineralized enamel samples treated with the 3 NSS peptide compared to samples without peptide treatment. PMID:25427512
Chung, Hsiu-Ying; Li, Cheng Che
Enzymatic methods for the determination of L-serine concentration and L-(/sup 14/C)serine specific radioactivity in blood plasma
Methods are described for the use of L-serine dehydratase purified from Clostridium acidiurici for the determination of L-serine concentrations and L(/sup 14/C)serine specific radioactivity in sheep plasma. A spectrophotometric assay using this enzyme accurately measured the concentration of L-serine in standard solutions and in a commerically available mixture of amino acids and related compounds. This assay was shown to be suitable for measurement of plasma L-serine concentrations in excess of 30 ..mu..M. The reverse isotope dilution method was used for plasma L-(/sup 14/C)serine specific radioactivity measurements. Carrier L-serine was added to plasma and separated from neutral and anionic compounds using ion-exchange chromatography. The L-serine was then converted to pyruvate with L-serine dehydratase and this was purified as the phenylhydrazone derivative. After recrystallization, drying and weighing, the derivative was assayed for radioactivity. The accuracy of this method was verified by adding L-(U-/sup 14/C)serine to plasma and comparing the experimentally determined L-(/sup 14/C)serine specific radioactivity with the calculated value. The method yielded a value which was 98.6 +/- 0.8% of this calculated value.
Cassady, A.I.; Reilly, P.E.B.
Evaluation of the effect of staphylococcal serine proteinase on phagocytosis.
Correlations between values of phagocytosis index and values of concentrations of staphylococcal serine proteinase were analysed. Preincubation of granulocytes with the proteinase stimulated phagocytosis of three bacterial strains: S. saprophyticus, S. aureus VS and S. aureus Smith diffuse. Significant correlations were also observed for S. saprophyticus strain in phagocytosis performed with or without bovine serum. Specific rabbit IgG anti-serine proteinase effected the increase of phagocytosis index only in the case of S. aureus Smith diffuse. Summary statistical analysis for all experimental conditions exhibits significant correlations also for S. aureus VS strain. No significant correlations were noted for the three remaining strains taken from patients. PMID:3508044
Miedzobrodzki, J; Tadeusiewicz, R; Porwit-B�br, Z
Serine/Threonine phosphatases: classification, roles and pharmacological regulation.
Phosphatases are important enzymes in a variety of biochemical pathways in different cells which they catalyze opposing reactions of phosphorylation and dephosphorylation, which may modulate the function of crucial signaling proteins in different cells. This is an important mechanism in the regulation of intracellular signal transduction pathways in many cells. Phosphatases play a key role in regulating signal transduction. It is known that phosphatases are specific for cleavage of either serine-threonine or tyrosine phosphate groups. To date, numerous compounds have been identified. This paper reviews the classification, roles and pharmacological of protein serine/threonine phosphates. PMID:25572726
Bastan, R; Eskandari, N; Sabzghabaee, A M; Manian, M
PS colloidal particles stabilized by graphene oxide.
Exfoliated graphene oxide (GO) sheets with hydrophilic functional groups on the surface were prepared by the oxidation of graphite. Because of the hydrophilic groups on the sheets and the hydrophobic carbon surface, GO sheets were located at the oil-water interface and could be used as a stabilizer in Pickering emulsions. After the Pickering emulsion polymerization of styrene, PS colloidal particles with GO sheets on the surface were prepared. The size of the GO sheets exerts an important influence on the preparation of PS colloidal particles. Small GO sheets located at the liquid-liquid interface and GO-stabilized PS colloidal particles were prepared; however, for large GO sheets, smaller PS colloidal particles prepared on the GO surface were observed. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS) were used to characterize the structure and morphology of the colloidal particles. TEM, SEM, and XPS results all suggest the successful preparation of GO-stabilized PS colloidal particles. PMID:21192694
Song, Xiaohui; Yang, Yongfang; Liu, Jinchuan; Zhao, Hanying
Proteolytic processing of presenilin-1 (PS1) is not associated with Alzheimer's disease with or without PS1 mutations
Cerebral presenilin-1 protein (PS-1) is normally composed of the amino-terminal fragment (NTF) with Mr 28 kDa and the carboxy-terminal fragment (CTF) with 18 kDa. We analyzed human PS-1 in brains with early-onset familial Alzheimer's disease (FAD) with and without PS-1 mutations to study whether mutated PS-1 was abnormally metabolized. Cerebral PS-1 were found to be cleaved into two fragments of
Masayasu Okochi; Kazuhiko Ishii; Mihoko Usami; Naruhiko Sahara; Fuyuki Kametani; Kikuko Tanaka; Peter E Fraser; Masaki Ikeda; Ann M Saunders; Lydia Hendriks; Shin-Ichi Shoji; Linda E Nee; Jean-Jacques Martin; Christine Van Broeckhoven; Peter H St. George-Hyslop; Allen D Roses; Hiroshi Mori
Mutagenesis of serine 40 of tyrosine hydroxylase: implications for the enzyme's regulatory mechanism
dissociation rate, thus lowering the catecholamine binding affinity. To understand more about how phosphorylation exerts this effect, amino acid substitutions were made at serine 40 of TYH. Tyrosine hydroxylase having serine 40 replaced by glutamate...
McCulloch, Ruth Irene
Dynamics simulation of the interaction between serine and water
NASA Astrophysics Data System (ADS)
Using the first principles density functional theory (DFT), we simulated the neutron scattering spectra of the hydration dynamics of serine. Experimental data analyses have shown that dissociative H2O molecules were more likely to form hydrogen bonds (H-bonds) with an -OH group in monohydrated serine and easily shift to a -NH_3 ^ + group at a higher hydration level [P. Zhang, Y. Zhang, S. H. Han, Q. W. Yan, R. C. Ford, and J. C. Li, J. Phys. Chem. A 110, 5000 (2006), 10.1021/jp0569741]. We set the 1:1 ratio hydrated compounds at the two positions and found that the H2O could be optimized to form H-bonds with -OH and -NH3+ separately. When the simulated phonon signals of the -OH�H2O and -NH3+�H2O combinations were summed on a 3:1 scale, the calculating spectra were in good agreement with the experimental results, especially for the peak at 423 cm-1 of the -OH�H2O combination and the peak at 367 cm-1 of the -NH3+�H2O combination, which mutually complemented the real spectrum. We confirm that H2O may break the intermolecular H-bonds of the interlaced binding -OH to form a new structure, and that with the skeleton deformation of serine, H2O forms stronger H-bonds more often with the -NH3+ side indicating the flexible dynamic mechanism of the serine hydration process.
Liu, Yang; Zhang, Peng; Lu, Ying-Bo; Han, Sheng-Hao; Yu, Hui
Activity of a newly identified serine protease in CNS demyelination
Summary We have identified a novel serine protease, myelen- cephalon-specific protease (MSP), which is preferen- tially expressed in the adult CNS, and therein, is abundant in both neurones and oligodendroglia. To determine the potential activity of MSP in CNS demye- lination, we examined its expression in multiple sclerosis lesions and in two animal models of multiple sclerosis: Theiler's murine encephalomyelitis
I. A. Scarisbrick; S. I. Blaber; C. F. Lucchinetti; C. P. Genain; M. Blaber; M. Rodriguez
Periportal expression of the serine dehydratase gene in rat liver
Summary��The mRNA for rat liver serine dehydratase, a gluconeogenic enzyme, exhibits a circadian rhythm with a maximum at the onset of darkness marking the end of the fasting period and a minimum at the onset of light that marks the end of the feeding period, when rats have free access to food and water. In situ hybridization with an antisense
Hirofumi Ogawa; Seiichi Kawamata
Evidence for a dimeric structure of rat liver serine dehydratase
Rat liver serine dehydratase (SDH) is known to be involved in gluconeogenesis. It has long been believed to be a dimeric protein with the subunit molecular weight (Mr) of 34,000. Recently, sheep liver SDH was reported to be a monomer with a Mr of 38,000. The native Mr of rat SDH was only determined by the ultracentrifugation method more than
Hirofumi Ogawa; Tomoharu Gomi; Fusao Takusagawa; Toru Masuda; Takuya Goto; Takanobu Kan; Num-Ho Huh
Localization of Serine Racemase and Its Role in the Skin
D-Serine is an endogenous coagonist of the N-methyl-D-aspartate (NMDA)�type glutamate receptor in the central nervous system and its synthesis is catalyzed by serine racemase (SR). Recently, the NMDA receptor has been found to be expressed in keratinocytes (KCs) of the skin and involved in the regulation of KC growth and differentiation. However, the localization and role of SR in the skin remain unknown. Here, using SR-knockout (SR-KO) mice as the control, we demonstrated the localization of the SR protein in the granular and cornified layer of the epidermis of wild-type (WT) mice and its appearance in confluent WT KCs. We also demonstrated the existence of a mechanism for conversion of L-serine to D-serine in epidermal KCs. Furthermore, we found increased expression levels of genes involved in the differentiation of epidermal KCs in adult SR-KO mice, and alterations in the barrier function and ultrastructure of the epidermis in postnatal day 5 SR-KO mice. Our findings suggest that SR in the skin epidermis is involved in the differentiation of epidermal KCs and the formation of the skin barrier. PMID:24441099
Inoue, Ran; Yoshihisa, Yoko; Tojo, Yosuke; Okamura, Chieko; Yoshida, Yuzo; Kishimoto, Jiro; Luan, Xinghua; Watanabe, Masahiko; Mizuguchi, Mineyuki; Nabeshima, Yuko; Hamase, Kenji; Matsunaga, Kenji; Shimizu, Tadamichi; Mori, Hisashi
Sequence Determinants of Function and Evolution in Serine Proteases
Serine proteases of the chymotrypsin family have maintained a common fold over an evolutionary span of more than one billion years. Notwithstanding modest changes in sequence, this class of enzymes has developed a wide variety of substrate specificities and important biological functions such as fibrinolysis, blood coagulation, and complement activation. Recently it has become apparent that the protease domain, especially
Maxwell M Krem; Thierry Rose; Enrico Di Cera
The Hunger Games: p53 regulates metabolism upon serine starvation.
Cancer cells reprogram their metabolism to support a high proliferative rate. A new study shows that, upon serine starvation, the tumor suppressor p53 activates p21 to shift metabolic flux from purine biosynthesis to glutathione production, which enhances cellular proliferation and viability by combating ROS (Maddocks et�al., 2013). PMID:23395165
Tavana, Omid; Gu, Wei
Purification and Characterization of Serine Racemase from a Hyperthermophilic Archaeon, Pyrobaculum islandicum
Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100�C. A pyridoxal 5-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr
Masato Ohnishi; Makoto Saito; Sadao Wakabayashi; Morio Ishizuka; Katsushi Nishimura; Yoko Nagata; Sabu Kasai
Identification and structural analysis of four serine proteases in a monotreme, the platypus, Ornithorhynchus anatinus
To study the emergence of the major subfamilies of serine proteases during vertebrate evolution, we present here the primary structure of four serine proteases expressed in the spleen of a monotreme, the platypus, Ornithorhynchus anatinus. Partial cDNA clones for four serine proteases were isolated by a PCR-based strategy. This strategy is based on the high level of sequence identity between
Maryam Poorafshar; Maria Aveskogh; Barry Munday; Lars Hellman
Beyond metric gravity: Progress on PS-200
The reconciliation of quantum mechanics and gravity on varying distance scales requires changes to General Relativity that may be testable implications. We briefly review the status of tests with matter of the inverse square law and the principle of equivalence, then report on progress on the drift-tube measurement section of PS- 200, the experiment to measure the gravitational acceleration of antiprotons.
Goldman, T.; Brown, R.E.; Camp, J.B.; Darling, T.; Dyer, P.; Holzscheiter, M.H.; Hughes, R.J.; Jarmie, N.; King, N.S.P.; Lizon, D.C.; Nieto, M.M.; Schauer, M.M.M.; Schecker, J.A. [Los Alamos National Lab., NM (United States); Cornford, S.; Hosea, K.; Kenefick, R.A. [Texas A and M Univ., College Station, TX (United States); Hoibraaten, S.; Midzor, M.M.; Parry, S.P.; Ristenen, R.A. [Colorado Univ., Boulder, CO (United States); Witteborn, F.C. [National Aeronautics and Space Administration, Moffett Field, CA (United States). Ames Research Center; Rochet, J. [European Organization for Nuclear Research, Geneva (Switzerland)
Beyond metric gravity: Progress on PS-200
The reconciliation of quantum mechanics and gravity on varying distance scales requires changes to General Relativity that may be testable implications. We briefly review the status of tests with matter of the inverse square law and the principle of equivalence, then report on progress on the drift-tube measurement section of PS- 200, the experiment to measure the gravitational acceleration of antiprotons.
Goldman, T.; Brown, R.E.; Camp, J.B.; Darling, T.; Dyer, P.; Holzscheiter, M.H.; Hughes, R.J.; Jarmie, N.; King, N.S.P.; Lizon, D.C.; Nieto, M.M.; Schauer, M.M.M.; Schecker, J.A. (Los Alamos National Lab., NM (United States)); Cornford, S.; Hosea, K.; Kenefick, R.A. (Texas A and M Univ., College Station, TX (United States)); Hoibraaten, S.; Midzor, M.M.; Parry, S.P.; Ristenen, R.A. (Colorado Univ., Boulder, CO (U
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Rumolo, Giovanni
John Adams parle de la pr�histoire du P.S. avec pr�sentation des dias. Le DG B.Gregory prend la parole. Les organisateurs pr�sentent sous la direction du "Prof.Ocktette"(?) un sketch tr�s humoristique (p.e.existence de Quark etc.....)
The 4 Ps as a Guiding Perspective
ERIC Educational Resources Information Center
A 4 Ps perspective addresses immediate needs: to help institutions gain traction in their retention strategies by framing and reframing the challenges and the possible responses, by challenging some of the traditional mental models about retention that can distract or dilute those strategies, and by offering focus and coherence to institutional�
Kalsbeek, David H.
Memantine improves spatial learning and memory impairments by regulating NGF signaling in APP/PS1 transgenic mice.
Memantine (MEM) is used for improving the cognitive impairments of the patients suffering from Alzheimer's disease (AD) by multiple neuroprotective mechanisms. However, it is still not clear whether nerve growth factor (NGF) signaling is involved in the mechanisms of MEM. The present study investigated the neuroprotective effects of MEM treatment on the cognitive performance and amyloidosis in APP/PS1 transgenic mice, and disclosed the NGF-related mechanism of MEM. We found that MEM treatment improved the cognitive performance by decreasing the escape latency and path length in the navigation test, by shortening the duration in target quadrant and reducing the frequency to pass through the target in probe trial, and by prolonging the latency and decreasing the frequencies of entering the dark compartment in passive avoidance test. The over-expressions of A?(1-42) and amyloid precursor protein (APP) were also decreased in the brains of APP/PS1 mice. Interestingly, MEM treatment improved the decreased NGF levels in APP/PS1 mice. Furthermore, NGF/TrkA signaling was activated by increasing the phosphorylation levels of tyrosine kinase (TrkA), proto-oncogene serine/threonine-protein kinase, Raf1 (c-Raf), extracellular regulated protein kinases (ERK)1/2 and cAMP-response element binding protein (CREB) after MEM treatment. Simultaneously, MEM also inhibited NGF/p75(NTR) signaling via decreasing the cleavage substrate of p75(NTR), increasing the JNK2 phosphorylation and decreasing the levels of p53 and cleaved-caspase 3. Therefore, the dual-regulation on NGF signaling was attributed to the improvements of cognitive deficits and A? depositions in APP/PS1 mice. In conclusion, MEM treatment activated the NGF/TrkA signaling, and inhibited the p75(NTR) signaling in APP/PS1 mice to ameliorate the behavioral deficits and amyloidosis, indicating that NGF signaling was a new potential target of MEM treatment for AD therapy. PMID:24846616
Liu, M Y; Wang, S; Yao, W F; Zhang, Z J; Zhong, X; Sha, L; He, M; Zheng, Z H; Wei, M J
Contrasting antiferromagnetic order between FePS3 and MnPS3
NASA Astrophysics Data System (ADS)
Transition-metal thiophosphates, MPS3 (M = Fe, Mn, etc.), make up a class of antiferromagnetic materials with quasi-two-dimensional magnetic behaviour. The metal atoms occupy a honeycomb lattice. In MnPS3, the first-neighbour interaction is 400 times stronger than the exchange between the planes and the Mn moments show little anisotropy. The antiferromagnetic order is not well established perpendicular to the planes. Here we present the first powder neutron diffraction patterns of FePS3. The absence of trailing edges on the magnetic Bragg peaks, such as have been observed in MnPS3, indicates that the magnetic order is three-dimensional in FePS3.
Rule, K. C.; Kennedy, S. J.; Goossens, D. J.; Mulders, A. M.; Hicks, T. J.
Antinociceptive Effect of Rat D-Serine Racemase Inhibitors, L-Serine-O-Sulfate, and L-Erythro-3-Hydroxyaspartate in an Arthritic Pain Model
N-methyl-D-aspartic acid receptor (NMDAr) activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5?-phosphate-dependent serine racemase (SR). D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS) and L-erythro-3-hydroxyaspartate (LEHA), among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100??g/10??L) and LEHA (100??g/10??L) in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300??g/10??L of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain. PMID:22536130
Laurido, Claudio; Hern�ndez, Alejandro; Pelissier, Teresa; Constandil, Luis
ZnO/PS/p-Si heterojunction properties
NASA Astrophysics Data System (ADS)
In this paper porous silicon (PS) has been prepared by electrochemical etching technique and then ZnO thin film deposition on PS by spray pyrolysis method, the study of AFM show improve the structural stability of the PS substrate with crystalline growth of ZnO thin film. PL spectra explained a blue-shifting in PS layer come from oxidation the surface of PS after coating with ZnO film, Raman measurement show quantum confinement in PS layers with decreasing in variation mode of ZnO film, and the J-V characteristic show increasing in resistivity of Al/ZnO/PS/c-Si/Al due to increasing in depletion layer junction compere with PS layer.
Muhsin Nayef, Uday; Waleed Muayad, Mohammed; Amer Khalaf, Haider
Construction of Escherichia coli strains producing L-serine from glucose.
L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 184.108.40.206) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt. PMID:22547037
Li, Yu; Chen, Gu-Kui; Tong, Xin-Wei; Zhang, Hui-Tu; Liu, Xiao-Guang; Liu, Yi-Han; Lu, Fu-Ping
Generation of iPS Cells from Granulosa Cells.
Various types of somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells. Somatic stem cells may generate iPS cells more efficiently than do differentiated cells. We show that granulosa cells exhibit characteristic of somatic stem cells and can be reprogrammed to iPS cells more efficiently or with few factors. Here, we describe generation of mouse and pig iPS cells from granulosa cells with high efficiency. PMID:25403467
Mao, Jian; Liu, Lin
Membrane-anchored serine proteases in health and disease.
Serine proteases of the trypsin-like family have long been recognized to be critical effectors of biological processes as diverse as digestion, blood coagulation, fibrinolysis, and immunity. In recent years, a subgroup of these enzymes has been identified that are anchored directly to plasma membranes, either by a carboxy-terminal transmembrane domain (Type I), an amino-terminal transmembrane domain with a cytoplasmic extension (Type II or TTSP), or through a glycosylphosphatidylinositol (GPI) linkage. Recent biochemical, cellular, and in vivo analyses have now established that membrane-anchored serine proteases are key pericellular contributors to processes vital for development and the maintenance of homeostasis. This chapter reviews our current knowledge of the biological and physiological functions of these proteases, their molecular substrates, and their contributions to disease. PMID:21238933
Antalis, Toni M; Bugge, Thomas H; Wu, Qingyu
Membrane-anchored serine proteases in health and disease
Serine proteases of the trypsin-like family have long been recognized to be critical effectors of biological processes as diverse as digestion, blood coagulation, fibrinolysis, and immunity. In recent years, a subgroup of these enzymes has been identified that are anchored directly to plasma membranes, either by a carboxy-terminal transmembrane domain (Type I), an amino-terminal transmembrane domain with a cytoplasmic extension (Type II or TTSP), or through a glycosyl-phosphatidylinositol (GPI) linkage. Recent biochemical, cellular, and in vivo analyses have now established that membrane-anchored serine proteases are key pericellular contributors to processes vital for development and the maintenance of homeostasis. This chapter will review our current knowledge of the biological and physiological functions of these proteases, their molecular substrates, and their contributions to disease. PMID:21238933
Bugge, Thomas; Wu, Qingyu
Why is D-serine nephrotoxic and alpha-aminoisobutyric acid protective?
D-Serine selectively causes necrosis of S(3) segments of proximal tubules in rats. This leads to aminoaciduria and glucosuria. Coinjection of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) prevents the tubulopathy. D-serine is selectively reabsorbed in S(3), thereby gaining access to peroxisomal D-amino acid oxidase (D-AAO). D-AAO-mediated metabolism produces reactive oxygen species. We determined the fractional excretion of amino acids and glucose in rats after intraperitoneal injection of d-serine alone or together with reduced glutathione (GSH) or AIB. Both compounds prevented the hyperaminoaciduria. We measured GSH concentrations in renal tissue before (control) and after D-serine injection and found that GSH levels decreased to approximately 30% of control. This decrease was prevented when equimolar GSH was coinjected with D-serine. To find out why AIB protected the tubule from D-serine toxicity, we microinfused D-[(14)C]serine or [(14)C]AIB (0.36 mmol/l) together with [(3)H]inulin in late proximal tubules in vivo and measured the radioactivity in the final urine. Fractional reabsorption of D-[(14)C]serine and [(14)C]AIB amounted to 55 and 70%, respectively, and 80 mmol/l of AIB or D-serine mutually prevented reabsorption to a great extent. D-AAO activity measured in vitro (using D-serine as substrate) was not influenced by a 10-fold higher AIB concentration. We conclude from these results that 1) D-AAO-mediated d-serine metabolism lowers renal GSH concentrations and thereby provokes tubular damage because reduction of reactive oxygen species by GSH is diminished and 2) AIB prevents d-serine-induced tubulopathy by inhibition of D-serine uptake in S(3) segments rather than by interfering with intracellular D-AAO-mediated D-serine metabolism. PMID:17429029
Krug, Alexander W; V�lker, Katharina; Dantzler, William H; Silbernagl, Stefan
Characterization of serine proteases in the monogenean Neobenedenia girellae
Proteases of Neobenedenia girellae were examined by using zymographic analysis. Zymography of N. girellae homogenate revealed proteases at approximately 88, 107, 149 and 167kDa for the adult worms and approximately 147 and 166kDa for the oncomiracidia, respectively. The enzyme activities were inhibited by Pefabloc SC but were not inhibited by E-64 and Pepstatin A, indicating that these proteases were serine
Noritaka Hirazawa; Naoko Umeda; Akimasa Hatanaka; Akashi Kuroda
Serine Phosphorylation, Insulin Resistance, and the Regulation of Androgen Synthesis
Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenemia and disordered gonadotropin secretion, often associated\\u000a with insulin resistance. It is likely that PCOS is a group of distinct diseases with similar clinical phenotypes but different\\u000a pathophysiological mechanisms, rather than being one disease caused by a single molecular defect. The serine phosphorylation hypothesis can potentially explain two major features of the syndrome:
Andrew A. Bremer; Walter L. Miller
Granules of cytolytic T-lymphocytes contain two serine esterases.
Cytoplasmic granules from cytolytic T-lymphocytes (CTL) contain two proteins which react with the serine esterase-specific affinity label diisopropylfluorophosphate (DFP). One of these is a trypsin-like esterase which consists of two disulfide-linked 35-kd subunits. The other consists of a single 29-kd chain. Both molecules are induced concomitantly with cytolytic activity and perforin activity in a CTL-derived T-cell hybrid. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:3488903
Masson, D; Nabholz, M; Estrade, C; Tschopp, J
Site-specific DNA Inversion by Serine Recombinases
Reversible site-specific DNA inversion reactions are widely distributed in bacteria and their viruses. They control a range of biological reactions that most often involve alterations of molecules on the surface of cells or phage. These programmed DNA rearrangements usually occur at a low frequency, thereby preadapting a small subset of the population to a change in environmental conditions, or in the case of phages, an expanded host range. A dedicated recombinase, sometimes with the aid of additional regulatory or DNA architectural proteins, catalyzes the inversion of DNA. RecA or other components of the general recombination-repair machinery are not involved. This chapter discusses site-specific DNA inversion reactions mediated by the serine recombinase family of enzymes and focuses on the extensively studied serine DNA invertases that are stringently controlled by the Fis-bound enhancer regulatory system. The first section summarizes biological features and general properties of inversion reactions by the Fis/enhancer-dependent serine invertases and the recently described serine DNA invertases in Bacteroides. Mechanistic studies of reactions catalyzed by the Hin and Gin invertases are then discussed in more depth, particularly with regards to recent advances in our understanding of the function of the Fis/enhancer regulatory system, the assembly of the active recombination complex (invertasome) containing the Fis/enhancer, and the process of DNA strand exchange by rotation of synapsed subunit pairs within the invertasome. The role of DNA topological forces that function in concert with the Fis/enhancer controlling element in specifying the overwhelming bias for DNA inversion over deletion and intermolecular recombination is emphasized.
Structural Basis for Catalytic Activation of a Serine Recombinase
Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 {angstrom} crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.
Keenholtz, Ross A.; Rowland, Sally-J.; Boocock, Martin R.; Stark, W. Marshall; Rice, Phoebe A. (Glasgow); (UC)
The HARP detector at the CERN PS
HARP is a high-statistics, large solid angle experiment to measure hadron production using proton and pion beams with momenta between 1.5 and 15GeV\\/c impinging on many different solid and liquid targets from low to high Z. The experiment, located in the T9 beam of the CERN PS, took data in 2001 and 2002. For the measurement of momenta of produced
M. G. Catanesi; M. T. Muciaccia; E. Radicioni; S. Simone; R. Edgecock; M. Ellis; S. Robbins; F. J. P. Soler; C. G��ling; M. Mass; S. Bunyatov; A. Chukanov; O. Klimov; I. Krasin; A. Krasnoperov; D. Kustov; B. Popov; V. Serdiouk; V. Tereshchenko; V. Carassiti; E. Di Capua; F. Evangelisti; G. Vidal-Sitjes; A. Artamonov; P. Arce; R. Brocard; G. Decreuse; B. Friend; S. Giani; S. Gilardoni; P. Gorbunov; A. Grant; A. Grossheim; P. Gruber; V. Ivanchenko; J.-C. Legrand; A. Kayis-Topaksu; J. Panman; I. Papadopoulos; J. Pasternak; E. Tcherniaev; I. Tsukerman; R. van der Vlugt; R. Veenhof; C. Wiebusch; P. Zucchelli; A. Blondel; S. Borghi; M. Campanelli; A. Cervera-Villanueva; M. C. Morone; G. Prior; R. Schroeter; I. Kato; U. Gastaldi; G. B. Mills; J. S. Graulich; G. Gr�goire; M. Bonesini; F. Chignoli; F. Ferri; F. Paleari; M. Kirsanov; V. Postoev; A. Bagulya; V. Grichine; N. Polukhina; V. Palladino; L. Coney; D. Schmitz; G. Barr; A. De Santo; C. Pattison; K. Zuber; G. Barichello; F. Bobisut; D. Gibin; A. Guglielmi; M. Laveder; A. Menegolli; M. Mezzetto; A. Pepato; J. Dumarchez; S. Troquereau; F. Vannucci; U. Dore; A. Iaciofano; M. Lobello; F. Marinilli; D. Orestano; D. Panayotov; M. Pasquali; F. Pastore; A. Tonazzo; L. Tortora; C. Booth; C. Buttar; P. Hodgson; L. Howlett; R. Nicholson; M. Bogomilov; K. Burin; M. Chizhov; D. Kolev; P. Petev; I. Rusinov; R. Tsenov; S. Piperov; P. Temnikov; M. Apollonio; P. Chimenti; G. Giannini; G. Santin; J. Burguet-Castell; J. J. G�mez-Cadenas; P. Novella; M. Sorel; A. Tornero
Intrinsic Viscosity Characterization of PS and PMMA
NSDL National Science Digital Library
In this experiment, you will use intrinsic viscosity measurements to determine the molecular weight of polystyrene, PS, or poly(methyl methacrylate), PMMA. After in-class presentation, completion of hands-on laboratory experiment and review of the information provided, you should be able to: � Identify several laboratory methods for molecular weight analysis of polymers. � Confidently discuss the differences between the methods of analysis for polymer molecular weight. � Discuss how polymer solution behavior affects molecular weight measurements.
DeRosa, Rebecca L.
L-Serine Catabolism via an Oxygen-Labile L-Serine Dehydratase Is Essential for Colonization of the Avian Gut by Campylobacter jejuni
Campylobacter jejuni is a microaerophilic, asaccharolytic bacterium. The identity of the carbon and energy sources used by C. jejuni in vivo is unknown, but the genome sequence of strain NCTC11168 indicates the presence of genes for catabolism of a limited range of amino acids, including serine. Specific omission of L-serine from a defined medium containing a mixture of amino acids
Jyoti Velayudhan; Michael A. Jones; Paul A. Barrow; David J. Kelly
Singly differential cross sections with exchange for Ps-fragmentation
Ps ionization in Ps-atom scattering is of fundamental importance. The singly differential cross sections (SDCS) provides more accurate information to test a theory than integrated or total ionization cross section since the averaging over one parameter is not required. We evaluate the SDCS for Ps-ionization with respect to the longitudinal energy distribution of the break-up positron and electron in Ps-H and Ps-He scattering and compare them with the recently available experimental and theoretical data.
Hasi Ray
Androgen Receptor Phosphorylation at Serine 308 and Serine 791 Predicts Enhanced Survival in Castrate Resistant Prostate Cancer Patients
We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone na�ve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR94), 308 (pAR308), 650(pAR650) and 791 (pAR791). No correlations with clinical parameters were observed for pAR94 or pAR650 in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR308 is significantly associated with a longer time to disease specific death (p = 0.011) and high pAR791 expression significantly associated with a longer time to disease recurrence (p = 0.018) in HNPC tumours and longer time to death from disease recurrence (p = 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p = 0.022) and low proliferating tumours (p = 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer. PMID:23945560
McCall, Pamela; Adams, Claire E.; Willder, Jennifer M.; Bennett, Lindsay; Qayyum, Tahir; Orange, Clare; Underwood, Mark A.; Edwards, Joanne
Storage and uptake of d-serine into astrocytic synaptic-like vesicles specify gliotransmission
Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signalling pathways. In particular, astrocytes have recently been shown to release small molecules such as the amino acids l-glutamate and d-serine as �gliotransmitters�, which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca2+-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles including the SNARE synaptobrevin 2 and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. While l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner. PMID:23426669
Martineau, Magalie; Shi, Ting; Puyal, Julien; Knolhoff, Ann M.; Dulong, J�r�me; Gasnier, Bruno; Klingauf, J�rgen; Sweedler, Jonathan V.; Jahn, Reinhard; Mothet, Jean-Pierre
PAN/PS elctrospun fibers for oil spill cleanup
NASA Astrophysics Data System (ADS)
A high-capacity oil sorbent was fabricated by electrospinning using PS/PAN blend. Morphology, contact angle and oil adsorption of PAN/PS fiber and PP nonwoven fabric were studied. It was found that the PAN/PS fiber had a smaller diameter than PP, and the maximum sorption capacities of the PAN/PS sorbent for pump oil, peanut oil, diesel, and gasoline were 194.85, 131.7, 66.75, and 43.38 g/g, which were far higher than those of PP. The sorbent PS/PAN fiber showed a contact angle of water144.32� and diesel oil 0�. The sorption kinetics of PAN/PS and PP sorbent were also investigated. Compared with the commercial PP fabric, the PAN/PS fiber seems to have the ability to be used in oil-spill cleanup application.
Ying, Qiao; Lili, Zhao; Haixiang, Sun; Peng, Li
Comparative Immunohistochemical Analysis of Ochratoxin A Tumourigenesis in Rats and Urinary Tract Carcinoma in Humans; Mechanistic Significance of p-S6 Ribosomal Protein Expression
Ochratoxin A (OTA) is considered to be a possible human urinary tract carcinogen, based largely on a rat model, but no molecular genetic changes in the rat carcinomas have yet been defined. The phosphorylated-S6 ribosomal protein is a marker indicating activity of the mammalian target of rapamycin, which is a serine/threonine kinase with a key role in protein biosynthesis, cell proliferation, transcription, cellular metabolism and apoptosis, while being functionally deregulated in cancer. To assess p-S6 expression we performed immunohistochemistry on formalin-fixed and paraffin-embedded tumours and normal tissues. Marked intensity of p-S6 expression was observed in highly proliferative regions of rat renal carcinomas and a rare angiosarcoma, all of which were attributed to prolonged exposure to dietary OTA. Only very small OTA-generated renal adenomas were negative for p-S6. Examples of rat subcutaneous fibrosarcoma and testicular seminoma, as well as of normal renal tissue, showed no or very weak positive staining. In contrast to the animal model, human renal cell carcinoma, upper urinary tract transitional cell carcinoma from cases of Balkan endemic nephropathy, and a human angiosarcoma were negative for p-S6. The combined findings are reminiscent of constitutive changes in the rat tuberous sclerosis gene complex in the Eker strain correlated with renal neoplasms, Therefore rat renal carcinogenesis caused by OTA does not obviously mimic human urinary tract tumourigenesis. PMID:23105973
Gazinska, Patrycja; Herman, Diana; Gillett, Cheryl; Pinder, Sarah; Mantle, Peter
Association between intrinsic disorder and serine/threonine phosphorylation in Mycobacterium tuberculosis
Serine/threonine phosphorylation is an important mechanism that is involved in the regulation of protein function. In eukaryotes, phosphorylation occurs predominantly in intrinsically disordered regions of proteins. Though serine/threonine phosphorylation and protein disorder are much less prevalent in prokaryotes, some bacteria have high levels of serine/threonine phosphorylation and disorder, including the medically important M. tuberculosis. Here I show that serine/threonine phosphorylation sites in M. tuberculosis are highly enriched in intrinsically disordered regions, indicating similarity in the substrate recognition mechanisms of eukaryotic and M. tuberculosis kinases. Serine/threonine phosphorylation has been linked to the pathogenicity and survival of M. tuberculosis. Thus, a better understanding of how its kinases recognize their substrates could have important implications in understanding and controlling the biology of this deadly pathogen. These results also indicate that the association between serine/threonine phosphorylation and disorder is not a feature restricted to eukaryotes. PMID:25648268
Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes
To investigate the importance of serine proteases in Leishmania amazonensis promastigotes, we analyzed the effects of classical serine protease inhibitors and a Kunitz-type inhibitor, obtained from\\u000a sea anemone Stichodactyla helianthus (ShPI-I), on the viability and morphology of parasites in culture. Classical inhibitors were selected on the basis of their\\u000a ability to inhibit L. amazonensis serine proteases, previously described. The N-tosyl-l-phenylalanine
R. E. Silva-Lopez; J. A. Morgado-D�az; S. Giovanni-De-Simone
New L-serine derivative ligands as cocatalysts for diels-alder reaction.
New L-serine derivative ligands were prepared and tested as cocatalyst in the Diels-Alder reactions between cyclopentadiene (CPD) and methyl acrylate, in the presence of several Lewis acids. The catalytic potential of the in situ formed complexes was evaluated based on the reaction yield. Bidentate serine ligands showed good ability to coordinate medium strength Lewis acids, thus boosting their catalytic activity. The synthesis of the L-serine ligands proved to be highly efficient and straightforward. PMID:24383009
Sousa, Carlos A D; Rodr�guez-Borges, Jos� E; Freire, Cristina
New L-Serine Derivative Ligands as Cocatalysts for Diels-Alder Reaction
New L-serine derivative ligands were prepared and tested as cocatalyst in the Diels-Alder reactions between cyclopentadiene (CPD) and methyl acrylate, in the presence of several Lewis acids. The catalytic potential of the in situ formed complexes was evaluated based on the reaction yield. Bidentate serine ligands showed good ability to coordinate medium strength Lewis acids, thus boosting their catalytic activity. The synthesis of the L-serine ligands proved to be highly efficient and straightforward. PMID:24383009
Sousa, Carlos A. D.; Rodr�guez-Borges, Jos� E.; Freire, Cristina
Kinetic, mutagenic, and structural homology analysis of l-serine dehydratase from Legionella pneumophila
A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe�4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology
Xiao Lan Xu; Shawei Chen; Gregory A. Grant
Roles and regulation of membrane-associated serine proteases.
Pericellular proteolytic activity affects many aspects of cellular behaviour, via mechanisms involving processing of the extracellular matrix, growth factors and receptors. The serine proteases have exquisitely sensitive regulatory mechanisms in this setting, involving both receptor-bound and transmembrane proteases. Receptor-bound proteases are exemplified by the uPA (urokinase plasminogen activator)/uPAR (uPAR receptor) plasminogen activation system. The mechanisms initiating the activity of this proteolytic system on the cell surface, a critical regulatory point, are poorly understood. We have found that the expression of the TTSP (type II transmembrane serine protease) matriptase is highly regulated in leucocytes, and correlates with the presence of active uPA on their surface. Using siRNA (small interfering RNA), we have demonstrated that matriptase specifically activates uPAR-associated pro-uPA. The uPA/uPAR system has been implicated in the activation of the plasminogen-related growth factor HGF (hepatocyte growth factor). However, we find no evidence for this, but instead that HGF can be activated by both matriptase and the related TTSP hepsin in purified systems. Hepsin is of particular interest, as the proteolytic cleavage sequence of HGF is an 'ideal substrate' for hepsin and membrane-associated hepsin activates HGF with high efficiency. Both of these TTSPs can be activated autocatalytically at the cell surface, an unusual mechanism among the serine proteases. Therefore these TTSPs have the capacity to be true upstream initiators of proteolytic activity with subsequent downstream effects on cell behaviour. PMID:17511657
Qiu, D; Owen, K; Gray, K; Bass, R; Ellis, V
Characterization of a chemostable serine alkaline protease from Periplaneta americana
Background Proteases are important enzymes involved in numerous essential physiological processes and hold a strong potential for industrial applications. The proteolytic activity of insects� gut is endowed by many isoforms with diverse properties and specificities. Thus, insect proteases can act as a tool in industrial processes. Results In the present study, purification and properties of a serine alkaline protease from Periplaneta americana and its potential application as an additive in various bio-formulations are reported. The enzyme was purified near to homogeneity by using acetone precipitation and Sephadex G-100 gel filtration chromatography. Enzyme activity was increased up to 4.2 fold after gel filtration chromatography. The purified enzyme appeared as single protein-band with a molecular mass of?~?27.8�kDa in SDS-PAGE. The optimum pH and temperature for the proteolytic activity for purified protein were found around pH�8.0 and 60�C respectively. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and compatibility towards detergents, oxidizing, reducing, and bleaching agents. In addition, enzyme also showed stability towards organic solvents and commercial detergents. Conclusion Several important properties of a serine protease from P. Americana were revealed. Moreover, insects can serve as excellent and alternative source of industrially important proteases with unique properties, which can be utilized as additives in detergents, stain removers and other bio-formulations. Properties of the P. americana protease accounted in the present investigation can be exploited further in various industrial processes. As an industrial prospective, identification of enzymes with varying essential properties from different insect species might be good approach and bioresource. PMID:24229392
Insulin resistance and muscle insulin receptor substrate?1 serine hyperphosphorylation
Abstract Insulin resistance in metabolic syndrome subjects is profound in spite of muscle insulin receptor and insulin?responsive glucose transporter (GLUT4) expression being nearly normal. Insulin receptor tyrosine kinase phosphorylation of insulin receptor substrate?1 (IRS?1) at Tyr896 is a necessary step in insulin stimulation of translocation of GLUT4 to the cell surface. Serine phosphorylation of IRS?1 by some kinases diminishes insulin action in mice. We evaluated the phosphorylation status of muscle IRS?1 in 33 subjects with the metabolic syndrome and seventeen lean controls. Each underwent euglycemic insulin clamps and a thigh muscle biopsy before and after 8 weeks of either strength or endurance training. Muscle IRS?1 phosphorylation at six sites was quantified by immunoblots. Metabolic syndrome muscle IRS?1 had excess phosphorylation at Ser337 and Ser636 but not at Ser307, Ser789, or Ser1101. Ser337 is a target for phosphorylation by glycogen synthase kinase 3 (GSK3) and Ser636 is phosphorylated by c?Jun N?terminal kinase 1 (JNK1). Exercise training without weight loss did not change the IRS?1 serine phosphorylation. These data suggest that baseline hyperphosphorylation of at least two key serines within muscle IRS?1 diminishes the transmission of the insulin signal and thereby decreases the insulin?stimulated translocation of GLUT4. Excess fasting phosphorylation of muscle IRS?1 at Ser636 may be a major cause of the insulin resistance seen in obesity and might prevent improvement in insulin responsiveness when exercise training is not accompanied by weight loss. PMID:25472611
Stuart, Charles A.; Howell, Mary E. A.; Cartwright, Brian M.; McCurry, Melanie P.; Lee, Michelle L.; Ramsey, Michael W.; Stone, Michael H.
Structural, mechanistic and regulatory studies of serine palmitoyltransferase.
SLs (sphingolipids) are composed of fatty acids and a polar head group derived from L-serine. SLs are essential components of all eukaryotic and many prokaryotic membranes but S1P (sphingosine 1-phosphate) is also a potent signalling molecule. Recent efforts have sought to inventory the large and chemically complex family of SLs (LIPID MAPS Consortium). Detailed understanding of SL metabolism may lead to therapeutic agents specifically directed at SL targets. We have studied the enzymes involved in SL biosynthesis; later stages are species-specific, but all core SLs are synthesized from the condensation of L-serine and a fatty acid thioester such as palmitoyl-CoA that is catalysed by SPT (serine palmitoyltransferase). SPT is a PLP (pyridoxal 5'-phosphate)-dependent enzyme that forms 3-KDS (3-ketodihydrosphingosine) through a decarboxylative Claisen-like condensation reaction. Eukaryotic SPTs are membrane-bound multi-subunit enzymes, whereas bacterial enzymes are cytoplasmic homodimers. We use bacterial SPTs (e.g. from Sphingomonas) to probe their structure and mechanism. Mutations in human SPT cause a neuropathy [HSAN1 (hereditary sensory and autonomic neuropathy type�1)], a rare SL metabolic disease. How these mutations perturb SPT activity is subtle and bacterial SPT mimics of HSAN1 mutants affect the enzyme activity and structure of the SPT dimer. We have also explored SPT inhibition using various inhibitors (e.g. cycloserine). A number of new subunits and regulatory proteins that have a direct impact on the activity of eukaryotic SPTs have recently been discovered. Knowledge gained from bacterial SPTs sheds some light on the more complex mammalian systems. In the present paper, we review historical aspects of the area and highlight recent key developments. PMID:22616865
Lowther, Jonathan; Naismith, James H; Dunn, Teresa M; Campopiano, Dominic J
Insulin resistance and muscle insulin receptor substrate-1 serine hyperphosphorylation.
Insulin resistance in metabolic syndrome subjects is profound in spite of muscle insulin receptor and insulin-responsive glucose transporter (GLUT4) expression being nearly normal. Insulin receptor tyrosine kinase phosphorylation of insulin receptor substrate-1 (IRS-1) at Tyr896 is a necessary step in insulin stimulation of translocation of GLUT4 to the cell surface. Serine phosphorylation of IRS-1 by some kinases diminishes insulin action in mice. We evaluated the phosphorylation status of muscle IRS-1 in 33 subjects with the metabolic syndrome and seventeen lean controls. Each underwent euglycemic insulin clamps and a thigh muscle biopsy before and after 8 weeks of either strength or endurance training. Muscle IRS-1 phosphorylation at six sites was quantified by immunoblots. Metabolic syndrome muscle IRS-1 had excess phosphorylation at Ser337 and Ser636 but not at Ser307, Ser789, or Ser1101. Ser337 is a target for phosphorylation by glycogen synthase kinase 3 (GSK3) and Ser636 is phosphorylated by c-Jun N-terminal kinase 1 (JNK1). Exercise training without weight loss did not change the IRS-1 serine phosphorylation. These data suggest that baseline hyperphosphorylation of at least two key serines within muscle IRS-1 diminishes the transmission of the insulin signal and thereby decreases the insulin-stimulated translocation of GLUT4. Excess fasting phosphorylation of muscle IRS-1 at Ser636 may be a major cause of the insulin resistance seen in obesity and might prevent improvement in insulin responsiveness when exercise training is not accompanied by weight loss. PMID:25472611
Stuart, Charles A; Howell, Mary E A; Cartwright, Brian M; McCurry, Melanie P; Lee, Michelle L; Ramsey, Michael W; Stone, Michael H
Characterization of a serine protease activity in Sarcocystis neurona merozoites.
Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo-(1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development. PMID:12760660
Barr, S C; Warner, K
Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii.
Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism. Disruption of the SHM 2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity. Determination of the enzyme's specific activity revealed a reduction from 3 m-units/mg of protein to 0.5 m-unit/mg protein. The remaining activity was due to an isoenzyme encoded by SHM 1, which is probably mitochondrial. A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM 2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine. Evidence for the correctness of that hypothesis was obtained from (13)C-labelling experiments. When 500 microM 99% [1-(13)C]threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity. More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase. Knockout of SHM 1 had no detectable effect on serine labelling, but disruption of SHM 2 led to a decrease in serine (2-5%) and an increase in glycine (59-67%) labelling, indicating a changed carbon flux. PMID:12350229
Schl�pen, Christina; Santos, Maria A; Weber, Ulrike; de Graaf, Albert; Revuelta, Jos� L; Stahmann, K-Peter
Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii.
Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism. Disruption of the SHM 2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity. Determination of the enzyme's specific activity revealed a reduction from 3 m-units/mg of protein to 0.5 m-unit/mg protein. The remaining activity was due to an isoenzyme encoded by SHM 1, which is probably mitochondrial. A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM 2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine. Evidence for the correctness of that hypothesis was obtained from (13)C-labelling experiments. When 500 microM 99% [1-(13)C]threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity. More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase. Knockout of SHM 1 had no detectable effect on serine labelling, but disruption of SHM 2 led to a decrease in serine (2-5%) and an increase in glycine (59-67%) labelling, indicating a changed carbon flux. PMID:12350229
Schl�pen, Christina; Santos, Maria A; Weber, Ulrike; de Graaf, Albert; Revuelta, Jos� L; Stahmann, K-Peter
Structural basis of substrate specificity in the serine proteases.
Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system. PMID:7795518
Perona, J. J.; Craik, C. S.
Phylogenetic analysis of serine proteases from Russell's viper (Daboia russelli siamensis) and Agkistrodon piscivorus leucostoma venom.
Serine proteases are widely found in snake venoms. They have variety of functions including contributions to hemostasis. In this study, five serine proteases were cloned and characterized from two different cDNA libraries: factor V activator (RVV-V), alpha fibrinogenase (RVAF) and beta fibrinogenase (RVBF) from Russell's viper (Daboia russelli siamensis), and plasminogen activator (APL-PA) and protein C activator (APL-C) from Agkistrodon piscivorus leucostoma. The snake venom serine proteases were clustered in phylogenetic tree according to their functions. K(A)/K(S) values suggested that accelerated evolution has occurred in the mature protein coding regions in cDNAs of snake venom serine proteases. PMID:21640745
Sukkapan, Pattadon; Jia, Ying; Nuchprayoon, Issarang; P�rez, John C
Pharmacokinetics of Oral d-Serine in d-Amino Acid Oxidase Knockout Mice
d-Amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-amino acids including d-serine, a full agonist at the glycine modulatory site of the N-methyl-d-aspartate (NMDA) receptor. To evaluate the significance of DAAO-mediated metabolism in the pharmacokinetics of oral d-serine, plasma d-serine levels were measured in both wild-type mice and transgenic mice lacking DAAO. Although d-serine levels were rapidly diminished in wild-type mice (t� = 1.2 h), sustained drug levels over the course of 4 h (t� > 10 h) were observed in mice lacking DAAO. Coadministration of d-serine with 6-chlorobenzo[d]isoxazol-3-ol (CBIO), a small-molecule DAAO inhibitor, in wild-type mice resulted in the enhancement of plasma d-serine levels, although CBIO seems to have only temporary effects on the plasma d-serine levels due to glucuronidation of the key hydroxyl group. These findings highlight the predominant role of DAAO in the clearance of d-serine from the systemic circulation. Thus, a potent DAAO inhibitor with a longer half-life should be capable of maintaining high plasma d-serine levels over a sustained period of time and might have therapeutic implications for the treatment of schizophrenia. PMID:22837388
Rais, Rana; Thomas, Ajit G.; Wozniak, Krystyna; Wu, Ying; Jaaro-Peled, Hanna; Sawa, Akira; Strick, Christine A.; Engle, Sandra J.; Brandon, Nicholas J.; Rojas, Camilo; Slusher, Barbara S.
Phylogenetic analysis of serine proteases from Russell's viper (Daboia russelli siamensis) and Agkistrodon piscivorus leucostoma venom
Serine proteases are widely found in snake venoms. They have variety of functions including contributions to hemostasis. In this study, five serine proteases were cloned and characterized from two different cDNA libraries: factor V activator (RVV-V), alpha fibrinogenase (RVAF) and beta fibrinogenase (RVBF) from Russell's viper (Daboia russelli siamensis), and plasminogen activator (APL-PA) and protein C activator (APL-C) from Agkistrodon piscivorus leucostoma. The snake venom serine proteases were clustered in phylogenetic tree according to their functions. KA/KS values suggested that accelerated evolution has occurred in the mature protein coding regions in cDNAs of snake venom serine proteases. PMID:21640745
Sukkapan, Pattadon; Jia, Ying; Nuchprayoon, Issarang; P�rez, John C.
Drosophila PS2 and PS3 integrins play distinct roles in retinal photoreceptors-glia interactions.
Cellular migration and differentiation are important developmental processes that require dynamic cellular adhesion. Integrins are heterodimeric transmembrane receptors that play key roles in adhesion plasticity. Here, we explore the developing visual system of Drosophila to study the roles of integrin heterodimers in glia development. Our data show that ?PS2 is essential for retinal glia migration from the brain into the eye disc and that glial cells have a role in the maintenance of the fenestrated membrane (Laminin-rich ECM layer) in the disc. Interestingly, the absence of glial cells in the eye disc did not affect the targeting of retinal axons to the optic stalk. In contrast, ?PS3 is not required for retinal glia migration, but together with Talin, it functions in glial cells to allow photoreceptor axons to target the optic stalk. Thus, we present evidence that ?PS2 and ?PS3 integrin have different and specific functions in the development of retinal glia. GLIA 2015;63:1155-1165. PMID:25731761
Tavares, L�gia; Pereira, Emiliana; Correia, Andreia; Santos, Mar�lia A; Amaral, Nuno; Martins, Torcato; Relvas, Jo�o B; Pereira, Paulo S
Amino acid changes in Drosophila alphaPS2betaPS integrins that affect ligand affinity.
We developed a ligand-mimetic antibody Fab fragment specific for Drosophila alphaPS2betaPS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the alphavbeta3 ligand-mimetic antibody WOW-1. The specificity of alphaPS2betaPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the betaPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the alphaPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the alphaPS2 GFFNR --> GFANA mutation. A mutation in the betaPS I domain (betaPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by alphaPS2betaPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin beta subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the betaPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because betaPS-DeltaPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system. PMID:16371365
Bunch, Thomas A; Helsten, Teresa L; Kendall, Timmy L; Shirahatti, Nikhil; Mahadevan, Daruka; Shattil, Sanford J; Brower, Danny L
NASA Astrophysics Data System (ADS)
Internal gas targets in AmPS A.P. Kaan, O. Postma, J.F.J. van den Brand, E. van Leeuwen, M. Doets, M. Kra= an National Institute for Nuclear Physics and High Energy Physics; Kruislaan 409; 1098 SJ Amsterdam; Holland In the Amsterdam Puls Stretcher/storage ring AmPS(1 GeV electrons), we designed a set-up in order to accommodate a gas target with a density of 1016 mol/cm2. The storage cell needed for this purpose is a aluminium tube with a length of 40 cm, a diameter of 15 mm and a wall thickness of 25 =B5m. Three sets of conductance limiters on both sides of the target, combined with dry turbopumps are designed to be used as differential pumping stations. These limiters cause discontinuities in the beam path and must therefor be retractable and radio frequency compatible in both positions. Low =B5 materials must be used because of the depolarisation effects of changing magnetic fields. The calculations show that the flow resistance's are sufficient to reduce the load of the getter pumps to a level with which the lifetime of the pump elements remain acceptable. The design of the mechanics and the vacuum system will be explained. Recent results from the measurements after installation in combination with the influence on the lifetime on the beam will be presented
Kaan, A. P.; Postma, O.; van den Brand, J. F. J.; van Leeuwen, E.; Doets, M.; Kraan, M.
Energy and expectation values of the PsH system
Close to converged energies and expectation values for PsH are computed using a ground state wave function consisting of 1800 explicitly correlated gaussians. The best estimate of the Ps{sup {infinity}}H energy was -0.789 196 740 hartree which is the lowest variational energy to date. The 2{gamma} annihilation rate for Ps{sup {infinity}}H was 2.471 78x10{sup 9} s{sup -1}.
Mitroy, J. [Faculty of Technology, Charles Darwin University, Darwin NT 0909 (Australia)
Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.
One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10?kDa, respectively, and under non-reducing conditions, 26?kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45?nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor. PMID:24090421
Odei-Addo, Frank; Frost, Carminita; Smith, Nanette; Ogawa, Tomohisa; Muramoto, Koji; Oliva, Maria Luiza Vilela; Gr�f, L�szl�; Naude, Ryno
Breaking the low barrier hydrogen bond in a serine protease.
The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water-miscible organic co-solvent such as N,N-dimethylformamide (DMF). However, in 50% DMF, the k(cat) for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the k(cat) for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397+1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 A resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 180 degrees around the Cbeta-Cgamma bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co-solvents. PMID:10048334
Kidd, R. D.; Sears, P.; Huang, D. H.; Witte, K.; Wong, C. H.; Farber, G. K.
Isolation of Serine:Glyoxylate Aminotransferase from Cucumber Cotyledons 1
Serine:glyoxylate aminotransferase, a marker enzyme for leaf peroxisomes, has been purified to homogeneity from cucumber cotyledons (Cucumis sativus cv Improved Long Green). The isolation procedure involved precipitation with polyethyleneimine, a two-step ammonium sulfate fractionation (35 to 45%), gel filtration on Ultrogel AcA 34, and ion exchange chromatography on diethylaminoethyl-cellulose, first in the presence of pyridoxal-5-phosphate, and then in its absence. The enzyme was purified approximately 690-fold to a final specific activity of 34.4 units per milligram. Electrophoresis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gels revealed two polypeptide bands with apparent molecular weights of approximately 47,000 and 45,000. Both polypeptides coeluted with enzyme activity under all chromatographic conditions investigated, both were localized to the peroxisome, and both accumulated in cotyledons as enzyme activity increased during development. The two polypeptides appear not to be structurally related, since they showed little immunological cross-reactivity and gave rise to different peptide fragments when subjected to partial proteolytic digestion. Antiserum raised against either the denatured enzyme or the 45,000-dalton polypeptide did not react with any other polypeptides present in a crude cotyledonary homogenate. The purified enzyme also had alanine:glyoxylate aminotransferase activity, but was about twice as active with serine as the amino donor. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:16664409
Hondred, David; Hunter, John McC.; Keith, Robert; Titus, David E.; Becker, Wayne M.
A novel Candida glabrata cell wall associated serine protease.
We set out to identify the Candida glabrata cell wall attached proteases which may play a role as virulence factors in candidosis, particularly in the immunocompromized host. We studied a clinical C. glabrata strain T-1639, which was isolated from a patient from the Helsinki University Central Hospital. With non-reducing 2-D electrophoresis using parallel fluorogenic gels and mass spectrometry we identified a novel appr. 25�kDa (192 aa in length) cell wall located protease with an estimated pI of 7.6. The LC-MS/MS peptides matched with the ORF of predicted C. glabrata CBS138 cell wall protein Cwp1.2p/pI 7.7/212 aa (http://cbi.labri.fr/G�nolevures/[NCBI access 49525604, UniProt access Q6FTZ7]), which is an ortholog to Saccharomyces cerevisiae cell wall protein Cwp1p (UniProt access P28319). The novel serine protease was released by ?-1,3-glucanase treatment from the cell wall. In contrast to previous predictions this protease has an enzymatic function instead of being merely a structural cell wall protein. The protease showed gelatinolytic activity and was inhibited by PMSF, a known serine protease inhibitor. Further characterization of the protease may give insight to its role in infections caused by C. glabrata and possibly aid in the development of new kinds of antifungal drugs. PMID:25617734
P�rn�nen, Pirjo; Meurman, Jukka H; Nikula-Ij�s, Pirjo
Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ? �
Airway mucins are the major molecular constituents of mucus. Mucus forms the first barrier to invading organisms in the airways and is an important defense mechanism of the lung. We confirm that mucin concentrations are significantly decreased in airway secretions of subjects with cystic fibrosis (CF) who have chronic Pseudomonas aeruginosa infection. In sputum from CF subjects without a history of P. aeruginosa, we found no significant difference in the mucin concentration compared to mucus from normal controls. We demonstrate that mucins can be degraded by synthetic human neutrophil elastase (HNE) and P. aeruginosa elastase B (pseudolysin) and that degradation was inhibited by serine proteases inhibitors (diisopropyl fluorophosphates [DFP], phenylmethylsulfonyl fluoride [PMSF], and 1-chloro-3-tosylamido-7-amino-2-heptanone HCl [TLCK]). The mucin concentration in airway secretions from CF subjects is similar to that for normal subjects until there is infection by P. aeruginosa, and after that, the mucin concentration decreases dramatically. This is most likely due to degradation by serine proteases. The loss of this mucin barrier may contribute to chronic airway infection in the CF airway. PMID:21646446
Henke, Markus O.; John, Gerrit; Rheineck, Christina; Chillappagari, Shashi; Naehrlich, Lutz; Rubin, Bruce K.
Antibacterial activity of silver nanoparticles synthesized from serine.
Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV-Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443nm. The emission spectrum of Ag NPs showed an emission band at 484nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO3 against Gram-positive and Gram-negative bacteria. PMID:25686955
Jayaprakash, N; Judith Vijaya, J; John Kennedy, L; Priadharsini, K; Palani, P
Distinct iPS Cells Show Different Cardiac Differentiation Efficiency
Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine. PMID:24367382
Yuasa, Shinsuke; Egashira, Toru; Seki, Tomohisa; Hashimoto, Hisayuki; Shimoji, Kenichiro; Kageyama, Toshimi; Tanaka, Tomofumi; Hattori, Fumiyuki; Murata, Mitsushige; Kimura, Kensuke; Fukuda, Keiichi
PS: A nonprocedural language with data types and modules
NASA Technical Reports Server (NTRS)
The Problem Specification (PS) nonprocedural language is a very high level language for algorithm specification. PS is suitable for nonprogrammers, who can specify a problem using mathematically-oriented equations; for expert programmers, who can prototype different versions of a software system for evaluation; and for those who wish to use specifications for portions (if not all) of a program. PS has data types and modules similar to Modula-2. The compiler generates C code. PS is first shown by example, and then efficiency issues in scheduling and code generation are discussed.
Gokhale, M. B.
The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis
-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1; Crucifereae; Papain; Subtilisin; Pepsin; Cysteine protease; Serine protease; Aspartic protease 1. Introduction
Jones, Alan M.
The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase (125 I)Fab dimer and monomer . Techniques were developed for the isolation of
Construction of an L-serine producing Escherichia coli via metabolic engineering.
L-Serine is a nonessential amino acid, but plays a crucial role as a building block for cell growth. Currently, L-serine production is mainly dependent on enzymatic or cellular conversion. In this study, we constructed a recombinant Escherichia coli that can fermentatively produce L-serine from glucose. To accumulate L-serine, sdaA encoding the L-serine dehydratase, iclR encoding the isocitrate lyase regulator, and arcA encoding the aerobic respiration control protein were deleted in turn. In batch fermentation, the engineered E. coli strain YF-5 exhibited obvious L-serine accumulation but poor cell growth. To restore cell growth, aceB encoding the malate synthase was knocked out, and the engineered strain was then transformed with plasmid that overexpressed serA (FR) , serB, and serC genes. The resulting strain YF-7 produced 4.5�g/L L-serine in batch cultivation and 8.34�g/L L-serine in fed-batch cultivation. PMID:24997624
Gu, Pengfei; Yang, Fan; Su, Tianyuan; Li, Fangfang; Li, Yikui; Qi, Qingsheng
Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease
Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease Madhavi. Herein, we demon- strate that Drosophila Omi (dOmi), a fly homologue of the serine protease Omi/HtrA2 to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC
Yin, Y. Whitney
Gene characterization of two digestive serine proteases in orange blossom wheat midge (Sitodiplosis mosellana)
Technology Transfer Automated Retrieval System (TEKTRAN)
Two full length cDNA sequences, encoding digestive serine proteases (designated as SmPROT-1 and SmPROT-2), were recovered from the midgut of the wheat midge, Sitodiplosis mosellana in an ongoing EST project. The deduced amino acid sequences shared homology with digestive serine proteases from insect...
Astrocytes are involved in trigeminal dynamic mechanical allodynia: potential role of D-serine.
Trigeminal neuropathic pain affects millions of people worldwide. Despite decades of study on the neuronal processing of pain, mechanisms underlying enhanced pain states after injury remain unclear. N-methyl-D-aspartate (NMDA) receptor-dependent changes play a critical role in triggering central sensitization in neuropathic pain. These receptors are regulated at the glycine site through a mandatory endogenous co-agonist D-serine, which is synthesized by astrocytes. Therefore, the present study was carried out to determine whether astrocytes are involved, through D-serine secretion, in dynamic mechanical allodynia (DMA) obtained after chronic constriction of the infraorbital nerve (CCI-IoN) in rats. Two weeks after CCI-IoN, an important reaction of astrocytes was present in the medullary dorsal horn (MDH), as revealed by an up-regulation of glial fibrillary acidic protein (GFAP) in allodynic rats. In parallel, an increase in D-serine synthesis, which co-localized with its synthesis enzyme serine racemase, was strictly observed in astrocytes. Blocking astrocyte metabolism by intracisternal delivery of fluorocitrate alleviated DMA. Furthermore, the administration of D-amino-acid oxidase (DAAO), a D-serine-degrading enzyme, or that of L-serine O-sulfate (LSOS), a serine racemase inhibitor, significantly decreased pain behavior in allodynic rats. These results demonstrate that astrocytes are involved in the modulation of orofacial post-traumatic neuropathic pain via the release of the gliotransmitter D-serine. PMID:23881719
Dieb, W; Hafidi, A
PS foams at high pressure drop rates
NASA Astrophysics Data System (ADS)
In this paper, we report data on PS foamed at 100 �C after CO2 saturation at 10 MPa in a new physical foaming batch that achieves pressure drop rates up to 120 MPa/s. Results show how average cell size of the foam nicely fit a linear behavior with the pressure drop rate in a double logarithmic plot. Furthermore, foam density initially decreases with the pressure drop rate, attaining a constant value at pressure drop rates higher than 40 MPa/s. Interestingly, furthermore, we observed that the shape of the pressure release curve has a large effect on the final foam morphology, as observed in tests in which the maximum pressure release rate was kept constant but the shape of the curve changed. These results allow for a fine tuning of the foam density and morphology for specific applications.
Tammaro, Daniele; De Maio, Attilio; Carbone, Maria Giovanna Pastore; Di Maio, Ernesto; Iannace, Salvatore
PS2004 Light-harvesting Systems Workshop
This special issue of the international scientific research journal Photosynthesis Research consists of 25 original peer-reviewed contributions from participants in the PS 2004 Lisht-Harvesting Systems Workshop. This workshop was held from 26-29, 2004 at Hotel Le Chantecler, Sainte-Adele, Quebec, Canada. The workshop was a satellite meeting of the XIII International Congress on Photosynthesis held August 29-September 3, 2004 in Montreal, Canada. The workshope dealt with all types of photosynthetic antenna systems and types of organisms, including anoxygenic photosynthetic bacteria, cyanobacteria, algae and higher plants, as well as in vitro studies of isolated pigments. This collection of papers is a good representation of the highly interdisciplinary nature of modern research on photosynthetic antenna complexes, utilizing techniques of advanced spectroscopy, biochemistry, molecular biology, synthetic chemistry and structural determination to understand these diverse and elegant molecular complexes.
Robert E. Blankenship
Serine proteases in the spiny lobster olfactory organ: their functional expression along a developmental axis, and the contribution of a CUB-serine protease.
Several serine proteases and protease inhibitors have been identified in the crustacean olfactory organ, which is comprised of the lateral flagellum of the antennule and its aesthetascs sensilla that house olfactory receptor neurons and their supporting cells. The function of these proteases in the olfactory organ is unknown, but may include a role in perireception (e.g., odor activation or inactivation) or in the development or survival of olfactory receptor neurons. To examine directly the function of proteases in the olfactory organ of the Caribbean spiny lobster Panulirus argus, we used different tissue fractions from the lateral flagellum in an enzyme activity assay with a variety of protease substrates and inhibitors. Trypsin-like serine protease activity occurs throughout the lateral flagellum but is enriched in the cell membranes from aesthetascs. Cysteine- and metalloprotease activities also occur in olfactory tissue, but are more abundant in tissue fractions other than aesthetascs. To assess the contribution of one of the olfactory serine proteases--CUB-serine protease (Csp)--Csp was immunoprecipitated using an antibody; results with the remaining fraction suggest that Csp accounts for at least 40% of the total serine protease activity in the olfactory organ. The amount of total serine protease activity follows a developmental axis in the lateral flagellum. Total protease activity is lowest in the proximal zone, which lacks aesthetascs, and the proliferation zone, where olfactory receptor neurons and associated cells are born, and highest in aesthetascs of the distally-located senescence zone, which has the oldest olfactory tissue. PMID:15389692
Johns, Malcolm E; Tai, Phang C; Derby, Charles D
Cloning and Expression of the Two Genes Coding for L-Serine Dehydratase from Peptostreptococcus asaccharolyticus: Relationship of the Iron-Sulfur Protein to Both L-Serine Dehydratases from Escherichia coli
L-Serine dehydratases and L-threonine dehydratases catalyze the irreversible overall deaminations of L-serine to pyruvate and L-threonine to 2-oxobutyrate. Most L-threonine dehydrata- ses have been shown to contain pyridoxal-59-phosphate as the prosthetic group. In contrast to L-threonine dehydratases, none of the bacterial L-serine dehydratases investigated to date has been conclusively proven to be dependent on pyridoxal-59- phosphate (6). An L-serine dehydratase
Serine is a natural ligand and allosteric activator of pyruvate kinase M2
Cancer cells exhibit several unique metabolic phenotypes that are critical for cell growth and proliferation. Specifically, they over-express the M2 isoform of the tightly regulated enzyme pyruvate kinase (PKM2), which controls glycolytic flux, and they are highly dependent on de novo biosynthesis of serine and glycine. Here we describe a novel rheostat-like mechanistic relationship between PKM2 activity and serine biosynthesis. We show that serine can bind to and activate human PKM2 and that following serine deprivation, PKM2 activity in cells is reduced. This reduction in PKM2 activity shifts cells to a fuel-efficient mode where more pyruvate is diverted to the mitochondria and more glucose derived carbon is channelled into serine biosynthesis to support cell proliferation. PMID:23064226
Zheng, Liang; Martin, Agn�s C.L.; Maddocks, Oliver D.K.; Chokkathukalam, Achuthanunni; Coyle, Joseph E; Jankevics, Andris; Holding, Finn P.; Vousden, Karen H.; Frezza, Christian; O�Reilly, Marc; Gottlieb, Eyal
Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms.
Envenomation by Bothrops species results, among other symptoms, in hemostatic disturbances. These changes can be ascribed to the presence of enzymes, primarily serine proteinases some of which are structurally similar to thrombin and specifically cleave fibrinogen releasing fibrinopeptides. A rapid, three-step, chromatographic procedure was developed to routinely purify serine proteinases from the venoms of Bothrops alternatus and Bothrops moojeni. The serine proteinase from B. alternatus displays an apparent molecular mass of ~32 kDa whereas the two closely related serine proteinases from B. moojeni display apparent molecular masses of ~32 kDa and ~35 kDa in SDS-PAGE gels. The partial sequences indicated that these enzymes share high identity with serine proteinases from the venoms of other Bothrops species. These proteins coagulate plasma and possess fibrinogenolytic activity but lack fibrinolytic activity. PMID:24140922
Fernandes de Oliveira, Liliane Maria; Ullah, Anwar; Masood, Rehana; Zelanis, Andr�; Spencer, Patrick J; Serrano, Solange M T; Arni, Raghuvir K
In vivo sulfhydryl modification of the ligand-binding site of Tsr, the Escherichia coli serine chemoreceptor.
The Escherichia coli chemoreceptor Tsr mediates an attractant response to serine. We substituted Cys for Thr-156, one of the residues involved in serine sensing. The mutant receptor Tsr-T156C retained serine- and repellent-sensing abilities. However, it lost serine-sensing ability when it was treated in vivo with sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). Serine protected Tsr-T156C from these reagents. We showed that [3H]NEM bound to Tsr-T156C and that binding decreased in the presence of serine. By pretreating cells with serine and cold NEM, Tsr-T156C was selectively labeled with radioactive NEM. These results are consistent with the location of Thr-156 in the serine-binding site. Chemical modification of the Tsr ligand-binding site provides a basis for simple purification and should assist further in vivo and in vitro investigations of this chemoreceptor protein. PMID:7721714
Iwama, T; Kawagishi, I; Gomi, S; Homma, M; Imae, Y
Safeguarding Nonhuman Primate iPS Cells With Suicide Genes
The development of technology to generate induced pluripotent stem (iPS) cells constitutes one of the most exciting scientific breakthroughs because of the enormous potential for regenerative medicine. However, the safety of iPS cell-related products is a major concern for clinical translation. Insertional mutagenesis, possible oncogenic transformation of iPS cells or their derivatives, or the contamination of differentiated iPS cells with undifferentiated cells, resulting in the formation of teratomas, have remained considerable obstacles. Here, we demonstrate the utility of suicide genes to safeguard iPS cells and their derivatives. We found suicide genes can control the cell fate of iPS cells in vitro and in vivo without interfering with their pluripotency and self-renewal capacity. This study will be useful to evaluate the safety of iPS cell technology in a clinically highly relevant, large animal model and further benefit the clinical use of human iPS cells. PMID:21587213
Zhong, Bonan; Watts, Korashon L; Gori, Jennifer L; Wohlfahrt, Martin E; Enssle, Joerg; Adair, Jennifer E; Kiem, Hans-Peter
A Novel Method with Ps Accuracy for Time Interval Measurement
High precise time interval measurement is widely used in time synchronization, satellite navigation, aerospace tracking telemetering, laser metering and nuclear electronics. The resolution and accuracy of the current used time counter is 25 ps and nearly 100 ps, respectively. A new time interval measurement method was put forward, in which, the signal under test is used to trigger a sampler
Xiangwei Zhu; Guangfu Sun; Shaowei Yong; Zhaowen Zhuang
Pan-STARRS PS1 Published Science Products Subsystem
NASA Astrophysics Data System (ADS)
This paper describes the requirements and design of the Pan-STARRS PS1 Published Science Products Subsystem (PSPS) that constitutes the primary distribution tool for the very large amount of science data products produced by the Pan-STARRS PS1 prototype telescope. The data management challenges are identified in terms of stressing characteristics: dynamic, fast, spatial, and large; these are countered by mitigating characteristics: simple and lenient. This combination of characteristics is not only distinctly more demanding than traditional survey astronomy data managers, but lies at the boundaries of current commercially available data management technology. The requirements imposed on the PSPS result in devising a design strategy at the boundaries of currently available data management technology. In particular, we describe the capabilities and characteristics of the four main PS1 PSPS components: the Web-Based Interface (WBI), the Data Retrieval Layer (DRL), the Object Data Manager (ODM), and the Solar System Data Manager (SSDM). Potential architectural strategies are examined in the context of the stressing and mitigating characteristics with the conclusion that the ODM should follow an architectural concept that emphasizes the pooling of application, processing, and storage resources. The PS1 PSPS is specifically designed to support the PS1 science mission (see K.C. Chambers et al., these proceedings) while at the same time providing substantial design direction for a future PSPS component of the final PS4 Pan-STARRS. Finally, the limitations and possible scalability of the PS1 design relative to PS4 are discussed.
Heasley, J.; Smith, W.; Eek, R.; Rosen, J.
L-Serine overproduction with minimization of by-product synthesis by engineered Corynebacterium glutamicum.
The direct fermentative production of L-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low L-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing L-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both L-serine production level (26.23�g/L) and its productivity (0.27�g/L/h). Compared to the parent strain, the by-products L-alanine and L-valine accumulation in the resulting strain were reduced by 87�% (from 9.80 to 1.23�g/L) and 60�% (from 6.54 to 2.63�g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards L-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as L-serine productivity. With addition of 2�g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72�%) and the L-serine productivity (67�%). In fed-batch fermentation, 42.62�g/L of L-serine accumulation was achieved with a productivity of 0.44�g/L/h and yield of 0.21�g/g sucrose, which was the highest production of L-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve L-serine productivity. PMID:25434811
Zhu, Qinjian; Zhang, Xiaomei; Luo, Yuchang; Guo, Wen; Xu, Guoqiang; Shi, Jinsong; Xu, Zhenghong
Design differences between the Pan-STARRS PS1 and PS2 telescopes
NASA Astrophysics Data System (ADS)
The PS2 telescope is the second in an array of wide-field telescopes that is being built for the Panoramic-Survey Telescope and Rapid Response System (Pan-STARRS) on Haleakala. The PS2 design has evolved incrementally based on lessons learned from PS1, but these changes should result in significant improvements in image quality, tracking performance in windy conditions, and reductions in scattered light. The optics for this telescope are finished save for their coatings and the fabrication for the telescope structure itself is well on the way towards completion and installation on-site late this year (2012). The most significant differences between the two telescopes include the following: secondary mirror support changes, improvements in the optical polishing, changes in the optical coatings to improve throughput and decrease ghosting, removal of heat sources inside the mirror cell, expansion of the primary mirror figure control system, changes in the baffle designs, and an improved cable wrap design. This paper gives a description of each of these design changes and discusses the motivations for making them.
Morgan, Jeffrey S.; Kaiser, Nicholas; Moreau, Vincent; Anderson, David; Burgett, William
Type II transmembrane serine protease (TTSP) deregulation in cancer.
The Type II transmembrane serine proteases (TTSP) are a relatively newly identified family of proteolytic enzymes that have become the subject of intense scrutiny in the field of cancer research. Advances in genome screening technology have enabled the identification of putative members and the further characterization of existing members. The TTSPs are involved in a diverse range of physiological functions and new roles continue to be discovered. A large majority of these proteases appear to play crucial roles in the development of disease, especially cancer development and progression. This review presents the current knowledge of the biological role of those TTSPs that have been identified in the development and progression of human cancers. PMID:21196187
Webb, Siobhan L; Sanders, Andrew J; Mason, Malcolm D; Jiang, Wen G
Type II transmembrane serine proteases in cancer and viral infections.
Regulated proteolysis of cellular factors is pivotal to tissue development and homeostasis, whereas uncontrolled proteolytic activity is linked to disease. Type II transmembrane serine proteases (TTSPs) are expressed at the cell surface and are thus ideally located to regulate cell-cell and cell-matrix interactions. Increasing evidence demonstrates that aberrant expression of TTSPs is a hallmark of several cancers and recent studies have defined molecular mechanisms underlying TTSP-promoted carcinogenesis. In addition, new findings suggest that influenza and other respiratory viruses could exploit TTSPs to promote their spread, making these proteases potential targets for intervention in cancer and viral infections. Here, we review the role of TTSPs in tumorigenesis and viral infection and discuss potential approaches to therapy. PMID:19581128
Choi, So-Young; Bertram, Stephanie; Glowacka, Ilona; Park, Young Woo; P�hlmann, Stefan
Type II transmembrane serine proteases in development and disease.
Recent advances in the mammalian genome projects have resulted in the identification of a surprisingly large number of genes encoding putative new members and even entire new families of proteolytic enzymes. In the past few years, one of these new families, type II transmembrane serine proteases (TTSPs), underwent a particularly rapid transformation from a group of predicted DNA and protein sequences into an established family of cell surface-associated proteases with important roles in the development and homeostasis of mammalian tissues such as heart, skin, inner ear, placenta, and digestive tract. Additionally, aberrant expression of TTSP genes appears to be involved in the aetiology of several human disorders, including cancer. This review presents our current knowledge of the biological functions of the individual TTSPs in mouse and human tissue development and disease. PMID:18191610
Szabo, Roman; Bugge, Thomas H
A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii
Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated �check-in� system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic ?-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states. PMID:23632025
Menyh�rd, D�ra K.; Kiss-Szem�n, Anna; Tichy-R�cs, �va; Hornung, Bal�zs; R�di, Krisztina; Szeltner, Zolt�n; Domokos, Klarissza; Szamosi, Ilona; N�ray-Szab�, G�bor; Polg�r, L�szl�; Harmat, Veronika
RAF protein-serine/threonine kinases: Structure and regulation
Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.
Roskoski, Robert, E-mail: firstname.lastname@example.com [Blue Ridge Institute for Medical Research, 3754 Brevard Road, Suite 116, Box 19, Horse Shoe, NC 28742 (United States)] [Blue Ridge Institute for Medical Research, 3754 Brevard Road, Suite 116, Box 19, Horse Shoe, NC 28742 (United States)
A 33 kDa serine proteinase from Scedosporium apiospermum.
An extracellular proteinase produced by the filamentous fungus Scedosporium apiospermum has been purified and characterized. Initially, in vitro conditions for enzyme synthesis were investigated. The highest yield of enzyme production was obtained when the fungus was cultivated in modified Czapek-Dox liquid medium supplemented with 0.1% bacteriological peptone and 1% (w/v) glucose as the nitrogen and carbon sources respectively. Purification to homogeneity of the proteinase was accomplished by (NH4)2SO4 precipitation, followed by gel filtration through Sephadex G-75 and finally affinity chromatography through immobilized phenylalanine. Analysis of the purified enzyme by SDS/PAGE revealed a single polypeptide chain with an apparent molecular mass of 33 kDa. Further investigation of its physical and biochemical properties disclosed numerous similarities with those of the previously described serine proteinase of Aspergillus fumigatus. The enzyme was not glycosylated and its pI was 9.3. Proteinase activity was optimum between 37 and 50 degrees C and at pH 9.0, but remained high within a large range of pH values between 7 and 11. The inhibition profile and N-terminal amino acid sequencing confirmed that this enzyme belongs to the subtilisin family of serine proteinases. In agreement with this, the specific synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide proved to be an excellent substrate for the proteinase with an estimated Km of 0.35 mM. Like the alkaline proteinase of A. fumigatus, this enzyme was able to degrade human fibrinogen, and thus may act as a mediator of the severe chronic bronchopulmonary inflammation from which cystic fibrosis patients suffer. PMID:8670095
Larcher, G; Cimon, B; Symoens, F; Tronchin, G; Chabasse, D; Bouchara, J P
D-Serine as a Neuromodulator: Regional and Developmental Localizations in Rat Brain Glia Resemble NMDA Receptors
D-Serine is localized in mammalian brain to a discrete popula- tion of glial cells near NMDA receptors, suggesting that D-serine is an endogenous agonist of the receptor-associated glycine site. To explore this possibility, we have compared the immu- nohistochemical localizations of D-serine, glycine, and NMDA receptors in rat brain. In the telencephalon, D-serine is concen- trated in protoplasmic astrocytes, which
Michael J. Schell; Roscoe O. Brady Jr; Mark E. Molliver; Solomon H. Snyder
Telomere Reprogramming and Maintenance in Porcine iPS Cells
Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells. PMID:24098638
Ji, Guangzhen; Ruan, Weimin; Liu, Kai; Wang, Fang; Sakellariou, Despoina; Chen, Jijun; Yang, Yang; Okuka, Maja; Han, Jianyong; Liu, Zhonghua; Lai, Liangxue; Gagos, Sarantis; Xiao, Lei; Deng, Hongkui; Li, Ning; Liu, Lin
Le Lait (1985), 65 (649-650), 103-121 Characterization of an L-serine dehydratase
Le Lait (1985), 65 (649-650), 103-121 Characterization of an L-serine dehydratase activity occurs via an L-serine dehydratase (EC.4.2.I.13), whose activity is maximal at the end of exponential inhibitors. L-serine dehydratase activity is strongly inhibited by o-phenanthraline. Pyridoxal phosphate
Paris-Sud XI, Universit� de
Serine integrase chimeras with activity in E. coli and HeLa cells
ABSTRACT In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC) and phiC31-TG1 (CT) hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{?1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies. PMID:25217617
Farruggio, Alfonso P.; Calos, Michele P.
PS W145A Syllabus file:///H|/Summer12/PS145A/syllabus1.html[4/11/2012 10:51:06 AM
PS W145A Syllabus file:///H|/Summer12/PS145A/syllabus1.html[4/11/2012 10:51:06 AM] Printer Friendly Version Course Information Please, note that this syllabus is subject to changes. Course Number: PS145A and armed conflicts; #12;PS W145A Syllabus file:///H|/Summer12/PS145A/syllabus1.html[4/11/2012 10:51:06 AM
Doudna, Jennifer A.
Crystal Structure of a Homolog of Mammalian Serine Racemase from Schizosaccharomyces pombe*
d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5?-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. The enzyme also catalyzes the dehydration of d- and l-serine. Both reactions are enhanced by Mg�ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 ? resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with l-serine and d-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique �lysino-d-alanyl� residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme. PMID:19640845
Goto, Masaru; Yamauchi, Takae; Kamiya, Nobuo; Miyahara, Ikuko; Yoshimura, Tohru; Mihara, Hisaaki; Kurihara, Tatsuo; Hirotsu, Ken; Esaki, Nobuyoshi
Acute D-serine treatment produces antidepressant-like effects in rodents.
Research suggests that dysfunctional glutamatergic signalling may contribute to depression, a debilitating mood disorder affecting millions of individuals worldwide. Ketamine, a N-methyl-D-aspartate (NMDA) receptor antagonist, exerts rapid antidepressant effects in approximately 70% of patients. Glutamate evokes the release of D-serine from astrocytes and neurons, which then acts as a co-agonist and binds at the glycine site on the NR1 subunit of NMDA receptors. Several studies have implicated glial deficits as one of the underlying facets of the neurobiology of depression. The present study tested the hypothesis that D-serine modulates behaviours related to depression. The behavioural effects of a single, acute D-serine administration were examined in several rodent tests of antidepressant-like effects, including the forced swim test (FST), the female urine sniffing test (FUST) following serotonin depletion, and the learned helplessness (LH) paradigm. D-serine significantly reduced immobility in the FST without affecting general motor function. Both D-serine and ketamine significantly rescued sexual reward-seeking deficits caused by serotonin depletion in the FUST. Finally, D-serine reversed LH behaviour, as measured by escape latency, number of escapes, and percentage of mice developing LH. Mice lacking NR1 expression in forebrain excitatory neurons exhibited a depression-like phenotype in the same behavioural tests, and did not respond to D-serine treatment. These findings suggest that D-serine produces antidepressant-like effects and support the notion of complex glutamatergic dysfunction in depression. It is unclear whether D-serine has a convergent influence on downstream synaptic plasticity cascades that may yield a similar therapeutic profile to NMDA antagonists like ketamine. PMID:21906419
Malkesman, Oz; Austin, Daniel R; Tragon, Tyson; Wang, Gang; Rompala, Gregory; Hamidi, Anahita B; Cui, Zhenzhong; Young, W Scott; Nakazawa, Kazu; Zarate, Carlos A; Manji, Husseini K; Chen, Guang
A role for serine-175 in modulating the molecular conformation of calponin.
Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin-myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175-->alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin-tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects: (a) structures of calponin are reconfigured by serine-175 modification, supporting the regulatory function of serine-175; (b) there are submolecular structures unaffected by modification of serine-175 in both free and thin filament-associated calponins, suggesting that the serine-175-based conformational modulation is a targeted allosteric effect; (c) significant conformational changes are detected between free and thin filament-associated calponins, indicating two functional states of the molecular conformation; and (d) the different epitope characteristics between thin filament-bound and free calponins suggest that calponin is a flexible molecule, and the modifications of serine-175 may also determine the structural flexibility to increase the epitope accessibility. These results provide novel information concerning the structure-function relationships of calponin and its regulation by phosphorylation. PMID:10947974
Jin, J P; Walsh, M P; Sutherland, C; Chen, W
Anti-staphylococcal serine proteinase and other serum factors in phagocytosis.
The interactions between polymorphonuclear cells (PMN), Staphylococcus saprophyticus cells and rabbit antibodies against Staphylococcus aureus V8 serine proteinase or normal rabbit serum proteins were investigated. The effect of opsonization on phagocytosis due to human peripheral polymorphonuclear cells was measured. The results were as follows: phagocytosis index values were relatively increased after the incubation of PMN cells with anti-serine proteinase gamma-globulin serum fraction, anti-serine proteinase IgG, non-immunized rabbit serum or with complement. PMID:1698965
Miedzobrodzki, J; Gorka, M; Wasniowska, A; Tadeusiewicz, R; Porwit-Bobr, Z
PP and PS interferometric images of near-seafloor sediments
I present interferometric processing examples from an ocean-bottom cable OBC dataset collected at a water depth of 800 m in the Gulf of Mexico. Virtual source and receiver gathers created through cross-correlation of full wavefields show clear PP reflections and PS conversions from near-seafloor layers of interest. Virtual gathers from wavefield-separated data show improved PP and PS arrivals. PP and PS brute stacks from the wavefield-separated data compare favorably with images from a non-interferometric processing flow. ?? 2011 Society of Exploration Geophysicists.
Haines, S.S.
Assessment of slope stability using PS-InSAR technique
NASA Astrophysics Data System (ADS)
In this research work, PS-InSAR approach is envisaged to monitor slope stability of landslides prone areas in Nainital and Tehri region of Uttarakhand, India. For the proposed work, Stanford Method for Persistent Scatterers (StaMPS) based PS-InSAR is used for processing ENVISAT ASAR C-Band data stacks of study area which resulted in a time series 1D-Line of Sight (LOS) map of surface displacement. StaMPS efficiently extracted the PS pixels on the unstable slopes in both areas and the time series 1D-LOS displacement map of PS pixels indicates that those areas in Nainital and Tehri region have measurement pixels with maximum displacement away from the satellite of the order of 22 mm/year and 17.6 mm/year respectively
Dwivedi, R.; Varshney, P.; Tiwari, A.; Singh, A. K.; Dikshit, O.
Lattice dynamics of lithium intercalated FePS3 compounds
NASA Astrophysics Data System (ADS)
The dynamics of Li-intercalated FePS3 has been studied by means of a force-constant model generated by a set of short-range two-body potentials, as already done for the pure material (see the preceding paper). The intercalated phases have been investigated for the three stoichiometric compositions Li0.5FePS3, LiFePS3, and Li1.5FePS3, with the aim of analyzing the evolution of the host-lattice normal modes as a function of the concentration, and finding the dispersion of the new phonon branches induced by lithium. The force constants are fitted to the infrared data and the phonon dispersion curves are calculated along the symmetry directions of the Brillouin zone.
Bernasconi, M.; Benedek, G.; Miglio, L.
Serine proteases in immune protection of the small intestine.
The gastrointestinal tract is subject to a huge antigenic load, which is especially significant in the intestinal lumen. Being the connecting link between the organism and the external environment, the small intestine fulfils not only digestive and transport functions, but also protective ones and acts as a selective barrier for the flow of nutrients. This review considers proteases of the protective system of small intestine cells, their biochemical properties and activation mechanisms, and involvement in biochemical processes responsible for normal functioning and defense reactions of the intestine. Serine proteases of intestinal immunity are multifunctional enzymes making proteolytic attack aimed to immediately exterminate aggressive elements of the intestinal contents (allergens, toxins), to activate (inactivate) zymogens, receptors, and peptide hormones, and to hydrolyze protein precursors and other biologically active factors. Proteases of intestinal immunity control the inflammatory response, proliferation of B-lymphocytes, apoptosis, and secretory and contractive activity of the intestine; they release neurogenic factors, inactivate biologically active substances, and are involved in degradation of the intercellular matrix and in tissue remodeling. PMID:23586713
Zamolodchikova, T S
New serine-derived gemini surfactants as gene delivery systems.
Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems. PMID:25513958
Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Silva, Sandra G; do Vale, M Lu�sa; Marques, Eduardo F; de Lima, Maria C Pedroso; Jurado, Am�lia S
Exploring a New Serine Protease from Cucumis sativus L.
Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed A? and B? subunits of fibrinogen, whereas it failed to degrade the ? subunit of fibrinogen even at a concentration as high as 100�?g and incubation time up to 4�h. AqFEC reduced the clotting time of citrated plasma by 87.65�%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process. PMID:25577345
Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda
Phosphorylation at serine 331 is required for Aurora B activation
Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora BS331A, in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation. PMID:22024163
Petsalaki, Eleni; Akoumianaki, Tonia; Black, Elizabeth J.; Gillespie, David A.F.
Serine palmitoyltransferase (SPT) deficient mice absorb less cholesterol?
Serine palmitoyltransferase (SPT) is the key enzyme for the biosynthesis of sphingolipids. It has been reported that oral administration of myriocin (an SPT inhibitor) decreases plasma sphingomyelin (SM) and cholesterol levels, and reduces atherosclerosis in apoE knockout (KO) mice. We studied cholesterol absorption in myriocin-treated WT or apoE KO animals and found that, after myriocin treatment, the mice absorbed significantly less cholesterol than controls, with no observable pathological changes in the small intestine. More importantly, we found that heterozygous Sptlc1 (a subunit of SPT) KO mice also absorbed significantly less cholesterol than controls. To understand the mechanism, we measured protein levels of Niemann-Pick C1-like 1 (NPC1L1), ABCG5, and ABCA1, three key factors involved in intestinal cholesterol absorption. We found that NPC1L1 and ABCA1 were decreased, whereas ABCG5 was increased in the SPT deficient small intestine. SM levels on the apical membrane were also measured and they were significantly decreased in SPT deficient mice, compared with controls. In conclusion, SPT deficiency might reduce intestinal cholesterol absorption by altering NPC1L1 and ABCG5 protein levels in the apical membranes of enterocytes through lowering apical membrane SM levels. This may be also true for ABCA1 which locates on basal membrane of enterocytes. Manipulation of SPT activity could thus provide a novel alternative treatment for dyslipidemia. PMID:19416652
Li, Zhiqiang; Park, Tae-Sik; Li, Yan; Pan, Xiaoyue; Iqbal, Jahangir; Lu, David; Tang, Weiqing; Yu, Liqing; Goldberg, Ira J.; Hussain, M. Mahmood; Jiang, Xian-Cheng
Preliminary Tuft Testing of Metallic Bristles Versus PS212, PS300, and HVOF300
NASA Technical Reports Server (NTRS)
Turbine engine brush seals are designed with sacrificial brushes and hard shaft coatings to minimize shaft wear and reduce the cost of engine overhauls. Replacing a worm seal is more cost and time effective than refinishing an engine shaft. However, this tribological design causes excessive brush wear and reduces long term seal efficiency. An alternative approach is to coat the shaft with a solid lubricant and allow the bristles to wear into the shaft coating similar to traditional abradable labyrinth seals. This approach can result in reduced seal leakage by forcing the leakage to flow through the seal bristle pack or through a more tortuous shaft wear track. Key to this approach is limiting the shaft wear to an acceptable level were surface refinishing would not be required during every engine overhaul. Included in this paper are brush seal tuft test results for four metallic bristles (nickel-chrome or cobalt-chrome based superalloys) tested against three solid lubricant coatings (NASA's PS212, PS300, and HVOF300). These test results are also compared to previous baseline tests conducted with plasma sprayed chrome carbide. Compared to the baseline results, no tribological benefit was achieved with the metallic bristle/solid lubricant tribopairs tested. To improve the performance of the solid lubricant coatings, issues regarding lubricant phase sizes (homogeneity), and composition need to be addressed.
Fellenstein, James A.; DellaCorte, Christopher
Small Molecule Activation of PKM2 in Cancer Cells Induces Serine Auxotrophy
SUMMARY Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress. PMID:22999886
Kung, Charles; Hixon, Jeff; Choe, Sung; Marks, Kevin; Gross, Stefan; Murphy, Erin; DeLaBarre, Byron; Cianchetta, Giovanni; Sethumadhavan, Shalini; Wang, Xiling; Yan, Shunqi; Gao, Yi; Fang, Cheng; Wei, Wentao; Jiang, Fan; Wang, Shaohui; Qian, Kevin; Saunders, Jeff; Driggers, Ed; Woo, Hin Koon; Kunii, Kaiko; Murray, Stuart; Yang, Hua; Yen, Katharine; Liu, Wei; Cantley, Lewis C.; Vander Heiden, Matthew G.; Su, Shinsan M.; Jin, Shengfang; Salituro, Francesco G.; Dang, Lenny
Membrane-Anchored Serine Proteases in Vertebrate Cell and Developmental Biology*
Analysis of vertebrate genome sequences at the turn of the millennium revealed that a vastly larger repertoire of enzymes execute proteolytic cleavage reactions within the pericellular and extracellular environments than was anticipated from biochemical and molecular analysis. Most unexpected was the unveiling of an entire new family of structurally unique multidomain serine proteases that are anchored directly to the plasma membrane. Unlike secreted serine proteases, which function primarily in tissue repair, immunity, and nutrient uptake, these membrane-anchored serine proteases regulate fundamental cellular and developmental processes, including tissue morphogenesis, epithelial barrier function, ion and water transport, cellular iron export, and fertilization. Here the cellular and developmental biology of this fascinating new group of proteases is reviewed. Particularly highlighted is how the study of membrane-anchored serine proteases has expanded our knowledge of the range of physiological processes that require regulated proteolysis at the cell surface. PMID:21721945
Szabo, Roman; Bugge, Thomas H.
Bacterial serine proteases secreted by the autotransporter pathway: classification, specificity and role in virulence
Serine proteases exist in eukaryotic and prokaryotic organisms and have emerged during evolution as the most abundant and functionally diverse group. In gram-negative bacteria, there is a growing family of high molecular weight serine proteases secreted to the external milieu by a fascinating and widely employed bacterial secretion mechanism, known as the autotransporter pathway. They were initially found in Neisseria, Shigella, and pathogenic Escherichia coli, but have now been also identified in Citrobacter rodentium, Salmonella, and Edwarsiella species. Here, we focus on proteins belonging to the Serine Protease Autotransporter of Enterobacteriaceae (SPATEs) family. Recent findings regarding the predilection of serine proteases to host intracellular or extracellular protein-substrates involved in numerous biological functions, such as those implicated in cytoskeleton stability, autophagy or innate and adaptive immunity, have helped provide a better understanding of SPATEs� contributions in pathogenesis. Here, we discuss their classification, substrate specificity, and potential roles in pathogenesis. PMID:23689588
Ruiz-Perez, Fernando; Nataro, James P.
Bacterial serine proteases secreted by the autotransporter pathway: classification, specificity, and role in virulence.
Serine proteases exist in eukaryotic and prokaryotic organisms and have emerged during evolution as the most abundant and functionally diverse group. In Gram-negative bacteria, there is a growing family of high molecular weight serine proteases secreted to the external milieu by a fascinating and widely employed bacterial secretion mechanism, known as the autotransporter pathway. They were initially found in Neisseria, Shigella, and pathogenic Escherichia coli, but have now also been identified in Citrobacter rodentium, Salmonella, and Edwardsiella species. Here, we focus on proteins belonging to the serine protease autotransporter of Enterobacteriaceae (SPATEs) family. Recent findings regarding the predilection of serine proteases to host intracellular or extracellular protein-substrates involved in numerous biological functions, such as those implicated in cytoskeleton stability, autophagy or innate and adaptive immunity, have helped provide a better understanding of SPATEs' contributions in pathogenesis. Here, we discuss their classification, substrate specificity, and potential roles in pathogenesis. PMID:23689588
Ruiz-Perez, Fernando; Nataro, James P
Enzymatic synthesis of isotopically labelled serine and tryptophan for application in peptide synthesis.
L-[1.2-13C2, 15N]Serine was prepared from [1,2-13C2, 15N]glycine on a gram scale by the use of the enzyme serine hydroxymethyltransferase. The reaction was monitored by 13C-NMR spectroscopy. This is the first simultaneously 13C- and 15N-labelled serine isotopomer so far reported. Part of the product was directly converted by tryptophan synthase to L-[1,2-13C2, 15N]tryptophan which could conveniently be purified and isolated as Boc-derivative in a yield of 71%. Most of the serine was isolated similarly but to remove remaining starting material in this case purification by column chromatography was required. PMID:9391911
Malthouse, J P; Fitzpatrick, T B; Milne, J J; Grehn, L; Ragnarsson, U
Sub20 ps silicon bipolar technology using selective epitaxial growth
A sub-20 ps silicon bipolar technology has been developed using selective epitaxial growth (SEG) for the active base and collector regions. This transistor concept allows the simultaneous reduction of base width and base\\/collector capacitance while maintaining low extrinsic base resistance. At a current of 0.8 mA a record CML gate delay time of 18 ps is achieved with devices showing
T. F. Meister; R. Stengl; H. W. Meul; R. Weyl; P. Packan; A. Felder; H. Klose; R. Schreiter; J. Popp; H. M. Rein; L. Treitinger
Lesions of the pedunculopontine tegmental nucleus reduce paradoxical sleep (PS) propensity: evidence from a short-term PS deprivation study in rats.
Cholinergic neurons in the mesopontine tegmentum are thought to play a critical role in the generation of paradoxical sleep (PS). However, no study has yet examined whether lesions of these neurons cause deficits of PS in the rat. We describe here the effects of lesions of the pedunculopontine tegmental nucleus (PPT) on spontaneous PS and on PS propensity, expressed during and after a short period of PS deprivation. Lesions were induced by bilateral injections of ibotenate. PS deprivation was performed manually by gently waking rats each time they showed polygraphic signs of PS. Two weeks after lesions, an 8-h baseline recording was performed; the following day, rats were PS deprived for 6 h and polygraphic recordings were then continued for 2 h, to examine recovery sleep. The same protocol was repeated 1 week later. Compared with controls and with rats with limited PPT lesions, rats bearing > 60% NADPH-diaphorase-positive cell loss within the PPT showed unaffected PS under baseline conditions. However, they made fewer attempts to enter PS during deprivation and they exhibited an attenuated rebound increase in PS time after deprivation. The number of PS attempts and the magnitude of PS rebound were negatively correlated with the percent loss of diaphorase-positive neurons within the PPT. Thus, PS propensity that accumulated as a result of PS deprivation was reduced after extensive PPT lesions. In summary, although spontaneous PS was found to be unaltered, the PS deprivation procedure used in this study demonstrated the dysfunctioning of PS caused by PPT lesions. PMID:11403690
Deurveilher, S; Hennevin, E
Glia-Derived d-Serine Controls NMDA Receptor Activity and Synaptic Memory
SUMMARY The NMDA receptor is a key player in excitatory transmission and synaptic plasticity in the cen- tral nervous system. Its activation requires the binding of both glutamate and a coagonist like D-serine to its glycine site. As D-serine is re- leased exclusively by astrocytes, we studied the physiological impact of the glial environ- ment on NMDA receptor-dependent activity and
Aude Panatier; Dionysia T. Theodosis; Jean-Pierre Mothet; Bastien Touquet; Loredano Pollegioni; Dominique A. Poulain; St�phane H. R. Oliet
Laparotomy Causes a Transient Induction of Rat Liver Serine Dehydratase mRNA
Rat liver serine dehydratase mRNA shows rhythmicity with a high level at the onset of dark (19:00) and a low level at the onset of light (07:00). We have examined the effect of stress (laparotomy) on the rhythm, Upon laparotomy at 09:00 or 17:00, a marked induction of serine dehydratase mRNA occurred 2 h after operation. The elevated mRNA level
H. Ogawa; S. Kawamata; T. Gomi; Y. Ansai; Y. Karaki
Serine dehydratase expression decreases in rat livers injured by chronic thioacetamide ingestion
Serine dehydratase (SerDH) is a gluconeogenic enzyme involved in the catabolism of serine, which is regulated by the composition of their diet and their hormonal status in rats. This study examines how chronic injury caused to the liver of rats by the ingestion of thioacetamide (TAA) affects SerDH protein, mRNA levels, enzyme kinetics and its tissue location. After 97 days�
Inmaculada L�pez-Flores; Juan B. Barroso; Raquel Valderrama; Francisco J. Esteban; Esther Mart�nez-Lara; Francisco Luque; M. �ngeles Peinado; Hirofumi Ogawa; Jos� A. Lupi��ez; Juan Perag�n
Purification and properties of l -serine dehydratase from Lactobacillus fermentum ATCC 14931
l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM\\u000al-cysteine in 50 mM phosphate buffer. Mr=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (Mr=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent Km forl-serine was 65 mM. Fe++ was required for the enzymatic activity,
Marta E. Far�as; Ana M. Strasser de Saad; A�da A. Pesce de Ruiz Holgado; Guillermo Oliver
Alzheimer�s disease victim fibroblasts transport choline and serine more slowly than do normal fibroblasts
To test whether defects in the transport of acetylcholine precursors are expressed in fibroblasts from Alzheimer victims,\\u000a choline and serine influx were studied using 6 lines of Alzheimer cells and 5 lines of age-and sex-matched normals grown in\\u000a cultures. Choline and serine are transported into fibroblasts with pseudo-zero order kinetics for about 20 and 200 minutes,\\u000a respectively. Influx kinetic analysis
Lewis Carl Mokrasch
Serine Protease Inhibitor Attenuates Ovalbumin Induced Inflammation in Mouse Model of Allergic Airway Disease
BackgroundSerine proteases promote inflammation and tissue remodeling by activating proteinase-activated receptors, urokinase, metalloproteinases and angiotensin. In the present study, 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) a serine protease inhibitor was evaluated for prophylactic and therapeutic treatment in mouse model of airway allergy.MethodsBALB\\/c mice were sensitized by i.p route and challenged with ovalbumin. They were treated i.n. with 2, 10 and 50 �g
Sanjay Saw; Sagar Laxman Kale; Naveen Arora
Serine-rich protein is a novel positive regulator for silicon accumulation in mangrove.
Silicon (Si) plays an important role in reducing plant susceptibility against a variety of different biotic and abiotic stresses; and also has an important regulatory role in soil to avoid heavy metal toxicity and providing suitable growing conditions for plants. A full-length cDNAs of 696bp of serine-rich protein was cloned from mangrove plant (Rhizophora apiculata) by amplification of cDNA ends from an expressed sequence tag homologous to groundnut (Arachis hypogaea), submitted to NCBI (KF211374). This serine-rich protein gene encodes a deduced protein of 223 amino acids. The transcript titre of the serine-rich protein was found to be strongly enriched in roots compared with the leaves of two month old mangrove plants and expression level of this serine-rich protein was found to be strongly induced when the mangrove seedlings were exposed to SiO2. Expression of the serine-rich protein transgenic was detected in transgenic Arabidopsis thaliana, where the amount of serine increased from 1.02 to 37.8mg/g. The same trend was also seen in Si content in the roots (14.3%) and leaves (7.4%) of the transgenic A. thaliana compared to the wild-type plants under Si treatment. The biological results demonstrated that the accumulation of the serine amino acid in the vegetative tissues of the transgenic plants enhanced their ability to absorb and accumulate more Si in the roots and leaves and suggests that the serine-rich protein gene has potential for use in genetic engineering of different stress tolerance characteristics. PMID:25479011
Sahebi, Mahbod; Hanafi, Mohamed M; Siti Nor Akmar, A; Rafii, Mohd Y; Azizi, Parisa; Idris, A S
Regulation of BAD phosphorylation at serine 112 by the Ras-mitogen-activated protein kinase pathway
The function of the pro-apoptotic molecule BAD is regulated by phosphorylation of two sites, serine-112 (Ser-112) and serine-136 (Ser-136). Phosphorylation at either site results in loss of the ability of BAD to heterodimerize with the survival proteins BCL-XL or BCL-2. Phosphorylated BAD binds to 14-3-3 and is sequestered in the cytoplasm. It has been shown that phosphorylation of BAD at
Xianjun Fang; Shuangxing Yu; Astrid Eder; Muling Mao; Robert C Bast; Douglas Boyd; Gordon B Mills
Purification and characterization of serine racemase from a hyperthermophilic archaeon, Pyrobaculum islandicum.
Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100 degrees C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95 degrees C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 micromol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 micromol/min/mg, respectively. PMID:17965169
Ohnishi, Masato; Saito, Makoto; Wakabayashi, Sadao; Ishizuka, Morio; Nishimura, Katsushi; Nagata, Yoko; Kasai, Sabu
Evolutionary Analysis of Novel Serine Proteases in the Venom Gland Transcriptome of Bitis gabonica rhinoceros
Background Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites. Methodology and Principal Findings Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189). Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. Conclusions and Significance Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites. PMID:21731776
Vaiyapuri, Sakthivel; Wagstaff, Simon C.; Harrison, Robert A.; Gibbins, Jonathan M.; Hutchinson, E. Gail
SPINK9: A Selective, Skin-Specific Kazal-Type Serine Protease Inhibitor
A previously unreported Kazal-type serine protease inhibitor, serine protease inhibitor Kazal type 9 (SPINK9), was identified in human skin. SPINK9 expression was strong in palmar epidermis, but not detectable or very low in non palmoplantar skin. Analysis of a human cDNA panel showed intermediate expression in thymus, pancreas, liver, and brain, and low or undetectable expression in other tissues. Using
Maria Brattsand; Kristina Stefansson; Thomas Hubiche; Stefan K Nilsson; Torbj�rn Egelrud
Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6
Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK
Tomasz Kantyka; Jan Fischer; Zhihong Wu; Wim Declercq; Karina Reiss; Jens-Michael Schr�der; Ulf Meyer-Hoffert
Fission yeast p107wee1 mitotic inhibitor is a tyrosine\\/serine kinase
THE fission yeast wee1+ gene product is a dose-dependent, negative regulator of entry into mitosis1,2. wee1+ encodes a protein of relative molecular mass 107,000 (Mr 107K), the C-terminal third of which has strong similarities with the serine\\/threonine protein kinase family2,3. Here we report that p107wee1 immune complexes phosphorylate p107wee1 equally on serine and tyrosine residues, and also phosphorylate an exogenous
Carol Featherstone; Paul Russell
The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum
Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in
Verena P�ll; Ursula Denk; Horng-Der Shen; Raphael C. Panzani; Oliver Dissertori; Peter Lackner; Wolfgang Hemmer; Adriano Mari; Reto Crameri; Friedrich Lottspeich; Raphaela Rid; Klaus Richter; Michael Breitenbach; Birgit Simon-Nobbe
Enzymatic characterization of purified NS3 serine proteinase of hepatitis C virus expressed in Escherichia coli
Non-structural protein 3 (NS3) of the hepatitis C virus (HCV) has been shown to be a serine proteinase which cleaves the HCV polyprotein thus activating its replicative machinery. To characterize enzymatic activities of NS3 serine proteinase, the proteinase region was expressed in Escherichia coli and purified. The purified proteinase specifically cleaved a purified fusion protein sandwiching the NS5A\\/5B cleavage sequence.
Akiko Moria; Kazunori Yamada; Junko Kimura; Tomoko Koide; Satoshi Yuasa; Ei Yamadaa; Tatsuo Miyamura
Copper(II)-mediated O-arylation of protected serines and threonines.
An effective protocol toward the O-arylation of ?-hydroxy-?-amino acid substrates serine and threonine has been developed via Chan-Lam cross-coupling. This Cu(II)-catalyzed transformation involves benign open-flask conditions that are well-tolerated with a variety of protected (Boc-, Cbz-, Tr-, and Fmoc-) serine and threonine derivatives and various potassium organotrifluoroborates and boronic acids. PMID:25208062
El Khatib, Mirna; Molander, Gary A
Detecting surface deformation by phase stacking based on the PS
NASA Astrophysics Data System (ADS)
In the surface deformation monitoring, synthetic aperture radar differential interferometry (D-InSAR) has the advantages of all-weather, large-scale and high accuracy, it is hard to form interferogram for limited factors such as spatial decorrelation, temporal decorrelation and atmospheric effect. For the reason, the method of PS-DInSAR was proposed. However, the method needs so many SAR images, more than twenty scenes. Therefore, the method based on the phase stacking of PS for surface deformation monitoring was proposed and verified. The PS-DInSAR model and D-InSAR model are combined and simplified under certain conditions that assume the phase error of atmospheric disturbances are random and equal in an interferogram and the deformation is linear. The optimal master image for interferometric combinations is selected by comprehensive correlation function model. Then the PS points are detected and the Delaunay triangle is established according to the PS. The Minimum Cost Flow is used based on the Delaunay triangle of PS to unwrap the phase. Then the deformation and deformation rate are obtained by the linear analysis for temporal series of interferograms. At last, nine ENVISAT images captured during 2003.6-2006.3 in Tianjin area were processed, and the mean subsidence rate of this area was obtained.
Hao, Ming; Deng, Kazhong; Fan, Hongdong
Electrical and thermal behavior of PS/ferrite composite
NASA Astrophysics Data System (ADS)
This work aims to study the effect of gamma radiation on the structure, thermal and electrical properties of PS/ferrite composite. The Ni0.6Cd0.4Fe2-xSmxO4 was prepared using a conventional sintering ceramic process. Ferrite powder and Styrene was mixed and achieve polymerization process by gamma irradiation at 50 kGy. The composite samples have single spinel phase structure. Stability of the crystalline structure and no phase transition due to irradiation are found. The bulk density decreases whereas X-ray density increases with increasing Sm contents for both ferrite and PS/ferrite. The tetrahedral radii rA remains constant with Sm content but octahedral radii rB increases for both ferrite and PS/ferrite composite. The grain size shows increasing trend for PS/ferrite composite. The PS nearly coat the grains and so their boundaries become faint and not sharp. The gamma radiation transfer Fe3+ to Fe2+ due to its ionizing effect.The Fe2+ occupy octahedral site and the stretching vibration of its bond with oxygen (Fe2+-O2-) gives absorption at about 392 cm-1, near octahedral absorption at 462 cm-1.The PS/Ni0.6Cd0.4SmxFe2-xO4 composite becomes thermally more stable than pure polystyrene. The activation energy of conduction E? has a small values and in the range of hopping conduction mechanism.
Ashour, A. H.; Hemeda, O. M.; Heiba, Z. K.; Al-Zahrani, S. M.
Identification of a Major Determinant for Serine-Threonine Kinase Phosphoacceptor Specificity
Summary Eukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the �DFG+1� residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence. PMID:24374310
Chen, Catherine; Ha, Byung�Hak; Th�venin, Anastasia�F.; Lou, Hua�Jane; Zhang, Rong; Yip, Kevin�Y.; Peterson, Jeffrey�R.; Gerstein, Mark; Kim, Philip�M.; Filippakopoulos, Panagis; Knapp, Stefan; Boggon, Titus�J.; Turk, Benjamin�E.
Kinetic, mutagenic, and structural homology analysis of L-serine dehydratase from Legionella pneumophila.
A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe-4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their ? and ? domains are on the same (Type 2) or separate (Type 1) polypeptides. The ? domains contain the catalytic iron-sulfur center while the ? domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 ? domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni-Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with K(i) values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron-sulfur complex. PMID:21878319
Xu, Xiao Lan; Chen, Shawei; Grant, Gregory A
The role of Serine Proteases and Serine Protease Inhibitors in the migration of Gonadotropin-Releasing Hormone neurons
Background Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH) neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes. Results Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1), and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction. Conclusions These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations. PMID:11872147
Drapkin, Paola T; Monard, Denis; Silverman, Ann-Judith
Profiling the microRNA Expression in Human iPS and iPS-derived Retinal Pigment Epithelium
The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore, we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs, RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129�5p may play a role in promoting differentiation, while down-regulated miRNAs such as miR-367, miR-18b, and miR-20b are implicated in cell proliferation. Subsequent miRNA�target and network analysis revealed that these miRNAs are involved in cellular development, cell cycle progression, cell death, and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis, eye differentiation and development. PMID:25392691
Wang, Heuy-Ching; Greene, Whitney A; Kaini, Ramesh R; Shen-Gunther, Jane; Chen, Hung-I H; Cai, Hong; Wang, Yufeng
Cellular responses to L-serine in Saccharomyces cerevisiae: roles of general amino acid control, compartmentalization, and aspartate synthesis.
In addition to its other roles, L-serine functions in one-carbon metabolism and is interconvertable with glycine via serine hydroxymethyltransferases. However, the transcriptional response by Saccharomyces cerevisiae to L-serine addition is markedly different from that to glycine, with L-serine acting as a nutrient source rather than one-carbon units. Following addition of excess L-serine, 743 genes showed significant expression changes. Induced functions included amino acid synthesis, some stress responses, and FeS metabolism, while ribosomal RNA processing, ribosome biogenesis and hexose transport were repressed. A co-regulated network of ten transcription factors could together control more than 90% of the induced and repressed genes forming a general response to changes induced by other amino acids or stresses and including the general amino acid control system usually activated in response to starvation for amino acids. A specific response to L-serine was induction of CHA1 encoding serine (threonine) dehydratase. L-serine addition resulted in a substantial transient increase in L-aspartate, which is, rather than L-glutamate, the major metabolite for short-term storage of ammonia derived from degradation of L-serine. L-aspartate synthesis was exclusively through mitochondrial metabolism of L-serine to pyruvate and ammonia, involving Cha1p, cytoplasmic pyruvate carboxylases Pyc1p and Pyc2p, and the cytoplasmic aspartate aminotransferase Aat2p. PMID:23837815
Lee, Johnny C-Y; Tsoi, Abraham; Kornfeld, Geoffrey D; Dawes, Ian W
VALIDATION OF PS HEIGHT ESTIMATES BY MEANS OF PHOTOGRAMMETRY Daniele Perissin
the two independent measures confirms the theoretical sub-metric accuracy of vertical positioning. A PS coherent benchmarks (the PS's) and reconstructing their displacement history. PS's are stable "radar of the PS technique (about 1m) is much higher than the system resolution of the used sensors (20m x 5m
Perissin, Daniele
Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues
The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.
Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.; (Monash); (Queensland Inst. of Med. Rsrch.)
Structural mechanisms of inactivation in scabies mite serine protease paralogues.
The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 A and 2.0 A resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical "canonical" fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite. PMID:19427318
Fischer, Katja; Langendorf, Christopher G; Irving, James A; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H P; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A; McGowan, Sheena; Whisstock, James C; Pike, Robert N; Kemp, David J; Buckle, Ashley M
Breast Cancer-Associated pS2 Protein: Synthesis and Secretion by Normal Stomach Mucosa
The human pS2 gene is specifically expressed under estrogen transcriptional control in a subclass of estrogen receptor-containing human breast cancer cells. The pS2 gene encodes an 84-amino acid protein that is secreted after signal peptide cleavage. The distribution of pS2 protein in normal human tissues was studied with antibodies to pS2; pS2 was specifically expressed and secreted by mucosa cells
M. C. Rio; J. P. Bellocq; J. Y. Daniel; C. Tomasetto; R. Lathe; M. P. Chenard; A. Batzenschlager; P. Chambon
EphrinBs regulate D-serine synthesis and release in astrocytes
There is growing evidence that astrocytes play critical roles in neuron-glial interactions at the synapse. Astrocytes are believed to regulate pre- and post-synaptic structures and functions, in part, by the release of gliotransmitters such as glutamate, ATP and D-serine; however, little is known of how neurons and astrocytes communicate to regulate these processes. Here, we investigated a family of transmembrane proteins called ephrins and Eph receptors that are expressed in the synapse and are known to regulate synaptic transmission and plasticity. In addition to their presence on CA1 hippocampal neurons, we determined that ephrins and Eph receptors are also expressed on hippocampal astrocytes. Stimulation of hippocampal astrocytes with soluble ephrinB3, known to be expressed on CA1 post-synaptic dendrites, enhanced D-serine synthesis and release in culture. Conversely, ephrinB3 had no effect on D-serine release from astrocytes deficient in EphB3 and EphA4, which are the primary receptors for ephrinB3. Eph receptors mediate this response through interactions with PICK1 and by dephosphorylating PKC? to activate the conversion of L-serine to D-serine by serine racemase. These findings are supported in vivo, where reduced D-serine levels and synaptic transmissions are observed in the absence of EphB3 and EphA4. These data support a role for ephrins and Eph receptors in regulating astrocyte gliotransmitters, which may have important implications on synaptic transmission and plasticity. PMID:21106840
Zhuang, Zhiye; Yang, Bing; Theus, Michelle H; Sick, Justin T; Bethea, John R; Sick, Thomas J; Liebl, Daniel J
Antimicrobial activity of a honeybee (Apis cerana) venom Kazal-type serine protease inhibitor.
Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03�nM) and proteinase K (Ki 91.53�nM), but not against ?-chymotrypsin or trypsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor. PMID:24076031
Kim, Bo Yeon; Lee, Kwang Sik; Zou, Feng Ming; Wan, Hu; Choi, Yong Soo; Yoon, Hyung Joo; Kwon, Hyung Wook; Je, Yeon Ho; Jin, Byung Rae
Gabapentin and (S)-pregabalin decrease intracellular D-serine concentrations in PC-12 cells.
The effects of gabapentin (GBP) and (S)-pregabalin (PGB) on the intracellular concentrations of d-serine and the expression of serine racemase (SR) in PC-12 cells were determined. Intracellular d-serine concentrations were determined using an enantioselective capillary electrophoresis assay with laser-induced fluorescence detection. Increasing concentrations of GBP, 0.1-20?M, produced a significant decrease in d-serine concentration relative to control, 22.9�6.7% at 20?M (*p<0.05), with an IC(50) value of 3.40�0.29?M. Increasing concentrations of PGB, 0.1-10?M, produced a significant decrease in d-serine concentration relative to control, 25.3�7.6% at 10?M (*p<0.05), with an IC(50) value of 3.38�0.21?M. The compounds had no effect on the expression of monomeric-SR or dimeric-SR as determined by Western blotting. The results suggest that incubation of PC-12 cells with GBP and PGB reduced the basal activity of SR, which is most likely a result of the decreased Ca(2+) flux produced via interaction of the drugs with the ?(2)-? subunit of voltage-gated calcium channels. d-Serine is a co-agonist of the N-methyl d-aspartate receptor (NMDAR) and reduced d-serine concentrations have been associated with reduced NMDAR activity. Thus, GBP and PGB may act as indirect antagonists of NMDAR, a mechanism that may contribute to the clinical effects of the drugs in neuropathic pain. PMID:23274708
Singh, Nagendra S; Paul, Rajib K; Torjman, Marc C; Wainer, Irving W
An Essential Signal Peptide Peptidase Identified in an RNAi Screen of Serine Peptidases of Trypanosoma brucei
The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival. PMID:25816352
Moss, Catherine X.; Brown, Elaine; Hamilton, Alana; Van der Veken, Pieter; Augustyns, Koen; Mottram, Jeremy C.
Allosteric activation and contrasting properties of L-serine dehydratase types 1 and 2.
Bacterial L-serine dehydratases differ from mammalian L- and D-serine dehydratases and bacterial D-serine dehydratases by the presence of an iron-sulfur center rather than a pyridoxyl phosphate prosthetic group. They exist in two forms, types 1 and 2, distinguished by their sequence and oligomeric configuration. Both types contain an ASB domain, and the type 1 enzymes also contain an ACT domain in a tandem arrangement with the ASB domain like that in type 1 D-3-phosphoglycerate dehydrogenases (PGDHs). This investigation reveals striking kinetic differences between L-serine dehydratases from Bacillus subtilis (bsLSD, type 1) and Legionella pneumophila (lpLSD, type 2). lpLSD is activated by monovalent cations and inhibited by monovalent anions. bsLSD is strongly activated by cations, particularly potassium, and shows a mixed response to anions. Flouride is a competitive inhibitor for lpLSD but an apparent activator for bsLSD at low concentrations and an inhibitor at high concentrations. The reaction products, pyruvate and ammonia, also act as activators but to different extents for each type. Pyruvate activation is competitive with L-serine, but activation of the enzyme is not compatible with it simply competing for binding at the active site and suggests the presence of a second, allosteric site. Because activation can be eliminated by higher levels of L-serine, it may be that this second site is actually a second serine binding site. This is consistent with type 1 PGDH in which the ASB domain functions as a second site for substrate binding and activation. PMID:22686449
Chen, Shawei; Xu, Xiao Lan; Grant, Gregory A
Identification and Characterization of Fusolisin, the Fusobacterium nucleatum Autotransporter Serine Protease
Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61�65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55�101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96�113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55�65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections. PMID:25357190
Ibrahim, Yara; Eini, Amir; Naor, Ronit; Rosen, Graciela; Bachrach, Gilad
Gabapentin and (S)-pregabalin decrease intracellular D-Serine concentrations in PC-12 cells
The effects of gabapentin (GBP) and (S)-pregabalin (PGB) on the intracellular concentrations of D-serine and the expression of serine racemase (SR) in PC-12 cells were determined. Intracellular D-serine concentrations were determined using an enantioselective capillary electrophoresis assay with laser-induced fluorescence detection. Increasing concentrations of GBP, 0.1 � 20 ?M, produced a significant decrease in D-serine concentration relative to control, 22.9 � 6.7% at 20 ?M (*p < 0.05), with an IC50 value of 3.40 � 0.29 ?M. Increasing concentrations of PGB, 0.1 � 10 ?M, produced a significant decrease in D-serine concentration relative to control, 25.3 � 7.6% at 10 ?M (*p < 0.05), with an IC50 value of 3.38 � 0.21 ?M. The compounds had no effect on the expression of monomeric-SR or dimeric-SR as determined by Western blotting. The results suggest that incubation of PC-12 cells with GBP and PGB reduced the basal activity of SR, which is most likely a result of the decreased Ca+2 flux produced via interaction of the drugs with the ?2-? subunit of voltage-gated calcium channels. D-serine is a co-agonist of the N-methyl D-aspartate receptor (NMDAR) and reduced D-serine concentrations have been associated with reduced NMDAR activity. Thus, GBP and PGB may act as indirect antagonists of NMDAR, a mechanism that may contribute to the clinical effects of the drugs in neuropathic pain. PMID:23274708
Singh, Nagendra S.; Paul, Rajib K.; Torjman, Marc C.; Wainer, Irving W.
Characterization of crosslinked polystyrene(PS) beads in SBR matrix
Monodisperse sized crosslinked polystyrene(PS) beads were prepared by a reaction of semibatch emulsion polymerization with styrene monomer, divinylbenzene(DVB) crosslinking agent and potassium persulfate(K{sub 2}S{sub 2}O{sub 9}) initiator in the absence of emulsifier. The glass transition temperature(T{sub g}) and the mean diameter of the beads were increased from 100{degrees}C to 135{degrees}C and from 402 nm to 532 nm, respectively, for an incorporation of 2 to 10 mol% DVB. Crosslinking density was also linearly increased with DVB content. SEM microphotographs of SBR composite filled with various contents of PS beads revealed that PS beads are relatively well dispersed without changing the spherical shape of the beads in all range of compositions. In stress-strain analysis, elongation at break and tensile strength of SBR composite were increased with the bead content. Applicability of the PS beads as a filler in SBR matrix is tested by plotting Mooney-Rivlin or Guth-Smallwood equations. However, mechanical properties of the composite with the beads were not so excellent as those of the composite with carbon black. Crosslinked PS beads are still tentative as a white color reinforcing filler on SBR matrix.
Cha, Yoon-Jong; Choe, Soonja [Inha Univ. (Korea, Republic of)
High-resolution PS-OCT of Enamel Remineralization
Previous studies have demonstrated that Polarization Sensitive Optical Coherence Tomography (PS-OCT) can be used to image the remineralization of early artificial caries lesions. However, the depth resolution of the imaging system employed in those previous studies was limited and the outer surface structure of the lesions were not resolved as clearly as desired. The purpose of this study was to repeat the earlier remineralization study using a broadband light-source of higher resolution to determine if there can be improved resolution of the remineralized surface zones of the lesions. An all polarization-maintaining fiber based PS-OCT system operating at 1310-nm was used to acquire polarization resolved images of bovine enamel surfaces exposed to a demineralizing solution at pH-4.9 followed by a fluoride containing remineralizing solution at pH-7.0 containing 2-ppm fluoride. The structure of the surface zones could be clearly resolved using PS-OCT in the samples that underwent remineralization. The PS-OCT measurements indicated a significant (p<0.05) reduction in the integrated reflectivity between the severity of the lesions that were exposed to the remineralization solution and those that were not. The lesion depth and mineral loss were also measured with polarized light microscopy and transverse microradiography after sectioning the enamel blocks. These results show that PS-OCT can be used to non-destructively monitor the remineralization potential of anti-caries agents. PMID:21909226
Can, Anna M.; Darling, Cynthia L.; Fried, Daniel
Lack of the alanine�serine�cysteine transporter 1 causes tremors, seizures, and early postnatal death in mice
The Na+-independent alanine�serine�cysteine transporter 1 (Asc-1) is exclusively expressed in neuronal structures throughout the central nervous system (CNS). Asc-1 transports small neutral amino acids with high affinity especially for d-serine and glycine (Ki: 8�12 ?M), two endogenous glutamate co-agonists that activate N-methyl-d-aspartate (NMDA) receptors through interacting with the strychnine-insensitive glycine binding-site. By regulating d-serine (and possibly glycine) levels in the
Xinmin Xie; Theodore Dumas; Lamont Tang; Thomas Brennan; Thadd Reeder; Winston Thomas; Robert D. Klein; Judith Flores; Bruce F. O'Hara; H. Craig Heller; Paul Franken
Isolation of PsPIN2 and PsAUX1 from etiolated pea epicotyls and their expression on a three-dimensional clinostat
NASA Astrophysics Data System (ADS)
We isolated novel cDNAs containing the complete open reading frames of a putative auxin influx carrier, PsAUX1, and a putative auxin efflux carrier, PsPIN2, from etiolated pea epicotyls. High levels of homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 (Accession No. AY222857) and AtPINs. Phylogenetic analyses based on deduced amino acid sequences revealed that PsPIN2 belonged to a subclade including AtPIN3, AtPIN4 and AtPIN7, while PsPIN1 belonged to the same clade as AtPIN1. The results were similar for PsAUX1 and AtAUX1, where PsAUX1 belongs to the same subclade as AtAUX1 and CS-AUX1. Expression of PsPIN1, PsPIN2 and PsAUX1 in pea epicotyl segments was promoted upon incubation of the segments with auxin (indole-3-acetic acid). In 3.5-d-old etiolated pea seedlings, relatively high expression of PsPIN1 and PsAUX1 was observed in the hook region, growing epicotyls and root tips as compared with those in mature regions of epicotyls and roots. Expression of PsPIN2 in roots was less than that in shoots. Simulated microgravity conditions on a three-dimensional clinostat remarkably increased gene expression of PsPIN1 and PsAUX1 in the hook and the internodes of pea epicotyls, but the increase in PsPIN2 was less. In contrast, polar auxin transport of pea epicotyls was substantially suppressed under simulated microgravity conditions on a 3D clinostat, similar to data from a space experiment on STS-95. These results suggest that PsPINs and PsAUX1 are auxin-inducible genes, and that the expression of PsPINs and PsAUX1 genes is sensitive to gravistimulation.
Hoshino, Tomoki; Hitotsubashi, Reiko; Miyamoto, Kensuke; Tanimoto, Eiichi; Ueda, Junichi
The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum.
Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in Cladosporium herbarum. Hence, a screening of a C. herbarum cDNA library was performed using the coding sequence of the Penicillium oxalicum vacuolar serine protease (Pen o 18) as hybridization probe, ending up with a full-length clone. Biochemical and immunological characterization of this clone revealed that C. herbarum vacuolar serine protease most likely is synthesized as a precursor with an N-terminal pro-enzyme sequence and represents a minor allergen (Cla h 9) with a prevalence of IgE-reactivity of 15.5%. Furthermore Cla h 9 specifically reacted with the two monoclonal antibodies FUM20 and PCM39, as do the vacuolar serine proteases from Aspergillus fumigatus and Penicillium species. Investigation of IgE-cross-reactivity between Cla h 9 and other fungal serine proteases revealed that cross-reactivity is higher between vacuolar than alkaline serine proteases. IgE-epitope mapping of Cla h 9 was done in order to test whether four Cla h 9-peptides having a high sequence homology to previously determined Pen ch 18-IgE-epitopes also harbour IgE-epitopes. Three-dimensional models of the vacuolar serine proteases from C. herbarum and Penicillium chrysogenum were generated for the three-dimensional localization of the Cla h 9- and Pen ch 18- IgE-reactive and -non-reactive peptides. Taken together a new C. herbarum allergen has been identified, which may be useful in a molecule-based approach of C. herbarum allergy-diagnosis and -therapy. Moreover, Cla h 9 represents a further member of the subtilisin-like serine protease allergen-family, which stresses the importance of these proteins with respect to fungal IgE-cross-reactivity. PMID:19162325
P�ll, Verena; Denk, Ursula; Shen, Horng-Der; Panzani, Raphael C; Dissertori, Oliver; Lackner, Peter; Hemmer, Wolfgang; Mari, Adriano; Crameri, Reto; Lottspeich, Friedrich; Rid, Raphaela; Richter, Klaus; Breitenbach, Michael; Simon-Nobbe, Birgit
The N-methyl D-aspartate receptor glycine site and D-serine metabolism: an evolutionary perspective.
The N-methyl D-aspartate (NMDA) type of glutamate receptor requires two distinct agonists to operate. Glycine is assumed to be the endogenous ligand for the NMDA receptor glycine site, but this notion has been challenged by the discovery of high levels of endogenous d-serine in the mammalian forebrain. I have outlined an evolutionary framework for the appearance of a glycine site in animals and the metabolic events leading to high levels of D-serine in brain. Sequence alignments of the glycine-binding regions, along with the scant experimental data available, suggest that the properties of invertebrate NMDA receptor glycine sites are probably different from those in vertebrates. The synthesis of D-serine in brain is due to a pyridoxal-5'-phosphate (B(6))-requiring serine racemase in glia. Although it remains unknown when serine racemase first evolved, data concerning the evolution of B(6) enzymes, along with the known occurrences of serine racemases in animals, point to D-serine synthesis arising around the divergence time of arthropods. D-Serine catabolism occurs via the ancient peroxisomal enzyme d-amino acid oxidase (DAO), whose ontogenetic expression in the hindbrain of mammals is delayed until the postnatal period and absent from the forebrain. The phylogeny of D-serine metabolism has relevance to our understanding of brain ontogeny, schizophrenia and neurotransmitter dynamics. PMID:15306409
Schell, Michael J
Positron annihilation in the MuPs system
The life-time of the four-body atomic system MuPs ($\\mu^{+} e^{-}_2 e^{+}$ or muonium-positronium) against positron annihilation has been evaluated as $\\tau = \\frac{1}{\\Gamma} \\approx 4.076453 \\cdot 10^{-10}$ $sec$. Various annihilation rates for MuPs are determined to a good numerical accuracy, e.g., $\\Gamma_{2 \\gamma} \\approx$ 2.446485$\\cdot 10^{9}$ $sec^{-1}$, $\\Gamma_{3 \\gamma} \\approx$ 6.62798$\\cdot 10^{6}$ $sec^{-1}$, $\\Gamma_{4 \\gamma} \\approx$ 3.61680$\\cdot 10^{3}$ $sec^{-1}$, $\\Gamma_{5 \\gamma} \\approx$ 6.32973 $sec^{-1}$. The hyperfine structure splitting for the ground state in the MuPs system has also been evaluated as $\\Delta$ = 23.078 $MHz$.
Alexei M. Frolov
Real-time cardiovascular MRI is a useful and challenging dynamic imaging application. The partial separability (PS) model enables reconstruction of dynamic cardiac images from highly undersampled (k, t)-space data. However, the underlying PS model-based reconstruction problem is ill-conditioned, so regularization is often necessary to stabilize its solution. It has been shown that ?1 regularization is useful for finding sparse solutions, and ?2 regularization is widely used to incorporate anatomical constraints. An important practical question is which regularization scheme to use for PS model-based cardiovascular imaging. We address this problem by implementing both schemes and evaluating their performances in terms of reconstruction error, image artifacts, image noise, computation time, and performance characterizability. The ?1-regularized results exhibit lower reconstruction error, artifact energy, and noise variance, while ?2 regularization is much faster and produces predictable reconstruction results. This study indicates that the ?1 scheme is preferable when image quality is the main concern. PMID:25283177
Christodoulou, Anthony G.; Zhao, Bo; Liang, Zhi-Pei
Pan-STARRS PS1 Observatory, Telescope and Instrument Control
NASA Astrophysics Data System (ADS)
An ultimate goal for the final Pan-STARRS array (PS4) is fully robotic facility operations. To facilitate that development, PS1 is a remotely operable observatory that includes many features of the future robotic Pan-STARRS. Both remote and robotic operational concepts require sufficient knowledge of environmental conditions to support survey scheduling, and this requires an auxiliary instrumentation suite to measure meteorological and atmospheric conditions in addition to the control systems for the observatory and telescope. For the Pan-STARRS PS1 prototype, the monitoring, control, and summit subsystem coordination are handled by the HW/SW within the Observatory, Telescope, and Instrumentation Subsystem (OTIS). In this talk, we present the functional capabilities of OTIS, and the HW and SW architectures designed to meet the subsystem operational requirements. OTIS performance through early commissioning is described as well as the future plans to incorporate the Pan-STARRS scheduler into system operations.
Pier, E.; Chambers, K.
The Cutting Edge: Membrane Anchored Serine Protease Activities in the Pericellular Microenvironment
Synopsis The serine proteases of the trypsin-like (S1) family play critical roles in many key biological processes including digestion, blood coagulation, and immunity. Recent studies have identified members of this family which contain amino- or carboxy-terminal domains that serve to tether the serine protease catalytic domain directly at the plasma membrane. These membrane anchored serine proteases are proving to be key components of the cell machinery for activation of precursor molecules in the pericellular microenvironment, playing vital functions in the maintenance of homeostasis. Substrates activated by membrane anchored serine proteases include peptide hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules and viral coat proteins. In addition, new insights into our understanding of the physiological functions of these proteases and their involvement in human pathology have come from animal models and patient studies. This review discusses emerging evidence for the diversity of this fascinating group of membrane serine proteases as potent modifiers of the pericellular microenvironment through proteolytic processing of diverse substrates. We also discuss the functional consequences of the activities of these proteases on mammalian physiology and disease. PMID:20507279
Antalis, Toni M.; Buzza, Marguerite S.; Hodge, Kathryn M.; Hooper, John D.; Netzel-Arnett, Sarah
Cell-type specific mechanisms of D-serine uptake and release in the brain.
Accumulating evidence during the last decade established that D-serine is a key signaling molecule utilized by neurons and astroglia in the mammalian central nervous system. D-serine is increasingly appreciated as the main physiological endogenous coagonist for synaptic NMDA receptors at central excitatory synapses; it is mandatory for long-term changes in synaptic strength, memory, learning, and social interactions. Alterations in the extracellular levels of D-serine leading to disrupted cell-cell signaling are a trademark of many chronic or acute neurological (i.e., Alzheimer disease, epilepsy, stroke) and psychiatric (i.e., schizophrenia) disorders, and are associated with addictive behavior (i.e., cocaine addiction). Indeed, fine tuning of the extracellular levels of D-serine, achieved by various molecular machineries and signaling pathways, is necessary for maintenance of accurate NMDA receptor functions. Here, we review the experimental data supporting the notion that astroglia and neurons use different pathways to regulate levels of extracellular D-serine. PMID:24910611
Martineau, Magalie; Parpura, Vladimir; Mothet, Jean-Pierre
Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma
Mistranslation from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences1-3. Throughout evolution, two editing-checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly1,2. One checkpoint is the AlaRS editing center, while the other is from widely distributed AlaXps�free-standing, genome-encoded editing proteins that clear Ser-tRNAAla. The paradox of misincorporating both a smaller (glycine) and a larger amino acid (serine) suggests a deep conflict for nature-designed AlaRS. To understand the chemical basis for this conflict, kinetic and mutational analysis, together with nine crystal structures, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the ?�amino group of the bound amino acid using an acidic residue. This design, of more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently not able to find better architecture for recognition of alanine, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery. PMID:20010690
Guo, Min; Chong, Yeeting E.; Shapiro, Ryan; Beebe, Kirk; Yang, Xiang-Lei; Schimmel, Paul
Bioinformatics analysis of the serine and glycine pathway in cancer cells
Serine and glycine are amino acids that provide the essential precursors for the synthesis of proteins, nucleic acids and lipids. Employing 3 subsequent enzymes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine phosphatase (PSPH), phosphoserine aminotransferase 1 (PSAT1), 3-phosphoglycerate from glycolysis can be converted in serine, which in turn can by converted in glycine by serine methyl transferase (SHMT). Besides proving precursors for macromolecules, serine/glycine biosynthesis is also required for the maintenance of cellular redox state. Therefore, this metabolic pathway has a pivotal role in proliferating cells, including cancer cells. In the last few years an emerging literature provides genetic and functional evidences that hyperactivation of serine/glycine biosynthetic pathway drives tumorigenesis. Here, we extend these observations performing a bioinformatics analysis using public cancer datasets. Our analysis highlighted the relevance of PHGDH and SHMT2 expression as prognostic factor for breast cancer, revealing a substantial ability of these enzymes to predict patient survival outcome. However analyzing patient datasets of lung cancer our analysis reveled that some other enzymes of the pathways, rather than PHGDH, might be associated to prognosis. Although these observations require further investigations they might suggest a selective requirement of some enzymes in specific cancer types, recommending more cautions in the development of novel translational opportunities and biomarker identification of human cancers. PMID:25436979
Morello, Maria; Minieri, Marilena; Melino, Gerry; Amelio, Ivano
Occurrence of a catabolic L-serine (L-threonine) deaminase in Saccharomyces cerevisiae.
Saccharomyces cerevisiae mutants lacking the anabolic L-threonine deaminase, the ilv1- mutants, have been found to exhibit a normal ability to grow, without auxotrophy towards isoleucine, on L-threonine of L-serine as only nitrogen nutrient. Starting from a strain carrying a ilv1- mutation, a new mutation affecting the ability to utilize L-threonine as nitrogen source was selected. This mutation, which also impairs the ability to utilize L-serine, has been denominated cha-, for 'catabolism of hydroxyamino acids' and was found to result in the lack of a catabolic L-serine (L-threonine) deaminase. This enzyme which, unlike the anabolic threonine deaminase, is more active towards serine than towards threonine, differs from the latter enzyme by a number of biochemical and regulatory properties. Whereas the anabolic enzyme is an allosteric enzyme sensitive to feedback inhibition by isoleucine, the catabolic enzyme exhibits Michaelian kinetics: no control of its activity has been detected. Its synthesis is induced by L-serine and L-threonine. These two enzymes, which thus can be easily differentiated by means of their regulations, display a limited ability to compensate for one another's absence and appear to play clearly distinct roles under normal physiological conditions. PMID:7042346
Ramos, F; Wiame, J M
Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.
A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 � 10(3) mM(-1) min(-1)vs. 1.3 � 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence. PMID:25386732
Ding, Xiaokang; Yang, Kun-Lin
Antibody-dependent tumour cytolysis by human neutrophils: effect of synthetic serine esterase inhibitors and substrates.
The requirement for serine esterase activity in antibody-dependent cellular cytotoxicity (ADCC) in human neutrophils against Raji target cells has been investigated. The lysis was prevented when the serine esterase inhibitors TPCK and TLCK (chloromethyl-ketone derivatives of tosylamino acids) were introduced into the system. Moreover, neutrophils pretreated with TPCK or TLCK and washed were inhibited as well, via a process unaffected by the presence of adequate amounts of enzymatic substrates. This suggests that the inhibition mediated by TPCK and TLCK is independent of serine esterase blockade, therefore implying the inactivation of some other step crucial to the lysis. The addition of synthetic chymotrypsin substrates (tyrosine and phenylalanine esters) impaired the Raji cell lysis in a dose-related manner without altering the constitution of neutrophil-target conjugates. Trypsin ester substrates were ineffective. These results are in agreement with the involvement of a serine esterase activity with chymotrypsin-like specificity, which should participate in the lysis at a post-binding step. We conclude that neutrophil-mediated ADCC, as developed in our model system, needs the intervention of a serine esterase or esterases, like other systems of cell-mediated cytotoxicity. PMID:3666787
Dallegri, F; Frumento, G; Ballestrero, A; Goretti, R; Torresin, A; Patrone, F
Proper Motions from the Pan-STARRS PS1 Survey
NASA Astrophysics Data System (ADS)
Preliminary studies of the PS1 astrometric capabilities have been presented at previous AMOS Technical Conferences. As of the 2009 AMOS deadline, PS1 is scheduled to go into routine survey operations in May 2009. Hence, large amounts of data should be available by the time of the conference, and detailed astrometric studies and results will be done on as much of the data as possible. All preliminary work suggests that with four months of epoch difference, proper motions as small as 0.1 arcsecond per year should be easily detectable.
Monet, D.; Ps1 Team
iPS Cell Modeling of Cardiometabolic Diseases
Cardiometabolic diseases encompass simple monogenic enzyme deficiencies with well-established pathogenesis and clinical outcomes to complex polygenic diseases such as the cardiometabolic syndrome. The limited availability of relevant human cell types such as cardiomyocytes has hampered our ability to adequately model and study pathway or drugs relevant to these diseases in the heart. The recent discovery of induced pluripotent stem (iPS) cell technology now offers a powerful opportunity to establish translational platforms for cardiac disease modeling, drug discovery and pre-clinical testing. In this article, we discuss the excitement and challenges of modeling cardiometabolic diseases using iPS cell and their potential to revolutionize translational research. PMID:23070616
Nakamura, Kenta; Hirano, Ken-ichi; Wu, Sean M.
TrpB2 enzymes are O-phospho-l-serine dependent tryptophan synthases.
The rapid increase of the number of sequenced genomes asks for the functional annotation of the encoded enzymes. We used a combined computational-structural approach to determine the function of the TrpB2 subgroup of the tryptophan synthase ? chain/? chain-like TrpB1-TrpB2 family (IPR023026). The results showed that TrpB2 enzymes are O-phospho-l-serine dependent tryptophan synthases, whereas TrpB1 enzymes catalyze the l-serine dependent synthesis of tryptophan. We found a single residue being responsible for the different substrate specificities of TrpB1 and TrpB2 and confirmed this finding by mutagenesis studies and crystallographic analysis of a TrpB2 enzyme with bound O-phospho-l-serine. PMID:25184516
Busch, Florian; Rajendran, Chitra; Mayans, Olga; L�ffler, Patrick; Merkl, Rainer; Sterner, Reinhard
Conservation of sequence and function in fertilization of the cortical granule serine protease in echinoderms.
Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding. PMID:24878526
Oulhen, Nathalie; Xu, Dongdong; Wessel, Gary M
Serine biosynthesis by photorespiratory and non-photorespiratory pathways: an interesting interplay with unknown regulatory networks.
Photorespiration is a primary metabolic pathway, which, given its energy costs, has often been viewed as a wasteful process. Despite having reached the consensus that one important function of photorespiration is the removal of toxic metabolite intermediates, other possible functions have emerged, and others could well emerge in the future. As a primary metabolic pathway, photorespiration interacts with other routes; however the nature of these interactions is not well known. One of these interacting pathways could be the biosynthesis of serine, since this amino acid is synthesised through photorespiratory and non-photorespiratory routes. At present, the exact contribution of each route to serine supply in different tissues and organs, their biological significance and how pathways are integrated and/or regulated remain unknown. Here, we review the non-photorespiratory serine biosynthetic pathways, their interactions with the photorespiratory pathway, their putative role in plants and their biotechnological interest. PMID:23199004
Ros, R; Cascales-Mi�ana, B; Segura, J; Anoman, A D; Toujani, W; Flores-Tornero, M; Rosa-Tellez, S; Mu�oz-Bertomeu, J
Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host
Dengue virus (DENV), a flavivirus of global importance, is transmitted to humans by mosquitoes. In this study, we developed in vitro and in vivo models of saliva-mediated enhancement of DENV infectivity. Serine protease activity in Aedes aegypti saliva augmented virus infectivity in vitro by proteolyzing extracellular matrix proteins, thereby increasing viral attachment to heparan sulfate proteoglycans and inducing cell migration. A serine protease inhibitor reduced saliva-mediated enhancement of DENV in vitro and in vivo, marked by a 100-fold reduction in DENV load in murine lymph nodes. A saliva-mediated infectivity enhancement screen of fractionated salivary gland extracts identified serine protease CLIPA3 as a putative cofactor, and short interfering RNA knockdown of CLIPA3 in mosquitoes demonstrated its role in influencing DENV infectivity. Molecules in mosquito saliva that facilitate viral infectivity in the vertebrate host provide novel targets that may aid in the prevention of disease. PMID:24131723
Conway, Michael J.; Watson, Alan M.; Colpitts, Tonya M.; Dragovic, Srdjan M.; Li, Zhiyong; Wang, Penghua; Feitosa, Fabiana; Shepherd, Denueve T.; Ryman, Kate D.; Klimstra, William B.; Anderson, John F.
Role of a conserved arginine residue during catalysis in serine palmitoyltransferase.
All sphingolipid-producing organisms require the pyridoxal 5'-phosphate (PLP)-dependent serine palmitoyltransferase (SPT) to catalyse the first reaction on the de novo sphingolipid biosynthetic pathway. SPT is a member of the alpha oxoamine synthase (AOS) family that catalyses a Claisen-like condensation of palmitoyl-CoA and L-serine to form 3-ketodihydrosphingosine (KDS). Protein sequence alignment across various species reveals an arginine residue, not involved in PLP binding, to be strictly conserved in all prokaryotic SPTs, the lcb2 subunits of eukaryotic SPTs and all members of the AOS family. Here we use UV-vis spectroscopy and site-directed mutagenesis, in combination with a substrate analogue, to show that the equivalent residue (R370) in the SPT from Sphingomonas wittichii is required to form the key PLP:L-serine quinonoid intermediate that condenses with palmitoyl-CoA and thus plays an essential role enzyme catalysis. PMID:21514297
Lowther, Jonathan; Charmier, Guillaume; Raman, Marine C; Ikushiro, Hiroko; Hayashi, Hideyuki; Campopiano, Dominic J
Modelling of tumour--host coexistence In vitro in the presence of serine protease inhibitors.
The activities of cell surface serine proteases are markedly enhanced in malignant tumours. Proteolytic degradation of the extracellular matrix and basal membrane of normal cells is an important event for tumour cell growth and invasion. Two well-known broad-spectrum inhibitors of serine protease, Foy-305 and Ono-3403, were evaluated for their ability to affect the growth rate and survival of MCF7 breast cancer cells co-cultured with MRC5 lung fibroblasts as feeder cells in the absence of serum. Flow cytometry and differential staining demonstrated that in the mixed culture, the rate of tumor growth was dependent upon the presence of the feeder MRC5 lung fibroblasts and could be obviated by the additional presence of the inhibitors of serine proteases. PMID:19779105
Engi, Helga; Gy�m�nt, N�ra; Ohkoshi, Motohiro; Amaral, Leonard; Moln�r, Joseph
Identification of the active-site serine in human lecithin: cholesterol acyltransferase
Lecithin:cholesterol acyltransferase (LCAT) from human plasma reacts stoichiometrically with diisopropylphosphorofluoridate (DFP) resulting in the complete loss of transacylase activity. Purified LCAT was covalently labeled with (TH) DFP and the labeled protein was reduced and carboxymethylated. Cyanogen bromide cleavage followed by gel permeation chromatography yielded a peptide of 4-5 KDa (LCAT CNBr-III) containing most of the radioactive label. Preliminary studies comparing the amino acid composition of the LCAT-CNBr-III with the sequence of LCAT indicate that this peptide corresponds to fragment 168-220. Automated Edman degradation of the radioactive peptide recovered a radioactive PTC-amino acid at cycle 14. Of all predicted CNBr fragments only peptide 168-220 contained a serine at residue 14 from the amino terminus of the peptide. The authors conclude that serine 181 is the active site serine of LCAT.
Farooqui, J.; Wohl, R.C.; Kezdy, F.J.; Scanu, A.M.
PS-b-PAA as a high ? polymer for directed self-assembly: a study of solvent and thermal annealing processes for PS-b-PAA
NASA Astrophysics Data System (ADS)
Poly(styrene)-b-poly(acrylic acid) copolymers (PS-b-PAA) was shown to be one promising material for achieving substantially smaller pitch patterns than PS-b-PMMA while still retaining high etch contrast and application for chemoepitaxy. Phase separation of acetone vapor annealed PS-b-PAA (Mw=16,000 g/mol with 50:50 volume ratio of PS: PAA) on PS brush achieved a lamellar morphology with a pattern pitch size (L0) of 30 nm. However the thermal annealing of the same PS-b-PAA generated a dramatically larger pitch size of 43 nm. SEM and GPC analysis revealed that the intermolecular crosslinking during thermal annealing process has increased the effective N (degree of polymerization), which suggests that even a small amount of crosslinking would lead to big pitch change. Thus, PS-b-PAA is not suitable for fast thermal annealing process as it loses pitch size control due to PAA crosslinking.
Cheng, Jing; Lawson, Richard A.; Yeh, Wei-Ming; Jarnagin, Nathan D.; Tolbert, Laren M.; Henderson, Clifford L.
Region-specific metabolic alterations in the brain of the APP/PS1 transgenic mice of Alzheimer's disease.
Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide, but its etiology is still not completely understood. The identification of underlying pathological mechanisms is becoming increasingly important for the discovery of biomarkers and therapies, for which metabolomics presents a great potential. In this work, we studied metabolic alterations in different brain regions of the APP/PS1 mice by using a high-throughput metabolomic approach based on the combination of gas chromatography-mass spectrometry and ultra-high performance liquid chromatography-mass spectrometry. Multivariate statistics showed that metabolomic perturbations are widespread, affecting mainly the hippocampus and the cortex, but are also present in regions not primarily associated with AD such as the striatum, cerebellum and olfactory bulbs. Multiple metabolic pathways could be linked to the development of AD-type disorders in this mouse model, including abnormal purine metabolism, bioenergetic failures, dyshomeostasis of amino acids and disturbances in membrane lipids, among others. Interestingly, region-specific alterations were observed for some of the potential markers identified, associated with abnormal fatty acid composition of phospholipids and sphingomyelins, or differential regulation of neurotransmitter amino acids (e.g. glutamate, glycine, serine, N-acetyl-aspartate), not previously described to our knowledge. Therefore, these findings could provide a new insight into brain pathology in Alzheimer's disease. PMID:25281826
Gonz�lez-Dom�nguez, Ra�l; Garc�a-Barrera, Tamara; Vitorica, Javier; G�mez-Ariza, Jos� Luis
Tau Phosphorylation at Serine 396 Residue Is Required for Hippocampal LTD
Tau is required for the induction of long-term depression (LTD) of synaptic transmission in the hippocampus. Here we probe the role of tau in LTD, finding that an AMPA receptor internalization mechanism is impaired in tau KO mice, and that LTD causes specific phosphorylation at the serine 396 and 404 residues of tau. Surprisingly, we find that phosphorylation at serine 396, specifically, is critical for LTD but has no role in LTP. Finally, we show that tau KO mice exhibit deficits in spatial reversal learning. These findings underscore the physiological role for tau at the synapse and identify a behavioral correlate of its role in LTD. PMID:25810511
Regan, Philip; Piers, Thomas; Yi, Jee-Hyun; Kim, Dong-Hyun; Huh, Seonghoo; Park, Se Jin; Ryu, Jong Hoon; Whitcomb, Daniel J.
Inhibition of monkey liver serine hydroxymethyltransferase by Cibacron Blue 3G-A.
Cibacron Blue 3G-A inhibited monkey liver serine hydroxymethyltransferase competitively with respect to tetrahydrofolate and non-competitively with respect to L-serine. NADH, a positive heterotropic effector, failed to protect the enzymes against inhibition by the dye and was unable to desorb the enzyme from Blue Sepharose CL-6B gel matrix. The binding of the dye to the free enzyme was confirmed by changes in the dye absorption spectrum. The results indicate that the dye probably binds at the tetrahydrofolate-binding domain of the enzyme, rather than at the 'dinucleotide fold'. PMID:6773521
Ramesh, K S; Appaji Rao, N
Petroleum Technology (AS) Curriculum Guide Student Name: PS#
Petroleum Technology (AS) Curriculum Guide Student Name: PS# GENERAL EDUCATION REQUIREMENTS ENG Introduction to Petroleum Industry PET 0102 Environment and Safety PET 0103 Introduction to Petroleum Geology PET 0201 Petroleum & Natural Gas Chemistry PET 0203 Oil & Gas Gathering & Transportation PET 0204 Well
Jiang, Huiqiang
T i ps to Avoid P hishing Scam s
UW-Madison T i ps to Avoid P hishing Scam s #12;This brochure provides tips to identify and avoid numbers, NetID, passwords, protected health information). � The message creates a sense of urgency the site's encryption certificate (if one is present). � Do use common sense. If you have any doubts, don
Sheridan, Jennifer
PS10 Solar Power Tower Xi Jing, Fang
PS10 Solar Power Tower Xi Jing, Fang #12;Overview Magnitudes , Cost & TechnologiesMagnitudes , Cost the turbine at 50% load #12;Concentrating Solar Power (CSP) Heliostats concentrates the Sun's rays to receiver the solar energy to the grid in 2007 Operating cash flow 1.4 millions in 2007.Operating cash flow 1
Prevedouros, Panos D.
The upper mantle discontinuities in western Canada from Ps conversions
We have investigated variations in the travel times ofPs converted phases from the upper mantle 410 and 660 km discontinuities recorded on the western stations of the Canadian National Seismograph Network using a variant of the technique introduced byVinnik (1977). Clear and unambiguous signals for both discontinuities are observed at 8 of the 11 stations considered and exhibit variations which
M. G. Bostock; J. F. Cassidy
A 100 ps gated x-ray spectrometer
Material opacities are of interest in many fields. We have developed a Bragg reflection spectrometer that is gated for imaging samples in a laser heated environment for opacity measurement. A micro-channel plate is coated with a photocathode material and a fast pulse is launched across it. Electrons are converted to photons in a phosphor and recorded on film. Optical gate pulse widths of 100 ps are achieved. Some optical pulse width and sensitivity enhancements are noted at launch and termination. Events of interest are 200 ps long. The framing window is approximately 250 ps in length. Timing jitter is a problem. The instrument timing networks have been examined, and the source of jitter is still unknown. Timing to 50 ps resolution is desired. Close in proximity to the laser-driven event leads to complications in shielding from hard x-rays, hot electrons and shock-driven damage. High Z materials provide shielding from hard x-rays. Magnets screen out hot electrons produced by laser-matter interactions Filters provide energy fiducials. PCD`s provide high resolution timing measurements. Data is recorded on film in a specially designed film pack. The instrument is designed to be used in the NOVA Laser Facility at Lawrence Livermore National Laboratory.
Walsh, P.J.; Blake, R.L.; Caldwell, S.; Hockaday, M.; Chrien, R.; Smith, R.C.
BioMaPS: A Roadmap for Success
ERIC Educational Resources Information Center
The manuscript outlines the impact that our National Science Foundation Interdisciplinary Training for Undergraduates in Biological and Mathematical Sciences program, BioMaPS, has had on the students and faculty at Murray State University. This interdisciplinary program teams mathematics and biology undergraduate students with mathematics and�
McCarthy, Maeve L.; Fister, K. Renee
Boiling treatment of ABS and PS plastics for flotation separation.
A new physical method, namely boiling treatment, was developed to aid flotation separation of acrylonitrile-butadiene-styrene (ABS) and polystyrene (PS) plastics. Boiling treatment was shown to be effective in producing a hydrophilic surface on ABS plastic. Fourier Transform Infrared analysis was conducted to investigate the mechanism of boiling treatment of ABS. Surface rearrangement of polymer may be responsible for surface change of boiling treated ABS, and the selective influence of boiling treatment on the floatability of boiling treated plastics may be attributed to the difference in the molecular mobility of polymer chains. The effects of flotation time, frother concentration and particle size on flotation behavior of simple plastic were investigated. Based on flotation behavior of simple plastic, flotation separation of boiling treatment ABS and PS with different particle sizes was achieved efficiently. The purity of ABS and PS was up to 99.78% and 95.80%, respectively; the recovery of ABS and PS was up to 95.81% and 99.82%, respectively. Boiling treatment promotes the industrial application of plastics flotation and facilitates plastic recycling. PMID:24602834
Wang, Chong-qing; Wang, Hui; Wu, Bao-xin; Liu, Qun
The four Ps in an undergraduate software engineering course
There has been much discussion in the past decade of more efficient ways to teach the practical contents of the introductory software engineering course. This paper introduces a teaching platform, the SE-class platform, which exposes the students to a special and stimulating learning environment. The concept behind the SE-class platform is the Four Ps in software engineering: people, project, product
Lili Hai
Framing Retention for Institutional Improvement: A 4 Ps Framework
ERIC Educational Resources Information Center
A 4 Ps framework for student retention strategy is a construct for reframing the retention discussion in a way that enables institutional improvement by challenging some conventional wisdom and prevailing perspectives that have characterized retention strategy for years. It opens new possibilities for action and improvement by suggesting that�
Kalsbeek, David H.
The Pan-STARRS PS4 telescope suite
NASA Astrophysics Data System (ADS)
The Pan-STARRS project is planning to build a suite of four telescopes (PS4) on the summit of Mauna Kea at the site of the current University of Hawaii 2.2-m telescope. These telescopes will have the goal of surveying the entire sky visible from a single site in 5 colors (g, r, i, z, and y) on the time scale of approximately 1 week at a spatial resolution limited primarily by the quality of the site. To accomplish this task each of these four telescopes will be equipped with a Giga-Pixel camera, a camera shutter, and a 6 filter mechanism. A prototype telescope for this project (PS1) that includes all of these subsystems is already under going commissioning. The project is currently involved in developing the Environmental Impact Statement that is required to build the PS4 array of telescopes. We give an overview here of the scientific goals, the instrumentation package, the telescope design, and the enclosure design for the PS4 system.
Morgan, Jeffrey S.; Burgett, William; Teran, Jose U.
PS145A Making Democracy Work: Lessons from India
PS145A Making Democracy Work: Lessons from India Course Information Course Instructor Dr. Pradeep fragmentation. When India gained independence from British Rule in 1947, observers noted that the likelihood of the new country remaining democratic was limited. Yet, India proved such observers wrong and remained one
Jacobs, Lucia
Syntheses, structures, and spectroscopic properties of K9Nd[PS4]4, K3Nd[PS4]2, Cs3Nd[PS4]2, and K3Nd3[PS4]4.
Four new quaternary alkali neodymium thiophosphates K 9Nd[PS 4] 4 ( 1), K 3Nd[PS 4] 2 ( 2), Cs 3Nd[PS 4] 2 ( 3), and K 3Nd 3[PS 4] 4 ( 4) were synthesized by reacting Nd with in situ formed fluxes of K 2S 3 or Cs 2S 3, P 2S 5 and S in appropriate molar ratios at 973 K. Their crystal structures are determined by single crystal X-ray diffraction. Crystal data: 1: space group C2/ c, a = 20.1894(16), b = 9.7679(5), c = 17.4930(15) A, beta = 115.66(1) degrees , and Z = 4; 2: space group P2 1/ c, a = 9.1799(7), b = 16.8797(12), c = 9.4828(7) A, beta = 90.20(1) degrees , and Z = 4; 3: space group P2 1/ n, a = 15.3641(13), b = 6.8865(4), c = 15.3902(13) A, beta = 99.19(1) degrees , and Z = 4; 4: space group C2/ c, a = 16.1496(14), b = 11.6357(7), c = 14.6784(11) A, beta = 90.40(1) degrees , and Z = 4. The structure of 1 is composed of one-dimensional (1) infinity{Nd[PS 4] 4} (9-) chains and charge balancing K (+) ions. Within the chains, eight-coordinated Nd (3+) ions, which are mixed with K (+) ions, are connected by [PS 4] (3-) tetrahedra. The crystal structures of 2 and 3 are characterized by anionic chains (1) infinity{Nd[PS 4] 2} (3-) being separated by K (+) or Cs (+) ions. Along each chain the Nd (3+) ions are bridged by [PS 4] (3-) anions. The difference between the structures of 2 and 3 is that in 2 the Nd (3+) ions are coordinated by four edge-sharing [PS 4] (3-) tetrahedra while in 3 each Nd (3+) ion is surrounded by one corner-sharing, one face-sharing, and two edge-sharing [PS 4] (3-) tetrahedra. The structure of 4 is a three-dimensional network with K (+) cations residing in tunnels running along [110] and [110]. The {Nd(1)S 8} polyhedra share common edges with four [PS 4] tetrahedra forming one-dimensional chains (1) infinity{Nd[PS 4] 2} (3-) running along [110] and [110]. The chains are linked by {Nd(2)S 8} polyhedra yielding the final three-dimensional network (3) infinity{Nd[PS 4] 2} (3-). The internal vibrations of both crystallographically independent [PS 4] (3-) anions of 2- 4 have been assigned in the range 200-650 cm (-1) by comparison of their corresponding far/mid infrared and Raman spectra (lambda exc = 488 nm) on account of locally imposed C 1 symmetry. In the Fourier-transform-Raman spectrum (lambda exc = 1064 nm) of 2- 4, very similar well-resolved electronic Raman (ER) transitions from the electronic Nd (3+) ground-state to two levels of the (4)I 9/2 ground manifold and to the six levels of the (4)I 11/2 manifold have been determined. Resonant Raman excitation via a B-term mechanism involving the (4)I 15/2 and (4)F 3/2 intermediate states may account for the significant intensity enhancement of the ER transitions with respect to the symmetric P-S stretching vibration nu 1. Broad absorptions in the UV/vis/NIR diffuse reflectance spectrum at 293 K in the range 5000-25000 cm (-1) of 2- 4 are attributed to spin-allowed excited quartet states [ (4)(I < F < S < G < D)] and spin-forbidden doublet states [ (2)(H < G < K < D < P)] of Nd (3+). A luminescense spectrum of 3 obtained at 15 K by excitation with 454.5 nm shows multiplets of narrow lines that reproduce the Nd (3+) absorptions. Sharp and intense luminescence lines are produced instead by excitation with 514.5 nm. Lines at 18681 ( (4)G 7/2), 16692 ( (4)G 5/2), 14489 ( (4)F 9/2), and 13186 cm (-1) ( (4)F 7/2) coincide with the corresponding absorptions. Hypersensitive (4)G 5/2 is split by 42 cm (-1). The most intense multiplet at about 16500 cm (-1) is assigned to the transition from (4)G 5/2 to the Stark levels of the ground manifold (4)I 9/2. PMID:18686946
Wu, Yuandong; Bensch, Wolfgang
Mapping the APP\\/Presenilin (PS) Binding Domains: The Hydrophilic N-Terminus of PS2 Is Sufficient for Interaction with APP and Can Displace APP\\/PS1 Interaction
Mutations in presenilin 1 and presenilin 2 (PS1 and PS2, respectively) genes cause the large majority of familial forms of early-onset Alzheimer's disease. The physical interaction between presenilins and APP has been recently described using coimmunoprecipitation. With a similar technique, we confirmed this interaction and have mapped the interaction domains on both PS2 and APP. Using several carboxy-terminal truncated forms
Laurent Pradier; Nathalie Carpentier; Laurence Delalonde; Nicole Clavel; Marie-Dominique Bock; Luc Bu�e; Luc Mercken; Bruno Tocqu�; Christian Czech
p53 Protein-mediated regulation of phosphoglycerate dehydrogenase (PHGDH) is crucial for the apoptotic response upon serine starvation.
Although p53 is frequently mutated in human cancers, about 80% of human melanomas retain wild-type p53. Here we report that PHGDH, the key metabolic enzyme that catalyzes the rate-limiting step of the serine biosynthesis pathway, is a target of p53 in human melanoma cells. p53 suppresses PHGDH expression and inhibits de novo serine biosynthesis. Notably, upon serine starvation, p53-mediated cell death is enhanced dramatically in response to Nutlin-3 treatment. Moreover, PHGDH has been found recently to be amplified frequently in human melanomas. We found that PHGDH overexpression significantly suppresses the apoptotic response, whereas RNAi-mediated knockdown of endogenous PHGDH promotes apoptosis under the same treatment. These results demonstrate an important role of p53 in regulating the serine biosynthesis pathway through suppressing PHGDH expression and reveal serine deprivation as a novel approach to sensitize p53-mediated apoptotic responses in human melanoma cells. PMID:25404730
Ou, Yang; Wang, Shang-Jui; Jiang, Le; Zheng, Bin; Gu, Wei
Expectation values of the e{sup +}PsH system
Close to converged energies and expectation values for e{sup +}PsH are computed using a ground-state wave function consisting of 1500 explicitly correlated Gaussians. The best estimate of the e{sup +}PsH{sup {infinity}} energy was -0.810 254 hartrees, which has a binding energy of 0.021 057 hartrees against dissociation into e{sup +}+PsH. The 2{gamma} annihilation rate was 2.7508x10{sup 9} s{sup -1}. Binding energies and annihilation rates are also given for the different finite-mass variants of e{sup +}PsH. Comparisons between expectation values for e{sup +}PsH and PsH provide compelling evidence that the e{sup +}PsH ground state can be regarded as consisting of a weakly bound positron orbiting the PsH ground state.
Zhang, J.-Y.; Mitroy, J. [Faculty of Technology, Charles Darwin University, Darwin, Northern Territory 0909 (Australia); ARC Center for Anti-Matter Studies, Faculty of Technology, Charles Darwin University, Darwin, Northern Territory 0909 (Australia)
Breast Cancer-Associated pS2 Protein: Synthesis and Secretion by Normal Stomach Mucosa
NASA Astrophysics Data System (ADS)
The human pS2 gene is specifically expressed under estrogen transcriptional control in a subclass of estrogen receptor-containing human breast cancer cells. The pS2 gene encodes an 84-amino acid protein that is secreted after signal peptide cleavage. The distribution of pS2 protein in normal human tissues was studied with antibodies to pS2; pS2 was specifically expressed and secreted by mucosa cells of the normal stomach antrum and body of both female and male individuals. Moreover, no estrogen receptor could be detected in these cells, indicating that pS2 gene expression is estrogen-independent in the stomach. The function of the pS2 protein in the gastrointestinal tract is unknown. However, the pS2 protein is similar in sequence to a porcine pancreatic protein that has been shown to inhibit gastrointestinal motility and gastric secretion.
Rio, M. C.; Bellocq, J. P.; Daniel, J. Y.; Tomasetto, C.; Lathe, R.; Chenard, M. P.; Batzenschlager, A.; Chambon, P.
Effect of lead on lipid peroxidation, phospholipids composition, and methylation in erythrocyte of human.
Lead (Pb) is one of the most abundant heavy metals on earth considered as number one environmental persistent toxin and health hazard affecting millions of people in all age groups. After entering bloodstream, 99% of Pb is accumulated in erythrocytes and causes poisoning. Toxic Pb effects on erythrocytes membrane's composition of phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), and sphingomyelin (SM), and phospholipids transmethylation were determined. Lipid peroxidation in Pb-exposed erythrocytes was evaluated as malondialdehyde (MDA) formation in presence of Fe and vitamin E to understand severity of Pb toxicity and its mitigation. Pb (0.5-5.0 ?M) degraded PS (12 to 31%, P < 0.05-0.001) and elevated SM (19-51%, P < 0.05-0.001). Composition of PC and PE were diminished (22%) and elevated (29%), respectively, with higher Pb exposure (5.0 ?M, P < 0.001). Pb toxicity suppressed (P < 0.001) transmethylation of phospholipids in membranes (34, 41, and 50%, respectively, with 0.5, 2.5, and 5.0 ?M). Pb-induced dose-related MDA production (P < 0.05-0.001) in erythrocytes was obtained, which was accentuated in presence of Fe (P < 0.05-0.001). The vitamin E mitigated (P < 0.05-0.01) the severity of Pb-induced lipid peroxidation. The ratio PS/SM showed maximum change of -27 (P < 0.01), -30 (P < 0.01), and -54% (P < 0.001), respectively at 0.5, 2.5, and 5.0 ?M Pb exposures. Ratios PC/SM and PS/PE were at the second, whereas PE/PS at the third order. The study suggests that the mechanisms underlying distortion of compositional phospholipids, inhibition of transmethylation, and exasperated phospholipid peroxidative damage are the active phenomena of Pb toxicity in erythrocytes. PMID:23846836
Changes in cholesterol metabolism are associated with PS1 and PS2 gene regulation in SK-N-BE.
Several lines of evidence suggest that the cholesterol content of neuronal membranes influences amyloid precursor protein (APP) processing; however, its role in transcriptional regulation of the cofactors for gamma-secretase, the key enzyme for the production of the Abeta peptide, is poorly understood. This study investigates whether the changes in cellular cholesterol metabolism modulate the expression of genes involved in the gamma-secretase complex function. The abundance of mRNA transcripts for presenilin 1 and 2 (PS1 and PS2), APP, and nicastrin were evaluated in neuroblastoma cells exposed either to serum-depleted medium or to low-density lipoproteins (LDL). Cholesterol esterification was markedly inhibited by mevinolin and U18666A, but was not significantly affected by any other of the tested treatments. gamma-Secretase genes and cofactors were not co-regulated and were not influenced by statin inhibition of cholesterol synthesis. Nicastrin and the APP isoforms showed constitutive expression. In the absence of exogenous lipids, cell PS1 and PS2 expression was induced by LDL and by lysosomal sequestration of cholesterol. However, a different pattern of induction of presenilin gene expression was observed in the latter condition, suggesting that lysosomal cholesterol levels are strong inducers of PS2 transcription. Taken together, these results indicate that lipid metabolism has a complex influence on gamma-secretase transcriptional pathways and, in particular, exogenous cholesterol and compartmentalization in neuroblastoma cells play a relevant role in regulating the transcription of presenilins, while modulation of the cholesterol biosynthesis pathway seems to exert a minor influence on the expression of gamma-secretase genes and cofactors. PMID:17401156
Crestini, A; Napolitano, M; Piscopo, P; Confaloni, A; Bravo, E
Kinetic evidence of a noncatalytic substrate binding site that regulates activity in Legionella pneumophila L-serine dehydratase.
The L-serine dehydratase from Legionella pneumophila (lpLSD) has recently been shown to contain a domain (? domain) that has a high degree of structural homology with the ASB domain of d-3-phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis. Furthermore, this domain has been shown by sequence homology to be present in all bacterial L-serine dehydratases that utilize an Fe-S catalytic center. In PGDH, L-serine binds to the ACT domain to inhibit catalytic activity. However, substrate must be bound to the ASB domain for serine to exert its effect. As such, the ASB domain acts as a codomain for the action of L-serine. Pre-steady-state kinetic analysis of L-serine binding to lpLSD demonstrates that L-serine binds to a second noncatalytic site and produces a conformational change in the enzyme. The rate of this conformational change is too slow for its participation in the catalytic cycle but rather occurs prior to catalysis to produce an activated form of the enzyme. That the conformational change must occur prior to catalysis is shown by a lag in the production of product that exhibits essentially the same rate constant as the conformational change. The second, noncatalytic site for L-serine is likely to be the ASB domain (? domain) of lpLSD that functions in a manner similar to that in PGDH. A mechanism whose overall effect is to keep L-serine levels from accumulating to high levels while not completely depleting the L-serine pool in the bacterial cell is proposed. PMID:22891658
Grant, Gregory A
Acceleration of the substrate Calpha deprotonation by an analogue of the second substrate palmitoyl-CoA in Serine Palmitoyltransferase.
Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of l-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at Calpha of l-serine is very slow in the SPT-l-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated alpha-deprotonation indicates that the alpha-deprotonation takes place before the Claisen-type C-C bond formation, which is consistent with the accepted mechanism of the alpha-oxamine synthase subfamily enzymes. Structural modeling of both the SPT-l-serine external aldimine complex and SPT-l-serine-palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-l-serine external aldimine so that the Calpha-H bond of l-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the alpha-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His(159) with l-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate. PMID:18167344
Ikushiro, Hiroko; Fujii, Shigeru; Shiraiwa, Yuka; Hayashi, Hideyuki
A tryptic peptide containing a unique serine phosphate residue in rabbit phosphoglucomutase.
(32)P-labelled phosphoglucomutase was digested with trypsin after denaturation and two peptides were isolated that contained the bulk of the radioactivity bound to peptides. Both peptides appeared to derive from an identical section of the molecule. Peptic and subtilisin digests of the tryptic peptides were prepared. The resulting radioactive peptides were purified and their sequences studied. The presence of a single serine [(32)P]phosphate residue was clearly established. Difficulties in purification and low yields, especially of the tryptic peptide, prevented exhaustive sequence studies, but a tentative sequence is proposed as:Ala-Ile-Gly-Gly-Ile-Ile-Leu-Thr-Ala-SerP-His-Asx-Pro-Gly-Gly-Pro-(Asx(2),Gly)-Phe-Gly-Ile-Lys(where SerP represents serine phosphate and Asx represents aspartic acid or asparagine). The results do not support the presence of two serine phosphate residues in the denatured enzyme, but confirm previous results of a unique sequence around a single serine phosphate residue. PMID:5669853
Milstein, C P; Milstein, C
Design of activated serine-containing catalytic triads with atomic level accuracy
A challenge in the computational design of enzymes is that multiple properties must be simultaneously optimized -- substrate-binding, transition state stabilization, and product release -- and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads, and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate-reactivity. Following optimization by yeast-display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest the designs could provide the basis for a new class of organophosphate captures agents. PMID:24705591
Rajagopalan, Sridharan; Wang, Chu; Yu, Kai; Kuzin, Alexandre P.; Richter, Florian; Lew, Scott; Miklos, Aleksandr E.; Matthews, Megan L.; Seetharaman, Jayaraman; Su, Min; Hunt, John. F.; Cravatt, Benjamin F.; Baker, David
Design of activated serine-containing catalytic triads with atomic-level accuracy.
A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents. PMID:24705591
Rajagopalan, Sridharan; Wang, Chu; Yu, Kai; Kuzin, Alexandre P; Richter, Florian; Lew, Scott; Miklos, Aleksandr E; Matthews, Megan L; Seetharaman, Jayaraman; Su, Min; Hunt, John F; Cravatt, Benjamin F; Baker, David
Latent Periodicity of Serine\\/Threonine and Tyrosine Protein Kinases and Other Protein Families
A method of noise decomposition has been developed. This method allows for the identification of a latent periodicity with symbol insertions and deletions that is specific for all or most amino acid sequences belonging to the same protein family or protein domain. The latent periodicity has been identified in catalytic domains of 85% of serine\\/threonine and tyrosine protein kinases. Similar
A. A. Laskin; Nikolay A. Kudryashov; Konstantin G. Skryabin; E. V. Korotkov
Mutations in serine protease inhibitor Kazal type 1 are strongly associated with chronic pancreatitis
Background: Although chronic pancreatitis is associated with risk factors such as alcoholism, hyperparathyroidism, and hypertriglyceridaemia, little is known of the actual aetiology of the disease. It is thought that inappropriate activation of trypsinogen causes pancreatitis, and indeed in cases of hereditary pancreatitis mutations of cationic trypsinogen (PRSS1) have been described. As serine protease inhibitor Kazal type 1 (SPINK1) is a
J P H Drenth; R te Morsche; J B M J Jansen
Expanding the complexity of the human degradome: polyserases and their tandem serine protease domains.
The large and growing number of protease genes identified in the human genome, more than 560, reflects the complexity and relevance of these enzymes in multiple biological processes. As part of our studies on the human degradome--which is defined as the complete set of human protease genes--we have recently identified and cloned three complex polyserine proteases called polyserases. Polyserase-1 is a member of the type-II transmembrane serine protease (TTSP) family of proteolytic enzymes that undergoes a series of post-translational processing events to generate three distinct and independent serine protease domains called serase-1, -2, and -3. Polyserase-2 is a secreted enzyme that also possesses three serine protease domains, but they remain as an integral part of the initial protein product. Finally, polyserase-3 is also a secreted enzyme that contains two serine protease domains embedded in the same polypeptide chain. Despite all three human polyserases share this complex molecular design characterized by the presence of several catalytic domains in their structure, they also exhibit distinctive features including unique expression patterns and different enzymatic properties. At present, the putative functional advantages derived from the complex structural organization of polyserases remain unknown, but the widespread occurrence of these enzymes in mammalian degradomes provides additional evidence about the complexity of proteolytic systems in these organisms. PMID:17485402
Cal, Santiago; Moncada-Pazos, Angela; Lopez-Otin, Carlos
Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi.
Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited ?-chymotrypsin to 9.4% residual activity and also inhibited ?-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania. PMID:24037187
Sigle, Leah Theresa; Ramalho-Ortig�o, Marcelo
Technology Transfer Automated Retrieval System (TEKTRAN)
Genetic variation in folate-regulating enzymes contributes to the risk of cardiovascular disease (CVD). The cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme is proposed to regulate a key metabolic intersection in folate metabolism. We hypothesized that a variant in cSHMT (cSHMT 1420CT) aff...
VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF eNOS. Zhuowei Li, Jacqueline D. Carter, Lisa A. Dailey, Joleen Soukup, Yuh-Chin T. Huang. CEMALB, University of North Carolina and ORD, US EPA, Chapel Hill, North Carolina V...
VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF eNOS. Zhuowei Li, Jacqueline D. Carter, Lisa A. Dailey, Joleen Soukup, Yuh-Chin T. Huang. CEMALB, University of North Carolina and NHEERL, US EPA, Chapel Hill, North Ca...
Cha4p of Saccharomyces Cerevisiae Activates Transcription via Serine/Threonine Response Elements
The CHA1 gene of Saccharomyces cerevisiae encodes the catabolic L-serine (L-threonine) deaminase responsible for the utilization of serine/threonine as nitrogen sources. Previously, we identified two serine/threonine response elements in the CHA1 promoter, UAS(CHA). We report isolation of a mutation, cha4-1, that impairs serine/threonine induction of CHA1 transcription. The cha4-1 allele causes noninducibility of a CHA1p-lacZ translational gene fusion, indicating that Cha4p exerts its action through the CHA1 promoter. Molecular and genetic mapping positioned the cha4 locus 17 cM centromere proximal to put1 on chromosome XII. The coding region of CHA4 predicts a 648-amino acid protein with a DNA-binding motif (residues 43-70) belonging to the Cys(6) zinc cluster class. Gel retardation employing a recombinant peptide, Cha4p(1-174), demonstrated that the peptide in vitro specifically binds UAS(CHA). Binding is abolished by a G-C to T-A mutation in the middle bases of the two CEZ-elements in UAS(CHA). The transcriptional activating ability of UAS(CHA) derivatives in vivo correlates with their ability to bind Cha4p(1-174) in vitro. We conclude that Cha4p is a positive regulator of CHA1 transcription and that Cha4p alone, or as part of a complex, is binding UAS(CHA). PMID:8889513
Holmberg, S.; Schjerling, P.
Regulation of human serine racemase activity and dynamics by halides, ATP and malonate.
D-Serine is a non-proteinogenic amino acid that acts as a co-agonist of the NMDA receptors in the central nervous system. D-Serine is produced by human serine racemase (hSR), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme that also catalyzes the physiologically relevant ?-elimination of both L- and D-serine to pyruvate and ammonia. After improving the protein purification yield and stability, which had so far limited the biochemical characterization of hSR, we found that the catalytic activity is affected by halides, in the order fluoride�>�chloride�>�bromide. On the contrary, iodide elicited a complete inhibition, accompanied by a modulation of the tautomeric equilibrium of the internal aldimine. We also investigated the reciprocal effects of ATP and malonate, an inhibitor that reversibly binds at the active site, 20�� away from the ATP-binding site. ATP increased ninefold the affinity of hSR for malonate and malonate increased 100-fold that of ATP, confirming an allosteric interaction between the two binding sites. To further investigate this allosteric communication, we probed the active site accessibility by quenching of the coenzyme fluorescence in the absence and presence of ATP. We found that ATP stabilizes a closed conformation of the external aldimine Schiff base, suggesting a possible mechanism for ATP-induced hSR activation. PMID:25331425
Marchetti, Marialaura; Bruno, Stefano; Campanini, Barbara; Bettati, Stefano; Peracchi, Alessio; Mozzarelli, Andrea
Interhelical Hydrogen Bonds and Spatial Motifs in Membrane Proteins: Polar Clamps and Serine Zippers
Interhelical Hydrogen Bonds and Spatial Motifs in Membrane Proteins: Polar Clamps and Serine hydrogen bond connections between TM helices. We find that almost all TM helices have interhelical hydrogen there are interhelical hydrogen bonds between them. We further describe several spatial motifs in the TM regions
Dai, Yang
Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases
Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases
Steffen Massberg; Lenka Grahl; Marie-Luise von Bruehl; Davit Manukyan; Susanne Pfeiler; Christian Goosmann; Volker Brinkmann; Michael Lorenz; Kiril Bidzhekov; Avinash B Khandagale; Ildiko Konrad; Elisabeth Kennerknecht; Katja Reges; Stefan Holdenrieder; Siegmund Braun; Christoph Reinhardt; Michael Spannagl; Klaus T Preissner; Bernd Engelmann
In-silico comparative study of inhibitory mechanism of Plant Serine Proteinase Inhibitors
The nematodes like root-knot and cyst are plant-parasitic pest found in horticultural and agricultural crops. They do damages in the roots of plants as a result losses million tons of production. High cost of nematicides and environment safety concern has necessitated finding of some alternative methods. Under Integrated Pest Management (IPM) such problems are solving significantly by means of target gene inhibition, agrobacterium mediated transformation etc. One of this strategy use Plant Proteinase Inhibitors (PIs) gene which are used to control the proteolysis mechanism of Pest by inhibiting gut Serine Proteinase (SP). Present work investigates the utility of computer aided methods to study the mechanism of Protein-Protein interactions and thereby inhibition of Serine Proteinase by PIs. Hence 3D models of Serine Proteinase as well as Serine Proteinase Inhibitors (SPIs) generated using homology modeling. Validations of constructed models have been done by PROCHECK, VERIFY3D, ERRAT and PROSA. Prediction of Protein interacting surface patches and site specific protein docking was performed by using ZDOCK Server. Backbone refinement of output protein complexes was executed in Fiber Dock server. Interaction study between SP and SPIs complexes shows their comparative inhibition efficacy, measured in terms of number of hydrogen bonds, Van dar wall attraction and docking energy. This work reported that Vigna marina and Phaseolus oligospermus are having better inhibition efficiency in comparison to other inhibitors. PMID:23055608
Siva Prasad, Chekkara Venkata Sathya; Gupta, Saurabh; Gaponenko, Alex; Dhar, Murli
Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata
NASA Astrophysics Data System (ADS)
The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.
Zhao, Chunling; Ju, Jiyu
Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone
Technology Transfer Automated Retrieval System (TEKTRAN)
Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...
[Kinetic and allosteric properties of L-threonine-L-serine dehydratase from human liver].
It was shown that low concentrations of ATP (1..10(-4)M) and 10-fold concentrations of AMP (1.10(-3)M) at three constant L-threonine concentrations activated the L-threonine dehydratase activity of L-threonine-L-serine dehydratase from human liver, but had no effect on the L-serine dehydratase activity of this enzyme. Higher concentrations of both nucleotides inhibited the enzyme. The effects of ATP and AMP were specific. The activating and inhibiting effects of various concentrations of ATP and AMP were revealed as changes in the shapes of the curves for the initial reaction rate of the L-threonine dehydratase reaction versus initial substrate concentration. For this reaction the curves were not hyperbolic and were characterized by intermediary plateaux. ATP and AMP also influenced the maximal rate of the enzymatic reaction. Using the desensitization method it was shown that the activating effects of ATP and AMP are of allosteric nature. Thus, human liver L-threonine-L-serine dehydratase is an allosteric enzyme, for which positive allosteric effectors are low concentrations of ATP and AMP and negative allosteric effectors are high concentrations of these nucleotides. A possible mechanism of allosteric regulation of the enzyme under catalysis of the L-threonine dehydratase reaction and the lack of regulation under catalysis of the L-serine dehydratase reaction as well as specificity of the allosteric sites of this enzyme to the two nucleotides and the physiological significance of this process are discussed. PMID:435568
Akopov, M A; Kagan, Z C; Berezov, T T; Filiptsev, P Ia
The VA, VCD, Raman and ROA spectra of tri-L-serine in aqueous solution
NASA Astrophysics Data System (ADS)
The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory with the Becke 3LYP hybrid exchange correlation (XC) functional and the PW91 GGA XC functional and the 6-31G* and aug-cc-pVDZ basis sets. Subsequently, the vibrational absorption, vibrational circular dichroism, Raman and Raman optical activity spectra were simulated in order to compare them to experimentally measured spectra. In addition, we compare to previously reported studies for both structural determination and spectral simulations and measurements. A comparison of the various ways to treat the effects of the environment and solvation on both the structure and the spectral properties is thoroughly investigated for one conformer, with the goal to determine which level of theory is appropriate to use in the systematic search of the conformational space. In addition, the effects of the counterion, here Cl- anion, are also investigated. Here we present the current state of the art in nanobiology, where the latest methods in experimental and theoretical vibrational spectroscopy are used to gain useful information about the coupling of the nuclear, electronic and magnetic degrees of freedom and structure of tri-L-serine and its capped peptide analog with the environment.
J�rgensen, V. W�rtz; Jalkanen, K.
Gelatinases and serine proteinase inhibitors of seminal plasma and the reproductive tract of turkey ( Meleagris gallopavo)
This study examined proteolytic enzymes and serine proteinase inhibitors in turkey seminal plasma with relation to their distribution within the reproductive tract and to yellow semen syndrome (YSS). Proteases of blood plasma, extracts from the reproductive tract, and seminal plasma were analyzed by gelatin zymography. We found a clear regional distribution of proteolytic enzymes in the turkey reproductive tract. Each
M. Kot?owska; R. Kowalski; J. Glogowski; J. Jankowski; A. Ciereszko
KM-based Project Team of CoPS in Manufacture Industry
Complex product system (CoPS) plays an important role in an organization, zone economy, and country economy. As prime part of CoPS, CoPS in manufacture industry is having more and more powerful effect to an organization's success and a country's economy. However, inherent characters of CoPS in manufacture industry, e.g. multi-discipline technology integration of product, cross-organization in management, and high risks,
Chen Zhan-duo
Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics.
The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the HCV-encoded proteinases. Images PMID:7966606
Lin, C; Pr�gai, B M; Grakoui, A; Xu, J; Rice, C M
Genomic imprinting is variably lost during reprogramming of mouse iPS cells
Derivation of induced pluripotent stem (iPS) cells is mainly an epigenetic reprogramming process. It is still quite controversial how genomic imprinting is reprogrammed in iPS cells. Thus, we derived multiple iPS clones from genetically identical mouse somatic cells. We found that parentally inherited imprint was variably lost among these iPS clones. Concurrent with the loss of DNA methylation imprint at the corresponding Snrpn and Peg3 imprinted regions, parental origin-specific expression of the Snrpn and Zim1 imprinted genes was also lost in these iPS clones. This loss of parental genomic imprinting in iPS cells was likely caused by the reprogramming process during iPS cell derivation because extended culture of iPS cells did not lead to significant increase in the loss of genomic imprinting. Intriguingly, one to several paternal chromosomes appeared to have acquired de novo methylation at the Snrpn and Zac1 imprinted regions in a high percentage of iPS clones. These results might have some implications for future therapeutic applications of iPS cells. Since DNA methylation imprint can be completely erased in some iPS clones at multiple imprinted regions, iPS cell reprogramming may also be employed to dissect the underlying mechanisms of erasure, reacquisition and maintenance of genomic imprinting in mammals. PMID:23832110
Shamis, Yulia; Wu, Tien-Yuan; Li, Xiajun
Functional and Structural Characterization of Vibrio cholerae Extracellular Serine Protease B, VesB*
The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na+ did not stimulate the activity of VesB, despite containing the Tyr250 signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed. PMID:24459146
Gadwal, Shilpa; Korotkov, Konstantin V.; Delarosa, Jaclyn R.; Hol, Wim G. J.; Sandkvist, Maria
An unorthodox sensory adaptation site in the Escherichia coli serine chemoreceptor.
The serine chemoreceptor of Escherichia coli contains four canonical methylation sites for sensory adaptation that lie near intersubunit helix interfaces of the Tsr homodimer. An unexplored fifth methylation site, E502, lies at an intrasubunit helix interface closest to the HAMP domain that controls input-output signaling in methyl-accepting chemotaxis proteins. We analyzed, with in vivo F�rster resonance energy transfer (FRET) kinase assays, the serine thresholds and response cooperativities of Tsr receptors with different mutationally imposed modifications at sites 1 to 4 and/or at site 5. Tsr variants carrying E or Q at residue 502, in combination with unmodifiable D and N replacements at adaptation sites 1 to 4, underwent both methylation and demethylation/deamidation, although detection of the latter modifications required elevated intracellular levels of CheB. These Tsr variants could not mediate a chemotactic response to serine spatial gradients, demonstrating that adaptational modifications at E502 alone are not sufficient for Tsr function. Moreover, E502 is not critical for Tsr function, because only two amino acid replacements at this residue abrogated serine chemotaxis: Tsr-E502P had extreme kinase-off output and Tsr-E502I had extreme kinase-on output. These large threshold shifts are probably due to the unique HAMP-proximal location of methylation site 5. However, a methylation-mimicking glutamine at any Tsr modification site raised the serine response threshold, suggesting that all sites influence signaling by the same general mechanism, presumably through changes in packing stability of the methylation helix bundle. These findings are consistent with control of input-output signaling in Tsr through dynamic interplay of the structural stabilities of the HAMP and methylation bundles. PMID:24272777
Han, Xue-Sheng; Parkinson, John S
Serine codon-usage bias in deep phylogenomics: pancrustacean relationships as a case study.
Phylogenomic analyses of ancient relationships are usually performed using amino acid data, but it is unclear whether amino acids or nucleotides should be preferred. With the 2-fold aim of addressing this problem and clarifying pancrustacean relationships, we explored the signals in the 62 protein-coding genes carefully assembled by Regier et al. in 2010. With reference to the pancrustaceans, this data set infers a highly supported nucleotide tree that is substantially different to the corresponding, but poorly supported, amino acid one. We show that the discrepancy between the nucleotide-based and the amino acids-based trees is caused by substitutions within synonymous codon families (especially those of serine-TCN and AGY). We show that different arthropod lineages are differentially biased in their usage of serine, arginine, and leucine synonymous codons, and that the serine bias is correlated with the topology derived from the nucleotides, but not the amino acids. We suggest that a parallel, partially compositionally driven, synonymous codon-usage bias affects the nucleotide topology. As substitutions between serine codon families can proceed through threonine or cysteine intermediates, amino acid data sets might also be affected by the serine codon-usage bias. We suggest that a Dayhoff recoding strategy would partially ameliorate the effects of such bias. Although amino acids provide an alternative hypothesis of pancrustacean relationships, neither the nucleotides nor the amino acids version of this data set seems to bring enough genuine phylogenetic information to robustly resolve the relationships within group, which should still be considered unresolved. PMID:22962005
Rota-Stabelli, Omar; Lartillot, Nicolas; Philippe, Herv�; Pisani, Davide
Functional and structural characterization of Vibrio cholerae extracellular serine protease B, VesB.
The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na(+) did not stimulate the activity of VesB, despite containing the Tyr(250) signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed. PMID:24459146
Gadwal, Shilpa; Korotkov, Konstantin V; Delarosa, Jaclyn R; Hol, Wim G J; Sandkvist, Maria
Four-frame gated optical imager with 120-ps resolution
In this paper we describe the operation and applications of a framing camera capable of four separate two-dimensional images with each frame having a 120-ps gate width. Fast gating of a single frame is accomplished by using a wafer image intensifier tube in which the cathode is capacitively coupled to an external electrode placed outside of the photocathode of the tube. This electrode is then pulsed relative to the microchannel plate by a narrow (120 ps), high-voltage pulse. Multiple frames are obtained by using multiple gated tubes which share a single bias supply and pulser with relative gate times selected by the cable lengths between the tubes and the pulser. A beamsplitter system has been constructed which produces a separate image for each tube from a single scene. Applications of the framing camera to inertial confinement fusion experiments are discussed.
Young, P.E.; Hares, J.D.; Kilkenny, J.D.; Phillion, D.W.; Campbell, E.M.
iPS cell derived neuronal cells for drug discovery.
Owing to the inherent disconnect between drug pharmacology in heterologous cellular models and drug efficacy in vivo, the quest for more predictive in vitro systems is one of the most urgent challenges of modern drug discovery. An improved pharmacological in vitro profiling would employ primary samples of the proper drug-targeted human tissue or the bona fide human disease-relevant cells. With the advent of induced pluripotent stem (iPS) cell technology the facilitated access to a variety of disease-relevant target cells is now held out in prospect. In this review, we focus on the use of human iPS cell derived neurons for high throughput pharmaceutical drug screening, employing detection technologies that are sufficiently sensitive to measure signaling in cells with physiological target protein expression levels. PMID:25096281
Heilker, Ralf; Traub, Stefanie; Reinhardt, Peter; Sch�ler, Hans R; Sterneckert, Jared
The four "P"s of marketing are dead.
For several decades marketing planning in the United States has relied upon the "four Ps" model. Product, price, place, and promotion were considered the foundation of the marketing mix. This model, however, has never been a comfortable fit for health care and, as the new century dawns, we find that a new marketing model--emphasizing the "four Rs"--is emerging. The foundations of the new model are relevance, response, relationships, and results. PMID:11183425
English, J
The Pan-STARRS PS4 telescope suite
The Pan-STARRS project is planning to build a suite of four telescopes (PS4) on the summit of Mauna Kea at the site of the current University of Hawaii 2.2-m telescope. These telescopes will have the goal of surveying the entire sky visible from a single site in 5 colors (g, r, i, z, and y) on the time scale of
Jeffrey S. Morgan; William Burgett; Jose U. Teran
Investigation of methylamine intercalated FePS 3
NASA Astrophysics Data System (ADS)
The 57Fe Mossbauer spectroscopic results of methylamine intercalated layered FePS 3 following the removal of the guest species from the initial intercalate in steps by heating at three different stages are presented. The initial intercalate described as [FePS 3- xO x] 2 x- [CH 3NH 3] 2 x+ (CH 3NH 2) y (H 2O) z with x=0.65 has the host lattice of FePS 3 modified through substitution of sulphur by oxygen due to intercalation of amine in aqueous medium and the neutral molecules, viz., water and methylamine solvating the ionised methylamine guest [CH 3NH 3] +. The present Mossbauer results show that when the guest species are removed by heating, 15% delocalised electrons present in the initial intercalate, get localised at the iron centres of the host. The most interesting feature of the M�ssbauer result is the detection of a new kind of intercalation process wherein in situ generation of large amount of ionised amines takes place when the initial intercalate is simply heated above 100�C.
Bhowmick, A.; Ganguli, S.; Bhattacharya, M.
Research of beam smoothing technologies using CPP, SSD, and PS
NASA Astrophysics Data System (ADS)
Precise physical experiments place strict requirements on target illumination uniformity in Inertial Confinement Fusion. To obtain a smoother focal spot and suppress transverse SBS in large aperture optics, Multi-FM smoothing by spectral dispersion (SSD) was studied combined with continuous phase plate (CPP) and polarization smoothing (PS). New ways of PS are being developed to improve the laser irradiation uniformity and solve LPI problems in indirect-drive laser fusion. The near field and far field properties of beams using polarization smoothing were studied and compared, including birefringent wedge and polarization control array. As more parameters can be manipulated in a combined beam smoothing scheme, quad beam smoothing was also studies. Simulation results indicate through adjusting dispersion directions of one-dimensional (1-D) SSD beams in a quad, two-dimensional SSD can be obtained. Experiments have been done on SG-III laser facility using CPP and Multi-FM SSD. The research provides some theoretical and experimental basis for the application of CPP, SSD and PS on high-power laser facilities.
Zhang, Rui; Su, Jingqin; Hu, Dongxia; Li, Ping; Yuan, Haoyu; Zhou, Wei; Yuan, Qiang; Wang, Yuancheng; Tian, Xiaocheng; Xu, Dangpeng; Dong, Jun; Zhu, Qihua
Physics with primary beams of the KEK-PS
NASA Astrophysics Data System (ADS)
The 12-GeV Proton Synchrotron (PS) at the National Laboratory for High Energy Physics (KEK) has provided great opportunities to high-energy-physics and related communities as a unique high-energy hadron machine, since its operation in 1976. Activities of the KEK-PS are indispensable for the rapid development in the field. Six experimental subjects are proposed in this report: (1) media effects in (phi) meson decay, (2) multifragmentation in high-energy reactions, (3) mechanism of high-energy reactions by means of radio-chemical methods, (4) physics with polarized high-energy neutrons, (5) physics with polarized high-energy deuterons, and (6) hypernucleus with high-energy heavy-ion beams. As a summary, new facilities (a new injector, a new beamline and a new experimental area) and physics programs with primary beams, proposed in this report are themselves unique and valuable. Moreover, technical developments and physics outcomes stimulated with those new facilities are indispensable for future plans of the KEK-PS.
Tanaka, Kazuhiro; Yoshii, Masahito
Anomalous high ionic conductivity of nanoporous -Li3PS4
Lithium-ion conducting solid electrolytes hold the promise for enabling high-energy battery chemistries and circumventing safety issues of conventional lithium batteries1-3. Achieving the combination of high ionic conductivity and broad electrochemical window in solid electrolytes is a grand challenge for the synthesis of battery materials. Herein we show an enhancement of room-temperature lithium-ion conductivity of 3 orders of magnitude by creating nanostructured Li3PS4. This material has a wide (5V) electrochemical window and superior chemical stability against lithium metal. The nanoporous structure of Li3PS4 reconciles two vital effects that enhance ionic conductivity: (1) The reduced dimension to nanometer-sized framework stabilizes the high conduction beta phase that occurs at elevated temperatures1,4; and (2) The high surface-to-bulk ratio of nanoporous -Li3PS4 promotes surface conduction5,6. Manipulating the ionic conductivity of solid electrolytes has far-reaching implications for materials design and synthesis in a broad range of applications such as batteries, fuel-cells, sensors, photovoltaic systems, and so forth3,7.
Liu, Zengcai [ORNL] [ORNL; Fu, Wujun [ORNL] [ORNL; Payzant, E Andrew [ORNL] [ORNL; Yu, Xiang [ORNL] [ORNL; Wu, Zili [ORNL] [ORNL; Dudney, Nancy J [ORNL] [ORNL; Kiggans, Jim [ORNL] [ORNL; Hong, Kunlun [ORNL] [ORNL; Rondinone, Adam Justin [ORNL; Liang, Chengdu [ORNL] [ORNL
Anomalous high ionic conductivity of nanoporous ?-Li3PS4.
Lithium-ion-conducting solid electrolytes hold promise for enabling high-energy battery chemistries and circumventing safety issues of conventional lithium batteries. Achieving the combination of high ionic conductivity and a broad electrochemical window in solid electrolytes is a grand challenge for the synthesis of battery materials. Herein we show an enhancement of the room-temperature lithium-ion conductivity by 3 orders of magnitude through the creation of nanostructured Li(3)PS(4). This material has a wide electrochemical window (5 V) and superior chemical stability against lithium metal. The nanoporous structure of Li(3)PS(4) reconciles two vital effects that enhance the ionic conductivity: (1) the reduction of the dimensions to a nanometer-sized framework stabilizes the high-conduction ? phase that occurs at elevated temperatures, and (2) the high surface-to-bulk ratio of nanoporous ?-Li(3)PS(4) promotes surface conduction. Manipulating the ionic conductivity of solid electrolytes has far-reaching implications for materials design and synthesis in a broad range of applications, including batteries, fuel cells, sensors, photovoltaic systems, and so forth. PMID:23305294
Liu, Zengcai; Fu, Wujun; Payzant, E Andrew; Yu, Xiang; Wu, Zili; Dudney, Nancy J; Kiggans, Jim; Hong, Kunlun; Rondinone, Adam J; Liang, Chengdu
STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells
Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western hemisphere, but its pathogenesis is still poorly understood. Constitutive tyrosine phosphorylation (p) of signal transducer and activator of transcription (STAT) 3 occurs in several solid tumors and hematologic malignancies. In CLL, however, STAT3 is constitutively phosphorylated on serine 727, not tyrosine 705, residues. Because the biologic significance of serine pSTAT3 in CLL is not known, we studied peripheral blood cells of 106 patients with CLL and found that, although tyrosine pSTAT3 was inducible, serine pSTAT3 was constitutive in all patients studied, regardless of blood count, disease stage, or treatment status. In addition, we demonstrated that constitutive serine pSTAT3 translocates to the nucleus by the karyopherin-? nucleocytoplasmic system and binds DNA. Dephosphorylation of inducible tyrosine pSTAT3 did not affect STAT3-DNA binding, suggesting that constitutive serine pSTAT3 binds DNA. Furthermore, infection of CLL cells with lentiviral STAT3-small hairpin RNA reduced the expression of several STAT3-regulated survival and proliferation genes and induced apoptosis, suggesting that constitutive serine pSTAT3 initiates transcription in CLL cells. Taken together, our data suggest that constitutive phosphorylation of STAT3 on serine 727 residues is a hallmark of CLL and that STAT3 be considered a therapeutic target in this disease. PMID:20154216
Hazan-Halevy, Inbal; Harris, David; Liu, Zhiming; Liu, Jie; Li, Ping; Chen, Xiaomin; Shanker, Sreejesh; Ferrajoli, Alessandra; Keating, Michael J.
Functional evidence for D-serine inhibition of non-N-methyl-D-aspartate ionotropic glutamate receptors in retinal neurons.
D-Serine is an important signaling molecule throughout the central nervous system, acting as an N-methyl-D-aspartate (NMDA) receptor coagonist. This study investigated the D-serine modulation of non-NMDA ionotropic glutamate receptors expressed by inner retinal neurons. We first identified that the degradation of endogenous retinal D-serine, by application of D-amino acid oxidase, caused an enhancement of kainate- and ?-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor-mediated calcium responses from the ganglion cell layer of the isolated rat retina and light-evoked responses obtained by multi-electrode array recordings from the guinea pig retina. Approximately 30-45% of cells were endogenously inhibited by D-serine, as suggested by the effect of D-amino acid oxidase. Conversely, bath application of D-serine caused a reduction in multi-electrode array recorded responses and decreased kainate, but not potassium-induced calcium responses, in a concentration-dependent manner (IC(50), 280 ?m). Using cultured retinal ganglion cells to reduce network influences, D-serine reduced kainate-induced calcium responses and AMPA induced whole-cell currents. Finally, the inhibitory effect of D-serine on the kainate-induced calcium response was abolished by IEM 1460, thereby identifying calcium-permeable AMPA receptors as a potential target for D-serine. To our knowledge, this is the first study to address specifically the effect of D-serine on AMPA/kainate receptors in intact central nervous system tissue, to identify its effect on calcium permeable AMPA receptors and to report the endogenous inhibition of AMPA/kainate receptors. PMID:22128843
Daniels, Bryan A; Wood, Leah; Tremblay, Fran�ois; Baldridge, William H
Generation of Partially Reprogrammed Cells and Fully Reprogrammed iPS Cells by Plasmid Transfection.
Induced pluripotent stem (iPS) cells can be directly generated from somatic cells by overexpression of defined transcription factors. iPS cells can perpetually self-renew and differentiate into all cell types of an organism. iPS cells were first generated through infection with retroviruses that contain reprogramming factors. However, development of an exogene-free iPS cell generation method is crucial for future therapeutic applications, because integrated exogenes result in the formation of tumors in chimeras and regain pluripotency after differentiation in vitro. Here, we describe a method to generate iPS cells by transfection of plasmid vectors and to convert partially reprogrammed cells into fully reprogrammed iPS cells by switching from mouse ESC culture conditions to KOSR-based media with bFGF. We also describe basic methods used to characterize fully reprogrammed iPS cells. PMID:25476445
Kim, Jong Soo; Choi, Hyun Woo; Hong, Yean Ju; Do, Jeong Tae
The midgut of Aedes albopictus females expresses active trypsin-like serine peptidases
Background Aedes albopictus is widely distributed across tropical and sub-tropical regions and is associated with the transmission of several arboviruses. Although this species is increasingly relevant to public health due its ability to successfully colonize both urban and rural habitats, favoring the dispersion of viral infections, little is known about its biochemical traits, with all assumptions made based on studies of A. aegypti. In previous studies we characterized the peptidase profile of pre-imaginal stages of A. albopictus and we reported the first proteomic analysis of the midgut from sugar-fed females of this insect species. Methods In the present work, we further analyzed the peptidase expression in the midgut of sugar-fed females using 1DE-substrate gel zymography, two-dimensional electrophoresis (2DE), mass spectrometry (MS), and protein identification based on similarity. Results The combination of zymography, in solution assays using fluorescent substrates and 2DE-MS/MS allowed us to identify the active serine peptidase �fingerprint� in the midgut of A. albopictus females. Zymographic analysis revealed a proteolytic profile composed of at least 13 bands ranging from ~25 to 250�kDa, which were identified as trypsin-like serine peptidases by using specific inhibitors of this class of enzymes. Concomitant use of the fluorogenic substrate Z-Phe-Arg-AMC and trypsin-like serine protease inhibitors corroborated the zymographic findings. Our proteomic approach allowed the identification of two different trypsin-like serine peptidases and one chymotrypsin in protein spots of the alkaline region in 2DE map of the A. albopictus female midgut. Identification of these protein coding genes was achieved by similarity to the A. aegypti genome sequences using Mascot and OMSSA search engines. Conclusion These results allowed us to detect, identify and characterize the expression of active trypsin-like serine peptidases in the midgut of sugar-fed A. albopictus females. In addition, proteomic analysis allowed us to confidently assign the expression of two trypsin genes and one chymotrypsin gene to the midgut of this mosquito. These results contribute to the gene annotation in this species of unknown genome and represent a small but important step toward the protein-level functional and localization assignment of trypsin-like serine peptidase genes in the Aedes genus. PMID:24886160
Rapid loss of perforin and serine protease RNA in cytotoxic lymphocytes exposed to sensitive targets.
We have previously reported that cytotoxic lymphocytes, when exposed to sensitive target cells, temporarily lose their lytic potential. The mechanism leading to this loss of lytic activity is still unknown but it is reversible and the lytic potency can be recovered when the effector cells are incubated with interleukin-2 (IL-2) for 12-14 hr. In this study, we have investigated the regulation of RNA coding for perforin and for two serine proteases, HSP1 and HSP2, in cytotoxic lymphocytes exposed to sensitive targets. Perforin and the two serine proteases are contained in granules of major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxic lymphocytes, but their exact role in the lytic mechanism is still debated. Here we used four different human cytotoxic lymphocytes (CTL) as effector cells: an MHC-restricted CTL (SG-CTL), a non-MHC-restricted CTL (IE6), a natural killer (NK)-like cell line (3.3) and lymphokine-activated killer (LAK) cells. In all effector cells we observed a rapid loss of perforin and of serine protease RNAs within 5 min following the addition of sensitive targets. The effector cells recovered the RNA messages as early as 30 min, although the kinetics of recovery was faster with CTL than with NK-like or LAK effector cells. When we exposed the effector cells to resistant targets we did not detect any loss of perforin or serine protease RNAs. Incubation of the effector cells with cycloheximide, prior to the addition of sensitive targets, did not block message loss, indicating that de novo protein synthesis was not required in this process. Cycloheximide treatment, however, inhibited the recovery of perforin and serine protease RNAs. Taken together, our results indicate that the target-mediated loss of lytic activity in cytotoxic lymphocytes may be a consequence of the down-regulation of perforin or of serine protease transcripts, or both. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1721042
Bajpai, A; Kwon, B S; Brahmi, Z
Human prostate-specific antigen: structural and functional similarity with serine proteases.
The complete amino acid sequence of the prostate-specific antigen (PA) from human seminal plasma has been determined from analyses of the peptides generated by cyanogen bromide, hydroxylamine, endoproteinases Arg-C and Lys-C. The single polypeptide chain of PA contains 240-amino acid residues and has a calculated Mr of 26,496. An N-linked carbohydrate side chain is predicted at asparagine-45, and O-linked carbohydrate side chains are possibly attached to serine-69, threonine-70, and serine-71. The primary structure of PA shows a high degree of sequence homology with other serine proteases of the kallikrein family. The active site residues of histidine, aspartic acid, and serine comprising the charge-relay system of typical serine proteases were found in similar positions in PA (histidine-41, aspartic acid-96, and serine-192). At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant interleukin 2, and--to a lesser extent--gelatin, myoglobin, ovalbumin, and fibrinogen. The cleavage sites of these proteins by PA were chemically analyzed as the alpha-carboxyl side of some hydrophobic residues, tyrosine, leucine, valine, and phenylalanine, and of basic residues histidine, lysine, and arginine. The chymotrypsin-like activity of PA exhibited with the chromogenic substrate N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide yielded a specific activity of 9.21 microM per min per mg of PA and Km and kcat values of 15.3 mM and 0.075s-1, respectively. "Trypsin-like" activity of PA was also detected with N alpha-benzoyl-DL-arginine p-nitroanilide and gave a specific activity of 1.98 microM per min per mg of PA. Protease inhibitors such as phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, L-1-tosylamido-2-phenylethyl chloromethyl ketone, aprotinin, leupeptin, soybean trypsin inhibitor as well as Zn2+ and spermidine were effective inhibitors of PA enzymatic activity. PMID:2422647
Watt, K W; Lee, P J; M'Timkulu, T; Chan, W P; Loor, R
KSR-Based Medium Improves the Generation of High-Quality Mouse iPS Cells
Induced pluripotent stem (iPS) cells from somatic cells have great potential for regenerative medicine. The efficiency in generation of iPS cells has been significantly improved in recent years. However, the generation of high-quality iPS cells remains of high interest. Consistently, we demonstrate that knockout serum replacement (KSR)-based medium accelerates iPS cell induction and improves the quality of iPS cells, as confirmed by generation of chimeras and all iPS cell-derived offspring with germline transmission competency. Both alkaline phosphatase (AP) activity assay and expression of Nanog have been used to evaluate the efficiency of iPS cell induction and formation of ES/iPS cell colonies; however, appropriate expression of Nanog frequently indicates the quality of ES/iPS cells. Interestingly, whereas foetal bovine serum (FBS)-based media increase iPS cell colony formation, as revealed by AP activity, KSR-based media increase the frequency of iPS cell colony formation with Nanog expression. Furthermore, inhibition of MAPK/ERK by a specific inhibitor, PD0325901, in KSR- but not in FBS-based media significantly increases Nanog-GFP+ iPS cells. In contrast, addition of bFGF in KSR-based media decreases proportion of Nanog-GFP+ iPS cells. Remarkably, PD can rescue Nanog-GFP+ deficiency caused by bFGF. These data suggest that MAPK/ERK pathway influences high quality mouse iPS cells and that KSR- and PD-based media could enrich homogeneous authentic pluripotent stem cells. PMID:25171101
Liu, Kai; Wang, Fang; Ye, Xiaoying; Wang, Lingling; Yang, Jiao; Zhang, Jingzhuo; Liu, Lin
M�ssbauer study of the transition metal phosphorus trichalcogenide FePS3 and spin glass Mn0.5Fe0.5PS3
NASA Astrophysics Data System (ADS)
The transition metal phosphorus trichalcogenides MnPS3 and the FePS3 are CdCl2 type layered compounds, where the transition metal ions form a hexagonal lattice. While these compounds order anti-ferromagnetically at low temperature, the magnetic structures are different. We have reported that these mixtures Mn0.5Fe0.5PS3 is a spin glass with a glass transition temperature T g=33.7 K. Then, in this work, we report that the results of the temperature variation of the 57Fe M�ssbauer spectra of FePS3 and Mn0.5Fe0.5PS3, in detail. In the anti-ferromagnetic state of FePS3, the hyperfine magnetic field H int increases with decreasing temperature and the Isomer shift I. S. increases slightly with decreasing temperature. However in Mn0.5Fe0.5PS3, the two broadened peaks are observed and the two peaks became a single peak with decreasing temperature at about 50.0 K, which is higher than T g=33.7 K. In the spin glass Mn0.5Fe0.5PS3, the M�ssbauer spectra suggest that the magnetic interactions exist far above T g.
Ban, Shuichi; Takano, Yoshiki; Ohkubo, Naoki; Hoya, Hisanobu; Takahashi, Yumiko; Takase, Kouichi; Sekizawa, Kazuko
Isolation and Nucleotide Sequence of the cDNA for Rat Liver Serine Dehydratase mRNA and Structures of the 5' and 3' Flanking Regions of the Serine Dehydratase Gene
Rat serine dehydratase cDNA clones were isolated from a lambda gt11 cDNA library on the basis of their reactivity with monospecific immunoglobulin to the purified enzyme. Using the cDNA insert from a clone that encoded the serine dehydratase subunit as a probe, additional clones were isolated from the same library by plaque hybridization. Nucleotide sequence analysis of the largest clone
Hirofumi Ogawa; Duncan A. Miller; Tracy Dunn; Yeu Su; James M. Burcham; Carl Peraino; Motoji Fujioka; Kay Babcock; Henry C. Pitot
Enhancement of cyanogen bromide cleavage yields for methionyl-serine and methionyl-threonine peptide bonds.
Cyanogen bromide (CNBr) is a common chemical used to hydrolyze peptide bonds C-terminal to methionine residues in peptides and proteins. In most cases, the efficiency of this bond cleavage is greater than 90% except in situations where a serine or threonine residue follows methionine in the amino acid sequence. We have explored the mechanism of the methionyl-serine and methionyl-threonine CNBr cleavage inefficiencies and have developed a simple methodology to more than double cleavage yields relative to standard literature conditions. This method entails increasing the concentration of water during the cleavage reaction either by reducing the formic acid concentration or by performing the cleavage in an acidic aqueous medium. This approach provides a more desirable methodology from the perspective of enhanced yields and greater ease of handling in cases of large-scale use. PMID:9887207
Kaiser, R; Metzka, L
Phosphorylation of adenylyl cyclase III at serine1076 does not attenuate olfactory response in mice
Feedback inhibition of adenylyl cyclase III (ACIII) via Ca2+-induced phosphorylation has long been hypothesized to contribute to response termination and adaptation of olfactory sensory neurons (OSNs). To directly determine the functional significance of this feedback mechanism for olfaction in vivo, we genetically mutated serine1076 of ACIII, the only residue responsible for Ca2+-induced phosphorylation and inhibition of ACIII (Wei et al., 1996; Wei et al., 1998), to alanine in mice. Immunohistochemistry and Western blot analysis showed that the mutation affects neither the cilial localization nor the expression level of ACIII in OSNs. Electroolfactogram analysis showed no differences in the responses between wildtype and mutant mice to single-pulse odorant stimulations or in several stimulation paradigms for adaptation. These results suggest that phosphorylation of ACIII on serine1076 plays a far less important role in olfactory response attenuation than previously thought. PMID:23077041
Cygnar, Katherine D; Collins, Sarah Ellen; Ferguson, Christopher H; Bodkin-Clarke, Chantal; Zhao, Haiqing
Characterization of the usage of the serine metabolic network in human cancer
The serine, glycine, one carbon (SGOC) metabolic network is implicated in cancer pathogenesis but its general functions are unknown. We carried out a computational reconstruction of the SGOC network and then characterized its expression across thousands of cancer tissues. Pathways including methylation and redox metabolism exhibited heterogeneous expression indicating a strong context dependency of their usage in tumors. From an analysis of coexpression, simultaneous up- or down-regulation of nucleotide synthesis, NADPH and glutathione synthesis was found to be a common occurrence in all cancers. Finally, we developed a method to trace the metabolic fate of serine using stable isotopes, high-resolution mass spectrometry and a mathematical model. Although the expression of single genes didn�t appear indicative of flux, the collective expression of several genes in a given pathway allowed for successful flux prediction. Together these findings identify expansive and heterogeneous functions for the SGOC metabolic network in human cancer. PMID:25456139
Mehrmohamadi, Mahya; Liu, Xiaojing; Shestov, Alexander A; Locasale, Jason W
Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6.
Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K(i) around 1nM, KLK4 with K(i)=27.3nM, KLK6 with K(i)=140nM, caspase-14 with a K(i) approximating 1?M and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members. PMID:21439340
Kantyka, Tomasz; Fischer, Jan; Wu, Zhihong; Declercq, Wim; Reiss, Karina; Schr�der, Jens-Michael; Meyer-Hoffert, Ulf
Preliminary characterization of a Kazal-type serine protease inhibitor from Caiman crocodilus yacare plasma.
Blood serine protease inhibitors are becoming better understood and increasingly applied in blood clotting, cancer and other diseases. Reptiles are suitable models for blood coagulation and related processes, moreover, caiman is a good comparative model of a non-poisonous reptile. Recently, we reported the purification of a kininogen, the presence of proteases involved in blood clotting, and a serine protease inhibitor in Caiman crocodilus yacare plasma. In this paper, we described the partial sequence of an inhibitor (CcTI). The inhibitor is an 80-kDa protein, and it inactivates trypsin and chymotrypsin the hydrolysis of specific chromogenic substrates and in the degradation of gelatin. The inhibitor is member of Kazal-type inhibitor family and consists of several domains, its putative reactive site is Arg-His. PMID:10615009
Araujo, M S; Nunes, V A; Gozzo, A J; Sampaio, M U; Auerswald, E; Ura, N; Shimamoto, K; Sampaio, C A
Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination.
Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity. PMID:18674905
Shiraishi, Takuya; Kadono, Shojiro; Haramura, Masayuki; Kodama, Hirofumi; Ono, Yoshiyuki; Iikura, Hitoshi; Esaki, Tohru; Koga, Takaki; Hattori, Kunihiro; Watanabe, Yoshiaki; Sakamoto, Akihisa; Yoshihashi, Kazutaka; Kitazawa, Takehisa; Esaki, Keiko; Ohta, Masateru; Sato, Haruhiko; Kozono, Toshiro
The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish
NASA Technical Reports Server (NTRS)
Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by th e 1998 Barrett nomenclature) has an unusual phylogenetic distribution , being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is larg ely restricted to the genus Streptomyces, although a few isolated occ urrences in other bacteria have been reported. The family may be enti rely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them ha ve been from early diverging phyla like Porifera or Cnidaria, We now report the existence of Group SlA serine proteases in a sponge (phylu m Porifera) and a jellyfish (phylum Cnidaria), making it safe to conc lude that all animal groups possess these enzymes.
Rojas, Ana; Doolittle, Russell F.
MicroRNA expression profiles of human iPS cells, retinal pigment epithelium derived from iPS, and fetal retinal pigment epithelium.
The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types. PMID:24999033
Greene, Whitney A; Mu�iz, Alberto; Plamper, Mark L; Kaini, Ramesh R; Wang, Heuy-Ching
Two potassium gadolinium(III) ortho-thiophosphates(V): K 3Gd 3[PS 4] 4 and K 9Gd[PS 4] 4
K3Gd3[PS4]4 and K9Gd[PS4]4 form by the reaction of gadolinium, red phosphorus and sulfur with appropriate portions of K2S at 950�C for 14 days in evacuated torch-sealed silica ampoules. Both structures contain isolated [PS4]3? tetrahedra (d(P�S)=201�208pm) and eightfold coordinated Gd3+ cations (d(Gd�S)=278�307pm). K3Gd3[PS4]4 which is isotypic with K3Ho3[PS4]4 crystallizes monoclinically in the space group C2\\/c (a=1601.87(9)pm, b=1162.48(6)pm, c=1452.39(8)pm, ?=90.773(5)�; Z=4). The
Theresa Komm; Sabine Strobel; Thomas Schleid
Effects of Enterococcus faecalis fsr Genes on Production of Gelatinase and a Serine Protease and Virulence
Three agr-like genes (fsrA, fsrB, and fsrC, for Enterococcus faecalis regulator) were found upstream of the previously reported gelatinase gene (gelE) and a downstream putative serine protease gene (sprE; accession number Z12296) of Enterococcus faecalis OG1RF. The deduced amino acid sequence of fsrA shows 26% identity and 38% similarity to Staphylococcus aureus AgrA (the response regulator of the accessory gene
Religiosin B, a milk-clotting serine protease from Ficus religiosa
A novel milk-clotting serine protease, named religiosin B, is purified from Ficus religiosa. The molecular mass of the protein is 63,000 with pI value of pH 7.6. The proteolytic activity of the enzyme is strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and chymostatin. Religiosin B acts optimally at pH 8.0�8.5 and temperature 55�C. The molar absorption coefficient of the enzyme is
Moni Kumari; Anurag Sharma; M. V. Jagannadham
Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity.
As a model to investigate the mechanism of caspase activation we have analysed the processing of pro-caspase-7 by serine proteases with varied specificities. The caspase-7 zymogen was rapidly activated by granzyme B and more slowly by subtilisin and cathepsin G, generating active enzymes with similar kinetic properties. Significantly, cathepsin G activated the zymogen by cleaving at a Gln-Ala bond, indicating that the canonical cleavage specificity at aspartic acid is not required for activation. PMID:9182691
Zhou, Q; Salvesen, G S
Neurobiology through the looking-glass: d-serine as a new glial-derived transmitter
d-Amino acids have been known to be present in bacteria for more than 50 years, but only recently they were identified in mammals. The occurrence of d-amino acids in mammals challenge classic concepts in biology in which only l-amino acids would be present or thought to play important roles. Recent discoveries uncovered a role of endogenous d-serine as a putative
Herman Wolosker; Rogerio Panizzutti; Joari De Miranda
Matriptase-3 is a novel phylogenetically preserved membrane-anchored serine protease with broad serpin reactivity.
We report in the present study the bioinformatic identification, molecular cloning and biological characterization of matriptase-3, a novel membrane-anchored serine protease that is phylogenetically preserved in fish, birds, rodents, canines and primates. The gene encoding matriptase-3 is located on syntenic regions of human chromosome 3q13.2, mouse chromosome 16B5, rat chromosome 11q21 and chicken chromosome 1. Bioinformatic analysis combined with cDNA cloning predicts a functional TTSP (type II transmembrane serine protease) with 31% amino acid identity with both matriptase/MT-SP1 and matriptase-2. This novel protease is composed of a short N-terminal cytoplasmic region followed by a transmembrane domain, a stem region with one SEA, two CUB and three LDLRa (low-density lipoprotein receptor domain class A) domains and a C-terminal catalytic serine protease domain. Transcript analysis revealed restricted, species-conserved expression of matriptase-3, with the highest mRNA levels in brain, skin, reproductive and oropharyngeal tissues. The full-length matriptase-3 cDNA directed the expression of a 90 kDa N-glycosylated protein that localized to the cell surface, as assessed by cell-surface biotin labelling. The purified activated matriptase-3 serine protease domain expressed in insect cells hydrolysed synthetic peptide substrates, with a strong preference for Arg at position P(1), and showed proteolytic activity towards several macromolecular substrates, including gelatin, casein and albumin. Interestingly, activated matriptase-3 formed stable inhibitor complexes with an array of serpins, including plasminogen activator inhibitor-1, protein C inhibitor, alpha1-proteinase inhibitor, alpha2-antiplasmin and antithrombin III. Our study identifies matriptase-3 as a novel biologically active TTSP of the matriptase subfamily having a unique expression pattern and post-translational regulation. PMID:15853774
Szabo, Roman; Netzel-Arnett, Sarah; Hobson, John P; Antalis, Toni M; Bugge, Thomas H
Matriptase-3 is a novel phylogenetically preserved membrane-anchored serine protease with broad serpin reactivity
We report in the present study the bioinformatic identification, molecular cloning and biological characterization of matriptase-3, a novel membrane-anchored serine protease that is phylogenetically preserved in fish, birds, rodents, canines and primates. The gene encoding matriptase-3 is located on syntenic regions of human chromosome 3q13.2, mouse chromosome 16B5, rat chromosome 11q21 and chicken chromosome 1. Bioinformatic analysis combined with cDNA cloning predicts a functional TTSP (type II transmembrane serine protease) with 31% amino acid identity with both matriptase/MT-SP1 and matriptase-2. This novel protease is composed of a short N-terminal cytoplasmic region followed by a transmembrane domain, a stem region with one SEA, two CUB and three LDLRa (low-density lipoprotein receptor domain class A) domains and a C-terminal catalytic serine protease domain. Transcript analysis revealed restricted, species-conserved expression of matriptase-3, with the highest mRNA levels in brain, skin, reproductive and oropharyngeal tissues. The full-length matriptase-3 cDNA directed the expression of a 90�kDa N-glycosylated protein that localized to the cell surface, as assessed by cell-surface biotin labelling. The purified activated matriptase-3 serine protease domain expressed in insect cells hydrolysed synthetic peptide substrates, with a strong preference for Arg at position P1, and showed proteolytic activity towards several macromolecular substrates, including gelatin, casein and albumin. Interestingly, activated matriptase-3 formed stable inhibitor complexes with an array of serpins, including plasminogen activator inhibitor-1, protein C inhibitor, ?1-proteinase inhibitor, ?2-antiplasmin and antithrombin III. Our study identifies matriptase-3 as a novel biologically active TTSP of the matriptase subfamily having a unique expression pattern and post-translational regulation. PMID:15853774
The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells
TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1�4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2. PMID:15537383
Structure of Soybean Serine Acetyltransferase and Formation of the Cysteine Regulatory Complex as a Molecular Chaperone*
Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75�3.0 ? resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms. The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His169 and Asp154 form a catalytic dyad for general base catalysis and that His189 may stabilize the oxyanion reaction intermediate. Glu177 helps to position Arg203 and His204 and the ?1c-?2c loop for serine binding. A similar role for ionic interactions formed by Lys230 is required for CoA binding. The GmSAT structures also identify Arg253 as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants. PMID:24225955
Yi, Hankuil; Dey, Sanghamitra; Kumaran, Sangaralingam; Lee, Soon Goo; Krishnan, Hari B.; Jez, Joseph M.
Occurrence of two l-threonine (l-serine) dehydratases in the thermophile Chloroflexus aurantiacus
The thermophilic phototrophic prokaryote, Chloroflexus aurantiacus was shown to contain high constitutive l-threonine (l-serine) deaminating activity. Separation of cellular proteins by DE 52-cellulose chromatography and by polyacrylamide gel electrophoresis with subsequent activity staining of the gels yielded two bands, one representing an isoleucine-sensitive, the other one an isoleucine-insensitive form of l-threonine dehydratase. Both enzymes had a molecular weight of 120,000
Gisela Laakmann-Ditges; Jobst-Heinrich Klemme
Phosphorylation of Two Serine Residues Regulates Human T-Cell Leukemia Virus Type 2 Rex Function
The function of the human T-cell leukemia virus (HTLV) Rex phosphoprotein is to increase the level of the viral structural and enzymatic gene products expressed from the incompletely spliced viral RNAs containing the Rex-responsive element. The phosphorylation of HTLV type 2 Rex (Rex-2), predominantly on serine residues, correlates with an altered conformation, as detected by a gel mobility shift, and is required for specific binding to its viral RNA target sequence. Thus, the phosphorylation state of Rex in the infected cell may be a switch that determines whether the virus exists in a latent or a productive state. A mutational analysis of Rex-2 that focused on serine and threonine residues was performed to identify regions or domains within Rex-2 important for function, with a specific emphasis on identifying Rex-2 phosphorylation mutants. We identified mutations near the carboxy terminus that disrupted a novel region or domain and abrogated Rex-2 function. Mutant M17 (with S151A and S153A mutations) displayed reduced phosphorylation that correlated with reduced function. Replacement of both serine residues 151 and 153 with phosphomimetic aspartic acid restored Rex-2 function and locked Rex-2 in a phosphorylated active conformation. A mutant containing threonine residues at positions 151 and 153 displayed a phenotype indistinguishable from that of wild-type Rex. Furthermore, this same mutant showed increased threonine phosphorylation and decreased serine phosphorylation, providing conclusive evidence that one or both of these residues are phosphorylated in vivo. Our results provide the first direct evidence that the phosphorylation of Rex-2 is important for function. Further understanding of HTLV Rex phosphorylation will provide insight into the regulatory control of HTLV replication and ultimately the pathobiology of HTLV. PMID:11507189
Narayan, Murli; Kusuhara, Koichi; Green, Patrick L.
SufA a novel subtilisin-like serine proteinase of Finegoldia magna
Finegoldia magna is an anaerobic Gram-positive bacterium and commensal, which is also associated with clinically important conditions such as skin and soft tissue infections. This study describes a novel subtilisin-like extracellular serine proteinase of F. magna, denoted SufA (subtilase of Finegoldia magna), which is believed to be the first subtilase described among Gram-positive anaerobic cocci. SufA is associated with the
Christofer Karlsson; Marie-Louise Andersson; Mattias Collin; Artur Schmidtchen; Lars Bjorck; Inga-Maria Frick
14-3-3 Inhibits Bad-Induced Cell Death through Interaction with Serine-136
on serine. At least three sites on Bad can be phosphorylated in vivo, including S112, S136, and S155 sites, S112 and S136, lie within potential 14-3-3 binding sites. S155 does not possess a known 14-3-3 binding motif. Indeed, mutation of S155 has not been shown to affect the 14-3-3/Bad interaction (Datta et
Datta, Sandeep Robert
Purification and Characterization of Myofibril-bound Serine Proteinase from Carp Cyprinus carpio Ordinary Muscle
1. A novel myofibril-bound serine proteinase (MBP) has been purified from ordinary muscle of the carp Cyprinus carpio. 2. It was solubilized from the myofibril fraction with acid treatment (under the conditions of 0.6 M KCl, pH 4.0), then purified by column chromatographic steps on Ultrogel AcA 54, and Arginine-Sepharose 4B. 3. The purified enzyme revealed a single protein band
Kiyoshi Osatomi; Hiroshi Sasai; Minjie Cao; Kenji Hara; Tadashi Ishihara
Malonate-based inhibitors of mammalian serine racemase: kinetic characterization and structure-based computational study.
Overactivation of NMDA receptors has been implicated in various neuropathological conditions, including brain ischaemia, neurodegenerative disorders and epilepsy. Production of d-serine, an NMDA receptor co-agonist, from l-serine is catalyzed in�vivo by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine racemase. Specific inhibition of this enzyme has been proposed as a promising strategy for treatment of neurological conditions caused by NMDA receptor dysfunction. Here we present the synthesis and activity analysis of a series of malonate-based inhibitors of mouse serine racemase (mSR). The compounds possessed IC50 values ranging from 40���11�mM for 2,2-bis(hydroxymethyl)malonate down to 57���1�?M for 2,2-dichloromalonate, the most effective competitive mSR inhibitor known to date. The structure-activity relationship of the whole series in the human orthologue (hSR) was interpreted using Glide docking, WaterMap analysis of hydration and quantum mechanical calculations based on the X-ray structure of the hSR/malonate complex. Docking into the hSR active site with three thermodynamically favourable water molecules was able to discern qualitatively between good and weak inhibitors. Further improvement in ranking was obtained using advanced PM6-D3H4X/COSMO semiempirical quantum mechanics-based scoring which distinguished between the compounds with IC50 better/worse than 2�mM. We have thus not only found a new potent hSR inhibitor but also worked out a computer-assisted protocol to rationalize the binding affinity which will thus aid in search for more effective SR inhibitors. Novel, potent hSR inhibitors may represent interesting research tools as well as drug candidates for treatment of diseases associated with NMDA receptor overactivation. PMID:25462239
Vorlov�, Barbora; Nachtigallov�, Dana; Jir�skov�-Van�?kov�, Jana; Ajani, Haresh; Jansa, Petr; Rez�?, Jan; Fanfrl�k, Jind?ich; Otyepka, Michal; Hobza, Pavel; Konvalinka, Jan; Lep��k, Martin
Mutants of the protein serine kinase PSKH1 disassemble the Golgi apparatus
We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring
Gaute Brede; Jorun Solheim; Espen Stang; Hans Prydz
Gene Cloning and Expression of an Alkaline Serine Protease with Dehairing Function from Bacillus pumilus
A new gene (named AP gene) encoding an alkaline serine protease with dehairing function was cloned from Bacillus pumilus UN-31-C-42 and its nucleotide sequence was determined. The expression of AP gene was induced with IPTG in Escherichia coli after the mature protease region was cloned into pET15b and SDS-PAGE showed expressed product clearly, but no alkaline protease activity was detected.
Jiao Pan; Qing Huang; YiZheng Zhang
Purification and Characterization of an Extracellular Alkaline Serine Protease with Dehairing Function from Bacillus pumilus
An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography,\\u000a ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal\\u000a activity at pH 10 and with a temperature of 55�C; the enzyme activity
Qing Huang; Yong Peng; Xin Li; Haifeng Wang; Yizheng Zhang
Soybean-milk-coagulating activity of Bacillus pumilus derives from a serine proteinase
A proteolytic enzyme from Bacillus pumilus strain TYO-67, which was able to coagulate the protein in soybean milk, was characterized enzymologically. The optimum pH\\u000a and temperature for its activities were 9.0 and 50?�C, respectively. The enzyme was strongly believed to be a serine proteinase\\u000a because it was completely inhibited by the addition of diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride. Hammerstein\\u000a milk
M. Aoyama; M. Yasuda; K. Nakachi; N. Kobamoto; H. Oku; F. Kato
Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase.
Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes a hydroxymethyl group transfer from l-serine to tetrahydrofolate (H4folate) to yield glycine and 5,10-methylenetetrahydrofolate (CH2-H4folate). SHMT is crucial for deoxythymidylate biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary complex mechanism. To define the kinetic mechanism of this catalysis, we explored the PvSHMT reaction by employing various methodologies including ligand binding, transient, and steady-state kinetics as well as product analysis by rapid-quench and HPLC/MS techniques. The results indicate that PvSHMT can bind first to either l-serine or H4folate. The dissociation constants for the enzyme�l-serine and enzyme�H4folate complexes were determined as 0.18 � 0.08 and 0.35 � 0.06 mm, respectively. The amounts of glycine formed after single turnovers of different preformed binary complexes were similar, indicating that the reaction proceeds via a random-order binding mechanism. In addition, the rate constant of glycine formation measured by rapid-quench and HPLC/MS analysis is similar to the kcat value (1.09 � 0.05 s(-1)) obtained from the steady-state kinetics, indicating that glycine formation is the rate-limiting step of SHMT catalysis. This information will serve as a basis for future investigation on species-specific inhibition of SHMT for antimalarial drug development. PMID:25678710
Maenpuen, Somchart; Amornwatcharapong, Watcharee; Krasatong, Pasupat; Sucharitakul, Jeerus; Palfey, Bruce A; Yuthavong, Yongyuth; Chitnumsub, Penchit; Leartsakulpanich, Ubolsree; Chaiyen, Pimchai
Regulation of Interferon Regulatory Factor3 by the Hepatitis C Virus Serine Protease
Persistent infections with hepatitis C virus (HCV) are likely to depend on viral inhibition of host defenses. We show that the HCV NS3\\/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3\\/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3
Eileen Foy; Kui Li; Chunfu Wang; Rhea Sumpter; Masanori Ikeda; Stanley M. Lemon; Michael Gale
Insights derived from molecular dynamics simulation into the molecular motions of serine protease proteinase K
Serine protease proteinase K, a member of the subtilisin family of enzymes, is of significant industrial, agricultural and\\u000a biotechnological importance. Despite the wealth of structural information about proteinase K provided by static X-ray structures,\\u000a a full understanding of the enzymatic mechanism requires further insight into the dynamic properties of this enzyme. Molecular\\u000a dynamics simulations and essential dynamics (ED) analysis were
Shu-Qun Liu; Zhao-Hui Meng; Yun-Xin Fu; Ke-Qin Zhang
Taraxalisin � a serine proteinase from dandelion Taraxacum officinale Webb s.l
Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp-Ala-Ala-Leu-pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose-Ala-Ala-Leu-mrp
G. N. Rudenskaya; A. M. Bogacheva; A. Preusser; A. V. Kuznetsova; Ya. E. Dunaevsky; B. N. Golovkin; V. M. Stepanov
The distribution of the serin ( Serinus serinus L., 1766) in the 16th century
All the sources of records of the serin ( Serinus serinus) in 16th century Europe are (re-)examined, both those already known and some that have been newly discovered. Interpretation of this more detailed information confirms the results which were published by Ernst Mayr in 1926 in his doctoral thesis: north of 48�N there were no free-living populations of Serinus serinus
Ragnar K. Kinzelbach
Alternaria-derived serine protease activity drives IL-33�mediated asthma exacerbations
Background The fungal allergen Alternaria alternata is implicated in severe asthma and rapid onset life-threatening exacerbations of disease. However, the mechanisms that underlie this severe pathogenicity remain unclear. Objective We sought to investigate the mechanism whereby Alternaria was capable of initiating severe, rapid onset�allergic inflammation. Methods IL-33 levels were quantified in wild-type and ST2?/? mice that lacked the IL-33 receptor given inhaled house dust mite, cat dander, or Alternaria, and the effect of inhibiting allergen-specific protease activities on IL-33 levels was assessed. An exacerbation model of allergic airway disease was established whereby mice were sensitized with house dust mite before subsequently being challenged with Alternaria (with or without serine protease activity), and inflammation, remodeling, and lung function assessed 24 hours later. Results Alternaria, but not other common aeroallergens, possessed intrinsic serine protease activity that elicited the rapid release of IL-33 into the airways of mice through a mechanism that was dependent upon the activation of protease activated receptor-2 and adenosine triphosphate signaling. The unique capacity of Alternaria to drive this early IL-33 release resulted in a greater pulmonary inflammation by 24 hours after challenge relative to the common aeroallergen house dust mite. Furthermore, this Alternaria serine protease�IL-33 axis triggered a rapid, augmented inflammation, mucus release, and loss of lung function in our exacerbation model. Conclusion Alternaria-specific serine protease activity causes rapid IL-33 release, which underlies the development of a robust TH2 inflammation and exacerbation of allergic airway disease. PMID:24636086
Snelgrove, Robert J.; Gregory, Lisa G.; Peir�, Teresa; Akthar, Samia; Campbell, Gaynor A.; Walker, Simone A.; Lloyd, Clare M.
Phosphorylation of serine 11 and serine 92 as new positive regulators of human Snail1 function: potential involvement of casein kinase-2 and the cAMP-activated kinase protein kinase A.
Snail1 is a major factor for epithelial-mesenchymal transition (EMT), an important event in tumor metastasis and in other pathologies. Snail1 is tightly regulated at transcriptional and posttranscriptional levels. Control of Snail1 protein stability and nuclear export by GSK3beta phosphorylation is important for Snail1 functionality. Stabilization mechanisms independent of GSK3beta have also been reported, including interaction with LOXL2 or regulation of the COP9 signalosome by inflammatory signals. To get further insights into the role of Snail1 phosphorylation, we have performed an in-depth analysis of in vivo human Snail1 phosphorylation combined with mutational studies. We identify new phosphorylation sites at serines 11, 82, and 92 and confirmed previously suggested phosphorylations at serine 104 and 107. Serines 11 and 92 participate in the control of Snail1 stability and positively regulate Snail1 repressive function and its interaction with mSin3A corepressor. Furthermore, serines 11 and 92 are required for Snail1-mediated EMT and cell viability, respectively. PKA and CK2 have been characterized as the main kinases responsible for in vitro Snail1 phosphorylation at serine 11 and 92, respectively. These results highlight serines 11 and 92 as new players in Snail1 regulation and suggest the participation of CK2 and PKA in the modulation of Snail1 functionality. PMID:19923321
MacPherson, Matthew Reid; Molina, Patricia; Souchelnytskyi, Serhiy; Wernstedt, Christer; Martin-P�rez, Jorge; Portillo, Francisco; Cano, Amparo
Serine-Threonine Ubiquitination Mediates Downregulation of BST-2/Tetherin and Relief of Restricted Virion Release by HIV-1 Vpu? �
The HIV-1 protein Vpu counteracts the antiviral activity of the innate restriction factor BST-2/tetherin by a mechanism that partly depends on its interaction with ?-TrCP, a substrate adaptor for an SCF (Skp-Cullin 1-F box) E3 ubiquitin ligase complex. This suggests that Vpu stimulates the ubiquitination of BST-2 and that this underlies the relief of restriction. Here, we show that Vpu stimulates ubiquitination of BST-2. Mutation of all potential ubiquitination sites in the cytoplasmic domain of BST-2, including lysines, cysteines, serines, and threonines, abrogates Vpu-mediated ubiquitination. However, a serine-threonine-serine sequence specifically mediates the downregulation of BST-2 from the cell surface and the optimal relief of restricted virion release. Serine-threonine ubiquitination of BST-2 is likely part of the mechanism by which Vpu counteracts innate defenses. PMID:20980512
Tokarev, Andrey A.; Munguia, Jason; Guatelli, John C.
Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation
Antigen recognition within immunological synapses triggers and sustains T cell activation by nucleating protein microclusters that gather T cell receptors (TCRs), kinases, and adaptors. Dissipation of these microclusters results in signal termination, but how this process is regulated is unclear. In this paper, we reveal that release of the adaptors SLP76 and GADS from signaling microclusters is induced by the serine/threonine protein kinase HPK1 and that phosphorylation of GADS plays a major role in this process. We found that HPK1 was recruited into microclusters and triggered their dissipation by inducing the phosphorylation of a threonine-containing motif of GADS, together with the previously described serine phosphorylation of SLP76. These events induced the cooperative binding of 14-3-3 proteins to SLP76�GADS complexes, leading to their uncoupling from the transmembrane adaptor LAT and consequently reducing microcluster persistence and activation-induced gene transcription. These results demonstrate that serine/threonine phosphorylation of multiple TCR-proximal effectors controls the stability of signaling microclusters, thereby determining the intensity of T cell responses. PMID:22105350
Lasserre, R�mi; Cuche, C�line; Blecher-Gonen, Ronnie; Libman, Evgeny; Biquand, Elise; Danckaert, Anne; Yablonski, Deborah; Alcover, Andr�s
Serine Protease MP2 Activates Prophenoloxidase in the Melanization Immune Response of Drosophila melanogaster
In arthropods, melanization plays a major role in the innate immune response to encapsulate and kill the invasive organisms. It is mediated by a serine protease cascade and is regulated by serpins. The identification of the molecular components of melanization and the regulation of those components are still unclear in Drosophila melanogaster, although some genetic research on the activation of melanization has been reported. Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. Overexpression or repression of MP2 in flies resulted in increased and decreased rates of cleavage, respectively, of prophenoloxidase-1. Moreover, serine protease inhibitor Spn27A formed SDS-stable complexes with MP2, both in vitro and in vivo. The amidase activity of MP2 was inhibited efficiently by Spn27A. Spn27A also prevented MP2 from cleaving prophenoloxidase-1. Taken together, these results indicate that under our experimental conditions MP2 functions as a prophenoloxidase-activating protease, and that this function is inhibited by Spn27A. MP2 and Spn27A thus constitute a regulatory unit in the prophenoloxidase activation cascade in Drosophila. The combination of genetic, molecular genetic and biochemical approaches should allow further advances in our understanding of the prophenoloxidase-activating cascade in insects and indirectly shed further light on protease-cascades in humans and other vertebrates. PMID:24260243
Chu, Yuan; Zhao, Zhangwu
Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia.
Abstract The genome of Tannerella forsythia, an etiological factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase [85 kDa, without its signal peptide (SP)] processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together, mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin, and the antimicrobial peptide LL-37. PMID:25391881
Ksiazek, Miroslaw; Karim, Abdulkarim Y; Bryzek, Danuta; Enghild, Jan J; Th�gersen, Ida B; Koziel, Joanna; Potempa, Jan
Serine protease inhibitors modulate smoke-induced chemokine release from human lung fibroblasts.
Smoking is associated with lung inflammation and a protease-antiprotease imbalance. We previously reported that cigarette smoke extract (CSE) stimulates human lung fibroblasts to release chemotactic cytokines. We hypothesized that serine protease inhibitors might modulate lung fibroblast release of chemotactic cytokines in response to CSE. To test this hypothesis, serine protease inhibitors (FK706, alpha1-antitrypsin, methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone, or Nalpha-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) from human fetal lung fibroblasts by the blind-well chemotactic chamber. Metalloproteinases and cysteine proteinases were not examined in this study. Similarly, the release and gene expression of chemokines and nuclear factor-kappaB (NF-kappaB) activation were measured by means of enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Release of NCA, MCA, chemotactic chemokines including interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor, and the expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA were attenuated by FK706. Furthermore, FK706 suppressed NF-kappaB activation. These data suggest that serine protease inhibitors attenuate the CSE-induced release of NCA and MCA from human fetal lung fibroblasts and that the inhibitory action of antiproteases might depend on NF-kappaB signaling pathway. PMID:12738688
Numanami, Hiroki; Koyama, Sekiya; Nelson, Dan K; Hoyt, Jeffrey C; Freels, Jon L; Habib, Michael P; Amano, Jun; Haniuda, Masayuki; Sato, Etsuro; Robbins, Richard A
HATL5: a cell surface serine protease differentially expressed in epithelial cancers.
Over the last two decades, cell surface proteases belonging to the type II transmembrane serine protease (TTSP) family have emerged as important enzymes in the mammalian degradome, playing critical roles in epithelial biology, regulation of metabolic homeostasis, and cancer. Human airway trypsin-like protease 5 (HATL5) is one of the few family members that remains uncharacterized. Here we demonstrate that HATL5 is a catalytically active serine protease that is inhibited by the two Kunitz type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and 2, as well as by serpinA1. Full-length HATL5 is localized on the cell surface of cultured mammalian cells as demonstrated by confocal microscopy. HATL5 displays a relatively restricted tissue expression profile, with both transcript and protein present in the cervix, esophagus, and oral cavity. Immunohistochemical analysis revealed an expression pattern where HATL5 is localized on the cell surface of differentiated epithelial cells in the stratified squamous epithelia of all three of these tissues. Interestingly, HATL5 is significantly decreased in cervical, esophageal, and head and neck carcinomas as compared to normal tissue. Analysis of cervical and esophageal cancer tissue arrays demonstrated that the squamous epithelial cells lose their expression of HATL5 protein upon malignant transformation. PMID:24498351
Miller, Gregory S; Zoratti, Gina L; Murray, Andrew S; Bergum, Christopher; Tanabe, Lauren M; List, Karin
TMPRSS4 is a type II transmembrane serine protease involved in cancer and viral infections.
Proteolytic enzymes are involved in almost all biological processes reflecting their importance in health and disease. The human genome contains nearly 600 protease-encoding genes forming more than 2% of the total human proteome. The serine proteases, with about 180 members, built the oldest and second largest family of human proteases. Ten years ago, a novel serine protease family named the type II transmembrane family (TTSP) was identified. This minireview summarizes the up-to-date knowledge about the still growing TTSPs, particularly focusing on the pathophysiological functions of the family member type II transmembrane serine protease (TMPRSS) 4. Recent studies provided important data on TMPRSS4 activity associated with the spreading of influenza viruses, mediated by the cleavage of hemagglutinin. Progression and metastatic potential of several cancers is concordant with an increased expression of TMPRSS4, though being a possible diagnostic marker. However, to benefit from TMPRSS4 as a therapeutic target, more data concerning its physiological relevance are needed, as done by a specific morpholino knockdown in zebrafish embryos. PMID:22944691
Ohler, Anke; Becker-Pauly, Christoph
HATL5: A Cell Surface Serine Protease Differentially Expressed in Epithelial Cancers
Over the last two decades, cell surface proteases belonging to the type II transmembrane serine protease (TTSP) family have emerged as important enzymes in the mammalian degradome, playing critical roles in epithelial biology, regulation of metabolic homeostasis, and cancer. Human airway trypsin-like protease 5 (HATL5) is one of the few family members that remains uncharacterized. Here we demonstrate that HATL5 is a catalytically active serine protease that is inhibited by the two Kunitz type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and 2, as well as by serpinA1. Full-length HATL5 is localized on the cell surface of cultured mammalian cells as demonstrated by confocal microscopy. HATL5 displays a relatively restricted tissue expression profile, with both transcript and protein present in the cervix, esophagus, and oral cavity. Immunohistochemical analysis revealed an expression pattern where HATL5 is localized on the cell surface of differentiated epithelial cells in the stratified squamous epithelia of all three of these tissues. Interestingly, HATL5 is significantly decreased in cervical, esophageal, and head and neck carcinomas as compared to normal tissue. Analysis of cervical and esophageal cancer tissue arrays demonstrated that the squamous epithelial cells lose their expression of HATL5 protein upon malignant transformation. PMID:24498351
Miller, Gregory S.; Zoratti, Gina L.; Murray, Andrew S.; Bergum, Christopher; Tanabe, Lauren M.; List, Karin
A novel serine protease cryptolepain from Cryptolepis buchanani: purification and biochemical characterization.
A novel protease is purified to homogeneity from the latex of a medicinally important plant Cryptolepis buchanani of family Apocynaceae (formerly Asclepiadaceae). The enzyme named cryptolepain has a molecular mass of 50.5 kDa. The isoelectric point and extinction coefficient (epsilon280nm1%) are 6.0 and 26.4, respectively. Cryptolepain contains 15 tryptophans, 41 tyrosines, and eight cysteine residues forming four disulfide bridges. The detectable carbohydrate moiety in the enzyme was found to be 6-7%. Cryptolepain hydrolyzes denatured natural substrates like casein, azocasein, and azoalbumin with high specific activity. The protease is exclusively inhibited by serine protease inhibitors phenylmethansulfonyl fluoride and diisopropyl fluorophosphate. Hydrolysis of azoalbumin by the cryptolepain is optimal in the pH range of 8-10 and temperatures of 65-75 degrees C. The enzyme shows high stability against pH (2.5-11.5), temperature (up to 80 degrees C), and chemical denaturants. The Km value of the enzyme was found to be 10 microM with azocasein as the substrate. The N-terminal sequence of cryptolepain is unique and shows only little homology to other known serine proteases, which makes this enzyme an ideal candidate for our ongoing biochemical and structure-function investigations of proteases. Easy availability of the latex and simple purification procedures make the enzyme a good system for exploring the biophysical chemistry of serine proteases as well as applications in the food industry. PMID:17177552
Pande, Monu; Dubey, Vikash K; Yadav, Subhash C; Jagannadham, Medicherla V
The host metabolite D-serine contributes to bacterial niche specificity through gene selection.
Escherichia coli comprise a diverse array of both commensals and niche-specific pathotypes. The ability to cause disease results from both carriage of specific virulence factors and regulatory control of these via environmental stimuli. Moreover, host metabolites further refine the response of bacteria to their environment and can dramatically affect the outcome of the host-pathogen interaction. Here, we demonstrate that the host metabolite, D-serine, selectively affects gene expression in E. coli O157:H7. Transcriptomic profiling showed exposure to D-serine results in activation of the SOS response and suppresses expression of the Type 3 Secretion System (T3SS) used to attach to host cells. We also show that concurrent carriage of both the D-serine tolerance locus (dsdCXA) and the locus of enterocyte effacement pathogenicity island encoding a T3SS is extremely rare, a genotype that we attribute to an 'evolutionary incompatibility' between the two loci. This study demonstrates the importance of co-operation between both core and pathogenic genetic elements in defining niche specificity. PMID:25526369
Connolly, James Pr; Goldstone, Robert J; Burgess, Karl; Cogdell, Richard J; Beatson, Scott A; Vollmer, Waldemar; Smith, David Ge; Roe, Andrew J
Involvement of a cytoplasmic-tail serine cluster in urotensin II receptor internalization
Most G-protein-coupled receptors that undergo agonist-dependent internalization require the presence of specific cytoplasmic-tail residues to initiate interactions with proteins of the endocytic machinery. Here we show that the UT receptor (urotensin II receptor) undergoes internalization, and that specific serine residues of the receptor's cytoplasmic tail participate in this process. We first observed a time-dependent increase in internalization of the UT receptor expressed in COS-7 cells following binding of the agonist urotensin II. This sequestration was significantly reduced in the presence of sucrose, demonstrating that the agonist-activated UT receptor is internalized in part by clathrin-coated pits. Moreover, the sequestered receptor was co-localized in endocytic vesicles with ?-arrestin1 and ?-arrestin2. To assess whether specific regions of the receptor's cytoplasmic tail were involved in internalization, five UT receptor mutants were constructed. In four constructs the receptor's cytoplasmic tail was truncated at various positions (UT?367, UT?363, UT?350 and UT?336), and in the other four adjacent serine residues at positions 364�367 were replaced by Ala (Mut4S). Each mutant, except UT?367, demonstrated a significantly reduced internalization rate, thereby revealing the importance of specific serine residues within the cytoplasmic tail of the UT receptor for its ability to be internalized efficiently. PMID:15458389
A CUB-serine protease in the olfactory organ of the spiny lobster Panulirus argus.
csp, a gene encoding a protein with high sequence identity to trypsinlike serine protease and CUB domains, was identified from a cDNA library from the olfactory organ (antennular lateral flagellum) of the spiny lobster Panulirus argus. The full-length cDNA sequence of csp is 1801 bp, encoding a protein of 50.25 kD, with three domains: signal peptide, trypsinlike serine protease, and CUB (named for a class of compounds including Complement subcomponents Clr/Cls, Uegf, and Bone morphogenic protein-1). RT-PCR, Northern blots, and immunoblots showed that csp is predominantly expressed in the lateral flagellum and eyestalk. Immunocytochemistry showed that Csp is present in olfactory (aesthetasc) sensilla around auxiliary cells (glia that surround the inner dendrites of olfactory receptor neurons, ORNs) and ORN outer dendrites. We propose that Csp is expressed and secreted by auxiliary cells, associates with ORN cell membranes or extracellular matrix via the CUB domain, and has trypsinlike activity. In the eyestalk, Csp is associated with cells surrounding axons between neuropils of the eyestalk ganglia. Possible functions in the olfactory organ and eyestalk are discussed. To our knowledge, this is the first report from any olfactory system of a gene encoding a protein with serine protease and CUB domains. PMID:11745665
Levine, M Z; Harrison, P J; Walthall, W W; Tai, P C; Derby, C D
Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues
Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells. PMID:25278935
Shi, Lei; Pigeonneau, Nathalie; Ravikumar, Vaishnavi; Dobrinic, Paula; Macek, Boris; Franjevic, Damjan; Noirot-Gros, Marie-Francoise; Mijakovic, Ivan
Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis
Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs) from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization) the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent. PMID:20936075
Hasson, S. S.; Mothana, R. A.; Sallam, T. A.; Al-balushi, M. S.; Rahman, M. T.; Al-Jabri, A. A.
Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory).
A new serine protease from the latex of Ipomoea carnea spp. fistulosa (Morning glory), belonging to the Convolvulaceae family, was purified to homogeneity by ammonium sulfate fractionation followed by cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24 kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. The pH and temperature optima for proteolytic activity were 6.5 and 65 degrees C, respectively. The extinction coefficient (epsilon2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35 tryptophan, 76 tyrosine, and seven cysteine residues. The effect of several inhibitors such as iodoacetic acid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsin inhibitor, HgCl2, 3S-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid, N-ethyl maleimide, ethylene glycol-bis(alpha-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and o-phenonthroline indicates that carnein belongs to the family of serine proteases. The enzyme is not prone to autolysis even at very low concentrations. The N-terminal sequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarity to those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilase family endopepetidases. PMID:17571896
Patel, Ashok Kumar; Singh, Vijay Kumar; Jagannadham, Medicherla V
Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom
Snake venom Kunitz\\/BPTI members are good tools for understanding of structure�functional relationship between serine proteases and their inhibitors. A novel dual Kunitz\\/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid
Ying-Ying He; Shu-Bai Liu; Wen-Hui Lee; Jin-Qiao Qian; Yun Zhang
Effects of feeding and lighting stimuli on the synthesis of ornithine aminotransferase and serine dehydratase in rat liver
This paper compares the circadian fluctuations in the rates of ornithine aminotransferase and serine dehydratase synthesis measured immunochemically in rats given a single 2-h daily feeding in conjunction with exposure to constant light or a 12-h light\\/12-h dark cycle. When the 2-hr feeding was administered to rats under constant light, reciprocal circadian oscillations in ornithine aminotransferase and serine dehydratase synthesis
K. B. Ekelman; C. Peraino
Purification and Functional Characterisation of Rhinocerase, a Novel Serine Protease from the Venom of Bitis gabonica rhinoceros
Background Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders. Methodology/Principal Findings In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading ? and ? chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate. Conclusions/Significance A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes. PMID:20300193
Vaiyapuri, Sakthivel; Harrison, Robert A.; Bicknell, Andrew B.; Gibbins, Jonathan M.; Hutchinson, Gail
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Williamson, John
UV inverse photoemission from FePS 3
NASA Astrophysics Data System (ADS)
Angle integrated inverse photoemission (IP) spectra from FePS 3 crystals cleaved in ultra high vacuum (UHV) were measured in the photon energy range between 10 and 25 eV. Two peaks are noticed above the Fermi energy: they are assigned to Fe 3 d derived states split by the crystal field, as in this energy region the IP spectra are expected to be very sensitive to d-symmetry orbitals. The photon energy dependence of the IP spectra allows a mixing between the iron 3 d derived states and the sulphur valence states to be pointed out. The experimental data are discussed in connection with the available calculations.
Puppin, E.; Scagliotti, M.; Chemelli, C.
Create and Publish a Hierarchical Progressive Survey (HiPS)
NASA Astrophysics Data System (ADS)
Since 2009, the CDS promotes a method for visualizing based on the HEALPix sky tessellation. This method, called �Hierarchical Progressive Survey" or HiPS, allows one to display a survey progressively. It is particularly suited for all-sky surveys or deep fields. This visualization method is now integrated in several applications, notably Aladin, the SiTools/MIZAR CNES framework, and the recent HTML5 �Aladin Lite". Also, more than one hundred surveys are already available in this view mode. In this article, we will present the progress concerning this method and its recent adaptation to the astronomical catalogs such as the GAIA simulation.
Fernique, P.; Boch, T.; Pineau, F.; Oberto, A.
T3PS: Tool for Parallel Processing in Parameter Scans
T3PS is a program that can be used to quickly design and perform parameter scans while easily taking advantage of the multi-core architecture of current processors. It takes an easy to read and write parameter scan definition file format as input. Based on the parameter ranges and other options contained therein, it distributes the calculation of the parameter space over multiple processes and possibly computers. The derived data is saved in a plain text file format readable by most plotting software. The supported scanning strategies include: grid scan, random scan, Markov Chain Monte Carlo, numerical optimization. Several example parameter scans are shown and compared with results in the literature.
Maurer, Vinzenz
BioMaPS: A Roadmap for Success
The manuscript outlines the impact that our National Science Foundation Interdisciplinary Training for Undergraduates in Biological and Mathematical Sciences program, BioMaPS, has had on the students and faculty at Murray State University. This interdisciplinary program teams mathematics and biology undergraduate students with mathematics and biology faculty and has produced research insights and curriculum developments at the intersection of these two disciplines. The goals, structure, achievements, and curriculum initiatives are described in relation to the effects they have had to enhance the study of biomathematics. PMID:20810948
Fister, K. Renee
Sub 100ps time resolution using silicon avalanche diodes
We have investigated silicon avalanche diodes (AVDs) as time of flight detectors for nuclear and particle physics applications. The signal is created by the large number of electron-hole pairs ({approx}100 pairs/mm) deposited along the path of a particle passing through the AVD. The electrons drift in a strong electric field to the diode junction, where they multiply in an avalanche region. A single diode time resolution of 65 ps has been obtained using fast leading-edge discriminators. This time resolution is comparable to the best obtained with the conventional alternative of plastic scintillator and photomultiplier tube. Further optimization appears possible.
Hauger, J.A.; Choi, Y.; Hirsch, A.S.; Porile, N.T. [and others
Integrated 13 GHz ps-pulse-source at 1064 nm
NASA Astrophysics Data System (ADS)
We present an integrated pulse source at 1064 nm and discuss design, fabrication and measurement results of the 3.5 mm long ridge-waveguide distributed Bragg reflector laser. The optical pulses with a peak power of 0.9 W, a temporal duration of 12.4 ps and a repetition frequency of 13 GHz are generated by passive mode-locking and are well suited for seeding master oscillator power amplifier systems for efficient green light generation with nonlinear frequency doubling in single pass configuration.
Brox, O.; Prziwarka, T.; Klehr, A.; Bugge, F.; Matalla, M.; Wenzel, H.; Erbert, G.
Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer.
Dysregulated proteolysis is a hallmark of cancer. Malignant cells require a range of proteolytic activities to enable growth, survival, and expansion. Serine proteases of the S1 or trypsin-like family have well recognized roles in the maintenance of normal homeostasis as well as in the pathology of diseases such as cancer. Recently a rapidly expanding subgroup of S1 proteases has been recognized that are directly anchored to plasma membranes. These membrane anchored serine proteases are anchored either via a carboxy-terminal transmembrane domain (Type I), a carboxy terminal hydrophobic region that functions as a signal for membrane attachment via a glycosyl-phosphatidylinositol linkage (GPI-anchored), or via an amino terminal proximal transmembrane domain (Type II or TTSP). The TTSPs also encode multiple domains in their stem regions that may function in regulatory interactions. The serine protease catalytic domains of these enzymes show high homology but also possess features indicating unique substrate specificities. It is likely that the membrane anchored serine proteases have evolved to perform complex functions in the regulation of cellular signaling events at the plasma membrane and within the extracellular matrix. Disruption or mutation of several of the genes encoding these proteases are associated with disease. Many of the membrane anchored serine proteases show restricted tissue distribution in normal cells, but their expression is widely dysregulated during tumor growth and progression. Diagnostic or therapeutic targeting of the membrane anchored serine proteases has potential as promising new approaches for the treatment of cancer and other diseases. PMID:12784999
Netzel-Arnett, Sarah; Hooper, John D; Szabo, Roman; Madison, Edwin L; Quigley, James P; Bugge, Thomas H; Antalis, Toni M
Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts.
The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases. PMID:22640634
Oppert, Brenda; Martynov, Alexander G; Elpidina, Elena N
Effects of feeding and lighting stimuli on the synthesis of ornithine aminotransferase and serine dehydratase in rat liver
This paper compares the circadian fluctuations in the rates of ornithine aminotransferase and serine dehydratase synthesis measured immunochemically in rats given a single 2-h daily feeding in conjunction with exposure to constant light or a 12-h light/12-h dark cycle. When the 2-hr feeding was administered to rats under constant light, reciprocal circadian oscillations in ornithine aminotransferase and serine dehydratase synthesis were observed regardless of the temporal location of the feeding interval. Ornithine aminotransferase synthesis began to increase after the feeding interval and reached a maximum 12 h later while serine dehydratase showed the opposite response. In rats maintained on both the restricted feeding regimen and a 12-h light/12-h dark cycle, however, retention of synthesis oscillations depended on the temporal location of the restricted feeding interval within the light-dark cycle. Rats fed for 2 h at the beginning of the dark phase exhibited circadian oscillations in serine dehydratase synthesis and a high nonoscillating level of ornithine aminotransferase synthesis, whereas rats fed for 2 h at the beginning of the light phase exhibited circadian oscillations in ornithine aminotransferase synthesis and a low nonoscillating level of serine dehydratase synthesis. These responses suggest the existence of meal-responsive and light-responsive regulators of ornithine aminotransferase and serine dehydratase synthesis. (JMT)
Ekelman, K.B.; Peraino, C.
Simultaneous measurement of d-serine dehydratase and d-amino acid oxidase activities by the detection of 2-oxo-acid formation with reverse-phase high-performance liquid chromatography
N-methyl-d-aspartate receptors (NMDARs) play critical roles in excitatory synaptic transmission in the vertebrate central nervous system. NMDARs need d-serine for their channel activities in various brain regions. In mammalian brains, d-serine is produced from l-serine by serine racemase and degraded by d-amino acid oxidase (DAO) to 3-hydroxypyruvate. In avian organs, such as the kidney, in addition to DAO, d-serine is
Hiroyuki Tanaka; Atsushi Yamamoto; Tetsuo Ishida; Kihachiro Horiike
Coexistence of WiFi and WiMAX Systems Based on PS-Request Protocols�
We introduce both the coexistence zone within the WiMAX frame structure and a PS-Request protocol for the coexistence of WiFi and WiMAX systems sharing a frequency band. Because we know that the PS-Request protocol has drawbacks, we propose a revised PS-Request protocol to improve the performance. Two PS-Request protocols are based on the time division operation (TDO) of WiFi system and WiMAX system to avoid the mutual interference, and use the vestigial power management (PwrMgt) bit within the Frame Control field of the frames transmitted by a WiFi AP. The performance of the revised PS-Request protocol is evaluated by computer simulation, and compared to those of the cases without a coexistence protocol and to the original PS-Request protocol. PMID:22163721
Kim, Jongwoo; Park, Suwon; Rhee, Seung Hyong; Choi, Yong-Hoon; Chung, Young-uk; Hwang, Ho Young
Coexistence of WiFi and WiMAX systems based on PS-request protocols.
We introduce both the coexistence zone within the WiMAX frame structure and a PS-Request protocol for the coexistence of WiFi and WiMAX systems sharing a frequency band. Because we know that the PS-Request protocol has drawbacks, we propose a revised PS-Request protocol to improve the performance. Two PS-Request protocols are based on the time division operation (TDO) of WiFi system and WiMAX system to avoid the mutual interference, and use the vestigial power management (PwrMgt) bit within the Frame Control field of the frames transmitted by a WiFi AP. The performance of the revised PS-Request protocol is evaluated by computer simulation, and compared to those of the cases without a coexistence protocol and to the original PS-Request protocol. PMID:22163721
Kim, Jongwoo; Park, Suwon; Rhee, Seung Hyong; Choi, Yong-Hoon; Chung, Young-uk; Hwang, Ho Young
Internal structure and positron annihilation in the four-body MuPs system
A large number of bound state properties of the four-body muonium-positronium system MuPs (or $\\mu^{+} e^{-}_2 e^{+}$) are determined to high accuracy. Based on these expectation values we predict that the weakly-bound four-body MuPs system has the `two-body' cluster structure Mu + Ps. The two neutral clusters Mu ($\\mu^{+} e^{-}$) and Ps ($e^{+} e^{-}$) interact with each other by the attractive van der Waals forces. By using our expectation values of the electron-positron delta-functions we evaluated the half-life $\\tau_a$ of the MuPs system against annihilation of the electron-positron pair: $\\tau_a = \\frac{1}{\\Gamma} \\approx 4.071509 \\cdot 10^{-10}$ $sec$. The hyperfine structure splitting of the ground state in the MuPs system evaluated with our expectation values is $\\Delta \\approx$ 23.064(5) $MHz$.
Alexei M. Frolov
Study on GASTOF - A 10 ps resolution timing detector
NASA Astrophysics Data System (ADS)
GASTOF (Gas Time Of Flight) is a type of fast-time detector affiliated to the HPS (High Precision Spectrometer) project which is a forward physics collaborator within CMS. It is a picosecond time resolution Cherenkov gas detector using very fast single anode micro-channel plate photomultiplier (Hamamatsu R3809U-50 or Photek 210) as a photon detector. We firstly measured characteristics of these two types of MCP-PMTs by a fast laser pulse in lab. Then two GASTOF detectors both equipped with a Hamamatsu R3809U-50 tube were studied in a beam test at CERN. According to the analysis of beam test data, the average number of photoelectrons (phe) was 2.0 for both phototubes. By making a cut on the number of photoelectrons such that the mean phe was 3.6 in one phototube and 3.2 in another, we obtained a time resolution of ?~11.7 picosecond (ps) and ?~8.2 ps.
Bonnet, Luc; Liao, Junhui; Piotrzkowski, Krzysztof
Unidirectional sub-100-ps magnetic vortex core reversal
NASA Astrophysics Data System (ADS)
We experimentally demonstrate that unidirectional reversal of the magnetic vortex core polarity is possible by excitation with sub-100-ps-long orthogonal monopolar magnetic pulse sequences in a wide range of pulse lengths and amplitudes. The application of such short digital pulses is a favorable excitation scheme for technological applications. Measured phase diagrams of this unidirectional, spin-wave mediated vortex core reversal are in good qualitative agreement with phase diagrams obtained from micromagnetic simulations. The time dependence of the reversal process, observed by time-resolved scanning transmission x-ray microscopy indicates a switching time of 100 ps and fits well with our simulations. The origin of the asymmetric response to clockwise and counterclockwise excitation which is a prerequisite for reliable unidirectional switching is discussed, based on the gyromode-spin-wave coupling. Situations are found in which a three-dimensional dynamics is important, because a vortex-antivortex pair starts to form close to the core of the original vortex in the lower part of the disk without completing the formation across the whole thickness so that it dissolves later on and does not lead to switching of the original vortex core.
Noske, Matthias; Gangwar, Ajay; Stoll, Hermann; Kammerer, Matthias; Sproll, Markus; Dieterle, Georg; Weigand, Markus; F�hnle, Manfred; Woltersdorf, Georg; Back, Christian H.; Sch�tz, Gisela
Properties of Extruded PS-212 Type Self-Lubricating Materials
NASA Technical Reports Server (NTRS)
Research has been underway at the NASA Lewis Research Center since the 1960's to develop high temperature, self-lubricating materials. The bulk of the research has been done in-house by a team of researchers from the Materials Division. A series of self-lubricating solid material systems has been developed over the years. One of the most promising is the composite material system referred to as PS-212 or PM-212. This material is a powder metallurgy product composed of metal bonded chromium carbide and two solid lubricating materials known to be self-lubricating over a wide temperature range. NASA feels this material has a wide potential in industrial applications. Simplified processing of this material would enhance its commercial potential. Processing changes have the potential to reduce processing costs, but tribological and physical properties must not be adversely affected. Extrusion processing has been employed in this investigation as a consolidation process for PM-212/PS-212. It has been successful in that high density bars of EX-212 (extruded PM-212) can readily be fabricated. Friction and strength data indicate these properties have been maintained or improved over the P.M. version. A range of extrusion temperatures have been investigated and tensile, friction, wear, and microstructural data have been obtained. Results indicate extrusion temperatures are not critical from a densification standpoint, but other properties are temperature dependent.
Waters, W. J.; Sliney, H. E.; Soltis, R. F.
Effect of D-serine on spermatogenesis and extracellular signal-regulated protein kinase (ERK) phosphorylation in the testis of the silkworm, Bombyx mori.
Although the pupae and larvae of Bombyx mori possess especially large amounts of free d-serine, the physiological role of the amino acid in the silkworm is unknown. We investigated the effect of d-serine on spermatogenesis. A lowered d-serine level throughout larval development caused a delay in spermatogenesis and resulted in reduced numbers of eupyrene sperm. Administration of d-serine transiently increased the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2; hereafter, ERK) by approximately 25% in the testis of day 3 fifth instar larvae. l-Serine had no effect on ERK activation, and other organs did not respond to d-serine. The effect of d-serine on ERK activation was confirmed by administering d-serine dehydratase, an enzyme that specifically degrades d-serine, and the enzyme's inhibitor, hydroxylamine. ERK phosphorylation in the testis was significantly inhibited by Go6983 and U0126, inhibitors of protein kinase C (PKC) and mitogen-associated protein kinase kinase 1/2 (MEK), respectively, but not by H-89, a protein kinase A (PKA) inhibitor, indicating that ERK was activated in the testis via PKC and MEK but not via PKA. The inhibition of ERK phosphorylation by Go6983 or U0126 was reduced by 20-30% by d-serine. Roughly 30% of c-Raf phosphorylation at an inhibitory site (Ser259) was decreased by the addition of d-serine. These results suggest that d-serine activates ERK in the testis of silkworms through a pathway including c-Raf but not PKC or MEK. Immunohistochemistry confirmed d-serine-induced ERK phosphorylation in the testis and revealed the presence of phospho-ERK in the nuclei of spermatocytes and spermatids. PMID:24971930
Suzuki, Chihiro; Tanigawa, Minoru; Tanaka, Hiroyuki; Horiike, Kihachiro; Kanekatsu, Rensuke; Tojo, Miki; Nagata, Yoko
Studies on an insulin-stimulated insulin receptor serine kinase activity: separation of the kinase activity from the insulin receptor and its reconstitution back to the insulin receptor.
In cells insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Recently we have achieved co-purification of the insulin receptor with insulin-stimulated insulin receptor serine kinase activity. We now show that the co-purified serine kinase activity can be removed by NaCl washing and reconstituted by adding back the NaCl eluate. Reconstitution enabled higher serine phosphorylation than achieved with the co-purified preparation. Myelin basic protein was discovered to be a potent substrate for insulin-stimulated serine phosphorylation by the co-purified preparation, with the activity responsible having similar properties to the serine kinase activity towards the receptor. Myelin basic protein was also phosphorylated on serine by the NaCl eluate. Myelin basic protein phosphorylated by the co-purified preparation or the NaCl eluate gave the same set of phosphoserine peptides. The major myelin basic protein serine kinase activity in the NaCl eluate co-purified exactly on Mono Q with the activity that restored insulin-stimulated insulin receptor serine phosphorylation. These results provide strong evidence for the true separation of the serine kinase from the insulin receptor and the distinctiveness of the serine kinase activity from the insulin receptor tyrosine kinase and mitogen-activated protein kinases. The procedures developed for the isolation of the serine kinase and the establishment of an effective in vitro substrate should allow purification of the kinase. The protocols also provide flexible systems for identifying the functions of the insulin-stimulated serine phosphorylations and the respective kinase(s). PMID:8948451
Asamoah, K A; Atkinson, P G; Carter, W G; Sale, G J
Performance Analysis of the ertPS Algorithm and Enhanced ertPS Algorithm for VoIP Services in IEEE 802.16e Systems
NASA Astrophysics Data System (ADS)
In this paper, we analyze the extended real-time Polling Service (ertPS) algorithm in IEEE 802.16e systems, which is designed to support Voice-over-Internet-Protocol (VoIP) services with data packets of various sizes and silence suppression. The analysis uses a two-dimensional Markov Chain, where the grant size and the voice packet state are considered, and an approximation formula for the total throughput in the ertPS algorithm is derived. Next, to improve the performance of the ertPS algorithm, we propose an enhanced uplink resource allocation algorithm, called the e2rtPS algorithm, for VoIP services in IEEE 802.16e systems. The e2rtPS algorithm considers the queue status information and tries to alleviate the queue congestion as soon as possible by using remaining network resources. Numerical results are provided to show the accuracy of the approximation analysis for the ertPS algorithm and to verify the effectiveness of the e2rtPS algorithm.
Kim, Bong Joo; Hwang, Gang Uk
How does Tg reduction affect the chain mobility in confined PS films?
NASA Astrophysics Data System (ADS)
It is well established that the glass transition temperature (Tg) of supported polystyrene (PS) thin films decrease with decreasing film thickness. This Tg reduction due to the free surface effect is associated with enhanced mobility. However, the correlation between the enhanced mobility and Tg reduction has not been studied yet. To understand the effect of Tg reduction on the vertical mobility of PS chains across the interfaces we have investigated the interdiffusion between PS and deuterated PS (dPS) films in bilayer and trilayer geometries using neutron reflectivity (NR). Bilayer films of 42 nm thick dPS bottom layer and 20 nm thick PS top layer are created in such a way to mimic the films where large Tg reductions has been demonstrated by recent fluorescence measurements. Trilayer films were created using the same bottom layer but floating a 10 nm thick PS middle layer and 10 nm thick dPS top layer to compare the mobilities at the interfaces between the top/middle and middle/bottom layers. NR results showed that there is almost no mixing between the layers up to 90-95 C for both bilayer and trilayer films which is not consistent with large Tg reductions observed in the literature. Our results also indicate no difference in the mobility of PS chains at the top/middle and middle/bottom interfaces in the trilayer film which argues against the enhanced mobility reported in the literature for the top 10 nm of PS thin films. Diffusion of PS chains across the interface gets faster as the MW decreases.
Akgun, Bulent; Dimitriou, Michael; Satija, Sushil K.
Continuous production of monoacylglycerols from palm olein in packed-bed reactor with immobilized lipase PS
A packed-bed reactor (PBR) system using immobilized lipase PS as biocatalyst was developed for continuous monoacylglycerols (MAG) production. The condition for continuous MAG production using immobilized lipase PS (IM-PS) of 1.5g (550U) in PBR (0.68cm i.d., 25cm long) was optimized. The effect of molar ratio of glycerol to palm olein, water content in glycerol and residence time on MAG production
Aran H-Kittikun; Wiphum Kaewthong; Benjamas Cheirsilp
Purified Mesenchymal Stem Cells Are an Efficient Source for iPS Cell Induction
Background Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by the forced expression of defined transcription factors. Although most somatic cells are capable of acquiring pluripotency with minimal gene transduction, the poor efficiency of cell reprogramming and the uneven quality of iPS cells are still important problems. In particular, the choice of cell type most suitable for inducing high-quality iPS cells remains unclear. Methodology/Principal Findings Here, we generated iPS cells from PDGFR?+ Sca-1+ (P?S) adult mouse mesenchymal stem cells (MSCs) and PDGFR?? Sca-1? osteo-progenitors (OP cells), and compared the induction efficiency and quality of individual iPS clones. MSCs had a higher reprogramming efficiency compared with OP cells and Tail Tip Fibroblasts (TTFs). The iPS cells induced from MSCs by Oct3/4, Sox2, and Klf4 appeared to be the closest equivalent to ES cells by DNA microarray gene profile and germline-transmission efficiency. Conclusions/Significance Our findings suggest that a purified source of undifferentiated cells from adult tissue can produce high-quality iPS cells. In this context, prospectively enriched MSCs are a promising candidate for the efficient generation of high-quality iPS cells. PMID:21412425
Niibe, Kunimichi; Kawamura, Yoshimi; Araki, Daisuke; Morikawa, Satoru; Miura, Kyoko; Suzuki, Sadafumi; Shimmura, Shigeto; Sunabori, Takehiko; Mabuchi, Yo; Nagai, Yasuo; Nakagawa, Taneaki; Okano, Hideyuki; Matsuzaki, Yumi
Application of iPS cells in dental bioengineering and beyond.
The stem-cell-based tissue-engineering approaches are widely applied in establishing functional organs and tissues for regenerative medicine. Successful generation of induced pluripotent stem cells (iPS cells) and rapid progress of related technical platform provide great promise in the development of regenerative medicine, including organ regeneration. We have previously reported that iPS cells could be an appealing stem cells source contributing to tooth regeneration. In the present paper, we mainly review the application of iPS technology in dental bioengineering and discuss the challenges for iPS cells in the whole tooth regeneration. PMID:24917330
Liu, Pengfei; Zhang, Yanmei; Chen, Shubin; Cai, Jinglei; Pei, Duanqing
Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells
Chinese hamster ovary (CHO-K1) cells were pulse labeled with ({sup 3}H)serine, and the synthesis of phosphatidyl({sup 3}H)ethanolamine from phosphatidyl({sup 3}H)serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 {mu}g of saponin per ml, there was no significant turnover of nascent phosphatidyl({sup 3}H)serine to form phosphatidyl({sup 3}H)ethanolamine during an ensuring chase. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl({sup 3}H)ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl({sup 3}H)serine during a subsequent 2-hr chase. Phosphatidyl({sup 3}H)ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl({sup 3}H)serine to phosphatidyl({sup 3}H)ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5{prime}-adenylyl imidodiphosphate, or apyrase plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins.
Voelker, D.R. (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO (USA))
Preliminary Evaluation of PS300: A New Self-Lubricating High Temperature Composite Coating for Use to 800 C
NASA Technical Reports Server (NTRS)
This paper introduces PS300, a plasma sprayed, self-lubricating composite coating for use in sliding contacts at temperatures to 800 C. PS300 is a metal bonded chrome oxide coating with silver and BaF2/CaF2 eutectic solid lubricant additives. PS300 is similar to PS200, a chromium carbide based coating, which is currently being investigated for a variety of tribological applications. In pin-on-disk testing up to 650 C, PS300 exhibited comparable friction and wear properties to PS200. The PS300 matrix, which is predominantly chromium oxide rather than chromium carbide, does not require diamond grinding and polishes readily with silicon carbide abrasives greatly reducing manufacturing costs compared to PS200. It is anticipated that PS300 has potential for sliding bearing and seal applications in both aerospace and general industry.
Dellacorte, C.; Edmonds, B. J.
Neural stem cells differentiated from iPS cells spontaneously regain pluripotency.
Differentiated somatic cells can be reprogrammed into pluripotent stem cells by transduction of exogenous reprogramming factors. After induced pluripotent stem (iPS) cells are established, exogenous genes are silenced. In the pluripotent state, retroviral genes integrated in the host genome are kept inactive through epigenetic transcriptional regulation. In this study, we tried to determine whether exogenous genes remain silenced or are reactivated upon loss of pluripotency or on differentiation using an in vitro system. We induced differentiation of iPS cells into neural stem cells (NSCs) in vitro; the NSCs appeared morphologically indistinguishable from brain-derived NSCs and stained positive for the NSC markers Nestin and Sox2. These iPS cell-derived NSCs (iPS-NSCs) were also capable of differentiating into all three neural subtypes. Interestingly, iPS-NSCs spontaneously formed aggregates on long-term culture and showed reactivation of the Oct4-GFP marker, which was followed by the formation of embryonic stem cell-like colonies. The spontaneously reverted green fluorescent protein (GFP)-positive (iPS-NSC-GFP(+) ) cells expressed high levels of pluripotency markers (Oct4 and Nanog) and formed germline chimeras, indicating that iPS-NSC-GFP(+) cells had the same pluripotency as the original iPS cells. The reactivation of silenced exogenous genes was tightly correlated with the downregulation of DNA methyltransferases (Dnmts) during differentiation of iPS cells. This phenomenon was not observed in doxycycline-inducible iPS cells, where the reactivation of exogenous genes could be induced only by doxycycline treatment. These results indicate that pluripotency can be regained through reactivation of exogenous genes, which is associated with dynamic change of Dnmt levels during differentiation of iPS cells. PMID:24898298
Choi, Hyun Woo; Kim, Jong Soo; Choi, Sol; Hong, Yean Ju; Kim, Min Jung; Seo, Han Geuk; Do, Jeong Tae
Generation of Endoderm derived Human iPS cells from Primary Hepatocytes
Recent advances in induced pluripotent stem (iPS) cell research significantly changed our perspective on regenerative medicine. Patient specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy. However, there have been insufficient data to prove that iPS cells are functionally equivalent to hES cells or safer than hES cells. There are several important issues which need to be addressed and foremost are the safety and efficacy of human iPS cells from different origins. Human iPS cells have been derived mostly from cells originated from mesoderm, with a few cases from ectoderm. So far there has been no report of endoderm derived human iPS cells, preventing comprehensive comparative investigations on the quality of human iPS cells from different origins. Here we show for the first time reprogramming of human endoderm derived cells (i.e. primary hepatocytes) to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells were able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. The technology to develop endoderm derived human iPS cell lines, together with other established cell lines, will provide a foundation to elucidate the mechanisms of cellular reprogramming and to study the safety and efficacy of differentially originated human iPS cells for cell therapy. For studying liver disease pathogenesis, this technology also provides a potentially more amenable system to generate liver disease specific iPS cells. PMID:20432258
Liu, Hua; Ye, Zhaohui; Kim, Yong-Hak; Sharkis, Saul; Jang, Yoon-Young
Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324
Background and Purpose Dysregulation of the thromboxane A2 (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis. Experimental Approach Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TP? serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TP? and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions. Key Results The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response. Conclusions and Implications In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity. PMID:24490858
Santhosh, K T; Sikarwar, A S; Hinton, M; Chelikani, P; Dakshinamurti, S
Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution
Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codon-switching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic �fossil record� of historical purifying selection against E1E2 intermediate phenotypes. PMID:24173227
Koutsoudakis, George; Urbanowicz, Richard A.; Mirza, Deeman; Ginkel, Corinne; Riebesehl, Nina; Calland, No�mie; Albecka, Anna; Price, Louisa; Hudson, Natalia; Descamps, V�ronique; Backx, Matthijs; McClure, C. Patrick; Duverlie, Gilles; Pecheur, Eve-Isabelle; Dubuisson, Jean; Perez-del-Pulgar, Sofia; Forns, Xavier; Steinmann, Eike; Tarr, Alexander W.; Pietschmann, Thomas
Role of Serine/Threonine Phosphatase (SP-STP) in Streptococcus pyogenes Physiology and Virulence*
Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis. PMID:21917918
Agarwal, Shivani; Agarwal, Shivangi; Pancholi, Preeti; Pancholi, Vijay
Indicain, a dimeric serine protease from Morus indica cv. K2.
A high molecular mass serine protease has been purified to homogeneity from the latex of Morus indica cv. K2 by the combination of techniques of ammonium sulfate precipitation, hydrophobic interaction chromatography, and size-exclusion chromatography. The protein is a dimer with a molecular mass of 134.5 kDa and with two monomeric subunits of 67.2 kDa and 67.3 (MALDI-TOF), held by weak bonds susceptible to disruption on exposure to heat and very low pH. Isoelectric point of the enzyme is pH 4.8. The pH and temperature optima for caseinolytic activity were 8.5 and 80 degrees C, respectively. The extinction coefficient (epsilon280(1%)) of the enzyme was estimated as 41.24 and the molecular structure consists of 52 tryptophan, 198 tyrosine and 42 cysteine residues. The enzyme activity was inhibited by phenylmethylsulfonylflouride, chymostatin and mercuric chloride indicating the enzyme to be a serine protease. The enzyme is fairly stable and similar to subtilases in its stability toward pH, strong denaturants, temperature, and organic solvents. Polyclonal antibodies specific to enzyme and immunodiffusion studies reveal that the enzyme has unique antigenic determinants. The enzyme has activity towards broad range of substrates comparable to those of subtilisin like proteases. The N-terminal residues of indicain (T-T-N-S-W-D-F-I-G-F-P) exhibited considerable similarity to those of other known plant subtilases, especially with cucumisin, a well-characterized plant subtilase. This is the first report of purification and characterization of a subtilisin like dimeric serine protease from the latex of M. indica cv. K2. Owing to these unique properties the reported enzyme would find applications in food and pharma industry. PMID:18561962
Singh, Vijay Kumar; Patel, Ashok Kumar; Moir, A J; Jagannadham, Medicherla V
Functional suppression of HAMP domain signaling defects in the E. coli serine chemoreceptor.
HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric Escherichia coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: unmodified E residues promote kinase-off output, and methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer 3 of the HAMP bundle and followed in vivo CheA activity with an assay based on F�rster resonance energy transfer. We found that an alanine or a serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E and S255A) shifted Tsr output toward the kinase-off state, but two others (S255G and L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations. PMID:25134756
Lai, Run-Zhi; Parkinson, John S
Analysis of serine codon conservation reveals diverse phenotypic constraints on hepatitis C virus glycoprotein evolution.
Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codon-switching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic "fossil record" of historical purifying selection against E1E2 intermediate phenotypes. PMID:24173227
Brown, Richard J P; Koutsoudakis, George; Urbanowicz, Richard A; Mirza, Deeman; Ginkel, Corinne; Riebesehl, Nina; Calland, No�mie; Albecka, Anna; Price, Louisa; Hudson, Natalia; Descamps, V�ronique; Backx, Matthijs; McClure, C Patrick; Duverlie, Gilles; Pecheur, Eve-Isabelle; Dubuisson, Jean; Perez-del-Pulgar, Sofia; Forns, Xavier; Steinmann, Eike; Tarr, Alexander W; Pietschmann, Thomas; Ball, Jonathan K
A novel fibrinolytic serine protease from the polychaete Nereis (Neanthes) virens (Sars): purification and characterization.
A novel fibrinolytic serine protease has been identified and purified to homogeneity from the coelomic fluid of polychaete Nereis (Neanthes) virens (Sars), and named N-V protease. N-V protease is a 29kDa single chain protein with an isoelectric point of pH 4.5. It hydrolyzes Aalpha-chain of fibrinogen with a high efficiency, and the Bbeta- and gamma-chains (Aalpha>Bbeta>gamma) with a lower efficiency. The proteolytic activity peaks at pH 7.8 is 45 degrees C. The activity is completely inhibited by serine protease inhibitors DFP (I(50)=5.8 x 10(-4)M) and PMSF (I(50)=5.5 x 10(-2)M), and almost completely by TLCK (I(50)=7.7 x 10(-1) M). But aprotinin, elastinal, SBTI, benzamidine, PCMB, EDTA, EGTA, iodoacetate, E64, and beta-mercaptoethanol have no effect on the protease activity. Therefore, N-V protease is identified as a serine protease. The primary amino acid sequence of N-V protease was determined by mass spectrometry (N-V protease, No. P83433). According to the MALDI-TOF MS analysis, there is no existing protein in the NCBI Non-redundant Protein Sequence Database that matches the N-V protease sequence. Therefore, N-V protease is a novel and special protein in N. virens. Furthermore, we have successfully established an expression cDNA library from the whole body of N. virens (data not shown). PMID:16950556
Zhang, Yunlong; Cui, Jiayue; Zhang, Rui; Wang, Yanpin; Hong, Min
Interaction of ortho-Phospho-l-serine with Hydroxyapatite: Formation of a Surface Complex
ortho-Phospho-l-serine (H2Psi, where Psi represents the serinephosphato ion), a constituent of salivary proteins, seems to play an important role in the mineralization of teeth. To understand the basic mechanism of this interaction, the uptake of o-phospho-l-serine from relatively concentrated aqueous solutions (up to 100 mmol/L) onto synthetic hydroxyapatite was studied. Previous studies have shown that in the dilute concentration range (<12.5 mmol/L) the uptake followed a regular Langmuirian adsorption plot. At higher concentrations the uptake curve increased steeply, but no formation of a separate phase in the reacted apatite was discernible, either by optical or by scanning electron microscopy. The dissolution of apatite released phosphate and calcium ions into the solution in amounts linearly related to the uptake of serine with P/Psi = 1 and Ca/Psi = 2. The charge and mass balance of the reaction can be reconciled with the formation of the surface complex (shown within brackets):Ca10(OH)2(PO4)6 + 6H2Psi --> [Ca6(HPsi)2(HPO4)2(PO4)2] + 4Ca2+ + 2HPsi1- + 2Psi2- + 2H2PO1-4 + 2H2O.The formation of two other surface complexes is possible; however, the complex shown above probably disrupts the apatite lattice the least. Traces of CaPsi�H2O precipitate out from the filtrates of highly concentrated solutions after 6 days. Copyright 1997 Academic Press. Copyright 1997Academic Press PMID:9367603
Sub-10ps monolithic and low-power photodetector readout
Recent advances in photon detectors have resulted in high-density imaging arrays that offer many performance and cost advantages. In particular, the excellent transit time spread of certain devices show promise to provide tangible benefits in applications such as Positron Emission Tomography (PET). Meanwhile, high-density, high-performance readout techniques have not kept on pace for exploiting these developments. Photodetector readout for next generation high event rate particle identification and time-resolved PET requires a highly-integrated, low-power, and cost-effective readout technique. We propose fast waveform sampling as a method that meets these criteria and demonstrate that sub-10ps resolution can be obtained for an existing device.
Varner, Gary S.; Ruckman, Larry L.
Development and characterization of sub-100 ps photomultiplier tubes
We describe the evaluation of a microchannel plate (MCP) photomultiplier tube (PMT), incorporating a 3 {mu}m pore MCP and constant voltage anode and cathode gaps. The use of the small pore size results in PMTs with response functions of the order of 85 ps full-width-half-maximum, while the constant electric field across the anode and cathode gaps produces a uniform response function over the entire operating range of the device. The PMT was characterized on a number of facilities and employed on gas Cherenkov detectors fielded on various deuterium tritium fuel (DT) implosions on the Omega Laser Facility at the University of Rochester. The Cherenkov detectors are part of diagnostic development to measure Gamma ray reaction history for DT implosions on the National Ignition Facility.
Horsfield, C. J.; Rubery, M. S. [AWE, Aldermaston, Reading RGR 4PR (United Kingdom); Mack, J. M.; Young, C. S.; Herrmann, H. W.; Caldwell, S. E.; Evans, S. C.; Sedilleo, T. J.; Kim, Y. H.; McEvoy, A. [Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States); Milnes, J. S.; Howorth, J. [Photek Ltd., 26 Castleham Rd., St. Leonards-on-Sea, East Sussex TN38 9NS (United Kingdom); Davis, B.; O'Gara, P. M.; Garza, I. [National Security Technologies, 2621 Losee Rd., North Las Vegas, Nevada 89030 (United States); Miller, E. K. [National Security Technologies, LLC, Santa Barbara, California 94551-0808 (United States); Stoeffl, W. [Lawrence Livermore National Laboratory, Livermore, California 93111 (United States); Ali, Z. [National Security Technologies, LO, Livermore, California 94551-2710 (United States)
Bacterial serine/threonine protein kinases in host-pathogen interactions.
In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701
Canova, Marc J; Molle, Virginie
Phosphorylation of insulin receptor substrate-1 serine 307 correlates with JNK activity in atrophic skeletal muscle
NASA Technical Reports Server (NTRS)
c-Jun NH(2)-terminal kinase (JNK) has been shown to negatively regulate insulin signaling through serine phosphorylation of residue 307 within the insulin receptor substrate-1 (IRS-1) in adipose and liver tissue. Using a rat hindlimb suspension model for muscle disuse atrophy, we found that JNK activity was significantly elevated in atrophic soleus muscle and that IRS-1 was phosphorylated on Ser(307) prior to the degradation of the IRS-1 protein. Moreover, we observed a corresponding reduction in Akt activity, providing biochemical evidence for the development of insulin resistance in atrophic skeletal muscle.
Hilder, Thomas L.; Tou, Janet C L.; Grindeland, Richard E.; Wade, Charles E.; Graves, Lee M.
Biochemical features, molecular biology and clinical relevance of the human 15-domain serine proteinase inhibitor LEKTI.
Based on the isolation of a 55 amino acid peptide from human hemofiltrate, we cloned the cDNA for a novel human 15-domain serine proteinase inhibitor termed LEKTI. A trypsin-inhibiting activity was demonstrated for three different domains. High levels of expression of the corresponding gene were detected in oral mucosa, followed by the tonsils, parathyroid glands, thymus, and trachea. Hovnanian and coworkers recently found that certain mutations within the LEKTI gene are linked to the severe congenital disease Netherton syndrome and atopic manifestations (including asthma). Thus, a future therapeutic use of LEKTI is conceivable. PMID:12437098
Walden, Michael; Kreutzmann, Peter; Dr�gem�ller, Katrin; John, Harald; Forssmann, Wolf-Georg; Hans-J�rgen, M�gert
A high throughput assay of the hepatitis C virus nonstructural protein 3 serine proteinase
A simple assay was developed based on intramolecular fluorescence resonance energy transfer for detection of the activity of hepatitis C virus (HCV) serine proteinase. Two quenched-fluorogenic substrates, (7-methoxycoumarin-4-yl)acetyl (Mca) Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-(2,4-dinitrophenyl, Dnp) Lys (Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Lys[Dnp], QF-1) and Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Lys(Dnp)-Arg-Arg (QF-2), which derived from the NS5A\\/5B junction of the HCV polyprotein, were designed. Kinetic studies revealed that QF-1 and QF-2 had high affinity for
Nobuko Kakiuchi; Satoshi Nishikawa; Masao Hattori; Kunitada Shimotohno
Immunization against a serine protease inhibitor reduces intensity of Plasmodium berghei infection in mosquitoes
The mosquito innate immune response is able to clear the majority of Plasmodium parasites. This immune clearance is controlled by a number of regulatory molecules including serine protease inhibitors (serpins). To determine whether such molecules could represent a novel target for a malaria transmission-blocking vaccine, we vaccinated mice with Anopheles gambiae serpin-2 (AgSRPN2). Antibodies against AgSRPN2 significantly reduced the infection of a heterologous Anopheles species (Anopheles stephensi) by Plasmodium berghei, however this effect was not observed with Plasmodium falciparum. Therefore, this approach of targeting regulatory molecules of the mosquito immune system may represent a novel approach to transmission-blocking malaria vaccines. PMID:23872520
Williams, Andrew R.; Zakutansky, Sara E.; Miura, Kazutoyo; Dicks, Matthew J. D.; Churcher, Thomas S.; Jewell, Kerry E.; Vaughan, Aisling M.; Turner, Alison V.; Kapulu, Melissa C.; Michel, Kristin; Long, Carole A.; Sinden, Robert E.; Hill, Adrian V. S.; Draper, Simon J.; Biswas, Sumi
Controlled Synthesis of Phosphorylcholine Derivatives of Poly(serine) and Poly(homoserine).
We report methods for the synthesis of polypeptides that are fully functionalized with desirable phosphorylcholine, PC, groups. Because of the inherent challenges in the direct incorporation of the PC group into ?-amino acid N-carboxyanhydride (NCA) monomers, we developed a synthetic approach that combined functional NCA polymerization with efficient postpolymerization modification. While poly(l-phosphorylcholine serine) was found to be unstable upon synthesis, we successfully prepared poly(l-phosphorylcholine homoserine) with controlled chain lengths and found these to be water-soluble with disordered chain conformations. PMID:25790104
Yakovlev, Ilya; Deming, Timothy J
Viscoelastic properties of pressure overload hypertrophied myocardium: effect of serine protease treatment
NASA Technical Reports Server (NTRS)
To determine whether and to what extent one component of the extracellular matrix, fibrillar collagen, contributes causally to abnormalities in viscoelasticity, collagen was acutely degraded by activation of endogenous matrix metalloproteinases (MMPs) with the serine protease plasmin. Papillary muscles were isolated from normal cats and cats with right ventricular pressure overload hypertrophy (POH) induced by pulmonary artery banding. Plasmin treatment caused MMP activation, collagen degradation, decreased the elastic stiffness constant, and decreased the viscosity constant in both normal and POH muscles. Thus, whereas many mechanisms may contribute to the abnormalities in myocardial viscoelasticity in the POH myocardium, changes in fibrillar collagen appear to play a predominant role.
Stroud, Jason D.; Baicu, Catalin F.; Barnes, Mary A.; Spinale, Francis G.; Zile, Michael R.
Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42
An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3�days at pH�10.5 and 37�C. Highest alkaline protease\\u000a activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate\\u000a by DEAE-cellulose chromatography followed by (NH4)2SO4 precipitation, with a yield of 58%. SDS-PAGE analysis
Dilek Kazan; Aziz Ak?n Denizci; Mine N. Kerimak �ner; Altan Erarslan
Serine, glycine and the one-carbon cycle: cancer metabolism in full circle
One carbon metabolism involving the folate and methionine cycle integrates carbon units from amino acids, including serine and glycine, and generates diverse outputs, such as the biosynthesis of lipids, nucleotides and proteins, the maintenance of redox status, and the substrates for methylation reactions. Long considered a �housekeeping� process, this pathway has been recently shown to have additional complexity. Recent genetic and functional evidence also suggests that hyperactivation of this pathway is a possible driver of oncogenesis and establishes links to cellular epigenetic status. Given the wealth of clinically available agents that target one carbon metabolism, these new findings could present opportunities for translation into precision cancer medicine. PMID:23822983
Locasale, Jason W
Protein Serine/Threonine Kinase StkP Positively Controls Virulence and Competence in Streptococcus pneumoniae
In the Streptococcus pneumoniae genome, stkP, encoding a membrane-associated serine/threonine kinase, is not redundant (L. Novakova, S. Romao, J. Echenique, P. Branny, and M.-C. Trombe, unpublished results). The data presented here demonstrate that StkP belongs to the signaling network involved in competence triggering in vitro and lung infection and bloodstream invasion in vivo. In competence, functional StkP is required for activation of comCDE upstream of the autoregulated ring orchestrated by the competence-stimulating peptide. This is the first description of positive regulation of comCDE transcription in balance with its repression by CiaRH. PMID:15039376
Echenique, Jose; Kadioglu, Aras; Romao, Susana; Andrew, Peter W.; Trombe, Marie-Claude
Regulation of Sulfate Assimilation by Light and O-Acetyl-l-Serine in Lemna minor L. 1
The effect of 0.5 millimolar O-acetyl-l-serine added to the nutrient solution on sulfate assimilation of Lemna minor L., cultivated in the light or in the dark, or transferred from light to the dark, was examined. During 24 hours after transfer from light to the dark the extractable activity of adenosine 5?-phosphosulfate sulfotransferase, a key enzyme of sulfate assimilation, decreased to 10% of the light control. Nitrate reductase (EC 184.108.40.206.) activity, measured for comparison, decreased to 40%. Adenosine 5?-triphosphate (ATP) sulfurylase (EC 220.127.116.11.) and O-acetyl-l-serine sulfhydrylase (EC 22.214.171.124.) activities were not affected by the transfer. When O-acetyl-l-serine was added to the nutrient solution at the time of transfer to the dark, adenosine 5?-phosphosulfate sulfotransferase activity was still at 50% of the light control after 24 hours, ATP sulfurylase and O-acetyl-l-serine sulfhydrylase activity were again not affected, and nitrate reductase activity decreased as before. Addition of O-acetyl-l-serine at the time of the transfer caused a 100% increase in acid-soluble SH compounds after 24 hours in the dark. In continuous light the corresponding increase was 200%. During 24 hours after transfer to the dark the assimilation of 35SO42? into organic compounds decreased by 80% without O-acetyl-l-serine but was comparable to light controls in its presence. The addition of O-acetyl-l-serine to Lemna minor precultivated in the dark for 24 hours induced an increase in adenosine 5?-phosphosulfate sulfotransferase activity so that a constant level of 50% of the light control was reached after an additional 9 hours. Cycloheximide as well as 6-methyl-purine inhibited this effect. In the same type of experiment O-acetyl-l-serine induced a 100-fold increase in the incorporation of label from 35SO42? into cysteine after additional 24 hours in the dark. Taken together, these results show that exogenous O-acetyl-l-serine has a regulatory effect on assimilatory sulfate reduction of L. minor in light and darkness. They are in agreement with the idea that this compound is a limiting factor for sulfate assimilation and seem to be in contrast to the proposed strict light control of sulf

subset: commoncrawl