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Isolation of a nitric oxide inhibitor from mammary tumor cells and its characterization as phosphatidyl serine
Macrophages from mice bearing large D1-DMBA-3 mammary tumors have a decreased capacity to kill tumor targets. This effect is due to an impaired ability to produce nitric oxide (NO) in response to lipopolysaccharide (LPS) stimulation. Here we report that the DA-3 tumor cell line, derived from the in vivo adenocarcinoma D1-DMBA-3, produces a factor that inhibits both NO production/release and cytotoxicity of LPS-activated peritoneal exudate macrophages (PEM). However, other complex macrophage functions such as phagocytosis, superoxide production, mitochondrial dehydrogenase activity, and synthesis of proteins were not reduced by this factor. The NO inhibitor has been found to be lipid in nature. Lipid extracts from DA-3 cell culture supernatants were purified by repeated silica gel column chromatography. The active molecule was unambiguously characterized as phosphatidyl serine (PS) by fast atom bombardment tandem mass spectrometry. Preliminary results indicate a lack of induced NO synthase (iNOS) activity in the lysates of LPS-activated PEM pretreated with PS. The ubiquity of PS in the inner leaflet of biological membranes and its NO inhibitory property, suggest that this phospholipid may be one of the long elusive molecules responsible for regulating physiological levels of NO in the host and hence preventing cellular dysfunction and/or tissue damage. Furthermore, the possible overexpression and shedding of PS by DA-3 tumor cells may represent a novel mechanism to impair macrophage cytotoxicity, a host function that contributes to the protection against developing neoplasms.
Phosphatidyl-tris rather than N-acylphosphatidylserine is synthesized by Rhodopseudomonas sphaeroides grown in tris-containing media
The authors have synthesized 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(N-oleoyl)serine (N-acyl-PS) and 1,2-dioleoyl-sn-glycero-3-phospho-Tris (phosphatidyl-Tris) and have characterized both phospholipids by their chemical and chromatographic properties, as well as by their IR, {sup 13}C NMR, and {sup 1}H NMR spectra. Comparison of these data with those reported for a phospholipid isolated from Rhodopseudomonas sphaeroides grown in Tris-supplemented media indicates that R. sphaeroides synthesizes phosphatidyl-Tris rather than N-acyl-PS.
Schmid, P.C.; Kumar, V.V.; Weis, B.K.; Schmid, H.H.O. (Univ. of Minnesota, Austin (USA))
Docosahexaenoic acid-deficient phosphatidyl serine and high alpha-tocopherol in a fetal mouse brain over-expressing Cu/Zn-superoxide dismutase.
The over-expressed Cu/Zn-superoxide dismutase (Cu/Zn-SOD) gene has been found in some circumstances phenotypically deleterious and associated with oxidative injury-mediated aberrations while in other studies it was considered neuroprotective. In this work we examine a number of biochemical markers in fetal and adult brain from transgenic (tg) mice expressing the human Cu/Zn-SOD gene, which may determine this dual characteristic. These markers include the polyunsaturated fatty acid (PUFA) profile in discrete phospholipid species, the alpha-tocopherol levels, a marker for lipid anti-oxidant status, and thiobarbituric acid reactive substance (TBARS), a marker for the tissue oxidative status. The PUFA profile in choline- and ethanolamine-phosphoglycerides was similar in tg and nontransgenic (ntg) animals of either fetal or adult brain. Serine-phosphoglycerides, however, showed a marked decrease from 20. 07+/-0.53 to 14.92+/-0.87 wt% and 14.52+/-1.15 wt% in docosahexaenoic acid (DHA; 22:6 n3), in the tg 51 and tg 69 fetal brains, respectively, but not in the comparable adult tissues. The alpha-tocopherol levels were significantly higher in the fetal compared to the adult brain. There were no differences in the anti-oxidant levels between the ntg and tg fetal brains, but there were differences in the adult animals; the tg mice were higher by at least two-fold than the control animals. The basal TBARS in the tg 51 fetal brain was 35% lower than that of ntg mouse and in the presence of Fe(2+), brain slices from the former released less TBARS (57% reduction) into the medium than the latter. These results suggest that higher dosages of Cu/Zn-SOD gene are compatible with increased alpha-tocopherol levels, reduced basal TBARS levels and a DHA deficiency in the fetal, but not the adult, tg brain. PMID:11018466
Glozman, S; Cerruti-Harris, C; Groner, Y; Yavin, E
Synthesis of the Naturally Occurring Phytanyl Diether Analogs of Phosphatidyl Glycerophosphate and Phosphatidyl Glycerol.
National Technical Information Service (NTIS)
The diastereomers of the diphytanyl glycerol ether analogs of phosphatidyl glycerophosphate and phosphatidyl glycerol have been synthesized chemically and compared with the respective natural isomer isolated from the extremely halophilic bacterium, Haloba...
C. N. Joo M. Kates
The Hydrolysis of Phosphatidyl-Alcohols by Phospholipases A 2
The ability of a variety of secretory phospholipases A2 (sPLA2: EC 18.104.22.168) to bind to and hydrolyse a series of phosphatidyl-alcohol substrates, in the absence of detergent, was explored by both fluorescence-based kinetic and interfacial binding assays. The enzymes used were sPLA2 from porcine pancreas, Naja naja venom and a recombinant human non-pancreatic enzyme. Four dioleoyl phosphatidyl-alcohols were used with
Adrian R Kinkaid; David C Wilton
Isolation and Characterization of Phosphatidyl Choline from Spinach Leaves.
ERIC Educational Resources Information Center
This inexpensive but informative experiment for undergraduate biochemistry students involves isolating phosphatidyl choline from spinach leaves. Emphasis is on introducing students to techniques of lipid extraction, separation of lipids, identification using thin layer chromatography, and identification of fatty acids. Three periods of three hours�
Devor, Kenneth A.
Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols
In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in
Shishir Jaikishan; Anders Bj�rkbom; J. Peter Slotte
Orientational changes in dipalmitoyl phosphatidyl glycerol Langmuir monolayers
Dipalmitoyl phosphatidyl glycerol (DPPG) as Langmuir monolayers at the air\\/water interface was investigated by means of surface pressure measurements in addition to Brewster angle microscopy (BAM) during film compression\\/expansion. A characteristic phase transition region appeared in the course of surface pressure�area (?�A) isotherms for monolayers spread on alkaline water or buffer subphase, while on neutral or acidic water the plateau
J. Mi�ones; P. Dynarowicz-??tka; J. M. Rodriguez Patino; E. Iribarnegaray
Orientational changes in dipalmitoyl phosphatidyl glycerol Langmuir monolayers.
Dipalmitoyl phosphatidyl glycerol (DPPG) as Langmuir monolayers at the air/water interface was investigated by means of surface pressure measurements in addition to Brewster angle microscopy (BAM) during film compression/expansion. A characteristic phase transition region appeared in the course of surface pressure-area (pi-A) isotherms for monolayers spread on alkaline water or buffer subphase, while on neutral or acidic water the plateau region was absent. This phase transition region was attributed to the ionization of DPPG monolayer. It has been postulated that the ionization of the phosphatidyl glycerol group leads to its increased solvation, which probably provokes both a change in the orientation of the polar group and its deeper penetration into bulk phase. Film compression along the transition region provokes the dehydration of polar groups and subsequent change of their conformation, thus causing the DPPG molecules to emerge up to the interface. Quantitative Brewster angle microscopy (BAM) measurements revealed that along the liquid-expanded to liquid-condensed phase transition the thickness of the ionized DPPG monolayer increases by 4.2 A as a result of the conformational changes of the ionized polar groups, which tend to emerge from the bulk subphase up to the surface. PMID:12962672
Mi�ones, J; Dynarowicz-?atka, P; Mi�ones, J; Rodriguez Patino, J M; Iribarnegaray, E
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 (1)H, (31)P, (13)C 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
Spectrophotometric determination of Sc in eriochrome cyanine R(chrome azurol S) - phosphatidyl choline system
Eriochrome cyanine R(chrome azurol S) is used as a color reagent to determine Sc in the presence of phosphatidyl choline, eta = 3.7 * 10⁴ (4.5 * 10⁴). This method has been connected to extraction separation to determine Sc in the presence of rare earth elements, and good results have been obtained. Phosphatidyl choline(PC) is a biochemical reagent, which can
Y. Xu; X. Chen; Z. Hu
Quantitation of phosphatidyl N-methyl- and N,N-Dimethylaminoethanol in rat liver
The contents of phosphatidyl N-methyl-and N,N-dimethylaminoethanol were determined in the liver of rats injected with (Me-C14) methionine. Total phospholipids were extracted from aliquots of the liver and fractionated by two-dimensional thin layer\\u000a chromatography after addition of carrier phosphatidyl-N-methyl-and N,N-dimethylaminoethanol. The radioactivity present in\\u000a the two phosphatide spots was determined and used to calculated total disintegrations per min\\/100 g body wt.
S. L. Katyal; B. Lombard
Quantitation of phosphatidyl N-methyl and N,N-dimethyl aminoethanol in liver and lung of N-methylaminoethanol fed rats
A diet supplemented with N-methylaminoethanol was fed to rats, and liver and lung phospholipids were resolved by a two dimensional\\u000a thin layere chromatographic method affording the separation of phosphatidyl N-methylaminoethanol and phosphatidyl N,N-dimethylaminoethanol,\\u000a as well as of the major phosphatides. Significant amounts of phosphatidyl N-methylaminoethanol and phosphatidyl N,N-dimethylaminoethanol\\u000a accumulated within 24 hr in liver and lung. The fatty acid composition
S. L. Katyal; B. Lombardi
Phosphatidyl choline-based colloidal systems for dermal and transdermal drug delivery.
In this study we have prepared various phosphatidyl choline based colloidal systems, namely liposomes, transfersomes, microemulsions and micelles, using similar excipients and compared their ability to deliver drugs into and through the skin under occlusive and non-occlusive conditions. Hydrophilic propranolol hydrochloride (PHCl) and lipophilic propranolol base (PB) were used as model drugs. All tested parameters, that is formulation composition, drug characteristics and testing conditions, influenced skin permeability and skin retention. A trend was observed showing that the skin permeation as well as skin retention decreases with the amount of phosphatidyl choline in the formulations for both tested model drugs (micelles > transfersomes > liposomes > microemulsion). The lipophilic model drug had higher skin permeability especially when incorporated into the systems containing mainly hydrophilic excipients. Skin retention, however, was not affected by the drug hydrophilicity to the same extent as skin permeability. Occlusion increased both skin retention and skin permeation for both model drugs. PMID:19863162
Ferderber, Kristina; Hook, Sarah; Rades, Thomas
Modulation of enzymatic PS synthesis by liposome membrane composition.
Phosphatidylserine (PS) is a phospholipid known to exert important physiological roles in humans. However, this phospholipid (PL) is poorly available as a natural source and hardly produced by the chemical route. In this work, PS was obtained by transphosphatidylation using phospholipase D (PLD) and PL self-assembled into liposomes as the substrates. The aim was to better understand how the liposome membrane composition could modulate PS yield. Three lecithins were used as PL substrates, one originated from a marine source providing a high amount of n-3 polyunsaturated fatty acids, and two issued from soya differing in their phosphatidylcholine (PC) content. Different parameters such as Ca(2+) content, enzyme and L-serine concentrations modulated PS synthesis. The presence of Ca(2+) increased PS conversion yield. The alcohol acceptor (L-serine) concentration positively acted on PL conversion, by governing the equilibrium between transphosphatidylation and hydrolysis. Beside these specific reaction conditions, it was demonstrated that the membrane composition of the liposomes modulated PS synthesis. A direct correlation between PS accumulation and the amount of cholesterol or ?-tocopherol incorporated into the soya lecithins was observed. This result was interpreted in terms of "head" spacers promoting PLD transphosphatidylation. On the whole, this work provided key parameters for the formulation of liposomes using enzymatic PLD technology, to produce lecithins enriched in different proportions of PS and esterified with various types of fatty acids depending on the initial lecithin source. PMID:24334268
Pinsolle, Alexandre; Roy, Philippe; Cansell, Maud
Spectrophotometric determination of Sc in eriochrome cyanine R(chrome azurol S) - phosphatidyl choline system
Eriochrome cyanine R(chrome azurol S) is used as a color reagent to determine Sc in the presence of phosphatidyl choline, eta = 3.7 * 10/sup 4/ (4.5 * 10/sup 4/). This method has been connected to extraction separation to determine Sc in the presence of rare earth elements, and good results have been obtained. Phosphatidyl choline(PC) is a biochemical reagent, which can be used as a surfactant. It has been reported that chrome azurol S(CAS) can be used to determine Be in the presence of PC but it has not been reported that eriochrome cyanine R(ECR) and CAS can been used to determine Sc in the presence of PC. This paper has put forward a method by which Sc can be determined. ECR (CAS) has been used as a color reagent and PC as a surfactant. Conditional experiments have been made and this method has been connected to extraction separation. Tributyl phosphate (TBP) extracts Sc from rare earth elements to make a determination and good results have been obtained.
Xu, Y.; Chen, X.; Hu, Z.
Lipoarabinomannans: characterization of the multiacylated forms of the phosphatidyl-myo-inositol anchor by NMR spectroscopy.
Lipoarabinomannans, which exhibit a large spectrum of immunological activities, emerge as the major antigens of mycobacterial envelopes. The lipoarabinomannan structure is based on a phosphatidyl-myo-inositol anchor whose integrity has been shown to be crucial for lipoarabinomannan biological activity and particularly for presentation to CD4/CD8 double-negative alphabetaT cells by CD1 molecules. In this report, an analytical approach was developed for high-resolution 31P-NMR analysis of native, i.e. multiacylated, lipoarabinomannans. The one-dimensional 31P spectrum of cellular lipoarabinomannans, from Mycobacterium bovis Bacillus Calmette-Gu�rin, exhibited four 31P resonances typifying four types of lipoarabinomannans. Two-dimensional 1H-31P heteronuclear multiple-quantum-correlation/homonuclear Hartmann-Hahn analysis of the native molecules showed that these four types of lipoarabinomannan differed in the number and localization of fatty acids (from 1 to 4) esterifying the anchor. Besides the three acylation sites previously described, i.e. positions 1 and 2 of glycerol and 6 of the mannosyl unit linked to the C-2 of myo-inositol, we demonstrate the existence of a fourth acylation position at the C-3 of myo-inositol. We report here the first structural study of native multiacylated lipoarabinomannans, establishing the structure of the intact phosphatidyl-myo-inositol anchor. Our findings would help gain more understanding of the molecular basis of lipoarabinomannan discrimination in the binding process to CD1 molecules.
Nigou, J; Gilleron, M; Puzo, G
US Patent & Trademark Office Database
The invention provides a human serine dehydratase homolog (SDHH) and polynucleotides which identify and encode SDHH. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of SDHH.
Serine metabolism in human pregnancy.
Serine plays an important role in intermediary metabolism as a source of one carbon pool for nucleotide biosynthesis, as a precursor for glycine and glucose, and as a contributor to cysteine biosynthesis. A unique serine-glycine cycling between the liver and the placenta has been demonstrated in the sheep fetus. We hypothesized that, because of serine's role in growth and development, significant changes in serine metabolism will occur in pregnancy with advancing gestation. The rate of appearance (R(a)) of serine and its metabolism were quantified in healthy women longitudinally through pregnancy with a [2-(15)N(13)C]serine tracer. The contribution of serine N to urea and the rate of oxidation of serine were measured using the precursor-product relation. Plasma serine concentrations and serine R(a) were lower in pregnant (P) women, in both early and late gestation, compared with nonpregnant (NP) women [plasma serine: NP, 113 +/- 24.5; P early, 71.9 +/- 6.2; P late, 68.5 +/- 9.6 micromol/l; serine R(a): NP (n = 7), 152.9 +/- 42.8; P early (n = 12), 123.7 +/- 21.5; P late (n = 8), 102.8 +/- 18.2 micromol x kg(-1) x h(-1)]. Serine contributed approximately 6% to urea N and 15-20% to the plasma glycine pool, and oxidation of serine represented approximately 8% of R(a). There was no significant difference between P and NP subjects. Glucose infusion, at 3 mg x kg(-1) x min(-1) in P subjects, resulted in a decrease in serine R(a) and an increase in oxidation. The decrease in serine turnover in pregnancy may represent a decrease in alpha-amino nitrogen turnover related to a decreased rate of branched-chain amino acid transamination and caused by pregnancy-related hormones aimed at nitrogen conservation and accretion. PMID:12488240
Kalhan, Satish C; Gruca, Lourdes L; Parimi, Prabhu S; O'Brien, Alicia; Dierker, Leroy; Burkett, Ed
Serine carboxypeptidases. A review
Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. Those characterized\\u000a until now can, dependent on their catalytic mechanism, be classified as either metallo carboxypeptidases or as serine carboxypeptidases.\\u000a Enzymes from the latter group are found in the vacuoles of higher plants and fungi and in the lysosomes of animal cells. Many\\u000a fungi, in addition, excrete
Klaus Breddam
Serine utilization by Klebsiella aerogenes.
Klebsiella aerogenes was found to contain a specific L-serine dehydrase that was induced by threonine, glycine or leucine, but not by its substrate. Cellular concentrations were sensitive to carbon rather than nitrogen sources in the growth medium. A nonspecific isoleucine-sensitive L-threonine dehydrase supplemented the specific L-serine dehydrase activity. K. aerogenes also contains a leucine-inducible L-threonine dehydrogenase which probably initiated a threonine-utilization pathway in which the serine-specific dehydrate participated. Strains that were altered in their ability to metabolize serine differed in either L-serine dehydrase or L-threonine dehydrase activity. Thus, K. aerogenes growing on L-serine as a sole nitrogen source relies upon two enzymes that metabolize the amino acid as subsidiary functions.
Vining, L C; Magasanik, B
Anti-phospholipid Antibodies in HIV Infection and SLE With or Without Anti-phospholipid Syndrome: Comparisons of Phospholipid Specificity, Avidity and Reactivity with ?2-GPI
Increased prevalence of anti-phospholipid antibodies (aPL) and increased levels of lipid peroxidation have been described in patients with HIV infection. To assess the binding specificity and avidity of aPL antibodies in HIV infection, sera from 44 HIV-1 infected patients were evaluated for antibodies to cardiolipin (aCL), phosphatidyl serine (aPS), phosphatidyl inositol (aPI) and phosphatidyl choline (aPC) using enzyme linked immunosorbent
C Petrovas; P. G Vlachoyiannopoulos; T Kordossis; H. M Moutsopoulos
NSDL National Science Digital Library
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Synthesis of Poly-O-Tert-Butyl-L-Serine and Poly-L-Serine.
National Technical Information Service (NTIS)
The synthesis of polymers containing O-tert-butyl-L-serine, O-tert-butyl-DL-serine, L-serine, and DL-serine is reported. The N-benzyloxycarbonyl-(L or DL)-serine benzyl ester was used to synthesize the corresponding N-benzyloxycarbonyl-O-tert-butyl-(L or ...
N. M. Tooney G. D. Fasman
Preparation and assay of recombinant serine racemase.
Serine racemase is a glial and neuronal enzyme that reversibly converts L-serine to D-serine, an endogenous co-agonist of N-methyl-D-aspartate receptor type glutamate receptors (NMDARs). Here we present methods to recombinantly express and purify serine racemase in bacteria and two complementary ways to determine D-serine levels in unknown samples. Furthermore, a detailed protocol of serine racemase activity assays is described that can be used to screen for activators and inhibitors in vitro. PMID:21956576
Baumgart, Florian; Aicart-Ramos, Clara; Rodriguez-Crespo, Ignacio
Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells
Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a range of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.
Johnsson, Anna-Karin; Karlsson, Roger, E-mail: email@example.com
Serine proteinase inhibitors in arthropod immunity
Arthropod hemolymph contains proteins with serine proteinase inhibitory activity. These inhibitors may exist in plasma or in hemocyte granules. Serine proteinase inhibitors from the Kazal, Kunitz, ?-macroglobulin, and serpin families have been identified in arthropod hemolymph and have been characterized biochemically. Two new families of low molecular weight serine proteinase inhibitors have recently been discovered: one in silkworms (the Bombyx
Michael R. Kanost
D-Serine inhibits serine palmitoyltransferase, the enzyme catalyzing the initial step of sphingolipid biosynthesis
Serine palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L-serine to produce 3-ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [3H]KDS from L-[3H]serine was performed using purified SPT. D-Serine inhibited [3H]KDS production as effectively as non-radioactive L-serine, whereas neither
Kentaro Hanada; Tomoko Hara; Masahiro Nishijima
Serine proteinase from rice bean.
A trypsin like serine-proteinase of M(r) 16,000 Da, optimally active at pH 8.4 on N-benzoyl-arginine ethyl ester (BAEE) was purified from 4-day old germinated seeds of rice bean, Vigna umbellata (Thunb), by ammonium sulphate precipitation, gel filtration, ion-exchange chromatography and by high performance liquid chromatography (HPLC). The purity of the enzyme was checked by polyacrylamide gel electrophoresis (PAGE). The enzyme activity was studied on natural substrates like casein, haemoglobin and vicilin, a rice bean storage protein. The activity of the enzyme was completely inhibited by phenylmethylsulfonyl fluoride, but not by iodoacetamide and HgCl2, suggesting it to be a serine protease. Loss of activity in presence of EDTA was reversed by addition of Ca2+. PMID:9219435
Basu, P S; Biswas, C; Majhi, R; Datta, T K
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.
An update on serine deficiency disorders.
Serine deficiency disorders are caused by a defect in one of the three synthesising enzymes of the L-serine biosynthesis pathway. Serine deficiency disorders give rise to a neurological phenotype with psychomotor retardation, microcephaly and seizures in newborns and children or progressive polyneuropathy in adult patients. There are three defects that cause serine deficiency of which 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency, the defect affecting the first step in the pathway, has been reported most frequently. The other two disorders in L-serine biosynthesis phosphoserine aminotransferase (PSAT) deficiency and phosphoserine phosphatase (PSP) deficiency have been reported only in a limited number of patients. The biochemical hallmarks of all three disorders are low concentrations of serine in cerebrospinal fluid and plasma. Prompt recognition of affected patients is important, since serine deficiency disorders are treatable causes of neurometabolic disorders. The use of age-related reference values for serine in CSF and plasma can be of great help in establishing a correct diagnosis of serine deficiency, in particular in newborns and young children. PMID:23463425
van der Crabben, S N; Verhoeven-Duif, N M; Brilstra, E H; Van Maldergem, L; Coskun, T; Rubio-Gozalbo, E; Berger, R; de Koning, T J
Structural study of the lipomannans from Mycobacterium bovis BCG: characterisation of multiacylated forms of the phosphatidyl-myo-inositol anchor.
A biosynthetic filiation is postulated between the mycobacterial phosphatidyl-myo-inositol mannosides (PIMs), the lipomannans (LMs) and the lipoarabinomannans (LAMs), the major antigens of the envelopes. Moreover, as the PI anchor is thought to play a role in the biological functions of the LAMs, we characterized the lipid moiety of the PI anchor from Mycobacterium bovis BCG cellular LMs. Their structure was investigated along with that of a purified tetra-acylated form of PIM2 (Ac4PIM2). A two-dimensional 1H-31P heteronuclear multiple quantum correlation homonuclear Hartmann-Hahn spectroscopy study of Ac4PIM2 unambiguously localised a fourth fatty acid on the C3 of the myo-Ins beside the fatty acids already described on the C1 and C2 position of the glycerol and on the C6 position of the mannose. This analytical strategy was extended to the structural study of the cellular LM anchor. Using an appropriate solvent system, the one dimensional 31P NMR spectrum exhibited four major resonances typifying the LM populations. These populations differed in number and location of the fatty acids. For one of these populations, we established the presence of an extra fatty acid on the C3 of the myo-Ins of the LM anchor. The fact that both types of molecules have an elaborated anchor in common, indicates that cellular LMs are multimannosylated forms of PIMs. In addition, the LM mannan core structure was analysed by two-dimensional NMR, pointing to a high level of branching by single alpha1-->2 Manp side-chains. PMID:9925791
Gilleron, M; Nigou, J; Cahuzac, B; Puzo, G
The effect of phospholipids on anaphylactic histamine release
1. Histamine release by antigen from three sensitized rat tissues is potentiated by phosphatidyl serine (PS). The effect is greatest with isolated peritoneal cells. Phosphatidyl inositol, ethanolamine, choline and phosphatidic acid are inactive. 2. PS greatly increases the rate of antigen-induced histamine release and only slightly prolongs the duration of the release process. 3. PS shows a concentration-dependent effect over the range 1-10 ?g/ml. At 10 ?g/ml it is active over a wide range of antigen concentrations. 4. Calcium ions are necessary for the potentiation by PS of antigen-induced histamine release from rat peritoneal cells.
Mongar, J. L.; Svec, P.
Metabolism of the neuromodulator D-serine.
Over the past years, accumulating evidence has indicated that D-serine is the endogenous ligand for the glycine-modulatory binding site on the NR1 subunit of N-methyl-D-aspartate receptors in various brain areas. D-Serine is synthesized in glial cells and neurons by the pyridoxal-5' phosphate-dependent enzyme serine racemase, and it is released upon activation of glutamate receptors. The cellular concentration of this novel messenger is regulated by both serine racemase isomerization and elimination reactions, as well as by its selective degradation catalyzed by the flavin adenine dinucleotide-containing flavoenzyme D-amino acid oxidase. Here, we present an overview of the current knowledge of the metabolism of D-serine in human brain at the molecular and cellular levels, with a specific emphasis on the brain localization and regulatory pathways of D-serine, serine racemase, and D-amino acid oxidase. Furthermore, we discuss how D-serine is involved with specific pathological conditions related to N-methyl-D-aspartate receptors over- or down-regulation. PMID:20195697
Pollegioni, Loredano; Sacchi, Silvia
STAT3 phosphorylation at tyrosine 705 and serine 727 differentially regulates mouse ESC fates.
STAT3 can be transcriptionally activated by phosphorylation of its tyrosine 705 or serine 727 residue. In mouse embryonic stem cells (mESCs), leukemia inhibitory factor (LIF) signaling maintains pluripotency by inducing JAK-mediated phosphorylation of STAT3 Y705 (pY705). However, the function of phosphorylated S727 (pS727) in mESCs remains unclear. In this study, we examined the roles of STAT3 pY705 and pS727 in regulating mESC identities, using a small molecule-based system to post-translationally modulate the quantity of transgenic STAT3 in STAT3(-/-) mESCs. We demonstrated that pY705 is absolutely required for STAT3-mediated mESC self-renewal, while pS727 is dispensable, serving only to promote proliferation and optimal pluripotency. S727 phosphorylation is regulated directly by fibroblast growth factor/Erk signaling and crucial in the transition of mESCs from pluripotency to neuronal commitment. Loss of S727 phosphorylation resulted in significantly reduced neuronal differentiation potential, which could be recovered by a S727 phosphorylation mimic. Moreover, loss of pS727 sufficed LIF to reprogram epiblast stem cells to na�ve pluripotency, suggesting a dynamic equilibrium of STAT3 pY705 and pS727 in the control of mESC fate. PMID:24302476
Huang, Guanyi; Yan, Hexin; Ye, Shoudong; Tong, Chang; Ying, Qi-Long
Polarizabilities of the Ps Negative Ion
NASA Technical Reports Server (NTRS)
We have calculated polarizabilities (alpha(sub l), beta(sub 1), gamma(sub l), alpha(sub 2), beta(sub 2), and gamma(sub 2)) of Ps(sup -) by the pseudostate method. These parameters can be used to calculate Rydberg states of Ps(sup -) in the presence of an external electron with high quantum numbers N and L. They are also of importance in a system containing Ps(sup -) bound to a proton [PsH], and also Rydberg states of Ps(sub 2).
Bhatia, A. K.; Drachman, Richard J.
Molecular markers of serine protease evolution
The evolutionary history of serine proteases can be accounted for by highly conserved amino acids that form crucial structural and chemical elements of the catalytic apparatus. These residues display non- random dichotomies in either amino acid choice or serine codon usage and serve as discrete markers for tracking changes in the active site environment and supporting structures. These markers categorize serine proteases of the chymotrypsin-like, subtilisin-like and ?/?-hydrolase fold clans according to phylogenetic lineages, and indicate the relative ages and order of appearance of those lineages. A common theme among these three unrelated clans of serine proteases is the development or maintenance of a catalytic tetrad, the fourth member of which is a Ser or Cys whose side chain helps stabilize other residues of the standard catalytic triad. A genetic mechanism for mutation of conserved markers, domain duplication followed by gene splitting, is suggested by analysis of evolutionary markers from newly sequenced genes with multiple protease domains.
Krem, Maxwell M.; Di Cera, Enrico
Rational Design of Peptide Serine Protease Inhibitors.
National Technical Information Service (NTIS)
Since the uncontrolled activity of serine protease enzymes is widely implicated in the pathology of disease states involving tissue destruction, there is considerable therapeutic potential for molecules designed specifically to inhibit these enzymes. In t...
K. S. Wibley S. Bansal D. J. Barlow
D-serine increases adult hippocampal neurogenesis
Adult hippocampal neurogenesis results in the continuous formation of new neurons and is a process of brain plasticity involved in learning and memory. The neurogenic niche regulates the stem cell proliferation and the differentiation and survival of new neurons and a major contributor to the neurogenic niche are astrocytes. Among the molecules secreted by astrocytes, D-serine is an important gliotransmitter and is a co-agonist of the glutamate, N-methyl-D-aspartate (NMDA) receptor. D-serine has been shown to enhance the proliferation of neural stem cells in vitro, but its effect on adult neurogenesis in vivo is unknown. Here, we tested the effect of exogenous administration of D-serine on adult neurogenesis in the mouse dentate gyrus. We found that 1 week of treatment with D-serine increased cell proliferation in vivo and in vitro and increased the density of neural stem cells and transit amplifying progenitors. Furthermore, D-serine increased the survival of newborn neurons. Together, these results indicate that D-serine treatment resulted in the improvement of several steps of adult neurogenesis in vivo.
Sultan, Sebastien; Gebara, Elias G.; Moullec, Kristell; Toni, Nicolas
BAP1 is phosphorylated at serine 592 in S-phase following DNA damage.
The human BAP1 deubiquitinating enzyme is a chromatin-bound transcriptional regulator and tumor suppressor. BAP1 functions in suppressing cell proliferation, yet its role in the DNA damage response pathway is less understood. In this study we characterized DNA damage-induced phosphorylation of BAP1 at serine 592 (pS592) and the cellular outcomes of this modification. In contrast to the majority of BAP1, pS592-BAP1 is predominantly dissociated from chromatin. Our findings support a model whereby stress induced phosphorylation functions to displace BAP1 from specific promoters. We hypothesize that this regulates the transcription of a subset of genes involved in the response to DNA damage. PMID:24211834
Eletr, Ziad M; Yin, Luming; Wilkinson, Keith D
Multitrack PS-InSAR datum connection
InSAR data acquired from independent overlapping tracks can be exploited for a reliability assessment of the Persistent Scatterer InSAR (PS-InSAR) technique. This is obtained by means of the datum connection of multiple tracks, simultaneously evaluating the misclosures between multi-track PS-InSAR estimates. Due to a different viewing geometry, many of the detected PS will physically not be the same. However, their
Gini Ketelaar; Freek van Leijen; Petar Marinkovic; Ramon Hanssen
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
Viscoelastic and Transport Properties of Sulfonated PS-PIB-PS Block Copolymers.
National Technical Information Service (NTIS)
Morphology, viscoelastic, and transport properties of the sulfonated polystyrene-polyisob utylene-polystyrene (PS-PIB-PS) block copolymer were investigated with respect to sulfonation level and counter-ion substitution. Dynamic mechanical analysis (DMA) w...
E. Napadensky D. Crawford J. Sloan N. B. Tan
Production and comparison of mature single-domain 'trefoil' peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58.
The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the 'trefoil' family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7
Chadwick, M P; May, F E; Westley, B R
The stability and structure of Li+Ps2 and Na+Ps2
The fixed-core stochastic variational method has been used to demonstrate the existence of an electronically stable ground state for the Li +Ps2 and Na+Ps2 systems. The stability of Li+Ps2 has been indicated previously in an ab initio calculation by Varga, but the present estimate of the binding energy is much closer to convergence. The Li+Ps2 system has a binding energy
J Mitroy; G G Ryzhikh
NASA Technical Reports Server (NTRS)
This Franklin PS-2 training glider is about to be towed aloft by the specially modified car in front. NACA researchers used the PS-2 in a study of ground effect on a towed glider. Langley flew two of the Franklin gliders in the late 1930s, but the Navy never really found a good use for training gliders.
L-serine dehydratase from Arthrobacter globiformis.
1. L-Serine dehydratase (EC 188.8.131.52) was purified 970-fold from glycine-grown Arthrobacter globiformis to a final specific activity of 660micronmol of pyruvate formed/min per mg of protein. 2. The enzyme is specific for L-serine; D-serine, L-threonine and L-cysteine are not attacked. 3. The time-course of pyruvate formation by the purified enzyme, in common with enzyme in crude extracts and throughout the purification, is non-linear. The reaction rate increases progressively for several minutes before becoming constant. The enzyme is activated by preincubation with L-serine and a linear time-course is then obtained. 4. The substrate-saturation curve for L-serine is sigmoid. The value of [S]0.5 varies with protein concentration, from 6.5mM at 23microng/ml to 20mM at 0.23microng/ml. The Hill coefficient remains constant at 2.9.5 The enzyme shows a non-specific requirement for a univalent or bivalent cation. Half-maximal activity is produced by 1.0mM-MgCl2 or by 22.5mM-KCl. 6. L-Cysteine and D-serine act as competitive inhibitors of L-serine dehydratase, with Ki values of 1.2 and 4.9mM respectively. L-Cysteine, at higher concentrations, also causes a slowly developing irreversible inhibition of the enzyme. 7. Inhibition by HgCl2 (5micronM)can be partially reversed in its initial phase by 1mM-L-cysteine, but after 10 min it becomes irreversible. 8. In contrast with the situation in all cell-free preparations, toluene-treated cells of A. globiformis form pyruvate from L-serine at a constant rate from the initiation of the reaction, show a hyperbolic substrate-saturation curve with an apparent Km of 7mM and do not require a cation for activity.
Gannon, F; Bridgeland, E S; Jones, K M
Viral Serine/Threonine Protein Kinases ?
Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.
Jacob, Thary; Van den Broeke, Celine; Favoreel, Herman W.
Rapid determination of free D-serine with chicken D-serine dehydratase.
We have developed a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free D-serine in tissue samples. This method uses chicken D-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. Pyridoxal 5'-phosphate (PLP), a cofactor of D-serine dehydratase, increased pyruvate formation by 28%. The presence of Zn(2+) or ethylenediaminetetraacetic acid (EDTA) did not have any effect on pyruvate formation under the present assay conditions. In addition, this method was not affected by the presence of a large excess of L-serine, nor by the presence of tissue extracts, and accurately determined concentrations of 2-30 ?M (200 pmol-3 nmol) of D-serine. The entire assay requires only 60 min. With this method, we determined the concentration of D-serine in various silkworm tissues. The results were in agreement with high performance liquid chromatography measurements. We found high concentrations of D-serine in silkworm larvae at day 3 of the fifth instar; specifically, 509 nmol g(-1) wet tissue in the midgut, 434 nmol g(-1) in the ovary, and 353 nmol g(-1) in the testis. PMID:21840271
Suzuki, Chihiro; Murakami, Masahito; Yokobori, Hirokazu; Tanaka, Hiroyuki; Ishida, Tetsuo; Horiike, Kihachiro; Nagata, Yoko
NASA Technical Reports Server (NTRS)
Franklin PS-2 (XPS-2) Glider: This Franklin PS-2 training glider is about to be towed aloft by the specially modified car in front. NACA researchers used the PS-2 in a study of ground effect on a towed glider. Langley flew two of the Franklin gliders in the late 1930s, but the Navy never really found a good use for training gliders.: This Franklin PS-2 training glider is about to be towed aloft by the specially modified car in front. NACA researchers used the PS-2 in a study of ground effect on a towed glider. Langley flew two of the Franklin gliders in the late 1930s, but the Navy never really found a good use for training gliders.
Interactions of cyclodextrins with dipalmitoyl, distearoyl, and dimyristoyl phosphatidyl choline liposomes. A study by leakage of carboxyfluorescein in inner aqueous phase of unilamellar liposomes.
The interaction of cyclodextrins (CDs) with L-alpha-dipalmitoyl phopsatidyl choline (DPPC), L-alpha-distearoyl phosphatidyl choline (DSPC), and L-alpha-dimyristoyl phosphatidyl choline (DMPC) unilamellar liposomes was investigated by the leakage of carboxylfluorescein (CF) entrapped in the inner aqueous phase of liposomes, at 25 degrees C (DPPC and DSPC liposomes) and at 5 degrees C (DMPC liposomes). The efficiency of CDs for CF leakage was remarkable in the order of heptakis (2,6-di-O-methyl)-beta-CD (DOM-beta-CD) > alpha-CD > heptakis (2,3,6-tri-O-methy)-beta-CD (TOM-beta-CD) from DPPC liposomes, in the order of DOM-beta-CD > TOM-beta-CD > alpha-CD from DSPC liposomes and in the order of alpha-CD > DOM-beta-CD > TOM-beta-CD from DMPC liposomes. The other CDs used in the present studies, beta-CD, 2-hydroxylpropyl beta-CD, and gamma-CD scarcely induced the CF leakage from above the three liposomes. From the profiles of % CF leakage, together with measurements of differential scanning calorimetry, it was found that hydrophobic DOM-beta-CD penetrates the matrix of the liposomes to interact with them as well as TOM-beta-CD, and that less hydrophobic alpha-CD exists at the surface of the membrane to interact with the liposomes. Further, it was found that the interaction of CDs with liposomes changes depending not only on the length of fatty acid chain of phospholipid (condensation force and hydrophobicity) but also the hydrophobicity and the cavity size of CD. PMID:10705474
Nishijo, J; Shiota, S; Mazima, K; Inoue, Y; Mizuno, H; Yoshida, J
The phosphatidyl-myo-inositol anchor of the lipoarabinomannans from Mycobacterium bovis bacillus Calmette Gu�rin. Heterogeneity, structure, and role in the regulation of cytokine secretion.
Lipoarabinomannans are major mycobacterial antigens capable of modulating the host immune response; however, the molecular basis underlying the diversity of their immunological properties remain an open question. In this study a new extraction and purification approach was successfully applied to isolate ManLAMs (lipoarabinomannans with mannosyl extensions) from bacillus Calmette Gu�rin leading to the obtention of two types of ManLAMs namely parietal and cellular. Structurally, they were found to differ by the percentage of mannooligosaccharide caps, 76 and 48%, respectively, and also, thanks to a new analytical method, by the structure of the phosphatidyl-myo-inositol anchor lipid moiety. A novel fatty acid in the mycobacterium genus assigned to a 12-O-(methoxypropanoyl)-12-hydroxystearic acid was the only fatty acid esterifying C-1 of the glycerol residue of the parietal ManLAMs, while the phosphatidyl unit of the cellular ManLAMs showed a large heterogeneity due to a combination of palmitic and tuberculostearic acid. Finally, parietal and cellular ManLAMs were found to differentially affect interleukin-8 and tumor necrosis factor-alpha secretion from human dendritic cells. We show that parietal but not cellular ManLAMs were able to stimulate tumor necrosis factor-alpha secretion from dendritic cells. From these studies we propose that the 1-[12-O-(methoxypropanoyl)-12-hydroxystearoyl]-sn-glycerol part is the major cytokine-regulating component of the ManLAMs. It seems likely that modification of the ManLAM lipid part, which may occur in hostile environments, could regulate macrophagic mycobacterial survival by altering cytokine stimulation. PMID:9287310
Nigou, J; Gilleron, M; Cahuzac, B; Boun�ry, J D; Herold, M; Thurnher, M; Puzo, G
Acid sphingomyelinase and inhibition by phosphate ion: role of inhibition by phosphatidyl-myo-inositol 3,4,5-triphosphate in oligodendrocyte cell signaling.
There is ample evidence that both acid (ASMase) and neutral (NSMase) sphingomyelinases play a role in cell death so inhibitors of either enzyme could have significant value as protectors against neurodegeneration. We used a fluorogenic sphingomyelinase substrate, 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine, and a [(14)C]choline-labeled sphingomyelin substrate to screen large numbers of phosphocompounds for inhibition of ASMase in extracts of human oligodendroglioma cells (HOG) and neonatal rat oligodendrocytes. Non-competitive inhibition was observed with inorganic phosphate and AMP, which was a more potent inhibitor of ASMase than cyclic AMP, ADP or ATP. However, other nucleotide phosphates, sugar phosphates, nucleotide sugars and glycerol phosphate did not inhibit ASMase. Our key finding was that phosphatidyl-myo-inositol 3,4,5-triphosphate [PtdIns (3,4,5)P(3)] was a much more potent inhibitor of ASMase than lysophosphatidic acid or phosphatidyl-myo-inositol 4,5-diphosphate [PtdIns(4,5)P(2)]. When PtdIns(3,4,5)P(3) was added to cultured cells we observed 50% inhibition of ASMase but no inhibition of other lysosomal hydrolases. After transfection of HOG cells with the tumor supressor phosphatase and tensin homolog protein (PTEN), which hydrolyses PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), we observed a two-fold increase in ASMase activity. Furthermore, the phosphatidylinositol-3-kinase inhibitor wortmannin (which reduces PtdIns(3,4,5)P(3) levels) also resulted in activation of ASMase. We propose that the small amount of ASMase activity associated with detergent-resistant cell membranes (Rafts) is regulated by PtdIns(3,4,5)P(3) and is most likely involved in receptor clustering and capping. PMID:15086520
Testai, F D; Landek, M A; Goswami, R; Ahmed, M; Dawson, G
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.
Sakinc, Turkan; Kline, Kimberly; Nielsen, Hailyn V.; Hultgren, Scott; Gatermann, Soren G.
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
d-Serine modulates neurogenic relaxation in rat corpus cavernosum.
d-Serine, an endogenous co-agonist for the N-methyl-d-aspartate (NMDA) receptor in mammals, is synthesized from l-serine by serine racemase. Although much attention has been focused on the role of d-serine within the central nervous system, the physiological role of d-serine in peripheral nerves such as corpus cavernosal nerves has not been investigated. The present study was aimed to study the expression, cellular localization and function of serine racemase/d-serine system in isolated rat corpus cavernosum. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis showed the expression of serine racemase in rat corpus cavernosum. Immunogold electron microscopy demonstrated the cellular localization of serine racemase in the cavernosal nerves' membrane of the tissue. The organ bath studies on isolated rat corpus cavernosum showed that d-serine increases the non-adrenergic non-cholinergic neurogenic relaxation of isolated rat corpus cavernosum in vitro. This effect of d-serine was inhibited by a variety of NMDA receptor antagonists (ketamine, MK 801 and ifenprodil), suggesting that NMDA receptors are involved in the effects of d-serine on the neurogenic relaxation of corporal tissue strips. These observations provide the first evidence for the role of d-serine in modulating the neurogenic relaxation of rat corpus cavernosum, and may open new therapeutic avenues for the treatment of impotence. PMID:20170643
Ghasemi, Mehdi; Rezania, Fatemeh; Lewin, Jackie; Moore, Kevin P; Mani, Ali R
Cross-talk between diverse serine integrases.
Phage-encoded serine integrases are large serine recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes. They are well suited for use in heterologous systems and for synthetic genetic circuits as the attP and attB attachment sites are small (<50 bp), there are no host factor or DNA supercoiling requirements, and they are strongly directional, doing only excisive recombination in the presence of a recombination directionality factor. Combining different recombinases that function independently and without cross-talk to construct complex synthetic circuits is desirable, and several different serine integrases are available. However, we show here that these functions are not reliably predictable, and we describe a pair of serine integrases encoded by mycobacteriophages Bxz2 and Peaches with unusual and unpredictable specificities. The integrases share only 59% amino acid sequence identity and the attP sites have fewer than 50% shared bases, but they use the same attB site and there is non-reciprocal cross-talk between the two systems. The DNA binding specificities do not result from differences in specific DNA contacts but from the constraints imposed by the configuration of the component half-sites within each of the attachment site DNAs. PMID:24161951
Singh, Shweta; Rockenbach, Kate; Dedrick, Rebekah M; VanDemark, Andrew P; Hatfull, Graham F
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
Changes in Properties of Serine 129 Phosphorylated ?-Synuclein with Progression of Lewy Type Histopathology in Human Brains
Modifications of ?-synuclein resulting in changes in its conformation are considered to be key pathological events for Lewy body diseases (LBD), which include Parkinson�s disease (PD) and dementia with Lewy bodies (DLB). We have previously described a histopathological Unified Staging System for LBD that classifies the spread of ?-synuclein phosphorylated at serine 129 (pS129-?-synuclein) from olfactory bulb to brainstem or limbic regions, and finally neocortex. Lewy bodies and Lewy neurites are highly enriched in pS129-?-synuclein. Increased formation of pS129-?-synuclein changes its solubility properties enhancing its tendency to aggregate and disrupt normal function. As in vitro and animal studies have shown that inhibiting formation of pS129-?-synuclein can prevent toxic consequences, this has become one of the therapeutic targets for LBD. However, detailed biochemical descriptions of the changes in pS129-?-synuclein properties in diseased human brains are needed to further our understanding of how these might contribute to molecular pathogenesis. In this study, we used 130 separate brain samples from cingulate cortex (limbic cortex) and 131 from temporal cortex (neocortex) that had been staged according to our Unified Staging System to examine progressive changes in properties of pS129-?-synuclein with the formation of progressively more severe histological Lewy-type pathology. The brain samples from these staged cases had been separated into cytosol-enriched, membrane-enriched (detergent soluble) and insoluble (ureas/SDS soluble) fractions. We also characterized the nature and appearance of higher molecular weight forms of pS129-?-synuclein. The major species was the 16 kD monomeric form; this accumulated with increasing stage with a large increase in Stage IV samples. By comparing two brain regions, we showed higher accumulation of insoluble pS129-?-synuclein in cingulate cortex, where histological deposits occur first, than in temporal cortex in samples with advanced (Stage IV) LB pathology.
Walker, Douglas G.; Lue, Lih-Fen; Adler, Charles H.; Shill, Holly A.; Caviness, John N.; Sabbagh, Marwan N.; Akiyama, Haruhiko; Serrano, Geidy E.; Sue, Lucia I.; Beach, Thomas G.
Human serine racemase: moleular cloning, genomic organization and functional analysis
High levels of d-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl d-aspartate type of glutamate receptors. d-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from l-serine . We now report cloning and expression of human serine racemase,
Joari De Miranda; Ana Santoro; Simone Engelender; Herman Wolosker
In vivo D-serine hetero-exchange through alanine-serine-cysteine (ASC) transporters detected by microelectrode biosensors.
D-serine, a co-agonist of N-methyl D-aspartate (NMDA) receptors, has been implicated in neurological and psychiatric disorders such as cerebral ischemia, lateral amyotrophic sclerosis, or schizophrenia. D-serine signaling represents an important pharmacological target for treating these diseases; however, the biochemical mechanisms controlling extracellular D-serine levels in vivo are still unclear. D-serine heteroexchange through small neutral amino acid transporters has been shown in cell cultures and brain slices and could provide a biochemical mechanism for the control of D-serine extracellular concentration in vivo. Alternatively, exocytotic D-serine release has also been proposed. In this study, the dynamics of D-serine release and clearance were explored in vivo on a second-by-second time scale using microelectrode biosensors. The rate of D-serine clearance in the rat frontal cortex after a microionophoretic injection revealed a transporter-mediated uptake mechanism. D-serine uptake was blocked by small neutral l-amino acids, implicating alanine-serine-cysteine (ASC) transporters, in particular high affinity Asc-1 and low affinity ASCT2 transporters. Interestingly, changes in alanine, serine, or threonine levels resulted in D-serine release through ASC transporters. Asc-1, but not ASCT2, appeared to release D-serine in response to changes in amino acid concentrations. Finally, neuronal silencing by tetrodotoxin increased D-serine extracellular concentration by an ASC-transporter-dependent mechanism. Together, these results indicate that D-serine heteroexchange through ASC transporters is present in vivo and may constitute a key component in the regulation of D-serine extracellular concentration. PMID:23581544
Maucler, Caroline; Pernot, Pierre; Vasylieva, Natalia; Pollegioni, Loredano; Marinesco, St�phane
NASA Technical Reports Server (NTRS)
Franklin PS-2 (XPS-2) Glider: This beefy-looking glider is a Franklin PS-2, a pair of which were operated by the NACA at Langley beginning in April 1936. The Navy only ordered half a dozen of these training gliders, which had a glide ratio of 15 feet forward for every foot down. The devices above the pilot's seat are venturi tubes, which gathered data for the instruments. Airfield lights are seen above the wing.
Determination of the PS I content of PS II core preparations using selective emission: a new emission of PS II at 780nm.
Routinely prepared PS II core samples are often contaminated by a significant (~1-5%) fraction of PS I, as well as related proteins. This contamination is of little importance in many experiments, but masks the optical behaviour of the deep red state in PS II, which absorbs in the same spectral range (700-730nm) as PS I (Hughes et al. 2006). When contamination levels are less than ~1%, it becomes difficult to quantify the PS I related components by gel-based, chromatographic, circular dichroism or EPR techniques. We have developed a fluorescence-based technique, taking advantage of the distinctively different low-temperature emission characteristics of PS II and PS I when excited near 700nm. The approach has the advantage of providing the relative concentration of the two photosystems in a single spectral measurement. A sensitivity limit of 0.01% PS I (or better) can be achieved. The procedure is applied to PS II core preparations from spinach and Thermosynechococcus vulcanus. Measurements made of T. vulcanus PS II preparations prepared by re-dissolving crystallised material indicate a low but measurable PS I related content. The analysis provides strong evidence for a previously unreported fluorescence of PS II cores peaking near 780nm. The excitation dependence of this emission as well as its appearance in both low PS I cyanobacterial and plant based PS II core preparations suggests its association with the deep red state of PS II. PMID:24055633
Morton, Jennifer; Hall, Jeremy; Smith, Paul; Akita, Fusamichi; Koua, Faisal Hammad Mekky; Shen, Jian-Ren; Krausz, Elmars
Neuroserpin, an axonally secreted serine protease inhibitor.
We have identified and chromatographically purified an axonally secreted glycoprotein of CNS and PNS neurons. Several peptides derived from it were microsequenced. Based on these sequences, a fragment of the corresponding cDNA was amplified and used as a probe to isolate a full length cDNA from a chicken brain cDNA library. Because the deduced amino acid sequence qualified the protein as a novel member of the serpin family of serine protease inhibitors, we called it neuroserpin. Analysis of the primary structural features further characterized neuroserpin as a heparin-independent, functional inhibitor of a trypsin-like serine protease. In situ hybridization revealed a predominantly neuronal expression during the late stages of neurogenesis and in the adult brain in regions which exhibit synaptic plasticity. Thus, neuroserpin might function as an axonally secreted regulator of the local extracellular proteolysis involved in the reorganization of the synaptic connectivity during development and synapse plasticity in the adult. Images
Osterwalder, T; Contartese, J; Stoeckli, E T; Kuhn, T B; Sonderegger, P
PS-6 and PS-7, new beta-lactam antibiotics. Isolation, physicochemical properties and structures.
Antibiotics PS-6 and PS-7 which are shown to be 5R,6R-3-(2-acetamido)ethylthio-6-isopropyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene- 2-carboxylic acid and 5R,6R-3-(E)-(2-acetamido) vinylthio-6-ethyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid respectively, are new beta-lactam compounds isolated from fermentation broths of Streptomyces cremeus subsp. auratilis A271, S. fulvoviridis A933, S. olivaceus ATCC 31126 and S. flavogriseus NRRL 8139. Fermentation, isolation, physicochemical properties and structures of antibiotics PS-6 and PS-7 are described. PMID:7451363
Shibamoto, N; Koki, A; Nishino, M; Nakamura, K; Kiyoshima, K; Okamura, K; Okabe, M; Okamoto, R; Fukagawa, Y; Shimauchi, Y; Ishikura, T; Lein, J
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
Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior
Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes�coagulation, inflammation, and digestion, for example�have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation.
Almonte, Antoine G.; Sweatt, J. David
Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis.
Many serine proteases play important regulatory roles in complex biological systems, but only a few have been linked directly with capillary morphogenesis and angiogenesis. Here we provide evidence that serine protease activities, independent of the plasminogen activation cascade, are required for microvascular endothelial cell reorganization and capillary morphogenesis in vitro. A homology cloning approach targeting conserved motifs present in all serine proteases, was used to identify candidate serine proteases involved in these processes, and revealed 5 genes (acrosin, testisin, neurosin, PSP and neurotrypsin), none of which had been associated previously with expression in endothelial cells. A subsequent gene-specific RT-PCR screen for 22 serine proteases confirmed expression of these 5 genes and identified 7 additional serine protease genes expressed by human endothelial cells, urokinase-type plasminogen activator, protein C, TMPRSS2, hepsin, matriptase/MT-SP1, dipeptidylpeptidase IV, and seprase. Differences in serine protease gene expression between microvascular and human umbilical vein endothelial cells (HUVECs) were identified and several serine protease genes were found to be regulated by the nature of the substratum, ie. artificial basement membrane or fibrillar type I collagen. mRNA transcripts of several serine protease genes were associated with blood vessels in vivo by in situ hybridization of human tissue specimens. These data suggest a potential role for serine proteases, not previously associated with endothelium, in vascular function and angiogenesis. PMID:12624642
Aimes, Ronald T; Zijlstra, Andries; Hooper, John D; Ogbourne, Steven M; Sit, Mae-Le; Fuchs, Simone; Gotley, David C; Quigley, James P; Antalis, Toni M
Accelerators for the PS neutrino beam
NASA Astrophysics Data System (ADS)
A recent memorandum for an experimental proposal [1] was discussed during the CERN PS and SPS experimental committee (SPSC) of April 2011 and at the Research Board of June 2011. The proposed experiment, with objective to investigate the anomalous ?? ? ?e oscillations, aims at re-using the discontinued CERN PS Neutrino Facility (PSNF) and experimental zones to install a 150 ton liquid argon time projection chamber (LArTPC) as near detector and a 600 ton LArTPC as far detector. This article will summarize the experimental needs, the proposed facility layout, a primary beam production scheme and the requirements for the reconstruction of the PSNF.
Steerenberg, R.; Calviani, M.; Gschwendtner, E.; Pardons, A.; Vincke, H.
Levels of D-serine in the brain and peripheral organs of serine racemase (Srr) knock-out mice.
D-Serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA) receptor, plays an important role in mammalian brain neurotransmission, via the NMDA receptor. D-Serine is synthesized from L-serine by the pyridoxal-5' phosphate-dependent enzyme serine racemase (SRR), and D-serine is metabolized by D-amino acid oxidase (DAAO). In this study, we measured levels of the neurotransmission related amino acids, d-serine, L-serine, glycine, glutamine and glutamate in the frontal cortex, hippocampus, striatum and cerebellum as well as in peripheral tissues of blood, heart, pancreas, spleen, liver, kidney, testis, epididymis, heart, lung, muscle and eyeball, in wild-type (WT) and Srr-knockout (Srr-KO) mice. Levels of D-serine in the frontal cortex, hippocampus, and striatum of Srr-KO mice were significantly lower than in WT mice, while levels in the cerebellum stayed the same. In contrast, levels of L-serine, glycine, glutamine and glutamate remained the same in all tested brain regions. In vivo microdialysis using free-moving mice showed that extracellular levels of D-serine in the hippocampus of Srr-KO mice were significantly lower than in WT mice while the other amino acid levels remained the same between mice. In peripheral organs, levels of D-serine in the kidney, testis, and muscle of Srr-KO mice were significantly lower than in WT mice. Tissue levels of the other tested amino acids in peripheral organs were not altered. These results suggest that SRR is the major enzyme responsible for D-serine production in the mouse forebrain, and that other pathways of d-serine production may exist in the brain and peripheral organs. PMID:21906644
Horio, Mao; Kohno, Mami; Fujita, Yuko; Ishima, Tamaki; Inoue, Ran; Mori, Hisashi; Hashimoto, Kenji
Effect of phospholipids on the transformation of amorphous calcium phosphate to hydroxyapatite in vitro
The conversion of amorphous calcium phosphate (ACP) to crystalline hydroxyapatite (HA) was studiedin vitro in the presence or absence of phosphatidyl serine (PS) and other phospholipids. ACP transformation and HA crystal growth\\u000a were monitored by electron microscopy, selected-area electron diffraction and X-ray diffraction, and by measuring supernatant\\u000a calcium and phosphate. PS, and other acidic phospholipids, had a significant stabilizing effect
R. E. Wuthier; E. D. Eanes
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
d-Serine: The right or wrong isoform?
Only recently, d-amino acids have been identified in mammals. Of these, d-serine has been most extensively studied. d-Serine was found to play an important role as a neurotransmitter in the human central nervous system (CNS) by binding to the N-methyl-d-aspartate receptor (NMDAr), similar to glycine. Therefore, d-serine may well play a role in all physiological and pathological processes in which
Sabine A. Fuchs; Ruud Berger; Tom J. de Koning
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.....)
Optimization on AmPS Chicane Magnets.
National Technical Information Service (NTIS)
During the design of the chicane part of the AmPS (Amsterdam Pulse Stretcher) extraction line, a choice for the construction of these magnets had to be made from many possibilities. Since a horizontal as well as a vertical displacement is needed, the numb...
R. Hoekstra
Isolated operation of PS and its application in modeling manufacturing process system
Based on the concept of polychromatic sets (PS), the basic mathematic expression of PS is introduced. According to the entity of PS, PS is classified into two types: disjunctive PS and conjunctive PS. The isolated operation of PS is studied thoroughly and the corresponding problems are analyzed elaborately. Together with manufacturing process system, the information model of conjunctive PS is
Xinqin Gao; Yan Li; Mingshun Yang; Xiaoqing Wu; Li Ba
Neonatal Disruption of Serine Racemase Causes Schizophrenia-Like Behavioral Abnormalities in Adulthood: Clinical Rescue by D-Serine
Background D-Serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA) receptor, is synthesized from L-serine by serine racemase (SRR). Given the role of D-serine in both neurodevelopment and the pathophysiology of schizophrenia, we examined whether neonatal disruption of D-serine synthesis by SRR inhibition could induce behavioral abnormalities relevant to schizophrenia, in later life. Methodology/Principal Findings Neonatal mice (7�9 days) were injected with vehicle or phenazine methosulfate (Met-Phen: 3 mg/kg/day), an SRR inhibitor. Behavioral evaluations, such as spontaneous locomotion, novel object recognition test (NORT), and prepulse inhibition (PPI) were performed at juvenile (5�6 weeks old) and adult (10�12 weeks old) stages. In addition, we tested the effects of D-serine on PPI deficits in adult mice after neonatal Met-Phen exposure. Finally, we assessed whether D-serine could prevent the onset of schizophrenia-like behavior in these mice. Neonatal Met-Phen treatment reduced D-serine levels in the brain, 24 hours after the final dose. Additionally, this treatment caused behavioral abnormalities relevant to prodromal symptoms in juveniles and to schizophrenia in adults. A single dose of D-serine improved PPI deficits in adult mice. Interestingly, chronic administration of D-serine (900 mg/kg/day from P35 to P70) significantly prevented the onset of PPI deficits after neonatal Met-Phen exposure. Conclusions/Significance This study shows that disruption of D-serine synthesis during developmental stages leads to behavioral abnormalities relevant to prodromal symptoms and schizophrenia, in later life. Furthermore, early pharmacological intervention with D-serine may prevent the onset of psychosis in adult.
Hagiwara, Hiroko; Iyo, Masaomi; Hashimoto, Kenji
The effect of PS porosity on the structure, optical and electrical properties of ZnS/PS
NASA Astrophysics Data System (ADS)
ZnS films were deposited on porous silicon (PS) substrates with different porosities by pulsed laser deposition (PLD). The crystalline structure, surface morphology of ZnS films on PS substrates and optical, electrical properties of ZnS/PS composites were studied. The results show that, ZnS films deposited on PS substrates were grown in preferred orientation along ?-ZnS (111) direction corresponding to crystalline structure of cubic phase. With the increase of PS porosity, the XRD diffraction peak intensity of ZnS films decreases. Some voids and cracks appear in the films. Compared with as-prepared PS, the PL peak of PS for ZnS/PS has a blueshift. The larger the porosity of PS, the greater the blueshift is. A new green light emission located around 550 nm is observed with increasing PS porosity, which is ascribed to defect-center luminescence of ZnS. The blue, green emission of ZnS combined with the red emission of PS, a broad photoluminescence band (450-750 nm) is formed. ZnS/PS composites exhibited intense white light emission. The I-V characteristics of ZnS/PS heterojunctions showed rectifying behavior. Under forward bias conditions, the current density is large. Under reverse bias conditions, the current density nearly to be zero. The forward current increases with increasing PS porosity. This work lay a foundation for the realization of electroluminescence of ZnS/PS and solid white light emission devices.
Wang, Cai-Feng; Hu, Bo; Yi, Hou-Hui; Li, Wei-Bing
Contributions of the D-serine pathway to schizophrenia.
The glutamate neurotransmitter system is one of the major candidate pathways for the pathophysiology of schizophrenia, and increased understanding of the pharmacology, molecular biology and biochemistry of this system may lead to novel treatments. Glutamatergic hypofunction, particularly at the NMDA receptor, has been hypothesized to underlie many of the symptoms of schizophrenia, including psychosis, negative symptoms and cognitive impairment. This review will focus on D-serine, a co-agonist at the NMDA receptor that in combination with glutamate, is required for full activation of this ion channel receptor. Evidence implicating D-serine, NMDA receptors and related molecules, such as D-amino acid oxidase (DAO), G72 and serine racemase (SRR), in the etiology or pathophysiology of schizophrenia is discussed, including knowledge gained from mouse models with altered D-serine pathway genes and from preliminary clinical trials with D-serine itself or compounds modulating the D-serine pathway. Abnormalities in D-serine availability may underlie glutamatergic dysfunction in schizophrenia, and the development of new treatments acting through the D-serine pathway may significantly improve outcomes for many schizophrenia patients. PMID:21295046
Labrie, Viviane; Wong, Albert H C; Roder, John C
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)
Rickettsia prowazekii requires host cell serine and glycine for growth.
The growth requirement of Rickettsia prowazekii for the amino acids serine and glycine was assessed in both wild-type cell lines and a mutant cell line. X-irradiated L929 cells supported the growth of R. prowazekii when the cells were incubated in Eagle minimal essential medium supplemented with serum. In contrast, in this medium, X-irradiated Vero cells did not support the growth of rickettsiae unless cycloheximide, serine, or glycine was added. Other nonessential amino acids, additional glucose, and potential products of host cell metabolism of serine and glycine were nonstimulatory. The concentration of serine or glycine required to support rickettsial growth had no effect on the doubling time of uninfected, unirradiated Vero cells. A comparison of intracellular amino acid pools indicated that the serine and glycine concentrations in mock-infected Vero cells were approximately 31 and 14% of the respective concentrations in mock-infected L929 cells. The pools of both amino acids in Vero cells increased markedly upon treatment of the cells with cycloheximide. Interconversion of serine and glycine catalyzed by serine hydroxymethyltransferase was detected in cell-free extracts of purified rickettsiae. However, this enzymatic activity did not permit rickettsial growth in a glycine-requiring clone (772-56d) of the Chinese hamster ovary cell CHO-K1 in the absence of glycine supplementation. These data indicate that R. prowazekii depends on the host cell for serine or glycine.
Austin, F E; Turco, J; Winkler, H H
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
Serine phosphorylation regulates paxillin turnover during cell migration
BACKGROUND: Paxillin acts as an adaptor protein that localizes to focal adhesion. This protein is regulated during cell migration by phosphorylation on tyrosine, serine and threonine residues. Most of these phosphorylations have been implicated in the regulation of different steps of cell migration. The two major phosphorylation sites of paxillin in response to adhesion to an extracellular matrix are serines
Nancy Abou Zeid; Ana-Maria Vall�s; Brigitte Boyer
Hydrolysis of polyesters by serine proteases.
The substrate specificity of alpha-chymotrypsin and other serine proteases, trypsin, elastase, proteinase K and subtilisin, towards hydrolysis of various polyesters was examined using poly(L-lactide) (PLA), poly(beta-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(epsilon-caprolactone) (PCL). alpha-Chymotrypsin could degrade PLA and PEA with a lower activity on PBS/A. Proteinase K and subtilisin degraded almost all substrates other than PHB. Trypsin and elastase had similar substrate specificities to alpha-chymotrypsin. PMID:15928850
Lim, Hyun-A; Raku, Takao; Tokiwa, Yutaka
Complement factor D, a novel serine protease.
Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of factor D activity is instead attained by a novel mechanism that depends on reversible conformational changes for expression and control of catalytic activity. These conformational changes are believed to be induced by the single natural substrate, C3bB, and to result in realignment of the catalytic triad, the specificity pocket, and the nonspecific substrate binding site, all of which have atypical conformations. Mutational studies have defined structural determinants responsible for these unique structural features of factor D and for the resultant low reactivity with synthetic esters.
Volanakis, J. E.; Narayana, S. V.
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.
Regulation of renal sodium handling through the interaction between serine proteases and serine protease inhibitors
Sodium balance, extracellular fluid volume, and ultimately blood pressure are maintained by precise regulation of the activity\\u000a of epithelial sodium channels (ENaC). Multiple mechanisms such as hormones, intracellular factors, and other regulatory factors\\u000a contribute to regulation of ENaC activity. Prostasin, a glycosylphosphatidylinositol-anchored serine protease, has been identified\\u000a as an activator of ENaC that increases its open probability. Furin cleaves ?ENaC
Kenichiro Kitamura; Kimio Tomita
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.
Zhong, Bonan; Watts, Korashon L; Gori, Jennifer L; Wohlfahrt, Martin E; Enssle, Joerg; Adair, Jennifer E; Kiem, Hans-Peter
Phosphorylation of Cdc6 at serine 74, but not at serine 106, drives translocation of Cdc6 to the cytoplasm.
Phosphorylation-dependent cytoplasmic translocation of human Cdc6 during S phase is sufficient to control its activity after origin firing. Export from the nucleus also serves as a mechanism for preventing re-replication in mammalian cells. Phosphorylation of the CDK consensus serine residues 54, 74, and 106 has been suggested to be involved in the cytoplasmic translocation of Cdc6. To determine the relative importance of the three phosphorylation sites, we have generated Cdc6 variants by substituting one or more of the three serine residues with alanine or aspartic acid and have assessed their cytoplasmic translocation behavior. Phosphorylation of serine 74 mainly contributes to the cytoplasmic translocation of Cdc6, while serine 54 phosphorylation provides a minor contribution. In contrast, phosphorylation at serine 106 does not affect the nuclear export of Cdc6. Comparative results were found in cells coexpressing the phosphorylation defective mutants of Cdc6 and cyclin A as well as in non-transfected cells synchronized by their release from a double thymidine block. We conclude that Cdk-mediated phosphorylation of Cdc6 at serine 74 is required for the cytoplasmic translocalization of Cdc6 during the cell cycle. Phosphorylation of Cdc6 at serine 54 plays a minor role and phosphorylation of serine 106 plays no role in the cytoplasmic localization of Cdc6. The phosphorylation of S74 in Cdc6 could be important for binding to the nuclear export protein for translocalization. PMID:23129444
Yim, Hyungshin; Park, Ji-Woong; Woo, Sang Uk; Kim, Seong-Taek; Liu, Linhua; Lee, Chul-Hoon; Lee, Seung Ki
Towards a Balanced View on iPS Cells
ABSTRACT In the past one and half years scientific community,and general public have been excited with the discovery of induced pluripotent stem (iPS) cells. However, how much truth is contained in the various claims made for iPS cells? What are these iPS cells? Are they really safe for therapeutic use? This review attempts to present a balanced view on iPS
Shi V. Liu
d-Serine in Glia and Neurons Derives from 3-Phosphoglycerate Dehydrogenase
d-Serine is an endogenous ligand for NMDARs generated from l-serine by the enzyme serine racemase (Srr). Both neuronal and glial localizations have been reported for d-serine and Srr. 3-Phosphoglycerate dehydrogenase is an exclusively astrocytic enzyme that catalyzes the first committed step of l-serine biosynthesis. Using transgenic mice expressing enhanced green fluorescent protein under the Srr promoter and mice with targeted deletion of Srr or 3-Phosphoglycerate dehydrogenase, we demonstrate predominantly neuronal sources of d-serine dependent on astrocytic supply of l-serine. These findings clarify the cellular basis for the regulation of NMDAR neurotransmission by d-serine.
Ehmsen, Jeffrey T.; Ma, Ting Martin; Sason, Hagit; Rosenberg, Dina; Ogo, Tadashi; Furuya, Shigeki
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)
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�0.3U/g protein) than cerebrum (0.19�0.03U/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
Phosphorylation of Activation Transcription Factor-2 at Serine 121 by Protein Kinase C Controls c-Jun-mediated Activation of Transcription*S?
Activation transcription factor-2 (ATF-2) is phosphorylated by various protein kinases, such as JNK/p38/ERK, calmodulin kinase IV, protein kinase A, and protein kinase C (PKC), in response to a variety of stimuli. However, the role of the phosphorylation of ATF-2 by PKC in vivo in the transcriptional control of genes that include the activation protein-1 (AP-1)/cyclic AMP-response element remains to be defined. Using antibodies against the phosphorylated serine residue (Ser(P)) at position 121 of ATF-2, we have demonstrated that PKC phosphorylates ATF-2 at Ser-121 and that phosphorylation of Ser-121 (to yield ATF-2pS121) becomes detectable at the late stage of the response of HeLa cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) and is maintained for more than 2 h. By contrast, phosphorylation of ATF-2 at threonine residues 69 and 71 (Thr-69/71, to yield ATF-2pT69/71) and at Ser-340 and Ser-367 (to yield ATF-2pS340 and ATF-2pS367) is detectable as an immediate early response. Unlike levels of ATF-2pT69/71 and ATF-2pS340, the level of ATF-2pS121 increases in the nuclei of HeLa cells in response to TPA. A serine-to-alanine mutation at position 121 of ATF-2 represses the c-Jun-dependent transcription of AP-1/cyclic AMP-response element reporter genes and also the p300-mediated activation of a Gal4-reporter gene in response to TPA. Our results suggest that the phosphorylation of ATF-2 at Ser-121 plays a key role in the c-Jun-mediated activation of transcription that occurs in response to TPA.
Yamasaki, Takahito; Takahashi, Akinori; Pan, Jianzhi; Yamaguchi, Naoto; Yokoyama, Kazunari K.
Pseudo-peptides derived from isomannide: inhibitors of serine proteases.
In this paper, we describe the synthesis of a novel class of pseudo-peptides derived from isomannide and several oxazolones as potential inhibitors of serine proteases as well as preliminary pharmacological assays for hepatitis C. Hepatitis C, dengue and West Nile fever are among the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes and are a primary target in the drug development field. Several pseudo-peptides were obtained in good yields from the reaction of isomannide and oxazolones, and their anti-HCV potential using the HCV replicon-based assay was shown. PMID:19330426
Barros, Thalita G; Pinheiro, Sergio; Williamson, J S; Tanuri, Am�lcar; Gomes, M; Pereira, Helena S; Brindeiro, R M; Neto, Jos� B A; Antunes, O A C; Muri, Estela M F
CDK8 as the STAT1 serine 727 kinase?
Whereas cytokine-induced tyrosine phosphorylation of STAT (signal transducer and activator of transcription) proteins by JAK kinases has been well studied, much less is known about STAT-specific serine kinases and their signal-dependent regulation. The paper by Joanna Bancerek and colleagues published recently in Immunity reports that upon interferon-? (IFN?) stimulation of cells the chromatin-associated cyclin-dependent kinase 8 (CDK8) phosphorylates the regulatory serine residue 727 in the transactivation domain of STAT1. The authors state that the CDK8 module of the Mediator complex is a key component in the STAT1 signal pathway, linking serine phosphorylation to gene-specific transcriptional events.
Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas
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 alpha,beta-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 [(3)H]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 [(3)H]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. PMID:20091774
Sikka, Pilleriin; Walker, Rosie; Cockayne, Rebecca; Wood, Matthew J A; Harrison, Paul J; Burnet, Philip W J
Theory of allosteric effects in serine proteases.
The classical Botts-Morales theory for the action of a modifier on the catalytic properties of an enzyme has been extended to deal with allosteric effects in serine proteases. The exact analytical solution derived for the linkage scheme at steady state provides a rigorous framework for the study of many biologically relevant systems, including enzymes activated by monovalent cations and cofactor-controlled protease-zymogen interactions in blood coagulation. When the enzyme obeys Michaelis-Menten kinetics, the exact solution of the kinetic linkage scheme simplifies considerably. Of particular importance for practical applications is a simple equation expressing the dependence of the specificity constant of the enzyme, kcat/Km, on the concentration of the modifier, from which the equilibrium binding constant for the formation of the enzyme-modifier complex can be estimated. Analysis of the allosteric changes in thrombin activity induced by thrombomodulin and Na+ in terms of this equation yields accurate determinations of the equilibrium binding constants for both effectors.
Di Cera, E; Hopfner, K P; Dang, Q D
The novel inhibitors of serine proteases.
Thirty optically active nonprotein alpha-amino acids and peptides based thereon have been screened for their ability to interact with bovine trypsin and proteinase K from Tritirachium album Limber, which belong to the group of serine proteases. Both structure-based drug design approach and determination of enzyme activity have been used to identify low molecular weight inhibitors of trypsin and proteinase K. Compounds have been selected that according to the docking analysis were able to interact with trypsin and proteinase K. Following the docking analysis measurement of enzymes activity (2R,3S)-beta-hydroxyleucine and (2S,3R)-beta-hydroxyleucine inhibited both enzymes activity, whereas (S)-alpha-methyl-beta-phenylalanine, (R)-alpha-methyl-beta-phenylalanine, (S)-allylglycine, (R)-allylglycine, (S)-alpha-allylalanine, (R)-alpha-allylalanine and allo-O-ethylthreonine inhibited only proteinase K; and N-formyl-(S)-methionyl-(2S,3R)-hydroxyleucine, N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine, N-formyl-(S)-methionyl-(S)-allylglycine and N-formyl-(S)-methionyl-(R)-allylglycine inhibited trypsin. It has been shown that inhibition of trypsin by (2R,3S)-beta-hydroxyleucine and N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine is of a competitive mode. PMID:19326044
Hovhannisyan, N; Harutyunyan, Sh; Hovhannisyan, A; Hambardzumyan, A; Chitchyan, M; Melkumyan, M; Oganezova, G; Avetisyan, N
Hunting for Serine 276-Phosphorylated p65
The transcription factor nuclear factor kappaB (NF-?B) is one of the central mediators of inflammatory gene expression. Several posttranslational modifications of NF-?B, regulating its transactivation ability, have been described. Especially phosphorylation of the NF-?B subunit p65 has been investigated in depth and several commercial phosphospecific antibodies, targeting selected p65 residues, are available. One of the p65 residues, that is subject to phosphorylation by protein kinase A (PKA) as well as by mitogen-stimulated kinase-1 (MSK-1), is the serine at position 276. Here, we have performed a detailed analysis of the performance of the most commonly used commercial anti-P-p65 Ser276 antibodies. Our findings indicate that at least three widely used anti-P-p65 Ser276 antibodies do not detect p65 in vivo via Western Blot, but instead crossreact with PKA-regulated proteins. As PKA is one of the main kinases responsible for phosphorylation of p65 at Ser276, this observation warrants cautious interpretation of data generated using the tested antibodies.
Spooren, Anneleen; Kolmus, Krzysztof; Vermeulen, Linda; Van Wesemael, Karlien; Haegeman, Guy; Gerlo, Sarah
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
Targeted disruption of serine racemase affects glutamatergic neurotransmission and behavior.
A subset of glutamate receptors that are specifically sensitive to the glutamate analog N-methyl-D-aspartate (NMDA) are molecular coincidence detectors, necessary for activity-dependent processes of neurodevelopment and in sensory and cognitive functions. The activity of these receptors is modulated by the endogenous amino acid D-serine, but the extent to which D-serine is necessary for the normal development and function of the mammalian nervous system was previously unknown. Decreased signaling at NMDA receptors has been implicated in the pathophysiology of schizophrenia based on pharmacological evidence, and several human genes related to D-serine metabolism and glutamatergic neurotransmission have been implicated in the etiology of schizophrenia. Here we show that genetically modified mice lacking the ability to produce D-serine endogenously have profoundly altered glutamatergic neurotransmission, and relatively subtle but significant behavioral abnormalities that reflect hyperactivity and impaired spatial memory, and that are consistent with elevated anxiety. PMID:19065142
Basu, A C; Tsai, G E; Ma, C-L; Ehmsen, J T; Mustafa, A K; Han, L; Jiang, Z I; Benneyworth, M A; Froimowitz, M P; Lange, N; Snyder, S H; Bergeron, R; Coyle, J T
Chemotaxis of Escherichia coli to L-serine
NASA Astrophysics Data System (ADS)
A novel experimental technique was used to quantify the motion of E. coli to varying serine concentrations and gradients so as to capture the spatial and temporal variation of the chemotactic response. The average run speed and the cell diffusivity are found to be dependent on the serine concentration. The measured diffusivities were in the range of 1.2-2.5 � 10 -10 m2 s-1. The study revealed that the rotational diffusivity of the cells, induced by the extracellular environment, also varies with the serine concentration. The drift velocity increased with serine gradients reaching a maximum value of ~5.5 �m s-1 at 1.6 �M �m-1 after which it decreased. Experimental analysis demonstrated the interdependence of run speed, rotational diffusivity and drift velocity that characterizes the motion. Further, the motion was found to critically depend on the oxygen concentration and energy level of the cells.
Vuppula, Rajitha R.; Tirumkudulu, Mahesh S.; Venkatesh, K. V.
Ion/molecule reactions of the protonated serine octamer.
The protonated homochiral octamer of serine exchanges all 33 of its labile hydrogens with CH(3)OD and undergoes ligand switching reactions with amines in a quadrupole ion trap mass spectrometer. PMID:15340613
Gronert, Scott; O'Hair, Richard A J; Fagin, Adelaide E
Two Proteases, Trypsin Domain-containing 1 (Tysnd1) and Peroxisomal Lon Protease (PsLon), Cooperatively Regulate Fatty Acid ?-Oxidation in Peroxisomal Matrix*
The molecular mechanisms underlying protein turnover and enzyme regulation in the peroxisomal matrix remain largely unknown. Trypsin domain-containing 1 (Tysnd1) and peroxisomal Lon protease (PsLon) are newly identified peroxisomal matrix proteins that harbor both a serine protease-like domain and a peroxisome-targeting signal 1 (PTS1) sequence. Tysnd1 processes several PTS1-containing proteins and cleaves N-terminal presequences from PTS2-containing protein precursors. Here we report that knockdown of Tysnd1, but not PsLon, resulted in accumulation of endogenous ?-oxidation enzymes in their premature form. The protease activity of Tysnd1 was inactivated by intermolecular self-conversion of the 60-kDa form to 15- and 45-kDa chains, which were preferentially degraded by PsLon. Peroxisomal ?-oxidation of a very long fatty acid was significantly decreased by knockdown of Tysnd1 and partially lowered by PsLon knockdown. Taken together, these data suggest that Tysnd1 is a key regulator of the peroxisomal ?-oxidation pathway via proteolytic processing of ?-oxidation enzymes. The proteolytic activity of oligomeric Tysnd1 is in turn controlled by self-cleavage of Tysnd1 and degradation of Tysnd1 cleavage products by PsLon.
Okumoto, Kanji; Kametani, Yukari; Fujiki, Yukio
Regulation of serine racemase activity by amino acids
The effects of various amino acids on the activity of serine racemase, purified from mouse brain, were examined. Those acting as inhibitors included compounds with electron withdrawing groups on the ?-carbon of alanine (?-halo-alanines and l-serine-O-sulfate), which can act as enzyme-activated inhibitors, and compounds containing ?-SH groups (cysteine and homocysteine) which react with enzyme-bound pyridoxal phosphate to form thiazolidine derivatives.
David S. Dunlop; Amos Neidle
d-Serine-induced nephrotoxicity: possible interaction with tyrosine metabolism
d-Serine selectively damages renal proximal tubule cells in rats by a mechanism that is not fully understood. Recent proteomic analysis identified that d-serine elevated plasma fumarylacetoacetate hydrolase (FAH). FAH is involved in tyrosine catabolism; hence, this pathway may be involved in mediating the toxicity. This work examines whether 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC), a potent inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) located
R. E Williams; E. A Lock
D-serine-induced nephrotoxicity: possible interaction with tyrosine metabolism.
D-serine selectively damages renal proximal tubule cells in rats by a mechanism that is not fully understood. Recent proteomic analysis identified that D-serine elevated plasma fumarylacetoacetate hydrolase (FAH). FAH is involved in tyrosine catabolism; hence, this pathway may be involved in mediating the toxicity. This work examines whether 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC), a potent inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) located upstream of FAH, modulates D-serine-induced nephrotoxicity. Rats were pretreated with NTBC (0.5 mg/kg p.o.) or corn oil and then 30 min later given either D-serine (250 mg/kg i.p.) or water. Urine was collected every 12 h until termination (48 h) and analysed by 1H NMR spectroscopy and principal component analysis (PCA). Markers of proximal tubule injury were evident in urine following treatment with D-serine and NTBC + D-serine. PCA could not distinguish between these urine samples suggesting that NTBC does not effect the development of nephrotoxicity. Clinical chemistry analysis of urine and terminal plasma samples and histopathological examination of the kidneys confirmed this. NTBC alone caused a marked increase in the excretion of 4-hydroxyphenylpyruvate (HPPA) and 4-hydroxyphenyllactate (HPLA); however, HPPA and HPLA excretion was minimal following NTBC + D-serine. Instead marked tyrosinuria was observed suggesting that D-serine-induced renal damage markedly affects the handling of increased levels of HPPA and HPLA resulting from the inhibition of HPPD. PMID:15297036
Williams, R E; Lock, E A
Undernutrition Decreases Serine Palmitoyltransferase Activity in Developing Rat Hypothalamus
Background\\/Aims: Undernutrition reduces the hypothalamic ganglioside concentration. This may be attributed to some modifications in the contents of precursors of sphingolipid biosynthesis in undernourished rats. The present study evaluated the serine palmitoyl transferase activity (SPT; EC 220.127.116.11) during the development of the rat hypothalamus. This work also shows the L-[3-14C]serine metabolic labeling of hypothalamic sphingolipids in normal and undernourished rats
Liane N. Rotta; Cristiani G. da Silva; Marcos L. S. Perry; Vera M. T. Trindade
The Structure of Serine Palmitoyltransferase; Gateway to Sphingolipid Biosynthesis
Sphingolipid biosynthesis commences with the condensation of L-serine and palmitoyl-CoA to produce 3-ketodihydrosphingosine (KDS). This reaction is catalysed by the PLP-dependent enzyme serine palmitoyltransferase (SPT; EC 126.96.36.199), which is a membrane-bound heterodimer (SPT1\\/SPT2) in eukaryotes such as humans and yeast and a cytoplasmic homodimer in the Gram-negative bacterium Sphingomonas paucimobilis. Unusually, the outer membrane of S. paucimobilis contains glycosphingolipid (GSL)
Beverley A. Yard; Lester G. Carter; Kenneth A. Johnson; Ian M. Overton; Mark Dorward; Huanting Liu; Stephen A. McMahon; Muse Oke; Daphn� Puech; Geoffrey J. Barton; James H. Naismith; Dominic J. Campopiano
Characterization of Serine Palmitoyltransferase in Normal Human Tissues
Sphingolipids serve as structural elements of cells and as lipid second messengers. They regulate cellular homeostasis, mitogenesis, and apoptosis. Sphingolipid signaling may also be important in various pathophysiologies such as vascular injury, inflammation, and cancer. Serine palmitoyltransferase (SPT) catalyzes the condensation of serine with palmitoyl-CoA, the first, rate-limiting step in de novo sphingolipid biosynthesis. This integral microsomal membrane protein consists
Ameesha D. Batheja; David J. Uhlinger; Jill M. Carton; George Ho; Michael R. DAndrea
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
Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism
The first step in the biosynthesis of sphingolipids is the condensation of serine and palmitoyl CoA, a reaction catalyzed by serine palmitoyltransferase (SPT) to produce 3-ketodihydrosphingosine (KDS). This review focuses on recent advances in the biochemistry and molecular biology of SPT. SPT belongs to a family of pyridoxal 5?-phosphate (PLP)-dependent ?-oxoamine synthases (POAS). Mammalian SPT is a heterodimer of 53-kDa
Kentaro Hanada
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.
Vaiyapuri, Sakthivel; Thiyagarajan, Nethaji; Hutchinson, E Gail; Gibbins, Jonathan M
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...
Pathogenic disruption of DISC1-serine racemase binding elicits schizophrenia-like behavior via D-serine depletion
Perturbation of Disrupted-In-Schizophrenia-1 (DISC1) and D-serine/NMDA receptor hypofunction have both been implicated in the pathophysiology of schizophrenia and other psychiatric disorders. In the present study, we demonstrate that these two pathways intersect with behavioral consequences. DISC1 binds to and stabilizes serine racemase (SR), the enzyme that generates D-serine, an endogenous co-agonist of the NMDA receptor. Mutant DISC1 fails to bind to SR, facilitating ubiquitination and degradation of SR and a decrease in D-serine production. To elucidate DISC1-SR interactions in vivo, we generated a mouse model of selective and inducible expression of mutant DISC1 in astrocytes, the main source of D-serine in the brain. Expression of mutant DISC1 down-regulates endogenous DISC1 and decreases protein but not mRNA levels of SR, resulting in diminished production of D-serine. In contrast, mutant DISC1 does not alter levels of ALDH1L1, connexins, GLT-1 or binding partners of DISC1 and SR, LIS1 or PICK1. Adult male and female mice with life-long expression of mutant DISC1 exhibit behavioral abnormalities consistent with hypofunction of NMDA neurotransmission. Specifically, mutant mice display greater responses to an NMDA antagonist, MK-801, in open field and pre-pulse inhibition of the acoustic startle tests and are significantly more sensitive to the ameliorative effects of D-serine. These findings support a model wherein mutant DISC1 leads to SR degradation via dominant-negative effects, resulting in D-serine deficiency that diminishes NMDA neurotransmission thus linking DISC1 and NMDA pathophysiologic mechanisms in mental illness.
Ma, T.M.; Abazyan, S.; Abazyan, B.; Nomura, J.; Yang, C.; Seshadri, S.; Sawa, A.; Snyder, S.H.; Pletnikov, M.V.
The best three-channel projectile-inelastic close-coupling approximation (CCA) is used to study the resonances in positronium (Ps) and hydrogen (H) scattering at the energy region below the inelastic threshold. The s-wave elastic phase shifts and s-wave elastic cross sections are studied using the static-exchange, two- and three-channel projectile-inelastic CCA for both the singlet (+) and triplet (-) channels. The singlet resonances detected using different CCA schemes confirm previous predictions [Drachman and Houston, Phys. Rev. A 12, 885 (1975); Page, J. Phys. B. 9, 1111 (1976)]. We report a resonance in the triplet channel too using the present three-channel CCA scheme.
Ray, Hasi [Department of Physics, Indian Institute of Technology Roorkee, Roorkee 247667, Uttaranchal (India)
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
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
Photoinactivation of PS2 secondary donors by PS2 cation radicals and superoxide radicals
Illumination of Mn- and Cl-depleted PS2 causes rapid irreversible inactivation of specific redox-active components on the donor side of the PS2 Reaction Center (RC). Under aerobic conditions, weak light preillumination of NH{sub 2}OH-PS2 causes rapid loss of Y{sub Z}{sup {plus_minus}} formation, Y{sub Z} {yields} P{sub 680}{sup +}, the A{sub T}-band thermoluminescence emission, the Y{sub Z}{sup +}-dependent (Site 1) photooxidation of exogenous e{sup {minus}} donors, and the capability to photoligate Mn{sup 2+} into the water oxidizing enzyme (photoactivation), all without significantly affecting P{sub 680}{sup +}/Q{sub A}{sup {minus}} charge separation. In contrast, aerobic high light preillumination of Mn-depleted PS2 promotes very rapid and parallel loss of photoactivation and A{sub T}-band emission capabilities significantly than loss of either Y{sub Z}{sup +}-formation or P{sub 680}{sup +}/Q{sub A}{sup {minus}} charge separation capabilities. These photodamages and those to Cl-depleted thylakoids (4,5) generally are believed to be caused by reactions between the highly oxidizing cation radicals (P{sub 680}{sup +}/Chl{sup +}) and nearby amino acid residues of D{sub 1}>D{sub 2}. The reported promotion of the photodamages by e{sup {minus}} acceptors of Q{sub A}{sup {minus}}/Q{sub B}{sup {minus}} their inhibition by e{sup {minus}} donors to Y{sub Z}{sup +} and their occurrence under strict anaerobic conditions all tend to support the idea of direct damage by P{sub 680}{sup +}/Chl{sup +}. Our studies lead us to conclude that the photodamages to the donor side components are caused minimally by a rapid mechanism requiring both superoxide and PS2 cation radicals; and by a slower mechanism driven by the PS2 cation radicals only.
Chen, G.X.; Cheniae, G.M. [Kentucky Univ., Lexington, KY (United States); Blubaugh, D.J. [Utah State Univ., Logan, UT (United States); Golbeck, J.H. [Nebraska Univ., Lincoln, NE (United States)
Gated MCP framing camera with 60-ps exposure time
NASA Astrophysics Data System (ADS)
High voltage pulse of 140 ps in width and 2.7 kV was generated to gate the multiframe images on a meander shape microstripline on MCP. The measurement time range was extended to 1.1 ns while the exposure time of each image is 60 ps. The measured spatial resolution of the framing camera is 25 lp/mm. New method to reduce the exposure time down to 10 ps was simulated numerically.
Chang, Zenghu; Shan, Bing; Liu, Xiouqin; Liu, Jinyuan; Zhu, Wenhua; Yang, Hongru; Ren, Y. A.; Gong, Maixia
Rise time of BC422 plastic scintillator < 20 ps
The rise time of the plastic scintillator BC-422 has been determined to be less than 20 ps. To make the measurement, scintillator excitation was produced by X-ray pulses generated by focusing 20-ps, 2.5-TW laser pulses onto gold targets. Scintillator output was recorded with an optical streak camera whose response is 15 ps. This fast rise time identifies BC-422 as a
R. A. Lerche; D. W. Phillion
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)
InSAR data acquired from independent overlapping tracks can be exploited for a reliability assessment of the Persistent Scatterer InSAR (PS-InSAR) tech- nique. Here we present a mathematical framework for the datum connection of multiple tracks, simul- taneously evaluating the precision and reliability of PS-InSAR estimates. This datum connection can be subdivided in two steps. Firstly, the PS loca- tions are
Gini Ketelaar; Freek van Leijen; Petar Marinkovic; Ramon Hanssen
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
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.
Crow, John P.; Marecki, John C.; Thompson, Misty
Effect of acute ethanol on serine biosynthesis in liver.
The effect of an acute intraperitoneal dose of ethanol (1 g/kg), glucose (7.2 g/kg), or the combination of the two on the metabolite pattern of the biosynthetic pathway of L-serine has been determined in rabbit liver in vivo as has the effect of 10 mM ethanol on the glucose-, fructose-, or pyruvate-stimulated accumulation of L-serine in rabbit hepatocytes in vitro. In vivo, the 50% increase in L-serine and 80% increase in L-phosphoserine content of liver following glucose injection was completely prevented by ethanol. In fact, the L-phosphoserine content fell to only 6% of the control value. In spite of these and other significant changes in the metabolite pattern of the pathway of L-serine biosynthesis (D-3-phosphoglycerate dehydrogenase, L-phosphoserine aminotransferase (PSAT), and L-phosphoserine phosphatase), the mass action ratio of the combined reactions of the first two steps remained close to their equilibrium position. As a consequence it is estimated that the tissue content of phosphohydroxypyruvate fell to less than 2% of the control value, to approximately 0.3% of its Km for the PSAT reaction. The conclusion that acute ethanol blocks L-serine biosynthesis (presumably by redox effects) was supported by the prevention or inhibition of L-serine accumulation in hepatocytes metabolizing glucose, fructose, or pyruvate. Because L-serine is an important source of one-carbon fragments, the inhibition of its biosynthesis may be another mechanism by which ethanol interferes with folate and one-carbon metabolism. PMID:3113336
LaBaume, L B; Merrill, D K; Clary, G L; Guynn, R W
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 genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively). Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.
Tripathi, Lokesh P; Sowdhamini, R
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
Ohno, Yohei; Yuasa, Shinsuke; Egashira, Toru; Seki, Tomohisa; Hashimoto, Hisayuki; Tohyama, Shugo; Saito, Yuki; Kunitomi, Akira; Shimoji, Kenichiro; Onizuka, Takeshi; Kageyama, Toshimi; Yae, Kojiro; Tanaka, Tomofumi; Kaneda, Ruri; Hattori, Fumiyuki; Murata, Mitsushige; Kimura, Kensuke; Fukuda, Keiichi
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.
Yuasa, Shinsuke; Egashira, Toru; Seki, Tomohisa; Hashimoto, Hisayuki; Shimoji, Kenichiro; Kageyama, Toshimi; Tanaka, Tomofumi; Hattori, Fumiyuki; Murata, Mitsushige; Kimura, Kensuke; Fukuda, Keiichi
Paradoxical roles of serine racemase and D-serine in the G93A mSOD1 mouse model of amyotrophic lateral sclerosis.
D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity, which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A Cu,Zn-superoxide dismutase (SOD1) mice--the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in the onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the alanine-serine-cysteine transporter 1 (Asc-1). Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the pre-symptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator. PMID:22117694
Thompson, Misty; Marecki, John C; Marinesco, Stephane; Labrie, Viviane; Roder, John C; Barger, Steven W; Crow, John P
Serine racemase is predominantly localized in neurons in mouse brain.
D-Serine is the endogenous ligand for the glycine binding site of the N-methyl-D-aspartate (NMDA)-type glutamate receptor (GluR) channel and is involved in the regulation of synaptic plasticity, neural network formation, and neurodegenerative disorders. D-Serine is synthesized from L-serine by serine racemase (SR), which was first reported to be localized in astrocytes. However, recently, SR mRNA and its protein have been detected in neurons. In this study, we examined the SR distribution in the brain during postnatal development and in cultured cells by using novel SR knockout mice as negative controls. We found that SR is predominantly localized in pyramidal neurons in the cerebral cortex and hippocampal CA1 region. Double immunofluorescence staining revealed that SR signals colocalized with those of the neuron-specific nuclear protein, but not with the astrocytic markers glial fibrillary acid protein and 3-phosphoglycerate dehydrogenase. In the striatum, we observed SR expression in gamma-aminobutyric acid (GABA)ergic medium-spiny neurons. Furthermore, in the adult cerebellum, we detected weak but significant SR signals in GABAergic Purkinje cells. From these findings, we conclude that SR is expressed predominantly in many types of neuron in the brain and plays a key role in the regulation of brain functions under physiological and pathological conditions via the production of the neuromodulator D-serine. PMID:18698599
Miya, Kazushi; Inoue, Ran; Takata, Yoshimi; Abe, Manabu; Natsume, Rie; Sakimura, Kenji; Hongou, Kazuhisa; Miyawaki, Toshio; Mori, Hisashi
Reelin is a serine protease of the extracellular matrix.
Reelin is an extracellular matrix protein that plays a pivotal role in development of the central nervous system. Reelin is also expressed in the adult brain, notably in the cerebral cortex, where it might play a role in synaptic plasticity. The mechanism of action of reelin at the molecular level has been the subject of several hypotheses. Here we show that reelin is a serine protease and that proteolytic activity is relevant to its function, since (i) Reelin expression in HEK 293T cells impairs their ability to adhere to fibronectin-coated surfaces, and adhesion to fibronectin is restored by micromolar concentrations of diisopropyl phosphorofluoridate, a serine hydrolase inhibitor; (ii) purified Reelin binds FP-Peg-biotin, a trap probe which irreversibly binds to serine residues located in active catalytic sites of serine hydrolases; (iii) purified Reelin rapidly degrades fibronectin and laminin, while collagen IV is degraded at a much slower rate; fibronectin degradation is inhibited by inhibitors of serine proteases, and by monoclonal antibody CR-50, an antibody known to block the function of Reelin both in vitro and in vivo. The proteolytic activity of Reelin on adhesion molecules of the extracellular matrix and/or receptors on neurons may explain how Reelin regulates neuronal migration and synaptic plasticity. PMID:11689558
Quattrocchi, Carlo C; Wannenes, Francesca; Persico, Antonio M; Ciafr�, Silvia Anna; D'Arcangelo, Gabriella; Farace, Maria G; Keller, Flavio
Cadherins Mediate Both the Association between PS1 and ?-Catenin and the Effects of PS1 on ?-Catenin Stability*
Presenilin1 (PS1), a protein involved in cellular development, forms functional complexes with ?-catenin, a regulator of Wnt signaling and cell-cell adhesion. In addition, both proteins have been shown to play important roles in disease including cancer and Alzheimer disease. Although PS1 and ?-catenin are found in the same complexes, it is not clear whether they bind directly to each other or a third complex component, like cadherin, may mediate their interactions. Here we show that PS1 and ?-catenin form no detectable complexes in cells that express no cadherin. In contrast, these complexes are readily found in E-cadherin containing cells. Furthermore, binding of both PS1 and ?-catenin to E-cadherin is necessary for the formation of PS1/?-catenin complexes. Importantly, our data show that binding of PS1 to cadherin mediates the effects of PS1 on the phosphorylation, ubiquitination, and destabilization of ?-catenin. Thus, cadherins mediate both the association of PS1 and ?-catenin and the effects of PS1 on the cellular levels of ?-catenin.
Serban, Geo; Kouchi, Zen; Baki, Lia; Georgakopoulos, Anastasios; Litterst, Claudia M.; Shioi, Junichi; Robakis, Nikolaos K.
Status and Early Science from the PS1 Science Mission
NASA Astrophysics Data System (ADS)
PS1, the Pan-STARRS Telescope No. 1 began the PS1 Science Mission May 13, 2009. Operations of the PS1 System, including the Observatory, Telescope, 1.4 Gigapixel Camera, Image Processing Pipeline , PSPS relational database and science specific software clients are presently funded for 2.5 years by PS1 Science Consortium. The PS1 Surveys include: (1) A 3pi Steradian Survey, (2) A Medium Deep survey of 11PS1 footprints spaced around the sky; (3) A solar system survey optimized for Near Earth Objects, (4) a Stellar Transit Survey; and (5) a Deep Survey of M31. As of October, 2010, the PS1 3pi Survey has covered half the sky above a declination of -30 in five bands with 2 or more images. By the time of the AAS meeting, PS1 will have covered 0.75 of the available sky, or about 20,000 square degrees. The coverage, cadence, and data quality of the surveys and the current performance of the PS1 System will be presented. Early science results will be outlined. Transient data release to the community will be described, as well as the eventual release of all PS1 data. The PS1 Science Consortium consists of The Institute for Astronomy at the University of Hawai'i in Manoa, the Max Planck Institute for Astronomy, Heidelberg and the Max Planck Institute for Extraterrestrial Physics, Garching, The Johns Hopkins University, the University of Durham, the University of Edinburgh, the Queen's University Belfast, the Harvard-Smithsonian Center for Astrophysics, the Los Cumbres Observatory Global Telescope Network Incorporated, the National Central University of Taiwan, and NASA. The Pan-STARRS construction project is led by the University of Hawaii Institute for Astronomy with funding support from the United States Air Force Research Laboratory and contributions from the Maui High Performance Computing Center and MIT Lincoln Laboratory."
Chambers, Kenneth C.
Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1 Mediates Phagocytosis of Aged\\/Apoptotic Cells in Endothelial Cells
Recognition of the exposure of phosphatidyl-serine (PS) on the outer surface of plasma membrane has been implicated in the phagocytosis of aged\\/apoptotic cells. Because oxidized low-density lipoprotein (OxLDL) has been reported to block the phagocytosis, here we examined whether lectin-like OxLDL receptor 1 (LOX-1), the OxLDL receptor in endothelial cells, mediates phagocytosis of aged\\/apoptotic cells by endothelial cells. Cultured bovine
Kozo Oka; Tatsuya Sawamura; Ken-Ichiro Kikuta; Shigekazu Itokawa; Noriaki Kume; Toru Kita; Tomoh Masaki
PS3 CELL Development for Scientific Computation and Research
The Cell processor is one of the most powerful processors on the market, and researchers in the earth sciences may find its parallel architecture to be very useful. A cell processor, with 7 cores, can easily be obtained for experimentation by purchasing a PlayStation 3 (PS3) and installing linux and the IBM SDK. Each core of the PS3 is capable
M. Christiansen; E. Sevre; S. M. Wang; D. A. Yuen; S. Liu; M. D. Lyness; M. Broten
Structural and photoluminescence properties of ZnO/PS heterojunction
NASA Astrophysics Data System (ADS)
Porous silicon templates were formed by electrochemical anodization on p-type (100) silicon wafers and transparent conducting Zinc oxide (ZnO) thin films were deposited on glass substrates and Porous silicon (PS) substrates by sol-gel dip coating technique, using Zinc acetate dehydrate, Monoethanoamine (MEA), ethanol and distilled water. In this process, the films were formed over the surface and also incorporated into the pores of PS and thereby making a shielding layer as well as a contacting workstation. Thus, the ZnO/PS/Si junctions were fabricated. The growth of ZnO on glass and PS has been thoroughly investigated by SEM and X-ray diffraction techniques. The results indicated that ZnO/PS nanocomposite films were polycrystalline in nature with a hexagonal wurtzite structure and the (002) oriented ZnO films had the best crystal quality. The grain size of the films on glass and PS were found to be around 60 nm. Photoluminescence (PL) of the ZnO/PS nanocomposite films were systematically investigated by fluorescence spectrophotometry. The influence on the PS interface was correlated with the luminescent behaviour of the resulting heterojunction structure, which is used for various potential applicarions.
Kulathuraan, Kavu; Natarajan, Balan
Regulation of serine racemase activity by amino acids.
The effects of various amino acids on the activity of serine racemase, purified from mouse brain, were examined. Those acting as inhibitors included compounds with electron withdrawing groups on the beta-carbon of alanine (beta-halo-alanines and L-serine-O-sulfate), which can act as enzyme-activated inhibitors, and compounds containing beta-SH groups (cysteine and homocysteine) which react with enzyme-bound pyridoxal phosphate to form thiazolidine derivatives. Glycine and a series of metabolites related to L-aspartic acid (L-aspartic acid, L-asparagine, and oxaloacetic acid) were also found to be competitive inhibitors of the racemase. The Ki values for glycine and aspartic acid inhibition were 0.15 and 1.9 mM, respectively, indicating that alterations in the concentrations of these amino acids might play a role in the regulation of D-serine synthesis. PMID:15710237
Dunlop, David S; Neidle, Amos
A proposed mechanism for IS607-family serine transposases
Background The transposases encoded by the IS607 family of mobile elements are unusual serine recombinases with an inverted domain order and minimal specificity for target DNA. Results Structural genomics groups have determined three crystal structures of the catalytic domains of IS607 family transposases. The dimers formed by these catalytic domains are very different from those seen for other serine recombinases and include interactions that usually only occur upon formation of a synaptic tetramer. Conclusions Based on these structures, we propose a model for how IS607-family transposases could form a synaptic tetramer. The model suggests that, unlike other serine recombinases, these enzymes carry out sequence-specific DNA binding and catalysis in trans: the DNA binding and catalytic domains of each subunit are proposed to interact with different DNA duplexes. The model also suggests an explanation for the minimal target DNA specificity.
Dosage Compensation of Serine-4 Transfer RNA in DROSOPHILA MELANOGASTER
A dosage series of the X chromosome site for serine-4 transfer RNA consisting of one of three copies in females and one to two in males was constructed to test whether transfer RNA expression is governed by dosage compensation. A dosage effect on the level of the serine-4 isoacceptor was observed in both females and males when the structural locus was varied. However, in males, each dose had a relatively greater expression so the normal one dose was slightly greater than the total female value and the duplicated male had the highest relative expression of all the types examined. Serine-4 levels in males and females from an isogenic Oregon-R stock were similar. Thus the transfer RNA levels conform to the expectations of dosage compensation.
Birchler, James A.; Owenby, R. Keith; Jacobson, K. Bruce
CDK8 as the STAT1 serine 727 kinase?
Whereas cytokine-induced tyrosine phosphorylation of STAT (signal transducer and activator of transcription) proteins by JAK kinases has been well studied, much less is known about STAT-specific serine kinases and their signal-dependent regulation. The paper by Joanna Bancerek and colleagues published recently in Immunity reports that upon interferon-? (IFN?) stimulation of cells the chromatin-associated cyclin-dependent kinase 8 (CDK8) phosphorylates the regulatory serine residue 727 in the transactivation domain of STAT1. The authors state that the CDK8 module of the Mediator complex is a key component in the STAT1 signal pathway, linking serine phosphorylation to gene-specific transcriptional events. PMID:24069555
Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas
Mosaic serine proteases in the mammalian central nervous system.
We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS. PMID:17981686
Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi
Annihilation of electron-positron pairs in the positronium ion Ps{sup -} and bipositronium Ps{sub 2}
Rates of the two-, three-, four-, and five-photon annihilations of the electron-positron pairs are determined numerically for the three-body positronium ion Ps{sup -}(e{sup -}e{sup +}e{sup -}) and four-body bipositronium ''molecule''Ps{sub 2}(e{sup -}e{sup +}e{sup -}e{sup +}). The values obtained in our computations are {gamma}{sub 2{gamma}}(Ps{sup -}){approx_equal}2.080 485 305 25x10{sup 9} s{sup -1}, {gamma}{sub 3{gamma}}(Ps{sup -}){approx_equal}5.636 415 155 0x10{sup 6} s{sup -1}, {gamma}{sub 4{gamma}}(Ps{sup -}){approx_equal}3.075x10{sup 3} s{sup -1}, {gamma}{sub 5{gamma}}(Ps{sup -}){approx_equal}5.383 s{sup -1}, and {gamma}{sub 2{gamma}}(Ps{sub 2}){approx_equal}4.438 595 2x10{sup 9} s{sup -1}, {gamma}{sub 3{gamma}}(Ps{sub 2}){approx_equal}1.202 497x10{sup 7} s{sup -1}, {gamma}{sub 4{gamma}}(Ps{sub 2}){approx_equal}6.562x10{sup 3} s{sup -1}, {gamma}{sub 5{gamma}}(Ps{sub 2}){approx_equal}11.484 s{sup -1}. The four- and five-photon annihilation rates are significantly smaller than the corresponding two- and three-photon annihilation rates known for these systems. We also determine the rates of one- and zero-photon annihilation for the Ps{sup -} ion and Ps{sub 2} system. The corresponding numerical values are {gamma}{sub 1{gamma}}(Ps{sup -}){approx_equal}3.824 91x10{sup -2} s{sup -1}, {gamma}{sub 1{gamma}}(Ps{sub 2}){approx_equal}1.941 88x10{sup -1} s{sup -1}, and {gamma}{sub 0{gamma}}(Ps{sub 2}){approx_equal}2.321 97x10{sup -9} s{sup -1}.
Frolov, Alexei M. [Department of Chemistry, University of Western Ontario, London, Ontario, N6A 5B7 (Canada)
Mutation modifying the serine pathway in methylotrophic bacteria.
Methylotrophic bacteria, Gram-positive, with the serine pathway, were shown to have their growth inhibited by 0.5% glycine. The effects of this amino acid on individual enzyme activities were studied in wild and mutant strains of Micrococcus varians and Bacillus licheniformis. The enzymes studied were glycerate dehydrogenase (EC 22.214.171.124), isocitrate lyase (EC 126.96.36.199), serine hydroxymethyltransferase (EC 188.8.131.52) and glycine--oxaloacetate aminotransferase (EC 188.8.131.52). The last-named enzyme was found to be inhibited, the kinetic constants having been determined for two strain types. PMID:6792008
Ratomahenina, R; Galzy, P
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.
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
pkaPS: prediction of protein kinase A phosphorylation sites with the simplified kinase-substrate binding model
Background Protein kinase A (cAMP-dependent kinase, PKA) is a serine/threonine kinase, for which ca. 150 substrate proteins are known. Based on a refinement of the recognition motif using the available experimental data, we wished to apply the simplified substrate protein binding model for accurate prediction of PKA phosphorylation sites, an approach that was previously successful for the prediction of lipid posttranslational modifications and of the PTS1 peroxisomal translocation signal. Results Approximately 20 sequence positions flanking the phosphorylated residue on both sides have been found to be restricted in their sequence variability (region -18...+23 with the site at position 0). The conserved physical pattern can be rationalized in terms of a qualitative binding model with the catalytic cleft of the protein kinase A. Positions -6...+4 surrounding the phosphorylation site are influenced by direct interaction with the kinase in a varying degree. This sequence stretch is embedded in an intrinsically disordered region composed preferentially of hydrophilic residues with flexible backbone and small side chain. This knowledge has been incorporated into a simplified analytical model of productive binding of substrate proteins with PKA. Conclusion The scoring function of the pkaPS predictor can confidently discriminate PKA phosphorylation sites from serines/threonines with non-permissive sequence environments (sensitivity of ~96% at a specificity of ~94%). The tool "pkaPS" has been applied on the whole human proteome. Among new predicted PKA targets, there are entirely uncharacterized protein groups as well as apparently well-known families such as those of the ribosomal proteins L21e, L22 and L6. Availability The supplementary data as well as the prediction tool as WWW server are available at . Reviewers Erik van Nimwegen (Biozentrum, University of Basel, Switzerland), Sandor Pongor (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy), Igor Zhulin (University of Tennessee, Oak Ridge National Laboratory, USA).
Neuberger, Georg; Schneider, Georg; Eisenhaber, Frank
Reversal of Solubility Characteristics of `Luxol' Dye-phospholipid Complexes
`LUXOL' fast blue G (Du Pont), the diarylguanidine salt of a sulphonated azo dye, forms complexes stoichiometrically with phospholipids1. These complexes have been found to be insoluble in a varying pattern in the lower alcohols. For this study phospholipid-dye complexes were prepared with the same dye and phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, or sphingomyelin. The complexes were
T. N. Salthouse
Two-dimensional thin-layer chromatography of rat liver phosphatides
SUMMARY A system of two-dimensional thin-layer chromatography was developed that separated rat liver phosphatides into several phosphate-positive spots in about 2 hr developing time. Characteristic hydrolysis products derived from phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl choline, sphingomyelin, and lysophos- phatidyl choline were identified. The hydrolytic products of \\
Feedback inactivation of D-serine synthesis by NMDA receptor-elicited translocation of serine racemase to the membrane
D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a major role in several NMDAR-dependent events. In this study we investigate mechanisms regulating D-serine production by the enzyme serine racemase (SR). We now report that NMDAR activation promotes translocation of SR to the plasma membrane, which dramatically reduces the enzyme activity. Membrane-bound SR isolated from rat brain is not extracted from the membrane by high detergent and salt concentration, indicating a strong association. Colocalization studies indicate that most membrane-bound SR is located at the plasma membrane and dendrites, with much less SR observed in other types of membrane. NMDAR activation promotes translocation of the cytosolic SR to the membrane, resulting in reduced D-serine synthesis, and this effect is averted by blockade of NMDARs. In primary neuronal cultures, SR translocation to the membrane is blocked by a palmitoylation inhibitor, indicating that membrane binding is mediated by fatty acid acylation of SR. In agreement, we found that SR is acylated in transfected neuroblastoma cells using [3H]palmitate or [3H]octanoic acid as precursors. In contrast to classical S-palmitoylation of cysteines, acylation of SR occurs through the formation of an oxyester bond with serine or threonine residues. In addition, we show that phosphorylation of Thr-227 is also required for steady-state binding of SR to the membrane under basal, nonstimulated condition. We propose that the inhibition of D-serine synthesis caused by translocation of SR to the membrane provides a fail-safe mechanism to prevent NMDAR overactivation in vicinal cells or synapses.
Balan, Livia; Foltyn, Veronika N.; Zehl, Martin; Dumin, Elena; Dikopoltsev, Elena; Knoh, Diana; Ohno, Yusuke; Kihara, Akio; Jensen, Ole N.; Radzishevsky, Inna S.; Wolosker, Herman
An Intergenic Stem-Loop Mutation in the Bacillus subtilis ccpA-motPS Operon Increases motPS Transcription and the MotPS Contribution to Motility�
A stem-loop mutation between ccpA and motP in the Bacillus subtilis ccpA-motPS operon increased motPS transcription and membrane-associated MotPS levels, motility, and number of flagella/cell when MotPS is the sole stator and the MotPS contribution to motility at high pH, Na+, and viscosity when MotAB is also present.
Terahara, Naoya; Fujisawa, Makoto; Powers, Benjamin; Henkin, Tina M.; Krulwich, Terry A.; Ito, Masahiro
An intergenic stem-loop mutation in the Bacillus subtilis ccpA-motPS operon increases motPS transcription and the MotPS contribution to motility.
A stem-loop mutation between ccpA and motP in the Bacillus subtilis ccpA-motPS operon increased motPS transcription and membrane-associated MotPS levels, motility, and number of flagella/cell when MotPS is the sole stator and the MotPS contribution to motility at high pH, Na+, and viscosity when MotAB is also present. PMID:16547058
Terahara, Naoya; Fujisawa, Makoto; Powers, Benjamin; Henkin, Tina M; Krulwich, Terry A; Ito, Masahiro
iPS cells: a source of cardiac regeneration.
For the treatment of heart failure, a new strategy to improve cardiac function and inhibit cardiac remodeling needs to be established. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent cells that can differentiate into cell types from all three germ layers both in vitro and in vivo. The therapeutic effect of ES/iPS cell-derived progeny was reported in animal model. Mouse and human somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the transduction of four transcription factors, Oct 3/4, Sox2, Klf4, and c-Myc. However, the low induction efficiency hinders the clinical application of iPS technology, and efforts have been made to improve the reprogramming efficiency. There are variations in the characteristics in ES/iPS cell lines, and the further understanding is necessary for the applications of ES/iPS cell technology. Some improvements were also made in the methods to induce cardiomyocytes from ES/iPS cells efficiently. This review article is focused on generation of iPS cells, cardiomyocyte differentiation from ES/iPS cells, and transplantation of derived cardiomyocytes.This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited". PMID:21040726
Yoshida, Yoshinori; Yamanaka, Shinya
Progress on the Construction of The PS2 Telescope
NASA Astrophysics Data System (ADS)
The PS2 telescope is the second in a series of 4 telescopes that are being fabricated for the Pan-STARRS project. Its fabrication is currently in progress and this talk will discuss the current state of this fabrication. The optics for this telescope consist of the primary and secondary mirrors along with 3 large corrector lenses. These have already been purchased from Rayleigh Optical Corporation and are mostly complete. We will show the interferometric measurements of the completed elements. The site and enclosure for PS2 have been chosen to be the old LURE north dome which sits adjacent to the current PS1 telescope on Haleakala, Maui. We will show design renderings for the renovations of this enclosure for the PS2 telescope. The design of the PS2 telescope has small, but significant differences that have been initiated by our experience with PS1. We will discuss these changes. Finally, we will discuss the fabrication schedule for PS2. The Pan-STARRS construction project is led by the University of Hawaii Institute for Astronomy with funding support from the United States Air Force AFRL and in partnership with the Maui High Performance Computing Center and MIT Lincoln Laboratory.
Morgan, Jeffrey S.
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
A serine/threonine protein kinase from Mycobacterium tuberculosis.
Genomic DNA sequencing in the vicinity of the pstA-1 gene from Mycobacterium tuberculosis allowed us to clone, sequence and identify a gene encoding a 70-kDa protein. The size of the protein was confirmed by in vitro coupled transcription/translation. Its N-terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase). The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring. The mbk gene was overexpressed in Escherichia coli and the gene product (Mbk) was purified as a fusion protein with gluthatione S-transferase. Recombinant Mbk was found to be autophosphorylated on threonine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese-dependent manner. The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases. A similar gene was found in Mycobacterium bovis BCG DNA by Southern blot analysis. Its expression was detected in cultures of M. bovis BCG by reverse transcriptase/PCR. Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria. PMID:9119030
Peirs, P; De Wit, L; Braibant, M; Huygen, K; Content, J
L-Serine-O-phosphate in the central nervous system.
L-serine-O-phosphate (L-SOP) is the immediate precursor to L-serine in the serine synthesis pathway and is also an agonist at the Group III metabotropic glutamate receptors (mGluRs). L-SOP is produced by the enzyme phosphoserine aminotransferase (PSAT) and metabolized to L-serine by phosphoserine phosphatase (PSP). Using a novel analytical procedure, we show that L-SOP is present in rat whole brain, and that in transfected cells, it is substantially more potent than L-glutamate at the mGluR4 receptor subtype. Immunocytochemical analyses showed that the distributions of PSAT and PSP in the cerebral cortex, hippocampus, and cerebellum were similar in the rat and macaque monkey brain. In the rat hippocampus, cells within the subgranular zone were co-labeled with anti-PSP and anti-PSA-NCAM, a marker for neurogenic cells. In the cerebellar cortex, Purkinje neurons expressed relatively high levels of both enzymes while robust expression of PSAT was also observed in the Bergmann glia. L-SOP released from Purkinje neurons or Bergmann glia could activate mGluR4 present on parallel fiber terminals. The presence of l-SOP in brain, its high potency at mGluR4, together with the restricted distributions of the synthetic and metabolic enzymes, suggest that L-SOP might act activate Group III metabotropic glutamate receptors in the CNS. PMID:19747453
Antflick, Jordan E; Vetiska, Sandra; Baizer, Joan S; Yao, Yi; Baker, Glen B; Hampson, David R
Serine palmitoyltransferase inhibitor suppresses HCV replication in a mouse model
Serine palmitoyltransferase (SPT) is a first-step enzyme in the sphingolipid biosynthetic pathway. Myriocin is an inhibitor of SPT and suppresses replication of the hepatitis C virus (HCV) replicon. However, it is still unknown whether this SPT inhibitor suppresses HCV replication in vivo. We investigated the anti-HCV effect of myriocin against intact HCV using chimeric mice with humanized liver infected with
Takuya Umehara; Masayuki Sudoh; Fumihiko Yasui; Chiho Matsuda; Yukiko Hayashi; Kazuaki Chayama; Michinori Kohara
Molecular genetics of serine and threonine catabolism in Saccharomyces cerevisiae.
The catabolic L-serine (L-threonine) deaminase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. A mutant, cha1 (catabolism of hydroxyamino acids), lacking this enzyme activity has been isolated. We have cloned the CHA1 gene by complementation of a cha1 mutation. Northern analysis showed that CHA1 mRNA has a size of about 1200 ribonucleotides. CHA1 is probably the structural gene for the enzyme; it is an abundant RNA in cells grown with serine and threonine as nitrogen source, whereas it is not detected when cells are grown on ammonium or proline, i.e., the transcription of the CHA1 gene is induced by serine or threonine. Under induced growth conditions haploid ilv1 CHA1 strains do not require isoleucine, i.e., the catabolic deaminase is able to substitute for the biosynthetic threnonine deaminase encoded by the ILV1 gene. We have identified a nuclear, recessive mutation, sil1, that suppresses ilv1 mutations by increased transcription of the CHA1 gene under growth conditions leading to partial induction. The sil1 mutation could exert its effect by increasing the effective pools of the hydroxyamino acids. Alternatively SIL1 may encode a negatively acting regulatory protein for CHA1. PMID:2841185
Petersen, J G; Kielland-Brandt, M C; Nilsson-Tillgren, T; Bornaes, C; Holmberg, S
Dynamics simulation of the interaction between serine and water.
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 -NH3 (+) 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)]. 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. PMID:23742519
Liu, Yang; Zhang, Peng; Lu, Ying-Bo; Han, Sheng-Hao; Yu, Hui
Identification of Serine Proteinases Involved in Breast Cancer Progression.
National Technical Information Service (NTIS)
The objective of the grant was to identify serine proteinases (a) upregulated in malignant breast epithelial T4-2 cells, and (b) for which specific inhibition by RNA interference (RNAi) results in suppression of the malignant phenotype, as assessed in thr...
E. Radisky
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.
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
Serine protease from midgut of Bombus terrestris males.
A serine protease was isolated from midguts of the bumblebee male Bombus terrestris by a combination of precipitation procedures with column chromatography. The purified enzyme exhibited two bands with molecular masses of 25 and 26 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These bands showed a proteolytic activity in zymography assay. Midgut enzymes showed optimum proteolytic activity at pH 9 and 35�C using N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanine 4-nitroanilide as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) were 0.55�0.042 mM and 0.714�0.056 ?mol p-nitroalanine produced min(-1) mg protein(-1) , respectively. Inhibition was affected by trypsin inhibitor, but not by phenylmethylsulfonyl fluoride and N-tosyl-L-phenylalanine chloromethyl ketone, which indicated the trypsin-like but not chymotrypsin-like specificity. The identity of the serine protease was confirmed by nanoliquid-tandem mass spectrometry. Eleven unique peptides of the B. terrestris serine protease were found. It shows high homology to a previously reported B. ignitus serine protease covering more than 65% of the protein amino acid sequence. PMID:23303700
Brabcov�, Jana; Kindl, Ji?�; Valterov�, Irena; Pichov�, Iva; Zarev�cka, Marie; Brabcov�, Jana; J�gr, Michal; Mik��k, Ivan
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
Calcium-induced inactivation of alamethicin in asymmetric lipid bilayers
This paper discusses a calcium-dependent inactivation of alamethicin- induced conductance in asymmetric lipid bilayers. The bilayers used were formed with one leaflet of phosphatidyl ethanolamine (PE) and one of phosphatidyl serine (PS). Calcium, initially confined to the neutral lipid (PE) side, can pass through the open alamethicin channel to the negative lipid (PS) side, where it can bind to the negative lipid and reduce the surface potential. Under appropriate circumstances, the voltage-dependent alamethicin conductance is thereby inactivated. We have formulated a model for this process based on the diffusion of calcium in the aqueous phases and we show that the model describes the kinetic properties of the alamethicin conductance under various circumstances. EGTA on the PS side of the membrane reduces the effects of calcium dramatically as predicted by the model.
Association study of serine racemase gene with methamphetamine psychosis.
Experimental studies have demonstrated that not only dopaminergic signaling but also glutamatergic/NMDA receptor signaling play indispensable roles in the development of methamphetamine psychosis. Our recent genetic studies provided evidence that genetic variants of glutamate-related genes such as DTNBP1, GLYT1, and G72, which are involved in glutamate release and regulation of co-agonists for NMDA receptors, conferred susceptibility to methamphetamine psychosis. Serine racemase converts l-serine to d-serine, which is an endogenous co-agonist for NMDA receptors. Three single nucleotide polymorphisms (SNPs) in the promoter region of the serine racemase gene (SRR), rs224770, rs3760229, and rs408067, were proven to affect the transcription activity of SRR. Therefore, we examined these SNPs in 225 patients with methamphetamine psychosis and 291 age- and sex-matched controls. There was no significant association between methamphetamine psychosis and any SNP examined or between the disorder and haplotypes comprising the three SNPs. However, rs408067 was significantly associated with the prognosis for methamphetamine psychosis and multi-substance abuse status. The patients with C-positive genotypes (CC or CG) of rs408067 showed better prognosis of psychosis after therapy and less abuse of multiple substances than the patients with GG genotypes. Because the C allele of rs408067 reduces the expression of SRR, a lower d-serine level or reduced NMDA receptor activation may affect the prognosis of methamphetamine psychosis and multiple substance abuse. Our sample size is, however, not large enough to eliminate the possibility of a type I error, our findings must be confirmed by replicate studies with larger samples. PMID:21886585
Yokobayashi, E; Ujike, H; Kotaka, T; Okahisa, Y; Takaki, M; Kodama, M; Inada, T; Uchimura, N; Yamada, M; Iwata, N; Iyo, M; Sora, I; Ozaki, N; Kuroda, S
Serine phosphorylation regulates paxillin turnover during cell migration
Background Paxillin acts as an adaptor protein that localizes to focal adhesion. This protein is regulated during cell migration by phosphorylation on tyrosine, serine and threonine residues. Most of these phosphorylations have been implicated in the regulation of different steps of cell migration. The two major phosphorylation sites of paxillin in response to adhesion to an extracellular matrix are serines 188 and 190. However, the function of this phosphorylation event remains unknown. The purpose of this work was to determine the role of paxillin phosphorylation on residues S188 and S190 in the regulation of cell migration. Results We used NBT-II epithelial cells that can be induced to migrate when plated on collagen. To examine the role of paxillin serines 188/190 in cell migration, we constructed an EGFP-tagged paxillin mutant in which S188/S190 were mutated into unphosphorylatable alanine residues. We provide evidence that paxillin is regulated by proteasomal degradation following polyubiquitylation of the protein. During active cell migration on collagen, paxillin is protected from proteasome-dependent degradation. We demonstrate that phosphorylation of serines 188/190 is necessary for the protective effect of collagen. In an effort to understand the physiological relevance of paxillin protection from degradation, we show that cells expressing the paxillin S188/190A interfering mutant spread less, have reduced protrusive activity but migrate more actively. Conclusion Our data demonstrate for the first time that serine-regulated degradation of paxillin plays a key role in the modulation of membrane dynamics and consequently, in the control of cell motility.
Abou Zeid, Nancy; Valles, Ana-Maria; Boyer, Brigitte
Serine racemase: Activation by glutamate neurotransmission via glutamate receptor interacting protein and mediation of neuronal migration
Serine racemase (SR), localized to astrocytic glia that ensheathe synapses, converts L-serine to D-serine, an endogenous ligand of the NMDA receptor. We report the activation of SR by glutamate neurotransmission involving -amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors via glutamate receptor interacting protein (GRIP) and the physiologic regulation of cerebellar granule cell migration by SR. GRIP physiologically binds SR, augmenting SR activity and D-serine
Paul M. Kim; Hiroyuki Aizawa; Peter S. Kim; Alex S. Huang; Sasrutha R. Wickramasinghe; Amir H. Kashani; Roxanne K. Barrow; Richard L. Huganir; Anirvan Ghosh; Solomon H. Snyder
Functional and molecular analysis of D-serine transport in retinal Muller cells
D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal Muller cells. RT-PCR and immunoblot- ting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 Muller cell line and primary
Y. Dun; B. Mysona; S. Itagaki; A. Martin-Studdard; V. Ganapathy; S. B. Smith
Functional and molecular analysis of d-serine transport in retinal M�ller cells
d-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for d-serine in retinal M�ller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for d-serine, is expressed in the rMC-1 M�ller cell line and primary cultures
Y. Dun; B. Mysona; S. Itagaki; A. Martin-Studdard; V. Ganapathy; S. B. Smith
Stat1 Serine Phosphorylation Occurs Independently of Tyrosine Phosphorylation and Requires an Activated Jak2 Kinase
Gamma interferon (IFN-g) induces both tyrosine and serine phosphorylation of Stat1. Stat1 serine phos- phorylation is required for maximal transcriptional activity of Stat1. In this report, we present evidence that Stat1 tyrosine phosphorylation is not a prerequisite for Stat1 serine phosphorylation, although an active Jak2 kinase is required for both phosphorylation events. Stat1 serine phosphorylation occurs with a more delayed
Serine Racemase: A Glial Enzyme Synthesizing D-Serine to Regulate Glutamate-N-Methyl-D-Aspartate Neurotransmission
Although D amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of D-serine have been found in mammalian brain where it activates glutamate\\/N-methyl-D-aspartate receptors by interacting with the \\
Herman Wolosker; Seth Blackshaw; Solomon H. Snyder
Effect of Hypoxic-Ischemic Injury on Serine Palmitoyltransferase Activity in the Developing Rat Brain
Background: Sphingolipid metabolism is strongly associated with central nervous system myelination. Ceramide is the most active of the sphingolipid metabolites. On the basis of ceramide biosynthesis indicated by serine palmitoyltransferase activity and cerebroside generated by ceramide, the evaluation of serine palmitoyltransferase activity in developing brains or hypoxic-ischemic damaged brains is worthwhile. Methods: Using a scintillation counter, we assessed serine palmitoyltransferase
Masaki Daigo; Yasuhiro Arai; Kyoichi Oshida; Yohei Kitamura; Masaharu Hayashi; Toshiaki Shimizu; Yuichiro Yamashiro
Serine esterases are required for pollen tube penetration of the stigma in Brassica
. We have investigated the diversity of serine esterases in pollen and stigma tissues of Brassica napus and the role of these enzymes in pollen germination and pollen tube penetration of the stigma. The serine esterase-specific inhibitor diisopropyl fluorophosphate was used as a probe in a tritiated form, [3H]-DIPF, to determine the number and diversity of serine esterases in crude
Simon J. Hiscock; Dominic Bown; Sarah J. Gurr; Hugh G. Dickinson
The title compound, potassium diniobium vanadium phospho�rus deca�sulfide, KNb1.75V0.25PS10, was obtained by reaction of the elements with a eutectic mixture of KCl/LiCl. It is isostructural with the quaternary KNb2PS10, but the Nb sites are occupied by statistically disordered Nb (87.5%) and V (12.5%) atoms. The structure is composed of anionic ? 1[M 2PS10]? chains (M = Nb/V) separated from each other by K+ ions. The chain is composed of [MS8] distorted bicapped trigonal prisms and [PS4] tetra�hedra. There are no inter�chain bonding inter�actions. The crystal used for the X-ray analysis was a racemic twin.
Yu, Jaemin; Yun, Hoseop
Doubly excited 3P(e) resonant states in Ps(-)
NASA Technical Reports Server (NTRS)
Doubly excited 3P(e) resonant states in Ps(-) are calculated using a method of complex-coordinate rotation. Resonance parameters (both resonance positions and widths) for doubly excited states associated with the n = 2, 3, 4, 5, and 6 thresholds of positronium atoms are evaluated using elaborate Hylleraas-type functions. In addition to ten Feshbach-type resonances lying below various Ps thresholds, three shape resonances were identified, one each lying above the n = 2, 4, and 6 Ps thresholds. It is further noted that the energy levels for the present 3P(e) states are nearly degenerate with respect to the previously calculated 1P(0) states. Such a symmetric character suggests that the highly and doubly excited Ps(-), similar to its counterpart in H(-), would exhibit rovibrational behaviors analogous to those of XYX triatomic molecules.
Ho, Y. K.; Bhatia, A. K.
Elementary Presentation of the PS ''Beam Control'' System.
National Technical Information Service (NTIS)
The PS synchrotron control system is explained in general terms, covering the topics of frequency control, beam transfer, damping, stability, conservation of longitudinal emittance, and second order problems. (ERA citation 12:008475)
D. Boussard
Development and operating characteristics of the PS132 thermal battery
NASA Astrophysics Data System (ADS)
The development and operating characterisitics of the PS132 thermal battery are described. The PS132 uses the electrochemical system Ca/LiCl-KCl eutectic-SiO2CACRO4, and it is one of the smallest batteries of its type ever built that can meet its electrical and envronmental requirements. A development program that included construction of approximately 400 PS132-like batteries showed that obtaining acceptable DEB electrolyte-cathode powders was a major problem. Most commercial DEB powders caused excessive amounts of CaLi2 molten metal to form in the operating thermal cells of the PS132. This molten metal then flowed from the cells and caused electrical short circuits.
Krieger, F. C.
Spectroscopic and thermal studies of PS\\/PVAc blends
Polystyrene and polyvinyl acetate (PS\\/PVAc) films were blended with different contents using casting method. The effect of PS content on PVAc blends was investigated by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), Ultra violet and visible studies (UV\\/VIS), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Significant changes in FT-IR, XRD and DSC analysis are observed which reveals an interactions
I. S. Elashmawi; N. A. Hakeem; E. M. Abdelrazek
ABSTRACT ThisstudydescribesamethodforInSAR\\/PS-InSAR processing using parallelization algorithms. The InSAR\\/PS-InSAR processing chain is analyzed from theaspectofthecomputationalload,whichresultsin a speciflc parallelization model. The theoretical con- cept for the porting of Delft Object-oriented Radar Interferometric Software (Doris) into a parallel com- putational environment is described. The features of the presented parallelization model include e?- cient interprocessor communications utilized by MPI (Multi Passage Interface) and a
Petar S. Marinkovic; Ramon F. Hanssen; Bert M. Kampes
PS InSAR (Permanent Scatters for Interferometric Synthetic Aperture Radar) was proposed at first by A.Ferretti in 2001(Ferretti, 2001). In this technique, stable natural reflectors or permanent scatters are detected and studied over long temporal series of interferometric SAR images, in order to detect and measure topographic changes. PS technique has already shown remarkable potentials and has proved to be a
Bai Jun; Veronique PRINET
Joint PP and PS AVO inversion based on Zoeppritz equations
NASA Astrophysics Data System (ADS)
Considering Zoeppritz equations, reflections of PP and PS are only the function of ratios of density and velocity. So the inversion results will be the same if the ratios are the same but values of density, velocities of P-wave and S-wave are different without strict constraint. This paper makes efforts to explore nonlinear simultaneous PP and PS inversion with expectation to reduce the ambiguity of AVO analysis by utilizing the redundancy of multi-component AVO measurements. Accurate estimation of ratio parameters depends on independence of input data. There are only two independent AVO attributes for PP reflectivity (i.e. intercept and gradient) and two for PS reflectivity (i.e. pseudo-intercept and pseudo-gradient or extreme amplitude), respectively. For individual PP and PS inversion, the values of least-squares objective function do not converge around a large neighborhood of chosen true model parameters. Fortunately for joint PP and PS inversion the values of the least-squares objective function show closed contours with single minima. Finally the power function fitting is used to provide a higher precision AVO attributes than traditional polynomial fitting. By using the four independent fitting attributes (two independent attributes for PP and PS respectively), the inversion of four ratio parameters (velocities and densities) would be estimated with less errors than that in traditional method.
Wei, Xiucheng; Chen, Tiansheng
Spectroscopic and thermal studies of PS/PVAc blends
NASA Astrophysics Data System (ADS)
Polystyrene and polyvinyl acetate (PS/PVAc) films were blended with different contents using casting method. The effect of PS content on PVAc blends was investigated by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), Ultra violet and visible studies (UV/VIS), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Significant changes in FT-IR, XRD and DSC analysis are observed which reveals an interactions between the two polymers and PS/PVAc blends had good or certain miscibility. XRD scans show some changes in the intensity and the height of the amorphous halos with increased PS. UV/VIS analysis revealed that the optical band gap decreases with increasing content of PS from 5 to 4.11 eV. A single glass transition temperature for each blend was observed, this DSC results supported that the miscibility existed in the blend. The apparent activation energy (E) of the blends was evaluated using TGA analysis. The value of E was increased with the increase of PS content.
Elashmawi, I. S.; Hakeem, N. A.; Abdelrazek, E. M.
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
Inner blood-retinal barrier mediates l-isomer-predominant transport of serine.
D-serine, a coagonist for N-methyl-D-aspartate-type glutamate receptors, which mediate visual signal transmission, is thought to be generated from L-serine via serine racemase in the retina. However, the source of L-serine and D-serine in the retina are yet to be determined. The purpose of the present study was to investigate the characteristics of the blood-to-retina transport of serine at the inner blood-retinal barrier (BRB). In vivo study revealed the blood-to-retina transport of [(3) H]L-serine with an influx clearance of 49.9 ?L/(min�g retina), which is greater than that of [(3) H]D-serine. This was consistent with the L-isomer-predominant uptake of serine by conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), an in vitro inner BRB model. [(3) H]L-Serine and [(3) H]D-serine uptake by TR-iBRB2 cells took place in an Na(+)-dependent and a concentration-dependent manner with Michaelis constant values of 97.5 ?M and 9.63 mM, respectively. The uptake process of [(3) H]L-serine and [(3) H]D-serine was significantly inhibited by system ASC (alanine-serine-cysteine) substrates. Polymerase chain reaction analysis and immunocytochemistry revealed the expression of ASC transporters ASCT1 and ASCT2 in TR-iBRB2 cells. These results suggest that the system ASC at the inner BRB is a potent pathway for supplying serine in the form of the L-isomer from the circulating blood to the retina. PMID:21590774
Tachikawa, Masanori; Okamoto, Masashi; Hirose, Shirou; Yoneyama, Daisuke; Akanuma, Shin-ichi; Terasaki, Tetsuya; Hosoya, Ken-ichi
Ischemic Acute Kidney Injury Perturbs Homeostasis of Serine Enantiomers in the Body Fluid in Mice: Early Detection of Renal Dysfunction Using the Ratio of Serine Enantiomers
The imbalance of blood and urine amino acids in renal failure has been studied mostly without chiral separation. Although a few reports have shown the presence of D-serine, an enantiomer of L-serine, in the serum of patients with severe renal failure, it has remained uncertain how serine enantiomers are deranged in the development of renal failure. In the present study, we have monitored serine enantiomers using a two-dimensional HPLC system in the serum and urine of mice after renal ischemia-reperfusion injury (IRI), known as a mouse model of acute kidney injury. In the serum, the level of D-serine gradually increased after renal IRI in parallel with that of creatinine, whereas the L-serine level decreased sharply in the early phase after IRI. The increase of D-serine was suppressed in part by genetic inactivation of a D-serine-degrading enzyme, D-amino acid oxidase (DAO), but not by disruption of its synthetic enzyme, serine racemase, in mice. Renal DAO activity was detected exclusively in proximal tubules, and IRI reduced the number of DAO-positive tubules. On the other hand, in the urine, D-serine was excreted at a rate nearly triple that of L-serine in mice with sham operations, indicating that little D-serine was reabsorbed while most L-serine was reabsorbed in physiological conditions. IRI significantly reduced the ratio of urinary D?/L-serine from 2.82�0.18 to 1.10�0.26 in the early phase and kept the ratio lower than 0.5 thereafter. The urinary D?/L-serine ratio can detect renal ischemia earlier than kidney injury molecule-1 (KIM-1) or neutrophil gelatinase-associated lipocalin (NGAL) in the urine, and more sensitively than creatinine, cystatin C, or the ratio of D?/L-serine in the serum. Our findings provide a novel understanding of the imbalance of amino acids in renal failure and offer a potential new biomarker for an early detection of acute kidney injury.
Sasabe, Jumpei; Suzuki, Masataka; Miyoshi, Yurika; Tojo, Yosuke; Okamura, Chieko; Ito, Sonomi; Konno, Ryuichi; Mita, Masashi; Hamase, Kenji; Aiso, Sadakazu
Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.
The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration. PMID:10744710
Beuvink, I; Hess, D; Flotow, H; Hofsteenge, J; Groner, B; Hynes, N E
[Serine-threonine protein phosphatases from Bacillus subtilis].
Gram-positive soil bacterium Bacillus subtilis possesses six eukaryotic-like serine-threonine protein phosphatases. These enzymes play an important role in the cell. The response to environmental or nutrional stress conditions are controlled by three Rsb phosphatases: RsbX, RsbU and RsbP. Phosphatases are also involved in endospore formation process (SpoIIE) and sugar transport (kinase/phosphatase Hpr). Moreover in the cell there are phosphatases with still unknown function (PrpC and PrpE). Cellular processes, presented here are regulated by serine/threonine protein phosphatases and very important for bacterial survival in natural environment. Protein phosphatases must act in cooperation with protein kinases and deserve the same attention as kinases. PMID:16209347
Obuchowski, Micha?
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.
Bugge, Thomas; Wu, Qingyu
Glutamatergic regulation of serine racemase via reversal of PIP2 inhibition.
D-serine is a physiologic coagonist with glutamate at NMDA-subtype glutamate receptors. As D-serine is localized in glia, synaptically released glutamate presumably stimulates the glia to form and release D-serine, enabling glutamate/D-serine cotransmission. We show that serine racemase (SR), which generates D-serine from L-serine, is physiologically inhibited by phosphatidylinositol (4,5)-bisphosphate (PIP2) presence in membranes where SR is localized. Activation of metabotropic glutamate receptors (mGluR5) on glia leads to phospholipase C-mediated degradation of PIP2, relieving SR inhibition. Thus mutants of SR that cannot bind PIP2 lose their membrane localizations and display a 4-fold enhancement of catalytic activity. Moreover, mGluR5 activation of SR activity is abolished by inhibiting phospholipase C. PMID:19193859
Mustafa, Asif K; van Rossum, Damian B; Patterson, Randen L; Maag, David; Ehmsen, Jeffrey T; Gazi, Sadia K; Chakraborty, Anutosh; Barrow, Roxanne K; Amzel, L Mario; Snyder, Solomon H
Serine\\/threonine phosphorylation in cytokine signal transduction
Over the past decade, the involvement of tyrosine kinases in signal transduction pathways evoked by cytokines has been intensively investigated. Only relatively recently have the roles of serine\\/threonine kinases in cytokine-induced signal transduction and anti-apoptotic pathways been examined. Cytokine receptors without intrinsic kinase activity such as interleukin-3 (IL-3), granulocyte�macrophage colony-stimulating factor (GM-CSF) and the interferons were thought to transmit their
JA McCubrey; W Stratford May; V Duronio; A Mufson
Targeting Protein Serine/Threonine Phosphatases for Drug Development
With the recent clinical success of drugs targeting protein kinase activity, drug discovery efforts are focusing on the role of reversible protein phosphorylation in disease states. The activity of protein phosphatases, enzymes that oppose protein kinases, can also be manipulated to alter cellular signaling for therapeutic benefits. In this review, we present protein serine/threonine phosphatases as viable therapeutic targets, discussing past successes, current challenges, and future strategies for modulating phosphatase activity.
McConnell, Jamie L.; Wadzinski, Brian E.
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
Zhiqiang Li; Tae-Sik Park; Yan Li; Xiaoyue Pan; Jahangir Iqbal; David Lu; Weiqing Tang; Liqing Yu; Ira J. Goldberg; M. Mahmood Hussain; Xian-Cheng Jiang
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
The role of scaffolding in standard mechanism serine proteinase inhibitors.
In single domain, "standard mechanism" protein inhibitors of serine proteinases, about a dozen residues make contact with the cognate enzyme. The remainder of the molecule, the scaffolding, holds the reactive site region of the inhibitor in a canonical conformation, improves the binding by about six orders of magnitude and protects it from proteolysis. However, the stability and global structure of the scaffolding is irrelevant to inhibition, provided that inhibition is measured much below the melting temperature, Tm. PMID:16029159
Kelly, Clyde A; Laskowski, Michael; Qasim, M A
Effects of Serine-to-Cysteine Mutations on ?-Lactamase Folding
B. licheniformis exo-small ?-lactamase (ESBL) has two nonsequential domains and a complex architecture. We replaced ESBL serine residues 126 and 265 with cysteine to probe the conformation of buried regions in each domain. Spectroscopic, hydrodynamic, and chemical methods revealed that the mutations do not alter the native fold but distinctly change stability (S-126C>wild-type>S-126\\/265C>S-265C ESBL) and the features of partially folded
Javier Santos; Valeria A. Risso; Mauricio P. Sica; Mario R. Erm�cora
A new serine protease from the leaves of Thespesia populnea.
A serine protease was purified 6.9-fold from the leaves of Thespesia populnea using ammonium sulfate fractionation followed by CM-cellulose and Sephadex G-100 chromatography. The purified enzyme was named populnein and was characterized. It was made up of a single polypeptide, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis showed that the enzyme had a molecular mass of 14,518 Da. Inhibition of enzyme activity by phenyl methane sulfonyl fluoride indicates that populnein belongs to the class of serine proteases. The enzyme had appreciable pH and temperature stability. The activity of the enzyme was optimal at pH 8.0 and temperature 40�C. The enzyme was thermostable and retained 85% of its activity at 70�C after 1 hr. The enzyme was also resistant to autodigestion. The stabilization of the membrane of red blood cells exhibited by the protease populnein was found to be higher than for diclofenac. More studies are necessary to investigate the biological activity and applications of serine protease of T. populnea. PMID:23215657
Ishwarya, S; Sangeetha, R
graal: a Drosophila gene coding for several mosaic serine proteases.
Serine proteases play vital roles in several biological processes such as development and immunity. We have characterized Graal, a large multi-domain serine protease from Drosophila. Graal is spliced in at least three transcripts that are present throughout development. The domains found in Graal proteins are: chitin-binding domains (CBD), scavenger receptor cysteine-rich (SRCR) domains, low density lipoprotein receptor cysteine-rich (LDLR-CR) domains, histidine and proline-rich domains, a NGGYQPP-repeat domain and a serine protease domain. The last 2370 nucleotides of these RNAs are identical and encode a His-rich domain, two SRCR domains, two LDLR-CR domains and a protease domain. The transcription of graal is upregulated after fungal or bacterial infection. Analysis of the Iso1 (y;cn,sp,bw) strain shows that graal transcription is impaired in this fly line due to the insertion of a retrotransposon in the sixth exon. However, no phenotype could be observed consecutive to the absence of graal full length transcripts, particularly in the context of an immune challenge. PMID:15475297
Munier, Anne Isabelle; Medzhitov, Ruslan; Janeway, Charles A; Doucet, Daniel; Capovilla, Maria; Lagueux, Marie
Serine racemase: a key player in apoptosis and necrosis
A fine balance between cell survival and cell death is required to sculpt the nervous system during development. However, an excess of cell death can occur following trauma, exposure to neurotoxins or alcohol, and some developmental and neurodegenerative diseases, such as Alzheimer's disease (AD). N-Methyl-D-aspartate receptors (NMDARs) support synaptic plasticity and survival of many neuronal populations whereas inappropriate activation may promote various forms of cell death, apoptosis, and necrosis representing the two extremes of a continuum of cell death processes both �in vitro� and �in vivo.� Hence, by identifying the switches controlling pro-survival vs. apoptosis and apoptosis vs. pro-excitotoxic outcome of NMDAR stimulation, NMDAR modulators could be developed that selectively block the cell death enhancing pro-survival signaling or synaptic plasticity mediated by NMDAR. Among these modulators, a role is emerging for the enzyme serine racemase (SR) that synthesizes D-serine, a key co-agonist with glutamate at NMDAR. This review summarizes the experimental evidence from �in vitro� neuronal cultures�with special emphasis on cerebellar granule neurons (CGNs)�and �in vivo� models of neurodegeneration, where the dual role of the SR/D-serine pathway as a master regulator of apoptosis and the apoptosis-necrosis shift will be discussed.
Canu, Nadia; Ciotti, Maria Teresa; Pollegioni, Loredano
Rubidium penta-aqua-(l-serine)cobalt(II) hexa-hydrogenhexa-molybdocobaltate(III) l-serine monosolvate deca-hydrate
The Co2+ ion in the title compound, Rb[Co(C3H7NO3)(H2O)5][H6CoMo6O24]�C3H7NO3�10H2O, is coordinated by five water mol�ecules and one O-monodentate l-serine ligand in a slightly distorted octahedral geometry. The Rb+ ion is irregularly coordinated by nine O atoms. In the crystal, the [H6CoIIIMo6O24]3? polyanions are stacked along the b-axis direction, mediated by bridging Rb�O bonds. N�H?O and O�H?O hydrogen bonds are observed involving the l-serine mol�ecules.
Iijima, Jun; Naruke, Haruo; Takiyama, Hiroshi
Inhibition of pan-class I phosphatidyl-inositol-3-kinase by NVP-BKM120 effectively blocks proliferation and induces cell death in diffuse large B-cell lymphoma.
Diffuse large B-cell lymphoma (DLBCL) is the most frequent aggressive lymphoma, with a great demand for novel treatments for relapsing and refractory disease. Constitutive activation of the phosphatidyl-inositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is often detected in this lymphoma. Inhibition of this signaling cascade with the pan-class I PI3K inhibitor NVP-BKM120 decreased cell proliferation and increased apoptotic cell death. DLBCL proliferation was further decreased if NVP-BKM120-induced autophagy was blocked. Treatment with NVP-BKM120 was associated with an increase of the pro-apoptotic BH3-only proteins Puma and Bim and down-regulation of the anti-apoptotic Bcl-xL and Mcl-1. Translation of Bcl-xL and Mcl-1 is facilitated by cap-dependent mRNA translation, a process that was partially inhibited by NVP-BKM120. Overall, we demonstrated here the potential of NVP-BKM120 for the treatment of DLBCL. PMID:23721513
Zang, Chuanbing; Eucker, Jan; Liu, Hongyu; Coordes, Annekatrin; Lenarz, Minoo; Possinger, Kurt; Scholz, Christian Wilfried
Defining substrate and blocker activity of alanine-serine-cysteine transporter 2 (ASCT2) Ligands with Novel Serine Analogs.
The neutral amino acid transporter alanine-serine-cysteine transporter 2 (ASCT2) belongs to the solute carrier 1 (SLC1) family of solute transporters and transports small, neutral amino acids across the membrane, including the physiologically important and ubiquitous amino acid glutamine. Our understanding of the involvement of ASCT2 in the physiological processes involving glutamine is hampered by a lack of understanding of its pharmacology and the absence of high-affinity inhibitors. In this study, we combined an in silico docking approach with experimental investigation of binding parameters to develop new ASCT2 inhibitors and substrates, a series of serine esters, and to determine structural parameters that govern their functional effects. The series of compounds was synthesized using standard methods and exhibited a range of properties, from inhibitors to partial substrates and full substrates. Our results suggest that amino acid derivatives with small side-chain volume and low side-chain hydrophobicity interact strongly with the closed-loop form of the binding site, in which re-entrant loop 2, the presumed extracellular gate for the substrate binding site, is closed off. However, these derivatives bind weakly to the open-loop form (external gate open to the extracellular side), acting as transported substrates. In contrast, inhibitors bind preferentially to the open-loop form. An aromatic residue in the side chain is required for high-affinity interaction. One of the compounds, the l-serine ester serine biphenyl-4-carboxylate reversibly inhibits ASCT2 function with an apparent affinity of 30 ?M. PMID:22113081
Albers, Thomas; Marsiglia, William; Thomas, Taniya; Gameiro, Armanda; Grewer, Christof
Defining Substrate and Blocker Activity of Alanine-Serine-Cysteine Transporter 2 (ASCT2) Ligands with Novel Serine Analogs
The neutral amino acid transporter alanine-serine-cysteine transporter 2 (ASCT2) belongs to the solute carrier 1 (SLC1) family of solute transporters and transports small, neutral amino acids across the membrane, including the physiologically important and ubiquitous amino acid glutamine. Our understanding of the involvement of ASCT2 in the physiological processes involving glutamine is hampered by a lack of understanding of its pharmacology and the absence of high-affinity inhibitors. In this study, we combined an in silico docking approach with experimental investigation of binding parameters to develop new ASCT2 inhibitors and substrates, a series of serine esters, and to determine structural parameters that govern their functional effects. The series of compounds was synthesized using standard methods and exhibited a range of properties, from inhibitors to partial substrates and full substrates. Our results suggest that amino acid derivatives with small side-chain volume and low side-chain hydrophobicity interact strongly with the closed-loop form of the binding site, in which re-entrant loop 2, the presumed extracellular gate for the substrate binding site, is closed off. However, these derivatives bind weakly to the open-loop form (external gate open to the extracellular side), acting as transported substrates. In contrast, inhibitors bind preferentially to the open-loop form. An aromatic residue in the side chain is required for high-affinity interaction. One of the compounds, the l-serine ester serine biphenyl-4-carboxylate reversibly inhibits ASCT2 function with an apparent affinity of 30 ?M.
Albers, Thomas; Marsiglia, William; Thomas, Taniya; Gameiro, Armanda
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.
McCall, Pamela; Adams, Claire E.; Willder, Jennifer M.; Bennett, Lindsay; Qayyum, Tahir; Orange, Clare; Underwood, Mark A.; Edwards, Joanne
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
Effect of interfaces on the melting of PEO confined in triblock PS-b-PEO-b-PS copolymers.
Block copolymers form nanostructures that have interesting physical properties because they combine, for a single compound, the complementary features brought by each block. However, in order to fully exploit these properties, the physical state of each kind of domain must be precisely controlled. In this work, triblock PS-b-PEO-b-PS copolymers consisting of a central poly(ethylene oxide) (PEO) block covalently bonded to polystyrene (PS) blocks were synthesized by Atom Transfer Radical Polymerization. Their morphology was investigated by X-ray scattering and TEM experiments whereas their thermodynamic behavior was characterized by DSC. A strong decrease of both the melting temperature and the degree of crystallinity of PEO, due to its confinement between the PS domains, was observed and analyzed with a modified Gibbs-Thomson equation, following the approaches used for fluids confined in porous media. The existence of an amorphous bound layer, a few nanometers thick, at the PEO/PS interface, that does not undergo any phase transition in the temperature range investigated, accounts for both the melting temperature depression and the decrease of crystallinity upon confinement. This interfacial layer may significantly affect the mechanical and transport properties of these block copolymers that find applications as solid polymer electrolytes in batteries for example. Moreover, the value obtained for the solid PEO/liquid PEO surface tension is lower than those previously published but is thermodynamically consistent with the surface tensions of polymers at the solid/vapor and liquid/vapor interfaces. PMID:23865710
Beaudoin, E; Phan, T N T; Robinet, M; Denoyel, R; Davidson, P; Bertin, D; Bouchet, R
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.
Nakamura, Kenta; Hirano, Ken-ichi; Wu, Sean M.
Tenectin is a novel ?PS2?PS integrin ligand required for wing morphogenesis and male genital looping in Drosophila
Morphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. We describe the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectin�s function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectin�s function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry.
Fraichard, Stephane; Bouge, Anne-Laure; Kendall, Timmy; Chauvel, Isabelle; Bouhin, Herve; Bunch, Thomas A.
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
Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease
A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8�10). In addition, the enzyme had an elevated optimum temperature (60�C). The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.
Majeed, Tanveer; Tabassum, Romana; Orts, William J.; Lee, Charles C.
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.
To the Teacher: P.S. Write Soon!
ERIC Educational Resources Information Center
Intended for teachers of grades 4 through 8 who want to adapt the activities listed in the publication "P.S. Write Soon!" for classroom use, this guide provides chapter by chapter examples of the kinds of classroom projects suggested by the book that can be incorporated into an existing unit on letter writing or used as the basis for letter�
Post Office Dept., Washington, DC.
Solvent annealing of Micropatterned PS-b-PEO copolymer films
NASA Astrophysics Data System (ADS)
Solvent annealing of block copolymer thin films have been known as an effective way to control both orientation of microdomains with respect to the surface and their registration into a well ordered periodic lattice structure. We have recently demonstrated hierarchically ordered microdomains in a thin poly(styrene-b-ethylene oxide)(PS-b-PEO) film combined with microcontact printing. The solvent annealing gave rise to well ordered spherical PEO microdomains in large area by the confined dewetting of thin PS-b-PEO films which had been micropatterned on chemically modified surface during solvent annealing. In this presentation, we intentionally prepare a micropatterned dewet film of PS-b-PEO by spincoating a block copolymer solution on a topographic PDMS pre-pattern. Convex lens shaped spherical caps of PS-b-PEO individually located on each PDMS mesa were successfully transferred to a Si substrate by a conventional transfer printing technique. We investigate the effect of solvent on not only film wettability but also formation of hierarchical nanostructures.
Kim, Tae Hee; Acharya, Himadri; Joeng, Hee June; Park, Cheolmin
Microproteins (miPs) - the next big thing
With iPS cells, sncRNAs, chromatin modification regulation and cancer stem cells already cooling off again, i.e. not being guaranteed publications in the 'ultimate' journals anymore, what will be very soon the new red-hot (or super-cool, i.e. anything but lukewarm) 'kid on the block'? We would vote for microproteins.
Further Comments on the Nature of iPS Cells
A criticism that induced pluripotent stem (iPS) cells may well be pre-existing stem cells was first accepted by the corresponding author of the original Nature publication but that confession was totally changed to a strong defiance after being guided by the Nature for a new response. The three reviews given by Nature actually contained an apparent duplication or \\
Shi V. Liu
Development and characterization of sub-100 ps photomultiplier tubes
We describe the evaluation of a microchannel plate (MCP) photomultiplier tube (PMT), incorporating a 3 mum 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
C. J. Horsfield; M. S. Rubery; J. M. Mack; C. S. Young; H. W. Herrmann; S. E. Caldwell; S. C. Evans; T. J. Sedilleo; Y. H. Kim; A. McEvoy; J. S. Milnes; J. Howorth; B. Davis; P. M. O'Gara; I. Garza; E. K. Miller; W. Stoeffl; Z. Ali
CERN's PS Booster LLRF renovation : plans and initial beam tests
In 2008 a project was started to renovate the CERN's PS Booster (PSB) low-level RF (LLRF). Required LLRF capabilities include frequency program, beam phase, radial and synchronization loops. The new LLRF will control the signals feeding the three RF cavities present in each ring; it will also shape the beam in a dual harmonic mode, operate a bunch splitting and
ME Angoletta; A Blas; A Butterworth; A Findlay; PM Leinonen; JC Molendijk; F Pedersen; J Sanchez-Quesada; M Schokker
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
Occurrence of Pyridoxal 5?-Phosphate-Dependent Serine Racemase in Silkworm, Bombyx mori
D-Serine is known to occur in the silkwormBombyx morias well as in the mammalian central nervous systems. We found that serine racemase occurs in the insect, catalyzing the conversion of L-serine to its antipode. The enzyme was partially purified from pupae of the insect, and was inactivated by treatment with hydroxylamine and reactivated with pyridoxal 5?-phosphate (PLP). L-Alanine was racemized
Takuma Uo; Tohru Yoshimura; Susumu Shimizu; Nobuyoshi Esaki
Functional and molecular analysis of D-serine transport in retinal M�ller cells.
D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal M�ller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 M�ller cell line and primary cultures of mouse M�ller cells (1 degrees MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. D-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for D-serine in M�ller cells. The Na+:substrate stoichiometry for the transport process was 1:1. D-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that D-serine transport in M�ller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in M�ller cells. Immunoblotting detected ASCT2 in neural retina and in 1 degrees MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1 degrees MCs. The efflux of D-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a M�ller cell line and in 1 degrees MCs and suggest that D-serine, synthesized in M�ller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons. PMID:17094966
Dun, Y; Mysona, B; Itagaki, S; Martin-Studdard, A; Ganapathy, V; Smith, S B
Functional and Molecular Analysis of D-Serine Transport in Retinal M?ller Cells
D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal M�ller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 M�ller cell line and primary cultures of mouse M�ller cells (1�MCs). The relative contributions of different amino acid transport systems to D-serine uptake were determined based on differential substrate specificities and ion dependencies. D-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for D-serine in M�ller cells. The Na+:substrate stoichiometry for the transport process was 1:1. D-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that D-serine transport in M�ller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in M�ller cells. Immunoblotting detected ASCT2 in neural retina and in 1�MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1�MCs. The efflux of D-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a M�ller cell line and in 1�MCs and suggest that D-serine, synthesized in M�ller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.
Dun, Y.; Mysona, B.; Itagaki, S.; Martin-Studdard, A.; Ganapathy, V.; Smith, S.B.
D-Amino Acids as Putative Neurotransmitters: Focus on D-Serine
Of the twenty amino acids in the mammalian body, only serine and aspartate occur in D-configuration as well as L-configuration in significant amount. D-serine is selectively concentrated in the brain, localized to protoplasmic astrocytes that ensheath synapses and distributed similarly to N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. D-serine has been found to function as an endogenous ligand for the �glycine�
Solomon H. Snyder; Paul M. Kim
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
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.
Schlupen, Christina; Santos, Maria A; Weber, Ulrike; de Graaf, Albert; Revuelta, Jose L; Stahmann, K-Peter
Enhancing the virulence of Paecilomyces lilacinus against Meloidogyne incognita eggs by overexpression of a serine protease.
To enhance the virulence of Paecilomyces lilacinus against Meloidogyne incognita eggs, a serine protease was overexpressed in P. lilacinus 9410 (PL9410). The cDNA of a mature serine protease gene was cloned from PL9410 and integrated into the genomes of PL9410 transformants through Agrobacterium tumefaciens-mediated transformation. Our results confirmed that the serine protease gene was overexpressed at the transcriptional level, and that the serine protease activities were enhanced in the transformants when compared to the parent strain. The bioassay results indicated that the relative parasitizing rates of the transformants against M. incognita eggs increased by about 20% in both conidial suspension and mycelium treatment groups. PMID:20473777
Wang, Jieping; Wang, Jiaxu; Liu, Fan; Pan, Cangsang
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.
Sukkapan, Pattadon; Jia, Ying; Nuchprayoon, Issarang; Perez, John C.
Ca2+ regulation of 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate formation and hydrolysis on sarcoplasmic-reticular Ca2+-transport ATPase. A new principle of phospholipid turnover regulation.
Lipid phosphorylation was shown to occur on the isolated sarcoplasmic-reticulum (SR) Ca2+-transport ATPase. More than 95% of the radioactivity incorporated on incubation of the SR ATPase with [gamma-32P]ATPMg can be extracted with acidic organic solvents and was identified as 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate (PtdIns4P) [Vars�nyi, Toelle, Heilmeyer, Dawson & Irvine (1983) EMBO J. 2, 1543-1548]. This lipid phosphorylation is only observed at nanomolar concentrations of free Ca2+; in the presence of micromolar free Ca2+ PtdIns4P disintegrates rapidly. Also, upon blockade of the kinase reaction PtdIns4P decomposes, indicating a PtdIns/PtdIns4P turnover. The PtdIns4P concentration is dependent on the free Ca2+ concentration, being half-maximal at 35 nM-Ca2+. PtdIns4P hydrolysis is catalysed by a PtdIns4P phosphomonoesterase; accordingly no diacylglycerol is formed, which would be a product of a phosphodiesteratic cleavage. Fluoride inhibits this phosphomonoesterase. Ca2+ does not influence directly either the PtdIns kinase or the PtdIns4P phosphomonoesterase. PtdIns4P forms a tight complex with the transport ATPase, from which it can be removed only by chromatography on heparin-agarose in the presence of Triton X-100. It is concluded that Ca2+ regulates the PtdIns/PtdIns4P turnover by availability of substrate, depending on the Ca2+-transport-ATPase conformation, which traps or exposes the respective lipid head groups.
Schafer, M; Behle, G; Varsanyi, M; Heilmeyer, L M
Cellular serine/threonine phosphatase activity during human cytomegalovirus infection
While the importance of cellular and viral kinases in HCMV replication has been demonstrated, relatively little is known about the activity of cellular phosphatases. We conducted a series of experiments designed to investigate the effect of HCMV infection on cellular serine/threonine phosphatase activity. We found that the abundance of two major cellular serine/threonine phosphatases, PP1 and PP2A, increases during HCMV infection. This was associated with an increase in threonine phosphatase activity in HCMV-infected cells. HCMV infection conferred resistance to the effects of the phosphatase inhibitors calyculin A (CA) and okadaic acid with regards to global protein hyperphosphorylation and the shutoff of protein synthesis. The protective effect of HCMV infection could be overcome at a high concentration of CA, suggesting that cellular phosphatase activity is required for critical cellular processes during HCMV infection. Specifically, phosphatase activity was required to limit the accumulation of phospho-eIF2?, but not phospho-PKR, during HCMV infection.
Hakki, Morgan; Geballe, Adam P.
Photoreversible inhibition of cholinesterases: catalytic serine-labeled caged butyrylcholinesterase.
The photoregulation of the catalytic activity of butyrylcholinesterase (BChE) was investigated by treating the enzyme with a newly developed carbamylating reagent, N-methyl-N-(2-nitrophenyl)carbamoyl chloride, which has proved to be an efficient photoremovable alcohol-protecting group. BChE was efficiently inhibited in a time- and concentration-dependent manner, and the enzyme could be protected against inhibition by active-site-specific ligands (that is, tacrine). The inactivation kinetics showed a biphasic character, which can be analyzed as the result of the existence of two conformational states in solution. Pseudo-irreversible inactivation of BChE, which results from catalytic serine carbamylation, was suggested by recovery of the enzyme activity after dilution and was demonstrated by X-ray crystallography. Remarkably, the 3D structure of the carbamylated BChE conjugate showed a nonambiguous carbamylation of the catalytic serine residue as the only chemical modification on the protein. The photoreversibility of the enzyme inactivation was analyzed by irradiating the inactivated enzyme at 365 nm and was shown to reach completion in some conditions. The efficient and specific "caging" of BChE, together with the availability of carbamylated BChE crystals, will offer a unique possibility to study the catalytic properties of this enzyme by kinetic crystallography after cryophotolytic uncaging of the enzyme conjugate crystals. PMID:12898628
Loudwig, Sandra; Nicolet, Yvain; Masson, Patrick; Fontecilla-Camps, Juan C; Bon, Suzanne; Nachon, Florian; Goeldner, Maurice
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.
Henke, Markus O.; John, Gerrit; Rheineck, Christina; Chillappagari, Shashi; Naehrlich, Lutz; Rubin, Bruce K.
The structure of serine palmitoyltransferase; gateway to sphingolipid biosynthesis.
Sphingolipid biosynthesis commences with the condensation of L-serine and palmitoyl-CoA to produce 3-ketodihydrosphingosine (KDS). This reaction is catalysed by the PLP-dependent enzyme serine palmitoyltransferase (SPT; EC 18.104.22.168), which is a membrane-bound heterodimer (SPT1/SPT2) in eukaryotes such as humans and yeast and a cytoplasmic homodimer in the Gram-negative bacterium Sphingomonas paucimobilis. Unusually, the outer membrane of S. paucimobilis contains glycosphingolipid (GSL) instead of lipopolysaccharide (LPS), and SPT catalyses the first step of the GSL biosynthetic pathway in this organism. We report here the crystal structure of the holo-form of S. paucimobilis SPT at 1.3 A resolution. The enzyme is a symmetrical homodimer with two active sites and a monomeric tertiary structure consisting of three domains. The PLP cofactor is bound covalently to a lysine residue (Lys265) as an internal aldimine/Schiff base and the active site is composed of residues from both subunits, located at the bottom of a deep cleft. Models of the human SPT1/SPT2 heterodimer were generated from the bacterial structure by bioinformatics analysis. Mutations in the human SPT1-encoding subunit have been shown to cause a neuropathological disease known as hereditary sensory and autonomic neuropathy type I (HSAN1). Our models provide an understanding of how these mutations may affect the activity of the enzyme. PMID:17559874
Yard, Beverley A; Carter, Lester G; Johnson, Kenneth A; Overton, Ian M; Dorward, Mark; Liu, Huanting; McMahon, Stephen A; Oke, Muse; Puech, Daphn�; Barton, Geoffrey J; Naismith, James H; Campopiano, Dominic J
RAF protein-serine/threonine kinases: structure and regulation.
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. PMID:20674547
Roskoski, Robert
Protein inhibitors of serine proteinases--mechanism and classification.
Protein proteinase inhibitors are widely distributed in plants, animals and microorganisms. They can be conveniently grouped since most frequently they inhibit proteinases belonging only to a single mechanistic class. Protein inhibitors of serine proteinases have been most extensively studied. They are strictly competitive inhibitors forming 1:1 complexes with the enzymes they inhibit. In these complexes, all activities of the enzyme are completely abolished. The inhibitors are substrates for the enzyme they inhibit at a unique peptide bond called the reactive site peptide bond (one for each inhibitory domain). However, compared to normal substrates where the enzyme-substrate and enzyme-product complexes dissociate very readily here, the complexes are very stable. Serine proteinase inhibitors can be divided into at least 13 families. Within each family the position of the reactive site and the closure of disulfide bridges can be inferred by homology. In enzyme-inhibitor complexes, about 10-15 residues of the inhibitor are in contact with the enzyme. Their specific nature strongly affects both the strength and the specificity of enzyme-inhibitor interaction. In all cases where the sequences of many inhibitors from the same family can be compared, the contact residues are not strongly conserved--instead, they are hypervariable. This raises major problems but also offers huge opportunities to those concerned with the role of inhibitors in biology and in medicine. PMID:3541508
Laskowski, M
The hydration of serine: multipole moments versus point charges.
Next-generation force fields must incorporate improved electrostatic potentials in order to increase the reliability of their predictions. A crucial decision toward this goal is to abandon point charges in favour of multipole moments centered on nuclear sites. Here we compare the geometries generated by quantum topological multipole moments with those generated by four popular point charge models (TAFF, OPLS-AA, MMFF94x and PFROSST) for a hydrated serine. A main feature of this study is the dual comparison made, both at static level (geometry optimisation via energy minimisation) and at dynamic level (via molecular dynamics and radial/spatial distribution function analysis). At static level, multipolar electrostatics best reproduces the ab initio reference geometry. At dynamic level, multipolar electrostatics produces more structure than point charge electrostatics does, over the whole range. From our previous work on liquid water [Int. J. Quantum. Chem., 2004, 99, 685], where agreement with experiment only occurs when using multipole moments, we deduce that our predictions for hydrated serine will also be closer to experiment when using multipolar electrostatics. The spatial distribution function shows that only multipolar electrostatics shows pronounced structure at long range. Even at short range there are many regions where waters appear in the system governed by multipolar electrostatics but not in that governed by point charges. PMID:24448691
Liem, Steven Y; Popelier, Paul L A
Functional and immunocytochemical characterization of D-serine transporters in cortical neuron and astrocyte cultures.
D-serine is an endogenous coagonist of N-methyl-D-aspartate (NMDA) receptors that plays an important role in synaptic function, neuronal development, and excitotoxicity. Mechanisms of D-serine transport are important in regulation of extracellular D-serine concentration and therefore of these critical processes. D-serine can be transported with low affinity through the Na(+)-dependent amino acid transporter termed ASCT2, whereas high-affinity D-serine uptake has been reported through the Na(+)-independent transporter termed asc-1. We investigated immunoreactivity for ASCT2 and asc-1 and D-serine transport kinetics in cultured cortical neurons and astrocytes to gain insight into how D-serine transporters regulate CNS D-serine levels. Both neurons and astrocytes exhibited low-affinity Na(+)-dependent D-serine uptake (K(T) > 1 mM) with broad substrate selectivity that was consistent with uptake through ASCT2. Both neurons and astrocytes also stained positively for ASCT2 in immunocytochemistry studies. Neurons but not astrocytes stained positively for the high-affinity D-serine transporter asc-1, but no evidence of functional asc-1 could be detected in neurons with conditions that produced such activity in cortical synaptosomes. These data support ASCT2 function in both neuron and astrocyte cultures and identify a discrepancy between observed asc-1 immunoreactivity and lack of functional asc-1 activity in neuron cultures. Together these findings further our knowledge of the processes that govern D-serine regulation. PMID:19382234
Shao, Zongjun; Kamboj, Amit; Anderson, Christopher M
Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an N-methyl-D-aspartate receptor co-agonist, in adult brain.
In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 126.96.36.199). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L- and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus. PMID:20966073
Yang, Jung Hoon; Wada, Akira; Yoshida, Kazuyuki; Miyoshi, Yurika; Sayano, Tomoko; Esaki, Kayoko; Kinoshita, Masami O; Tomonaga, Shozo; Azuma, Norihiro; Watanabe, Masahiko; Hamase, Kenji; Zaitsu, Kiyoshi; Machida, Takeo; Messing, Albee; Itohara, Shigeyoshi; Hirabayashi, Yoshio; Furuya, Shigeki
Spectra and relaxation dynamics of the pseudohalide (PS) vibrational bands for Ru(bpy)2(PS)2 complexes, PS = CN, NCS and N3
NASA Astrophysics Data System (ADS)
Static and transient infrared spectroscopy were used to investigate cis-Ru(bpy)2(N3)2 (bpy = 2,2'-bipyridine), cis-Ru(bpy)2(NCS)2, and cis-Ru(bpy)2(CN)2 in solution. The NC stretching IR band for cis-Ru(bpy)2(NCS)2 appears at higher frequency (�2106 cm-1 in DMSO) than for the free NCS- anion while the IR bands for the azide and cyanide complexes are closer to those of the respective free anions. The vibrational energy relaxation (VER) lifetime for the azide complex is found to be much shorter (�5 ps) than for either the NCS or CN species (both �70 ps in DMSO) and the lifetimes resemble those for each corresponding free anion in solution. However, for cis-Ru(bpy)2(N3)2, it is determined that the transition frequency depends more on the solvent than the VER lifetime implying that intramolecular vibrational relaxation is predominant over solvent energy-extracting interactions. These results are compared to the behavior of other related metal complexes in solution.
Compton, Ryan; Gerardi, Helen K.; Weidinger, Daniel; Brown, Douglas J.; Dressick, Walter J.; Heilweil, Edwin J.; Owrutsky, Jeffrey C.
Functional Characterization of Calcineurin Homologs PsCNA1/PsCNB1 in Puccinia striiformis f. sp. tritici Using a Host-Induced RNAi System
Calcineurin plays a key role in morphogenesis, pathogenesis and drug resistance in most fungi. However, the function of calcineurin genes in Puccinia striiformis f. sp. tritici (Pst) is unclear. We identified and characterized the calcineurin genes PsCNA1 and PsCNB1 in Pst. Phylogenetic analyses indicate that PsCNA1 and PsCNB1 form a calcium/calmodulin regulated protein phosphatase belonging to the calcineurin heterodimers composed of subunits A and B. Quantitative RT-PCR analyses revealed that both PsCNA1 and PsCNB1 expression reached their maximum in the stage of haustorium formation, which is one day after inoculation. Using barely stripe mosaic virus (BSMV) as a transient expression vector in wheat, the expression of PsCNA1 and PsCNB1 in Pst was suppressed, leading to slower extension of fungal hyphae and reduced production of urediospores. The immune-suppressive drugs cyclosporin A and FK506 markedly reduced the germination rates of urediospores, and when germination did occur, more than two germtubes were produced. These results suggest that the calcineurin signaling pathway participates in stripe rust morphogenetic differentiation, especially the formation of haustoria during the early stage of infection and during the production of urediospores. Therefore PsCNA1 and PsCNB1 can be considered important pathogenicity genes involved in the wheat-Pst interaction.
Zhang, Hong; Guo, Jun; Voegele, Ralf T.; Zhang, Jinshan; Duan, Yinghui; Luo, Huaiyong; Kang, Zhensheng
Functional characterization of calcineurin homologs PsCNA1/PsCNB1 in Puccinia striiformis f. sp. tritici using a host-induced RNAi system.
Calcineurin plays a key role in morphogenesis, pathogenesis and drug resistance in most fungi. However, the function of calcineurin genes in Puccinia striiformis f. sp. tritici (Pst) is unclear. We identified and characterized the calcineurin genes PsCNA1 and PsCNB1 in Pst. Phylogenetic analyses indicate that PsCNA1 and PsCNB1 form a calcium/calmodulin regulated protein phosphatase belonging to the calcineurin heterodimers composed of subunits A and B. Quantitative RT-PCR analyses revealed that both PsCNA1 and PsCNB1 expression reached their maximum in the stage of haustorium formation, which is one day after inoculation. Using barely stripe mosaic virus (BSMV) as a transient expression vector in wheat, the expression of PsCNA1 and PsCNB1 in Pst was suppressed, leading to slower extension of fungal hyphae and reduced production of urediospores. The immune-suppressive drugs cyclosporin A and FK506 markedly reduced the germination rates of urediospores, and when germination did occur, more than two germtubes were produced. These results suggest that the calcineurin signaling pathway participates in stripe rust morphogenetic differentiation, especially the formation of haustoria during the early stage of infection and during the production of urediospores. Therefore PsCNA1 and PsCNB1 can be considered important pathogenicity genes involved in the wheat-Pst interaction. PMID:23139840
Zhang, Hong; Guo, Jun; Voegele, Ralf T; Zhang, Jinshan; Duan, Yinghui; Luo, Huaiyong; Kang, Zhensheng
PS3 CELL Development for Scientific Computation and Research
NASA Astrophysics Data System (ADS)
The Cell processor is one of the most powerful processors on the market, and researchers in the earth sciences may find its parallel architecture to be very useful. A cell processor, with 7 cores, can easily be obtained for experimentation by purchasing a PlayStation 3 (PS3) and installing linux and the IBM SDK. Each core of the PS3 is capable of 25 GFLOPS giving a potential limit of 150 GFLOPS when using all 6 SPUs (synergistic processing units) by using vectorized algorithms. We have used the Cell's computational power to create a program which takes simulated tsunami datasets, parses them, and returns a colorized height field image using ray casting techniques. As expected, the time required to create an image is inversely proportional to the number of SPUs used. We believe that this trend will continue when multiple PS3s are chained using OpenMP functionality and are in the process of researching this. By using the Cell to visualize tsunami data, we have found that its greatest feature is its power. This fact entwines well with the needs of the scientific community where the limiting factor is time. Any algorithm, such as the heat equation, that can be subdivided into multiple parts can take advantage of the PS3 Cell's ability to split the computations across the 6 SPUs reducing required run time by one sixth. Further vectorization of the code can allow for 4 simultanious floating point operations by using the SIMD (single instruction multiple data) capabilities of the SPU increasing efficiency 24 times.
Christiansen, M.; Sevre, E.; Wang, S. M.; Yuen, D. A.; Liu, S.; Lyness, M. D.; Broten, M.
iPS cells produce viable mice through tetraploid complementation
Since the initial description of induced pluripotent stem (iPS) cells created by forced expression of four transcription factors in mouse fibroblasts, the technique has been used to generate embryonic stem (ES)-cell-like pluripotent cells from a variety of cell types in other species, including primates and rat. It has become a popular means to reprogram somatic genomes into an embryonic-like pluripotent
Xiao-Yang Zhao; Wei Li; Zhuo Lv; Lei Liu; Man Tong; Tang Hai; Jie Hao; Chang-Long Guo; Qing-Wen Ma; Liu Wang; Fanyi Zeng; Qi Zhou
iPS cells: A source of cardiac regeneration
For the treatment of heart failure, a new strategy to improve cardiac function and inhibit cardiac remodeling needs to be established. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent cells that can differentiate into cell types from all three germ layers both in vitro and in vivo. The therapeutic effect of ES\\/iPS cell-derived progeny was reported
Yoshinori Yoshida; Shinya Yamanaka
Opportunities for Human iPS Cells in Predictive Toxicology
Toxicity assessment is a major challenge for cost-effective drug development, and there is great need for better tools to accurately predict adverse drug reactions. Technological advances are empowering new cell-based assays for predictive toxicology, and these assays are critically dependent on the cell source. Here we describe the properties of human induced pluripotent stem (iPS) cells that make them a promising cell source for toxicity assessment and highlight progress to date and important roadblocks remaining.
Anson, Blake D.; Kolaja, Kyle; Kamp, Timothy J.
Spaceborne SAR Differential interferometry (DInSAR) has been used to monitor some geological disasters such as ground subsidence. This paper applies two Permanent Scatterer InSAR(PS-InSAR) techniques to measure long-term and short-term subsidence in urban area of Shanghai urban. A great number of archieved ERS data are utilized to measure long-term subsidence during 1993-2000 year. A small set of ASAR data are
Lijun Lu; Mingsheng Liao
The structure of mammalian serine racemase: evidence for conformational changes upon inhibitor binding.
Serine racemase is responsible for the synthesis of D-serine, an endogenous co-agonist for N-methyl-D-aspartate receptor-type glutamate receptors (NMDARs). This pyridoxal 5'-phosphate-dependent enzyme is involved both in the reversible conversion of L- to D-serine and serine catabolism by alpha,beta-elimination of water, thereby regulating D-serine levels. Because D-serine affects NMDAR signaling throughout the brain, serine racemase is a promising target for the treatment of disorders related to NMDAR dysfunction. To provide a molecular basis for rational drug design the x-ray crystal structures of human and rat serine racemase were determined at 1.5- and 2.1-A resolution, respectively, and in the presence and absence of the orthosteric inhibitor malonate. The structures revealed a fold typical of beta-family pyridoxal 5'-phosphate enzymes, with both a large domain and a flexible small domain associated into a symmetric dimer, and indicated a ligand-induced rearrangement of the small domain that organizes the active site for specific turnover of the substrate. PMID:20106978
Smith, Myron A; Mack, Volker; Ebneth, Andreas; Moraes, Isabel; Felicetti, Brunella; Wood, Michael; Schonfeld, Dorian; Mather, Owen; Cesura, Andrea; Barker, John
Activities of serine palmitoyltransferase (3-ketosphinganine synthase) in microsomes from different rat tissues
Serine palmitoyltransferase (EC 220.127.116.111 catalyzes the first unique reaction of sphingolipid biosynthesis. To deter- mine whether or not different rat tissues are capable of initiating this pathway, its activity was determined for microsomes from rat liver, lung, brain, kidney, intestine, spleen, muscle, heart, pancreas, testes, ovary, and stomach. Serine palmitoyltrans- ferase was found in every tissue, and, when compared to
Alfred H. Merrill; Daniel W. Nixon; Robert D. Williams
Free Amino Acids in Serine-Antagonized Cells of Tetrahymena pyriformis1
Wragg, June B. (Agricultural Research Service, Beltsville, Md.), Howard Reynolds, and Michael J. Pelczar, Jr. Free amino acids in serine-antagonized cells of Tetrahymena pyriformis. J. Bacteriol. 90:748�754. 1965.�Growth inhibition of Tetrahymena pyriformis by l-serine in a chemically defined medium was reversed by l-arginine in a manner which resembled competitive antagonism. Composition of the free amino acid pools from cells grown in either a balanced amino acid mixture or a mixture with serine concentrations which inhibited growth suggested an antagonism by serine with energy-yielding reactions. Growth in media with excess serine resulted in the accumulation of higher concentrations of free cellular amino acids and an apparent increase in the rate of conversion of arginine to ornithine, as compared with growth in the balanced medium. The results suggested that serine or a metabolic product of serine interferes with the formation of pyruvic acid. In the presence of high levels of serine, arginine appeared to be metabolized more rapidly and to be spared when alanine, aspartic acid, or glutamic acid was added to the unbalanced medium.
Wragg, June B.; Reynolds, Howard; Pelczar, Michael J.
Neurotrypsin, a Novel Multidomain Serine Protease Expressed in the Nervous System
We have cloned a novel murine cDNA encoding a multidomain serine protease, termed neurotrypsin, which exhibits an unprecedented domain composition. The deduced amino acid sequence defines a mosaic protein of 761 amino acids consisting of a kringle domain, followed by three scavenger receptor cysteine-rich repeats, and a serine protease domain. Based on comparisons of the primary structure, the protease domain
Thomas P. Gschwend; Stefan R. Krueger; Serguei V. Kozlov; David P. Wolfer; Peter Sonderegger
Aromatase is phosphorylated in situ at Serine-118
Phosphorylation of the cytochrome P450 aromatase has been proposed as a switch to rapidly modulate enzymatic activity and estrogen biosynthesis. Herein, we demonstrate that aromatase serine-118 is a potential phosphorylation site in mammalian cells. The amino acid context surrounding S118 is highly conserved among diverse animal species and suggests that an AGC-like kinase may phosphorylate aromatase. Mutation of S118 to Ala blocked phosphorylation. Mutation of S118 to either Ala or Asp destabilized aromatase, indicating an important structural role for S118. The phosphomimetic S118D mutant showed decreased specific enzymatic activity, decreased Vmax, and increased Km, while the S118A phospho-inhibiting mutant showed opposite effects. Our findings suggest that phosphorylation of S118 may decrease aromatase activity, presenting a mechanism whereby kinase signaling may modulate estrogen production and hormone balance.
Miller, Todd W.; Shin, Incheol; Kagawa, Norio; Evans, Dean B.; Waterman, Michael R.; Arteaga, Carlos L.
[Progress in the research of D-serine on neuron-glia communication].
D-serine is an important gliotransmitter in CNS. As an endogenous ligand for glycine-bind site in NR1 subunit of NMDA glutamate receptors, D-serine is more potent than glycine at activating the site. It is synthesized from L-serine via racemization of serine racemase, which is regulated by many factors. D-serine participates in many physiological and pathological progresses, including synaptic plasticity, sensory information transmission, neural development and neurotoxicity, and is supposed as potential therapeutic target for the treatment of nervous system disease like Alzheimer disease. Here, we provide an overview of recent findings on the mechanisms of its synthesis, degradation, release and physiological and pathological functions in CNS. PMID:21417027
He, Wen-Juan; Ruan, Huai-Zhen
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
Large serine recombinase domain structure and attachment site binding.
Large serine recombinases (LSRs) catalyze the movement of DNA elements into and out of bacterial chromosomes using site-specific recombination between short DNA "attachment sites". The LSRs that function as bacteriophage integrases carry out integration between attachment sites in the phage (attP) and in the host (attB). This process is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded recombination directionality factor, nor does recombination typically occur between other pairings of attachment sites. Although the mechanics of strand exchange are reasonably well understood through studies of the closely related resolvase and invertase serine recombinases, many of the fundamental aspects of the LSR reactions have until recently remained poorly understood on a structural level. In this review, we discuss the results of several years worth of biochemical and molecular genetic studies of LSRs in light of recently described structural models of LSR-DNA complexes. The focus is understanding LSR domain structure, how LSRs bind to the attP and attB attachment sites, and the differences between attP-binding and attB-binding modes. The simplicity, site-selectivity and strong directionality of the LSRs has led to their use as important tools in a number of genetic engineering applications in a wide variety of organisms. Given the important potential role of LSR enzymes in genetic engineering and gene therapy, understanding the structure and DNA-binding properties of LSRs is of fundamental importance for those seeking to enhance or alter specificity and functionality in these systems. PMID:23980849
Van Duyne, Gregory D; Rutherford, Karen
Genome Sequence of the Bacillus subtilis Biofilm-Forming Transformable Strain PS216
Bacillus subtilis PS216, a strain isolated in Slovenia, has been sequenced. PS216 is transformable and forms robust biofilms, making it useful for the study of competence regulation in an undomesticated bacterium.
Durrett, Russell; Miras, Mathieu; Mirouze, Nicolas; Narechania, Apurva; Mandic-Mulec, Ines
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)
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
Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila alpha PS2 beta PS integrins.
Genetic and other studies of Drosophila integrins have implicated these extracellular matrix receptors in various morphogenetic events, but identification of their endogenous ligands has been elusive. We report the biochemical purification and cloning of tiggrin, a novel extracellular matrix protein from Drosophila. This 255 x 10(3) M(r) polypeptide contains the potential integrin recognition sequence Arg-Gly-Asp (RGD) and 16 repeats of a novel 73-77 amino acid motif. The tiggrin gene is at chromosome locus 26D1-2 and is expressed by embryonic hemocytes and fat body cells. Tiggrin protein is detected in matrices, especially at muscle attachment sites that also strongly express integrins. Tiggrin-coated surfaces support primary embryo cell culture and provide excellent substrates for alpha PS2 beta PS integrin-mediated cell spreading. Soluble RGD-peptides inhibit this cell spreading. PMID:7924982
Fogerty, F J; Fessler, L I; Bunch, T A; Yaron, Y; Parker, C G; Nelson, R E; Brower, D L; Gullberg, D; Fessler, J H
ATP binding to human serine racemase is cooperative and modulated by glycine.
The N-methyl D-aspartate (NMDA) receptors play a key role in excitatory neurotransmission, and control learning, memory and synaptic plasticity. Their activity is modulated by the agonist glutamate and by the co-agonists d-serine and glycine. In the human brain, d-serine is synthesized from l-serine by the dimeric pyridoxal 5'-phosphate-dependent enzyme serine racemase, which also degrades l- and d-serine to pyruvate and ammonia. The dependence of l- and d-serine ?-elimination and l-serine racemization activities on ATP concentration was characterized, and was found to be strongly cooperative, with Hill coefficients close to 2 and apparent ATP dissociation constants ranging from 0.22 to 0.41 mm. ATP binding to the holo-enzyme, monitored by the fluorescence changes of the coenzyme, was also determined to be cooperative, with an apparent dissociation constant of 0.24 mm. Glycine, an active-site ligand, increased the serine racemase affinity for ATP by ~ 22-fold, abolishing cooperativity. Conversely, ATP increased the non-cooperative glycine binding 15-fold. These results indicate cross-talk between allosteric and active sites, leading to the stabilization of two alternative protein conformations with ATP affinities of ~ 10 ?M and 1.8 mm, as evaluated within the Monod, Wyman and Changeux model. Therefore, intracellular ATP and glycine control d-serine homeostasis, and, indirectly, NMDA receptor activity. Because hyper- and hypo-activation of NMDA receptors are associated with neuropathologies, the development of allosteric drugs modulating serine racemase activity is a promising therapeutic strategy. PMID:23992455
Marchetti, Marialaura; Bruno, Stefano; Campanini, Barbara; Peracchi, Alessio; Mai, Nicole; Mozzarelli, Andrea
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 signaling pathways. In particular, astrocytes have been shown recently 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 Ca(2+)-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 (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) 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. Whereas 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
PsbS genotype in relation to coordinated function of PS II and PS I in Arabidopsis leaves
Application of multiple probes to systems that carry specific mutations provides a powerful means for studying how known regulators of light utilization interact in vivo. Two lines of Arabidopsis thaliana were studied, each carrying a unique lesion in the nuclear psbS gene encoding a 22-kDa pigment-binding protein (PS II-S) essential for full expression of photoprotective, rapid-phase, nonphotochemical quenching of chlorophyll
Richard B. Peterson
Mutations in the Drosophila ?PS2 integrin subunit uncover new features of adhesion site assembly
The Drosophila ?PS2?PS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the ?PS2 gene that cause a subset of the null phenotype. One mutation changes a residue in ?PS2 that is equivalent to the residue in ?V that contacts the arginine of RGD. This change severely reduced ?PS2?PS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the ?PS2 ?-propeller, plus a fourth that eliminated a late phase of ?PS2 expression, all led to a strong decrease in ?PS2?PS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires ?PS2?PS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.
Devenport, Danelle; Bunch, Thomas A.; Bloor, James W.; Brower, Danny L.; Brown, Nicholas H.
Characterization of rapid and high-affinity uptake of L-serine in neurons and astrocytes in primary culture
The non-essential amino acid L-serine was shown to be required to support the survival of rat cerebellar Purkinje neurons because of lack of the expression of the L-serine biosynthesis enzyme 3-phosphoglycerate dehydrogenase in them. In the present study, we investigated L-[3H]serine uptake in primary cultures of neurons and astrocytes from the rat telencephalon. In both neurons and astrocytes, L-[3H]serine uptake
Toshifumi Yamamoto; Itone Nishizaki; Shigeki Furuya; Yoshio Hirabayashi; Kenzo Takahashi; Shigeru Okuyama; Hideko Yamamoto
New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina
Background Subtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors. Within Pezizomycotina, nematode-trapping fungi are unique because they capture soil nematodes using specialized trapping devices. Increasing evidence suggests subtilisin-like serine proteases from nematode-trapping fungi are involved in the penetration and digestion of nematode cuticles. Here we also conduct positive selection analysis on the subtilisin-like serine protease genes from nematode-trapping fungi. Results Phylogenetic analysis of 189 subtilisin-like serine protease genes from Pezizomycotina suggests five strongly-supported monophyletic clades. The subtilisin-like serine protease genes previously identified or presumed as endocellular proteases were clustered into one clade and diverged the earliest in the phylogeny. In addition, the cuticle-degrading protease genes from entomopathogenic and nematode-parasitic fungi were clustered together, indicating that they might have overlapping pathogenic mechanisms against insects and nematodes. Our experimental bioassays supported this conclusion. Interestingly, although they both function as cuticle-degrading proteases, the subtilisin-like serine protease genes from nematode-trapping fungi and nematode-parasitic fungi were not grouped together in the phylogenetic tree. Our evolutionary analysis revealed evidence for positive selection on the subtilisin-like serine protease genes of the nematode-trapping fungi. Conclusions Our study provides new insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. Pezizomycotina subtilisins most likely evolved from endocellular to extracellular proteases. The entomopathogenic and nematode-parasitic fungi likely share similar properties in parasitism. In addition, our data provided better understanding about the duplications and subsequent functional divergence of subtilisin-like serine protease genes in Pezizomycotina. The evidence of positive selection detected in the subtilisin-like serine protease genes of nematode-trapping fungi in the present study suggests that the subtilisin-like serine proteases may have played important roles during the evolution of pathogenicity of nematode-trapping fungi against nematodes.
The isolation and characterization of a novel collagenolytic serine protease allergen (Der p 9) from the dust mite Dermatophagoides pteronyssinus
Background: Dust mites have been shown to contain a serine protease distinct from the previously reported trypsin and chymotrypsin. The latter enzymes have been shown to be allergens, but the allergenic importance of the former is unknown. Objective: This study was performed to isolate and characterize the novel mite serine protease and determine its allergenicity. Methods: The mite serine protease
Cecile King; Richard J. Simpson; Robert L. Moritz; Gaven E. Reed; Philip J. Thompson; Geoffrey A. Stewart
Essential Role of STAT3 in Postnatal Survival and Growth Revealed by Mice Lacking STAT3 Serine 727 Phosphorylation
A large number of extracellular polypeptides bound to their cognate receptors activate the transcription factor STAT3 by phosphorylation of tyrosine 705. Supplemental activation occurs when serine 727 is also phosphorylated. STAT3 deletion in mice leads to embryonic lethality. We have produced mice with alanine substituted for serine 727 in STAT3 (the SA allele) to examine the function of serine 727
Yuhong Shen; Karni Schlessinger; Xuejun Zhu; Eric Meffre; Fred Quimby; David E. Levy; J. E. Darnell
Effect of Composition on the Surface Finish of PS400: A New High Temperature Solid Lubricant Coating.
National Technical Information Service (NTIS)
A new composite, multi-constituent, solid lubricant coating, NASA PS400, developed for high temperature tribological applications, exhibits a smoother surface finish after grinding and polishing than its predecessors PS200 and PS300. In this paper, the ba...
B. J. Edmonds C. DellaCorte F. Thomas K. Stanford
AMPA receptor mediated D-serine release from retinal glial cells
The NMDA receptor coagonist D-serine is important in a number of different processes in the central nervous system, ranging from synaptic plasticity to disease states, including schizophrenia. D-serine appears to be the major coagonist acting on retinal ganglion cell NMDA receptors, but the cell type from which it originates and whether its release can be modulated by activity are unknown. In this study, we utilized a mutant mouse line with elevated D-serine to investigate this question. Direct measurements of extracellular D-serine using capillary electrophoresis demonstrate that D-serine can be released from the intact mouse retina through an AMPA receptor dependent mechanism. AMPA-evoked D-serine release persisted in the presence of a cocktail of neural inhibitors but was abolished after administration of a glial toxin. These findings provide the first evidence that extracellular D-serine levels in the retina can be modulated, and that such modulation is contingent upon glial cell activity.
Sullivan, Steve J.; Miller, Robert F.
D-serine as a gliotransmitter and its roles in brain development and disease
The development of new techniques to study glial cells has revealed that they are active participants in the development of functional neuronal circuits. Calcium imaging studies demonstrate that glial cells actively sense and respond to neuronal activity. Glial cells can produce and release neurotransmitter-like molecules, referred to as gliotransmitters, that can in turn influence the activity of neurons and other glia. One putative gliotransmitter, D-serine is believed to be an endogenous co-agonist for synaptic N-methyl-D-aspartate receptors (NMDARs), modulating synaptic transmission and plasticity mediated by this receptor. The observation that D-serine levels in the mammalian brain increase during early development, suggests a possible role for this gliotransmitter in normal brain development and circuit refinement. In this review we will examine the data that D-serine and its associated enzyme serine racemase are developmentally regulated. We will consider the evidence that D-serine is actively released by glial cells and examine the studies that have implicated D-serine as a critical player involved in regulating NMDAR-mediated synaptic transmission and neuronal migration during development. Furthermore, we will consider how dysregulation of D-serine may play an important role in the etiology of neurological and psychiatric diseases.
Horn, Marion R. Van; Sild, Mari; Ruthazer, Edward S.
Endocytosis by the asialoglycoprotein receptor is independent of cytoplasmic serine residues.
The human asialoglycoprotein (ASGP) receptor, like most other plasma membrane receptors, has previously been shown to be phosphorylated at serine residues within the cytoplasmic domain. Phorbol esters, which activate protein kinase C, cause hyperphosphorylation and down-regulation of the ASGP receptor in HepG2 cells. To test the importance of serine residues for receptor traffic and function, we have mutated all the cytoplasmic serines of the two receptor subunits H1 (at positions 16 and 37) and H2 (at positions 12, 13, and 55) to alanines or glycines. Stable transfected fibroblast cell lines expressing either mutant H1 alone or both mutant subunits together were created and compared to cell lines expressing the respective wild-type proteins. Mutant and wild-type subunits were found to have very similar distributions between the cell surface and intracellular compartments. Constitutive internalization of H1 alone and ligand uptake and degradation by cells expressing both receptor subunits were not affected by the mutations. Cytoplasmic serines and serine phosphorylation are thus not essential for receptor function and intracellular traffic. Analysis of individual serine mutations identified serine-12 of subunit H2 as the major site of phosphorylation in the ASGP receptor. Images
Geffen, I; Fuhrer, C; Spiess, M
Overdose of D-serine Induces Movement Disorder and Neuromuscular Changes of Zebrafish Larvae
D-serine is a well-known activator of N-methyl-D-aspartate receptors; however, little is known about the teratogenic effects of D-serine overdose during early embryonic development. Here, we used zebrafish as a model to test toxicity and teratogenicity, since they have transparent eggs, making the organogenesis of zebrafish embryos easier to be observed. After D-serine injection (100�1000 ppm), the most evident defective phenotypes were bent trunk phenotypes, including malformed somite boundary, twisted body axis and shorter body length. As the injection dosages increased, the rates of embryos with bent trunk phenotypes decreased (0% for 0 ppm, n=573; 59.9~84.3% for 100�1000 ppm of D-serine, n=383�451). In addition, D-serine-injected embryos exhibited significantly reduced the frequencies of spontaneous in-chorion contraction (21.7 for 0 ppm vs. 18.3�0.9 for 100�1000 ppm D-serine, n=30) in comparison with mock-treated controls (0 ppm). Subtle changes are easily observed by staining with specific monoclonal antibodies F59, Znp1, Zn5 and ?-bungarotoxin to detect morphological changes in muscle fibers, primary motor axons, secondary motor axon projections and neuromuscular junctions, respectively. Our data show that overdose of D-serine leads to misalignment of muscle fibers and motor neuron defects, especially secondary motor neuron axonal growth defects.
Chen, Xing-Guang; Wang, Yun-Hsin; Wen, Chi-Chung; Chen, Yau-Hung
Human iPS Cell-Based Therapy: Considerations Before Clinical Applications
Generation of induced pluripotent stem (iPS) cells has revolutionized the field of regenerative medicine. With the exponential increase in iPS cell research in the past three years, human iPS cells have been derived with different technologies and from various cell types. From a translational perspective, however, a number of issues must be addressed before safe and high quality patient-specific iPS cells can be derived for clinical applications. In addition, iPS cell-based therapies also need to be thoroughly evaluated in pre-clinical animal models before they can be applied to human subjects.
Sun, Ning; Longaker, Michael T.; Wu, Joseph C.
Cell selective conditional null mutations of serine racemase demonstrate a predominate localization in cortical glutamatergic neurons.
D-serine, which is synthesized by the enzyme serine racemase (SR), is a co-agonist at the N-methyl-D-aspartate receptor (NMDAR). Crucial to an understanding of the signaling functions of D-serine is defining the sites responsible for its synthesis and release. In order to quantify the contributions of astrocytes and neurons to SR and D-serine localization, we used recombinant DNA techniques to effect cell type selective suppression of SR expression in astrocytes (aSRCKO) and in forebrain glutamatergic neurons (nSRCKO). The majority of SR is expressed in neurons: SR expression was reduced by ~65% in nSRCKO cerebral cortex and hippocampus, but only ~15% in aSRCKO as quantified by western blots. In contrast, nSRCKO is associated with only modest decreases in D-serine levels as quantified by HPLC, whereas D-serine levels were unaffected in aSRCKO mice. Liver expression of SR was increased by 35% in the nSRCKO, suggesting a role for peripheral SR in the maintenance of brain D-serine. Electrophysiologic studies of long-term potentiation (LTP) at the Schaffer collateral-CA1 pyramidal neuron synapse revealed no alterations in the aSRCKO mice versus wild-type. LTP induced by a single tetanic stimulus was reduced by nearly 70% in the nSRCKO mice. Furthermore, the mini-excitatory post-synaptic currents mediated by NMDA receptors but not by AMPA receptors were significantly reduced in nSRCKO mice. Our findings indicate that in forebrain, where D-serine appears to be the endogenous co-agonist at NMDA receptors, SR is predominantly expressed in glutamatergic neurons, and co-release of glutamate and D-serine is required for optimal activation of post-synaptic NMDA receptors. PMID:22362148
Benneyworth, Michael A; Li, Yan; Basu, Alo C; Bolshakov, Vadim Y; Coyle, Joseph T
Combustion of PMMA, PE, and PS in a ramjet
This paper reports the combustion behavior of polymethylmetharcrylate (PMMA), polyethylene (PE), and polystyrene (PS) with air investigated in a connected pipe test facility; spectroscopy showed the presence of OH, C{sub 2}, and CH and temperatures between 1300 and 3000 K during combustion. Particular attention was focused on regression rate and combustion efficiency and the role of temperature and soot production. The present investigation gives an understanding of the most important phenomena that control (or emanate from) the combustion of a cylindrical solid fuel with a rearward facing step, and this has application for solid fuel ramjets, the safe burning of toxic waste, and hot gas generators. The results are summarized.
van der Geld, C.W.M. (Eindhoven University of Technology, Faculty of Mechanical Engineering, 5600 MB Eindhoven (NL)); Korting, P.A.O.G. (Prins Maurits Lab. TNO, Rijswijk (Netherlands)); Wijchers, T. (Delft University of Technology (NL))
Genetic transformation in the methanogen Methanococcus voltae PS
Mutations causing requirements for histidine, purine, and vitamin B/sub 12/ were obtained in strain PS of Methanococcus voltae (archaebacteria) upon irradiation with UV or gamma rays. The first two mutations were shown to revert at low frequencies and were sued to demonstrate the occurrence of transformation with homologous, wild-type DNA. The transformation rates obtained for these presumably chromosomal markers were in the range of 2 to 100 transformants per ..mu..g of DNA. Mutants resistant to 2-bromoethanesulfonate and to 5-methyl-DL-tryptophan were also isolated.
Bertani, G.; Baresi, L.
Genetic transformation in the methanogen Methanococcus voltae PS
NASA Technical Reports Server (NTRS)
Mutations causing requirements for histidine, purine, and vitamin B12 were obtained in strain PS of Methanococcus voltae (archaebacteria) upon irradiation with UV or gamma rays. The first two mutations were shown to revert at low frequencies and were used to demonstrate the occurrence of transformation with homologous, wild-type DNA. The transformation rates obtained for these presumably chromosomal markers were in the range of 2 to 100 transformants per microgram of DNA. Mutants resistant to 2-bromoethanesulfonate and to 5-methyl-DL-tryptophan were also isolated.
Bertani, G.; Baresi, L.
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.
Fister, K. Renee
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.
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
McCarthy, Maeve L; Fister, K Renee
Radio observations of PS1-12fo (=CSS120121)
NASA Astrophysics Data System (ADS)
PS1-12fo is an ultra-luminous supernova of Type Ic at z=0.175 (ATel #3918), belonging to the SN class identified by Quimby et al. (2011, Nature, 474, 487). We searched for radio continuum emission from this SN with the EVLA on 2012 Feb 12.3 UT at a frequency of 5.9 GHz, yielding a non-detection of 2 � 7 microJy. This is consistent with the non-detection of radio emission from other objects in this class (Chomiuk et al.
Chomiuk, L.; Soderberg, A.; Margutti, R.; Berger, E.; Milisavljevic, D.; Sanders, N.
The effects of a low protein diet on amino acids and enzymes in the serine synthesis pathway in mice.
L-serine is required for cellular and tissue growth and is particularly important in the immature brain where it acts as a crucial neurotrophic factor. In this study, the levels of amino acids and enzymes in the L-serine biosynthetic pathway were examined in the forebrain, cerebellum, liver, and kidney after the exposure of mice to protein-restricted diets. The levels of L-serine, D-serine, and L-serine-O-phosphate were quantified by HPLC and quantitative Western blotting was used to measure changes in protein levels of five enzymes in the pathway. The L-serine biosynthetic enzyme phosphoserine phosphatase was strongly upregulated, while the serine degradative enzymes serine racemase and serine dehydratase were downregulated in the livers and kidneys of mice fed low (6%) or very low (2%) protein diets for 2 weeks compared with mice fed a normal diet (18% protein). No changes in these enzymes were seen in the brain. The levels of L-serine increased in the livers of mice fed 2% protein; in contrast, D-serine levels were reduced below the limit of detection in the livers of mice given either the 6 or 2% diets. D-Serine is a co-agonist at the NMDA class of glutamate receptors; no alterations in NMDA-R1 subunit expression were observed in liver or brain after protein restriction. These findings demonstrate that the expression of L-serine synthetic and degradative enzymes display reciprocal changes in the liver and kidney to increase L-serine and decrease D-serine levels under conditions of protein restriction, and that the brain is insulated from such changes. PMID:19921396
Antflick, Jordan E; Baker, Glen B; Hampson, David Richard
Crystal Structure of Serine Racemase that Produces Neurotransmitter d-Serine for Stimulation of the NMDA Receptor
NASA Astrophysics Data System (ADS)
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. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 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 binds the substrate serine in the active site, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.
Goto, Masaru
Synthesis of d-erythro-Sphinganine through Serine-Derived ?-Amino Epoxides.
A total synthesis of d-erythro-sphinganine [(2S,3R)-2-aminooctadecane-1,3-diol] starting from commercial N-tert-butyloxycarbonyl-l-serine methyl ester is described. The approach is based on the completely stereoselective preparation of an ?-amino epoxide obtained by treating a protected l-serinal derivative with dimethylsulfoxonium methylide. The oxirane synthon is obtained with an anti configuration fitting the (2S,3R) stereochemistry of the 2-amino-1,3-diol polar head of d-erythro-sphinganine. The synthetic procedure afforded the target compound in a 68% overall yield based on the initial amount of the starting l-serine material. PMID:24807867
Siciliano, Carlo; Barattucci, Anna; Bonaccorsi, Paola; Di Gioia, Maria Luisa; Leggio, Antonella; Minuti, Lucio; Romio, Emanuela; Temperini, Andrea
Intramolecular Modulation of Serine Protease Inhibitor Activity in a Marine Cyanobacterium with Antifeedant Properties
Extracts of the Floridian marine cyanobacterium Lyngbya cf. confervoides were found to deter feeding by reef fish and sea urchins (Diadema antillarum). This antifeedant activity may be a reflection of the secondary metabolite content, known to be comprised of many serine protease inhibitors. Further chemical and NMR spectroscopic investigation led us to isolate and structurally characterize a new serine protease inhibitor 1 that is formally derived from an intramolecular condensation of largamide D (2). The cyclization resulted in diminished activity, but to different extents against two serine proteases tested. This finding suggests that cyanobacteria can endogenously modulate the activity of their protease inhibitors.
Matthew, Susan; Ratnayake, Ranjala; Becerro, Mikel A.; Ritson-Williams, Raphael; Paul, Valerie J.; Luesch, Hendrik
Neutron spectroscopic study of hydrogen bonding dynamics in L-serine.
Inelastic incoherent neutron scattering (IINS) spectra were obtained at 10 K for normal and deuterated L-serine. The geometry of L-serine molecule was optimized for the zwitterion form using ab initio HF, MP2 and DFT (B3LYP) levels with 6-31G* and 6-311 + +G4** basis sets. The theoretical frequencies of normal and d4-L-serine were compared with IINS spectra. Normal coordinate analysis and band assignments based on ab initio calculations and experimental data were presented. IINS frequencies due to the out-of-plane gamma(N-H...O) hydrogen bond motions were observed and identified. PMID:12477035
Pawlukoj?, A; Leciejewicz, J; Tomkinso, J; Parker, S F
Letter: some more aspects of formation and stability of the protonated serine octamer cluster.
The formation of protonated serine octamer clusters from homochiral and heterochiral monomer solutions was investigated. The well-established preference for homochiral cluster formation was found to originate from collision-induced dissociation of the less stable ion population B prior to reaching the mass spectrometer's analyzer cell. In addition, collision-induced dissociation experiments were undertaken to investigate the relative stabilities of populations A and B and infrared multi-photon dissociation experiments addressed the relative stabilities of the protonated serine octamer cluster and its metaclusters. Isotopically-labeled serine was used throughout the experiments. PMID:16531652
Mazurek, Ulf
Interactome Analyses of Mature ?-Secretase Complexes Reveal Distinct Molecular Environments of Presenilin (PS) Paralogs and Preferential Binding of Signal Peptide Peptidase to PS2*
?-Secretase plays a pivotal role in the production of neurotoxic amyloid ?-peptides (A?) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature ?-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the ?-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H+-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing ?-secretase complexes and was observed to influence A? production.
Jeon, Amy Hye Won; Bohm, Christopher; Chen, Fusheng; Huo, Hairu; Ruan, Xueying; Ren, Carl He; Ho, Keith; Qamar, Seema; Mathews, Paul M.; Fraser, Paul E.; Mount, Howard T. J.; St George-Hyslop, Peter; Schmitt-Ulms, Gerold
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.
Structure-guided reprogramming of serine recombinase DNA sequence specificity
Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences >�10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with >�80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.
Gaj, Thomas; Mercer, Andrew C.; Gersbach, Charles A.; Gordley, Russell M.; Barbas III, Carlos F.
Regulating the regulators: serine/arginine-rich proteins under scrutiny.
Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation. PMID:22941908
Risso, Guillermo; Pelisch, Federico; Quaglino, Ana; Pozzi, Berta; Srebrow, Anabella
Pramipexole reduces phosphorylation of ?-synuclein at serine-129.
?-Synuclein is a central component of the pathogenesis of Parkinson's disease (PD). Phosphorylation at serine-129 represents an important post-translational modification and constitutes the major form of the protein in Lewy bodies. Several kinases have been implicated in the phosphorylation of ?-synuclein. The targeting of kinase pathways as a potential to influence the pathogenesis of PD is an important focus of attention, given that mutations of specific kinases (LRRK2 and PINK1) are causes of familial PD. Pramipexole (PPX) is a dopamine agonist developed for the symptomatic relief of PD. Several in vitro and in vivo laboratory studies have demonstrated that PPX exerts neuroprotective properties in model systems of relevance to PD. The present study demonstrates that PPX inhibits the phosphorylation of ?-synuclein and that this is independent of dopamine receptor activation. PPX blocks the increase in phosphorylated ?-synuclein induced by inhibition of the ubiquitin proteasomal system. The phosphorylation of ?-synuclein occurs in part at least through casein kinase 2, and PPX in turn reduces the phosphorylation of this enzyme, thereby inhibiting its activity. Thus, PPX decreases the phosphorylation of ?-synuclein, and this mechanism may contribute to its protective properties in PD models. PMID:23681749
Chau, Kai-Yin; Cooper, J Mark; Schapira, Anthony Henry V
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
NMDA- and beta-amyloid1-42-induced neurotoxicity is attenuated in serine racemase knock-out mice.
D-Serine is detected in the brain and acts as a coagonist at the "glycine-site" of the NMDA-type glutamate receptor. Although D-serine can be directly produced from L-serine by serine racemase (SR), the relative contribution of SR in D-serine formation in vivo is not known. Pathological roles of brain D-serine mediating NMDA receptor overactivation are suggested in studies using in vitro culture systems. However, we have recently demonstrated the differential SR protein expression in vivo and in culture. Here, we reported an approximately 90% decrease in forebrain D-serine content in SR knock-out (KO) mice. We also found a reduced neurotoxicity induced by NMDA- and Abeta(1-42)- peptide injections into the forebrain in SR KO mice. These results suggest that SR is the major enzyme for D-serine production in the brain, D-serine is the predominant endogenous coagonist of the NMDA receptor in the forebrain, and D-serine may be involved in controlling the extent of NMDA receptor-mediated neurotoxic insults observed in disorders including Alzheimer's disease. The control of SR activity and D-serine level in the brain may lead to a novel strategy for neuroprotection against various neurodegenerative diseases. PMID:19118183
Inoue, Ran; Hashimoto, Kenji; Harai, Tomomi; Mori, Hisashi
Effects of Chronic D-Serine Elevation on Animal Models of Depression and Anxiety-Related Behavior
NMDA receptors are activated after binding of the agonist glutamate to the NR2 subunit along with a co-agonist, either L-glycine or D-serine, to the NR1 subunit. There is substantial evidence to suggest that D-serine is the most relevant co-agonist in forebrain regions and that alterations in D-serine levels contribute to psychiatric disorders. D-serine is produced through isomerization of L-serine by serine racemase (Srr), either in neurons or in astrocytes. It is released by astrocytes by an activity-dependent mechanism involving secretory vesicles. In the present study we generated transgenic mice (SrrTg) expressing serine racemase under a human GFAP promoter. These mice were biochemically and behaviorally analyzed using paradigms of anxiety, depression and cognition. Furthermore, we investigated the behavioral effects of long-term administration of D-serine added to the drinking water. Elevated brain D-serine levels in SrrTg mice resulted in specific behavioral phenotypes in the forced swim, novelty suppression of feeding and olfactory bulbectomy paradigms that are indicative of a reduced proneness towards depression-related behavior. Chronic dietary D-serine supplement mimics the depression-related behavioral phenotype observed in SrrTg mice. Our results suggest that D-serine supplementation may improve mood disorders.
Otte, David-Marian; Barcena de Arellano, Maria Luisa; Bilkei-Gorzo, Andras; Albayram, Onder; Imbeault, Sophie; Jeung, Haang; Alferink, Judith; Zimmer, Andreas
Improvement in regional CBF by L-serine contributes to its neuroprotective effect in rats after focal cerebral ischemia.
To investigate the mechanisms underlying the neuroprotective effect of L-serine, permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery while monitoring cerebral blood flow (CBF). Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1) reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2) improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3) increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca(2+)-activated K(+) channels on the cerebral blood vessel endothelium. PMID:23825613
Ren, Tao-Jie; Qiang, Ren; Jiang, Zheng-Lin; Wang, Guo-Hua; Sun, Li; Jiang, Rui; Zhao, Guang-Wei; Han, Le-Yang
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
New Class of Serine and Cysteine Protease Inhibitor with Chemotherapeutic Potential.
National Technical Information Service (NTIS)
Metastasis of cancer is dependent on a variety of biologically active compounds including serine proteases. Inhibition of proteases such as plasmin is a potential route for both suppression of metastatic lesions and control of primary tumor growth. Synthe...
T. C. Sanders
Epithelial-Derived, Integral Membrane, Kunitz-Type Serine Protease Inhibitor in Breast Cancer.
National Technical Information Service (NTIS)
HAI-1 was initially identified as cognate inhibitor of matriptase, a membrane-bound serine protease. Paradoxically HAI-1 is also required for matriptase activation, a process that requires sphingosine 1-phosphate (S1P)- mediated translocation of the prote...
C. Lin
Neurotrypsin, a novel multidomain serine protease expressed in the nervous system.
We have cloned a novel murine cDNA encoding a multidomain serine protease, termed neurotrypsin, which exhibits an unprecedented domain composition. The deduced amino acid sequence defines a mosaic protein of 761 amino acids consisting of a kringle domain, followed by three scavenger receptor cysteine-rich repeats, and a serine protease domain. Based on comparisons of the primary structure, the protease domain belongs to the subfamily of trypsin-like serine proteases. In situ hybridization revealed that the expression of neurotrypsin in the adult murine nervous system is confined to distinct subsets of neurons. The most prominent expression was found in the cerebral cortex, the hippocampus, and the amygdala. Le., structures engaged in the processing and storage of learned behaviors and memories. Together with the recently obtained evidence that extracellular serine proteases play a role in neural plasticity, this expression pattern suggests that the extracellular proteolytic action of neurotrypsin subserves structural reorganizations associated with learning and memory operations. PMID:9245503
Gschwend, T P; Krueger, S R; Kozlov, S V; Wolfer, D P; Sonderegger, P
Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish.
National Technical Information Service (NTIS)
Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic 'chymotrypsin family' (Group S1A by the 1998 Barrett nomenclature) has an unusual phylogenetic distribution, being especially common in animals, entirely ...
A. Rojas R. F. Doolittle
[Induction and characterization of induced pluripotent stem (iPS) cells: a review].
The somatic cells can be induced into ES-like stem cells when retrovirally infected the defined transcription factors including Oct4, Sox2, Klf4 and c-Myc. These ES-like cells are named induced pluripotent stem (iPS) cells and this method is called iPS technology. Until the end of 2009, iPS cell lines have been generated in various animal species, such as mouse, human, rhesus monkey, rat and pig. Mouse iPS cells are also used to generate chimera mice and viable mice through the tetraploid complementation. Although iPS cells are extremely similar to ES cells in both morphology and growth features, to generate iPS cells do need the defined culture procedures. Based on the update global iPS technology development and the iPS studies in our laboratory, this paper focused on the establishment of iPS cell lines and improvement of iPS cell culture condition. PMID:20575428
Cheng, De; Lei, Lei; Lu, Zhijuan; Li, Zhen; Wang, Huayan
Identification and purification of O ?-acetyl- l -serine sulphhydrylase in Penicillium chrysogenum
We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 188.8.131.52) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence\\u000a of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this\\u000a microorganism. Only O-acetyl-l-serine sulphhydrylase and
S. �stergaard; H. B. Aa. Theilgaard; J. Nielsen
Interactions between ?2-syntrophin and a family of microtubule-associated serine\\/threonine kinases
A screen for proteins that interact with ?2-syntrophin led to the isolation of MAST205 (microtubule-associated serine\\/threonine kinase-205 kD) and a newly identified homologue, SAST (syntrophin-associated serine\\/threonine kinase). Binding studies showed that ?2-syntrophin and MAST205\\/SAST associated via a PDZ�PDZ domain interaction. MAST205 colocalized with ?2-syntrophin and utrophin at neuromuscular junctions. SAST colocalized with syntrophin in cerebral vasculature, spermatic acrosomes and neuronal
Carey Lumeng; Stephanie Phelps; Gregory E. Crawford; Paul D. Walden; Kate Barald; Jeffrey S. Chamberlain
Characterization of an extracellular chymostatin-sensitive serine protease preferentially expressed in young plant tissues
Intercellular washing fluids from leaves of all tested higher plant species contained a serine-type protease which efficiently cleaved the artificial fluorogenic substrate MCA-Pro-Leu-Gly-Leu-Dnp-Ala-Arg (MCA). The activity varied between the species. The classification as serine protease was based on the sensitivity towards chymostatin and phenylmethylsulfonyl fluoride. MCA protease activity strongly declined with leaf age and was also detected in stems, roots
David Messdaghi; Karl-Josef Dietz
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 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�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 ?mol/min/mg, respectively, while the corresponding values of the dehydratase activity of l-serine were 2.2 mM and 80.4 ?mol/min/mg, respectively.
Ohnishi, Masato; Saito, Makoto; Wakabayashi, Sadao; Ishizuka, Morio; Nishimura, Katsushi; Nagata, Yoko; Kasai, Sabu
Novel neuroglial and glioglial relationships mediated by L-serine metabolism.
L-Serine is a non-essential amino acid that can be synthesized in the body. It derives from an intermediate of the glycolytic pathway, 3-phosphoglycerate, and utilized for the syntheses of proteins, other amino acids, membrane lipids, heme, and nucleotides. Emerging evidence indicates that L-serine functions as a glia-derived trophic factor, which strongly promotes the survival and differentiation of cultured neurons. L-Serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH) and small neutral amino acid transporter ASCT1 have been revealed to be expressed preferentially in the radial glia-astrocyte lineage and olfactory ensheathing glia of both adult and developing rodent brains. In contrast, these biosynthetic and transporter molecules for L-serine are faint or undetectable in neurons and phagocytic cells. In this review, we summarize recent progress to propose that L-serine synthesis in these glial cells and its supply to nearby neurons and other glia constitute a novel metabolic unit in the brain. Based on these neuroglial and glioglial relationships, glucose in neurons and phogocytes can be strategically used for energy production, while a variety of L-serine-derived biomolecules required for their proliferaton, survival, differentiation, and function are synthesized in and supplied from the radial glia-astrocyte lineage and olfactory ensheathing glia. A transient capillary expression of ASCT1 in fetal and neonatal brains further suggests that, in addition to the glia-borne L-serine, an active transport of blood-borne L-serine would play an essential role in neural development. PMID:12846552
Furuya, Shigeki; Watanabe, Masahiko
Serine proteinase from Bacillus brevis : lytic action on intact yeast cells
A serine proteinase which showed lytic acitivity against either intact cell or cell wall preparations of Candida utilis has been isolated from Bacillus brevis culture filtrate by affinity chromatography on bacitracin-silochrome and phenylboronale-Sepharose. Both its proteolytic and lytic activities were completely abolished by inhibitors of serine proteinases, including phenylmethylsul-phonylfluoride, the inhibitor from Actinomyces janthinus, and duck ovomucoid. The optimum pH
Tatyana S. Kalebina; Galina N. Rudenskaya; Irina O. Selyakh; Olga M. Khodova; Galina G. Chestukhina; Valentin M. Stepanov; Igor S. Kulaev
In-cell Selectivity Profiling of Serine Protease Inhibitors by Activity-based Proteomics
Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases
Ludovic C. J. Gillet; Kenji Namoto; Alexandra Ruchti; Sjouke Hoving; Danielle Boesch; Bruno Inverardi; Dieter Mueller; Michele Coulot; Patrick Schindler; Patrick Schweigler; Anna Bernardi; Shirley Gil-Parrado
Ontogeny of serine hydroxymethyltransferase isoenzymes in fetal sheep liver, kidney, and placenta.
Serine is an amino acid that is not transported from the placenta to the ovine fetus. Thus, fetal plasma serine levels may be controlled by flux through their relevant biosynthetic pathways. This study was designed to determine, in fetal sheep tissues, the ontogeny of the three key enzymes in the biosynthetic pathway for serine, the cytosolic (c) and mitochondrial (m) isoforms of serine hydroxymethyltransferase (SHMT), phosphoglycerate dehydrogenase (PGD), and phosphoserine aminotransferase (PSAT). PGD and PSAT activity did not vary during gestation in either liver (PSAT, 9.4 +/- 1.3 nmol/min/mg cytosolic protein; and PGD, 76 +/- 10 mU/mg protein) or placenta (PGD, 8.0 +/- 3.6 mU/mg protein). In the liver, cSHMT activity was low early in gestation (0.6 +/- 0.5 nmol/min/mg protein at 45 days), rose in the last one-third of gestation, and peaked in the newborn period (25 +/- 3 nmol/min/mg protein at 1 week of age). Hepatic cSHMT RNA levels parallel the activity pattern. Mitochondrial SHMT was stable throughout gestation and with low constant mSHMT RNA levels. In contrast, the kidney and placenta had high mSHMT and steady low cSHMT activity throughout gestation. These data support the possible role of SHMT in the fetal control of plasma serine levels. While cSHMT may contribute to fetal hepatic serine production, its activity pattern does not support a primary role in the control of fetal hepatic serine biosynthesis. In the placenta, mSHMT may be important for glycine production from serine. PMID:10607477
Narkewicz, M R; Moores, R R; Battaglia, F C; Frerman, F 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
Jonathan Lowther; Guillaume Charmier; Marine C. Raman; Hiroko Ikushiro; Hideyuki Hayashi; Dominic J. Campopiano
Bacterial serine palmitoyltransferase: a water-soluble homodimeric prototype of the eukaryotic enzyme
Serine palmitoyltransferase (SPT, EC 18.104.22.168) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to 3-ketodihydrosphingosine (KDS). We found that the gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395T have significant SPT activity, and purified SPT to homogeneity. Unlike eukaryotic enzymes, this enzyme was a water-soluble homodimeric protein. We isolated the
Hiroko Ikushiro; Hideyuki Hayashi; Hiroyuki Kagamiyama
Molecular Characterization of Membrane-Associated Soluble Serine Palmitoyltransferases from Sphingobacterium multivorum and Bdellovibrio stolpii
Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decar- boxylative condensation of L-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5-phosphate (PLP)-dependent -oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et
Hiroko Ikushiro; Mohammad Mainul Islam; Hiromasa Tojo; Hideyuki Hayashi
Comparison of Serine Palmitoyltransferase in Morris Hepatoma 7777 and Rat Liver1
Serine palmitoyltransferase (EC 22.214.171.124) catalyzes an initial and regulatory reaction of sphingolipid biosynthesis, the forma tion of a homologue of 3-ketosphinganine from L-serine and a fatty acyl coenzyme A thioester. We have demonstrated that this enzyme exists in microsomes from Morris hepatoma 7777 and, moreover, found that its specific activity was 199 ±23 pmol\\/min\\/mg of microsomal protein, which was significantly
Robert D. Williams; Daniel W. Nixon; Alfred H. Merrill
Partial Characterization of Matrix-Associated Serine Protease Inhibitors from Human Skin Cells
Serine protease inhibitors have important regulatory roles in angiogenesis, intravascular fibrinolysis, wound healing, and cell migration. In this study, the extracellular matrix secreted by cultured human keratinocytes, foreskin fibroblasts, and SV-40-transformed human skin fibroblasts was analyzed for serine protease inhibitors by substrate reverse zymography. We found that the extracellular matrix deposited by these cells contained three inhibitors (Mr 33,000, 31,000,
C. N. Rao; Christina L. Peavey; YueYing Liu; Jean-Christophe Lapiere; David T. Woodley
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.
Vaiyapuri, Sakthivel; Wagstaff, Simon C.; Harrison, Robert A.; Gibbins, Jonathan M.; Hutchinson, E. Gail
Cloning, genomic organization, chromosomal assignment and expression of a novel mosaic serine proteinase: epitheliasin
We report the isolation of a cDNA encoding a novel murine serine proteinase, epitheliasin. The cDNA spans 1753 bp and encodes a mosaic protein with a calculated molecular mass of 53?529 Da. Its domains include a cytoplasmic tail, a type II transmembrane domain, a low-density lipoprotein receptor class A domain, a cysteine rich scavenger receptor-like domain and a serine proteinase
Eric Jacquinet; Narayanam V. Rao; Gopna V. Rao; John R. Hoidal
Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA
CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to
L. Csanady; Donna Seto-Young; Kim W. Chan; Cristina Cenciarelli; Benjamin B. Angel; Jun Qin; Derek T. McLachlin; Andrew N. Krutchinsky; Brian T. Chait; Angus C. Nairn; David C. Gadsby
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.
Drapkin, Paola T; Monard, Denis; Silverman, Ann-Judith
Impaired D-serine-mediated cotransmission mediates cognitive dysfunction in epilepsy.
The modulation of synaptic plasticity by NMDA receptor (NMDAR)-mediated processes is essential for many forms of learning and memory. Activation of NMDARs by glutamate requires the binding of a coagonist to a regulatory site of the receptor. In many forebrain regions, this coagonist is d-serine. Here, we show that experimental epilepsy in rats is associated with a reduction in the CNS levels of d-serine, which leads to a desaturation of the coagonist binding site of synaptic and extrasynaptic NMDARs. In addition, the subunit composition of synaptic NMDARs changes in chronic epilepsy. The desaturation of NMDARs causes a deficit in hippocampal long-term potentiation, which can be rescued with exogenously supplied d-serine. Importantly, exogenous d-serine improves spatial learning in epileptic animals. These results strongly suggest that d-serine deficiency is important in the amnestic symptoms of temporal lobe epilepsy. Our results point to a possible clinical utility of d-serine to alleviate these disease manifestations. PMID:23926260
Klatte, Katharina; Kirschstein, Timo; Otte, David; Pothmann, Leonie; M�ller, Lorenz; Tokay, Tursonjan; Kober, Maria; Uebachs, Mischa; Zimmer, Andreas; Beck, Heinz
Serine racemase as a prime target for age-related memory deficits.
The learning and memory deficits associated with non-pathological ageing mainly result from alterations to the plasticity of neuronal network dynamics within the hippocampus. In addition to the broad spectrum of changes that affect the morphology and function of hippocampal excitatory circuits in the ageing brain, the impaired activation of the N-methyl-D-aspartate subtype of glutamate receptors (NMDA-R) is a typical feature, altering the induction and maintenance of long-term potentiation, a major form of synaptic plasticity. In addition to glutamate, the binding of a co-agonist at the strychnine-insensitive glycine-binding site is required for NMDA-R activation. This review presents recent evidence that: (i) the amino acid D-serine is an endogenous co-agonist of synaptic NMDA-R and necessary for long-term potentiation expression, (ii) reduced d-serine levels in the hippocampus contribute to synaptic plasticity and memory deficits in normal ageing, and (iii) age-related oxidative stress selectively targets hippocampal serine racemase to impact D-serine availability in neuronal networks. These results emphasize the critical role of the hippocampal d-serine-dependent pathway in changes affecting neuronal network dynamics in physiological ageing that underlie memory deficits. In addition, the central role of serine racemase in these changes opens new perspectives in the search for relevant therapeutic strategies aimed at reducing age-related memory defects. PMID:23773062
Billard, J-M
Thermodynamic characteristics of protolytic equilibria of L-serine in aqueous solutions
NASA Astrophysics Data System (ADS)
The heat effects of the reaction of aqueous solution of L-serine with aqueous solutions of HNO3 and KOH were determined by calorimetry at temperatures of 288.15, 298.15, and 308.15 K, and ionic strength values of 0.2, 0.5, and 1.0 (background electrolyte, KNO3). Standard thermodynamic characteristics (?r H o, ?r G o, ?r S o, ? C {/p o}) of the acid-base reactions in aqueous solutions of L-serine were calculated. The effect of the concentration of background electrolyte and temperature on the heats of dissociation of amino acid was considered. The combustion energy of L-serine by bomb calorimetry in the medium of oxygen was determined. The standard combustion and formation enthalpies of crystalline L-serine were calculated. The heats of dissolution of crystalline L-serine in water and solutions of potassium hydroxide at 298.15 K were measured by direct calorimetry. The standard enthalpies of formation of L-serine and products of its dissociation in aqueous solution were calculated.
Kochergina, L. A.; Volkov, A. V.; Khokhlova, E. A.; Krutova, O. N.
Contribution of the d-Serine-Dependent Pathway to the Cellular Mechanisms Underlying Cognitive Aging
An association between age-related memory impairments and changes in functional plasticity in the aging brain has been under intense study within the last decade. In this article, we show that an impaired activation of the strychnine-insensitive glycine site of N-methyl-d-aspartate receptors (NMDA-R) by its agonist d-serine contributes to deficits of synaptic plasticity in the hippocampus of memory-impaired aged rats. Supplementation with exogenous d-serine prevents the age-related deficits of isolated NMDA-R-dependent synaptic potentials as well as those of theta-burst-induced long-term potentiation and synaptic depotentiation. Endogenous levels of d-serine are reduced in the hippocampus with aging, that correlates with a weaker expression of serine racemase synthesizing the amino acid. On the contrary, the affinity of d-serine binding to NMDA-R is not affected by aging. These results point to a critical role for the d-serine-dependent pathway in the functional alterations of the brain underlying memory impairment and provide key information in the search for new therapeutic strategies for the treatment of memory deficits in the elderly.
Potier, B.; Turpin, F. R.; Sinet, P.-M.; Rouaud, E.; Mothet, J.-P.; Videau, C.; Epelbaum, J.; Dutar, P.; Billard, J.-M.
A novel high throughput screening assay for HCV NS3 serine protease inhibitors.
Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors. PMID:12505640
Berdichevsky, Yevgeny; Zemel, Romy; Bachmatov, Larisa; Abramovich, Alex; Koren, Ruth; Sathiyamoorthy, Peramachi; Golan-Goldhirsh, Avi; Tur-Kaspa, Ran; Benhar, Itai
Phosphorylation of serine 212 confers novel activity to human estrogen receptor ?
Estrogen receptor ? (ER?) can be phosphorylated at various residues, one of which is serine 212 in the DNA binding domain. The majority of human nuclear receptors conserves, as a motif, this serine residue within their DNA binding domain. Among these nuclear receptors, phosphorylation of the corresponding threonine 38 in the nuclear receptor CAR is essential for determining its activity [9]. Here, we have investigated the role of phosphorylated serine 212 in the regulation of ER? activity by comparing it with serine 236, another potential phosphorylation site within the DNA binding domain, and demonstrated that phosphorylation of serine 212 confers upon ER? a distinct activity regulating gene expression in Huh-7 cells. In Western blot analysis, wild type ER? and mutants ER? S212A, ER? S212D, ER? S236A and ER? S236D were equally expressed in the nucleus, thus indicating that phosphorylation does not determine nuclear localization of ER?. ER? S212D, but not ER? S236D, retained its capability of activating an ERE-reporter gene in luciferase assays. Similar results were also obtained for human ER?; the ER? S176D mutant retained its trans-activation activity, but the ER? S200D mutant did not. cDNA microarray and Ingenuity Pathway Analysis, employed on Huh-7 cells ectopically expressing either ER? S212A or ER? S212D, revealed that phosphorylation of serine 212 enabled ER? to regulate a unique set of genes and cellular functions.
Shindo, Sawako; Sakuma, Tsutomu; Negishi, Masahiko; Squires, James
Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration
Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called �classical� catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of �nonclassical� serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.
Ekici, Ozlem Dogan; Paetzel, Mark; Dalbey, Ross E.
STAT3 serine 727 phosphorylation influences clinical outcome in glioblastoma
Besides STAT3 tyrosine 705 phosphorylation (pTyr705-STAT3), phosphorylation of STAT3 at serine 727 (pSer727-STAT3) is shown to contribute to tumorigenesis and be closely related with resistance to radiotherapy and chemotherapy in glioma, but there is currently no study regarding its relevance to prognosis in glioblastoma (GBM). Here, the expression of phosphorylated STAT3 was detected in tumor specimens from 88 patients with newly diagnosed GBM by immunohistochemistry, the Kaplan-Meier survival curve and COX proportional hazards regression model were applied to estimate its influences on progression-free survival (PFS) and overall survival (OS). Immunohistochemical assay showed elevated expression of pSer727-STAT3 in GBM compared with normal brain tissue. Univariate analysis indicated significant correlations of high percentage of pSer727-STAT3 positive tumor cells with shorter PFS (P = 0.006) and OS (P = 0.002). In multivariate analysis, high pSer727-STAT3 expression was demonstrated as an independent unfavorable prognostic indicator for PFS (HR 1.830, P = 0.022) and OS (HR 1.797, P = 0.040). And patients with high expression of both pTyr705-STAT3 and pSer727-STAT3 had a poorer prognosis compared with the remainder (P < 0.005). In conclusion, the high proportion of pSer727-STAT3 positive neoplastic cells in GBM is an independent unfavorable prognostic factor, and increased expression of both pTyr705-STAT3 and pSer727-STAT3 is predictive of poorer clinical outcome, thereby adding to the growing evidence that STAT3 inhibition may be a potential therapeutic strategy in glioblastoma.
Lin, Guo-Shi; Chen, Yu-Peng; Lin, Zhi-Xiong; Wang, Xing-Fu; Zheng, Zong-Qing; Chen, Long
Serine-71 Phosphorylation of Rac1 Modulates Downstream Signaling
The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.
Schwarz, Janett; Proff, Julia; Havemeier, Anika; Ladwein, Markus; Rottner, Klemens; Barlag, Britta; Pich, Andreas; Tatge, Helma; Just, Ingo; Gerhard, Ralf
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.
Magnetic Properties of Layered Compound MnPS3
NASA Astrophysics Data System (ADS)
Magnetic properties of a layered compound MnPS3 were investigated by means of magnetic susceptibility, high field magnetization and electron spin resonance. Temperature and angular dependence of EPR line width are characteristic of 2D-antiferromagnet. The main exchange interaction in the ab-plane is estimated to be J/k{=}-9.5� 0.2 K. High field magnetization process and AFMR measurements at low temperatures below TN{=}78 K showed a spin-flop transition at Hc{=}38.0 kOe. They are satisfactorily explained by assuming the uniaxial anisotropy energy K{=}1.52� 0.10� 105 erg/cm3 perpendicular to the ab-plane.
Okuda, Kiichi; Kurosawa, Ko; Saito, Shozo; Honda, Makoto; Yu, Zhihong; Date, Muneyuki
OCT/PS-OCT imaging of brachial plexus neurovascular structures
NASA Astrophysics Data System (ADS)
Introduction: Optical coherence tomography (OCT) allows high-resolution imaging (less than 10 microns) of tissue structures. A pilot study with OCT and polarization-sensitive OCT (PS-OCT) was undertaken to image ex-vivo neurovascular structures (vessels, nerves) of the canine brachial plexus. Methods: OCT is an interferometry-based optical analog of B-mode ultrasound, which can image through non-transparent biological tissues. With approval of the USC Animal Care and Use Committee, segments of the supra- and infraclavicular brachial plexus were excised from euthanized adult dogs, and the ex-vivo specimens were placed in cold pH-buffered physiologic solution. An OCT beam, in micrometer translational steps, scanned the fixed-position bisected specimens in transverse and longitudinal views. Two-dimensional images were obtained from identified arteries and nerves, with specific sections of interest stained with hematoxylin-eosin for later imaging through a surgical microscope. Results: with the beam scan direction transverse to arteries, the resulting OCT images showed an identifiable arterial lumen and arterial wall tissue layers. By comparison, transverse beam OCT images of nerves revealed a multitude of smaller nerve bundles contained within larger circular-shaped fascicles. PS-OCT imaging was helpful in showing the characteristic birefringence exhibited by arrayed neural structures. Discussion: High-resolution OCT imaging may be useful in the optical identification of neurovascular structures during attempted regional nerve blockade. If incorporated into a needle-shaped catheter endoscope, such a technology could prevent intraneural and intravascular injections immediately prior to local anesthetic injection. The major limitation of OCT is that it can form a coherent image of tissue structures only to a depth of 1.5 - 2 mm.
Raphael, David T.; Zhang, Jun; Zhang, Yaoping; Chen, Zhongping; Miller, Carol; Zhou, Li
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.
Kim, Jongwoo; Park, Suwon; Rhee, Seung Hyong; Choi, Yong-Hoon; Chung, Young-uk; Hwang, Ho Young
Calibration of PS09, PS10, and PS11 trans-Alaska pipeline system strong-motion instruments, with acceleration, velocity, and displacement records of the Denali fault earthquake, 03 November 2002
In September, 2003, the Alyeska Pipeline Service Company (APSC) and the U.S. Geological Survey (USGS) embarked on a joint effort to extract, test, and calibrate the accelerometers, amplifiers, and bandpass filters from the earthquake monitoring systems (EMS) at Pump Stations 09, 10, and 11 of the Trans-Alaska Pipeline System (TAPS). These were the three closest strong-motion seismographs to the Denali fault when it ruptured in the MW 7.9 earthquake of 03 November 2002 (22:12:41 UTC). The surface rupture is only 3.0 km from PS10 and 55.5 km from PS09 but PS11 is 124.2 km away from a small rupture splay and 126.9 km from the main trace. Here we briefly describe precision calibration results for all three instruments. Included with this report is a link to the seismograms reprocessed using these new calibrations: http://nsmp.wr.usgs.gov/data_sets/20021103_2212_taps.html Calibration information in this paper applies at the time of the Denali fault earthquake (03 November 2002), but not necessarily at other times because equipment at these stations is changed by APSC personnel at irregular intervals. In particular, the equipment at PS09, PS10, and PS11 was changed by our joint crew in September, 2003, so that we could perform these calibrations. The equipment stayed the same from at least the time of the earthquake until that retrieval, and these calibrations apply for that interval.
Evans, John R.; Jensen, E. Gray; Sell, Russell; Stephens, Christopher D.; Nyman, Douglas J.; Hamilton, Robert C.; Hager, William C.
Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to
Akitsu Hotta; Aaron Y L Cheung; Natalie Farra; Kausalia Vijayaragavan; Cheryle A S�guin; Jonathan S Draper; Peter Pasceri; Irina A Maksakova; Dixie L Mager; Janet Rossant; Mickie Bhatia; James Ellis
PS-IIDQ: a supported coupling reagent for efficient and general amide bond formation
Polystyrene-IIDQ, a polymer-supported coupling reagent, was synthesized in three steps from Merrifield resin in 86% overall conversion. This reagent efficiently coupled carboxylic acids to amines in good yields and high purities, required no pre-activation step, and was tolerant of the order of reagent addition. PS-IIDQ was observed to be more efficient than polymer-supported carbodiimides (PS-EDC and PS-DCC) and gave higher
Eric Valeur; Mark Bradley
Although Persistent Scatterer (PS) InSAR deformation measurements may be very precise, this does not necessarily imply a reliable estimation of the parameters of interest. PS-InSAR deformation measurements may be caused by different de- formation regimes, like gas extraction, shallow compaction or structural instabilities making unambiguous interpretation difficult. This research investigates the use of geo-information technology for the interpretation of PS-InSAR
S. Gehlot; V. B. H. Ketelaar; E. Verbree; R. F. Hanssen
Recursive data processing and data volume minimization for PS-InSAR
PS-InSAR has proven to be an accurate and ef- ficient technique for the joint estimation of topographic and displacement signal from stacked interferometric combinations. In this contribution a new method for PS-Insert processing is introduced, which enables the recursive estimation of parameters of interest. The method is based on the ILSQ PS-InSAR concept and makes use of the estimation vector
Petar S. Marinkovic; Freek van Leijen; Gini Ketelaar; Ramon F. Hanssen
Neutron Reflectivity Studies of Thin Films of Symmetric Block Copolymer and PS-Modified C60
NASA Astrophysics Data System (ADS)
C60 has a miscibility limit of approximately 1wt% in polystyrene as indicated by wide angle x-ray scattering and molecular dynamics simulations. In order to use block copolymers as a template for creating ordered arrays of fullerenes, it necessary to increase the concentration of C60 while maintaining dispersion. C60 grafted with six polystyrene arms (C60PS6) was shown to be miscible with PS up to 80wt% as C60 aggregation was not observed. Because C60PS6 offers improved miscibility with PS, we have investigated C60PS6 with a symmetric poly(deuterated styrene-b-methyl methacrylate) block copolymer (dPS-PMMA) using neutron reflectivity (NR). NR was used to examine the effects of nanoparticle concentration and size, annealing conditions, and thin film thickness on the location and formation of a C60PS6 layer and block copolymer phase behavior. Reflectivity results indicate the formation of dPS-PMMA lamellae oriented parallel to the silicon substrate after thermal annealing for films prepared from 4wt% solution at all concentrations of C60PS6 investigated. Similar results were seen for films prepared at different thicknesses and annealed at constant temperature.
Campbell, Katie; Kincer, Ryan; Bucknall, David; Thio, Yonathan; Beckham, Haskell
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
D-Serine-induced nephrotoxicity: a HPLC-TOF/MS-based metabonomics approach.
HPLC-MS-based metabonomic analysis was used to investigate urinary metabolic perturbations associated with D-serine-induced nephrotoxicity. D-Serine causes selective necrosis of the proximal straight tubules in the rat kidney accompanied by aminoaciduria, proteinuria and glucosuria. Alderely Park (Wistar-derived) rats were dosed with either D-serine (250 mg/kg ip) or vehicle (deionised water) and urine was collected at 0-12, 12-24, 24-36 and 36-48 h post-dosing. Samples were analysed using a Waters Alliance HT 2795 HPLC system coupled to a Waters Micromass Q-ToF-micro equipped with an electrospray source operating in either positive or negative ion mode. Changes to the urinary profile were detected at all time points compared to control. In negative ion mode, increases were observed in serine (m/z=103.0077), m/z=104.0376 (proposed to be hydroxypyruvate) and glycerate (m/z=105.0215), the latter being metabolites of D-serine. Furthermore, an increase in tryptophan, phenylalanine and lactate and decreases in methylsuccinic acid and sebacic acid were observed. Positive ion analysis revealed a decrease in xanthurenic acid, which has previously been assigned and reported using HPLC-MS following exposure to mercuric chloride and cyclosporine A. A general aminoaciduria, including proline, methionine, leucine, tyrosine and valine was also observed as well as an increase in acetyl carnitine. Investigation of additional metabolites altered as a result of exposure to D-serine is on-going. Thus, HPLC-MS-based metabonomic analysis has provided information concerning the mechanism of D-serine-induced renal injury. PMID:15596249
Williams, R E; Major, H; Lock, E A; Lenz, E M; Wilson, I D
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 presynaptic and postsynaptic 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 ephrinBs 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 postsynaptic 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 (protein interacting with C-kinase) and by dephosphorylating protein kinase C ? 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
High-power passively mode-locked Nd:YVO4 oscillator with adjustable pulse duration between 46 ps and 12 ps
We report on a high-power, passively mode-locked, TEM00 Nd:YVO4 oscillator with adjustable pulse duration between 46 and 12ps. The laser is end-pumped by an 888nm laser diode and mode-locking is achieved with a semiconductor saturable absorber mirror (SESAM). The laser has a repetition rate of 91MHz and the M2 beam quality factor is better than 1.2 at 15ps. At the
Marie-Christine Nadeau; St�phane Petit; Philippe Balcou; Romain Czarny; S�bastien Montant; Christophe Simon-Boisson
Adaptational modification of serine and threonine metabolism in the liver to essential amino acid deficiency in rats
It is known that plasma serine and threonine concentrations are elevated in rats chronically fed an essential amino acid deficient\\u000a diet, but the underlying mechanisms including related gene expressions or serine and threonine concentrations in liver remained\\u000a to be elucidated. We fed rats lysine or valine deficient diet for 4�weeks and examined the mRNA expressions of serine synthesising\\u000a (3-phosphoglycerate dehydrogenase,
Kenji Nagao; Makoto Bannai; Shinobu Seki; Masato Mori; Michio Takahashi
Induction of release and up-regulated gene expression of interleukin (IL)-8 in A549 cells by serine proteinases
BACKGROUND: Hypersecretion of cytokines and serine proteinases has been observed in asthma. Since protease-activated receptors (PARs) are receptors of several serine proteinases and airway epithelial cells are a major source of cytokines, the influence of serine proteinases and PARs on interleukin (IL)-8 secretion and gene expression in cultured A549 cells was examined. RESULTS: A549 cells express all four PARs at
Haiyan Wang; Yanshan Zheng; Shaoheng He
Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase.
Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D-serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca(2+) produced dilation of penetrating arterioles in a manner attenuated by scavenging D-serine with D-amino acid oxidase, deleting the enzyme responsible for D-serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D-serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling. PMID:23386721
Stobart, Jillian L LeMaistre; Lu, Lingling; Anderson, Hope D I; Mori, Hisashi; Anderson, Christopher M
A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins.
A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins). PMID:1637313
Stepanov, V M; Rudenskaya, G N; Revina, L P; Gryaznova, Y B; Lysogorskaya, E N; Filippova IYu; Ivanova, I I
Serine protease activity contributes to control of Mycobacterium tuberculosis in hypoxic lung granulomas in mice
The hallmark of human Mycobacterium tuberculosis infection is the presence of lung granulomas. Lung granulomas can have different phenotypes, with caseous necrosis and hypoxia present within these structures during active tuberculosis. Production of NO by the inducible host enzyme NOS2 is a key antimycobacterial defense mechanism that requires oxygen as a substrate; it is therefore likely to perform inefficiently in hypoxic regions of granulomas in which M. tuberculosis persists. Here we have used Nos2�/� mice to investigate host-protective mechanisms within hypoxic granulomas and identified a role for host serine proteases in hypoxic granulomas in determining outcome of disease. Nos2�/� mice reproduced human-like granulomas in the lung when infected with M. tuberculosis in the ear dermis. The granulomas were hypoxic and contained large amounts of the serine protease cathepsin G and clade B serine protease inhibitors (serpins). Extrinsic inhibition of serine protease activity in vivo resulted in distorted granuloma structure, extensive hypoxia, and increased bacterial growth in this model. These data suggest that serine protease activity acts as a protective mechanism within hypoxic regions of lung granulomas and present a potential new strategy for the treatment of tuberculosis.
Reece, Stephen T.; Loddenkemper, Christoph; Askew, David J.; Zedler, Ulrike; Schommer-Leitner, Sandra; Stein, Maik; Mir, Fayaz Ahmad; Dorhoi, Anca; Mollenkopf, Hans-Joachim; Silverman, Gary A.; Kaufmann, Stefan H.E.
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
Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana.
Serine biosynthesis in plants proceeds by two pathways; the glycolate pathway which is associated with photorespiration and the pathway from 3-phosphoglycerate which is presumed to take place in the plastids. The 3-phosphoglycerate pathway (phosphorylated pathway) involves three enzymes catalyzing three sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT) and 3-phosphoserine phosphatase (PSP). cDNA and genomic clones encoding these three enzymes from spinach and Arabidopsis thaliana were isolated by means of heterologous probe screening, homologous EST clones and genetic complementation in an Escherichia coli mutant. The identity of the isolated cDNAs was confirmed by functional complementation of serine auxotrophy in E. coli mutants and/or the detection of catalytic activity in the recombinant enzymes produced in E. coli. Northern blot analyses indicated the most preferential expression of these three genes in light-grown roots. In contrast, the mRNAs of two proteins involved in the glycolate pathway (H-protein of glycine decarboxylase multienzyme complex and serine hydroxymethyltransferase) accumulated to high levels in light-grown shoots. Environmental stresses, such as high salinity, flooding and low temperature, induced changes in mRNA levels of enzymes in the plastidic phosphorylated serine biosynthetic pathway but not in that of the glycolate pathway. These results indicate that the plastidic 3-phosphoglycerate pathway plays an important role in supplying serine in non-photosynthetic tissues in plants and under environmental stresses. PMID:11354602
Ho, C L; Saito, 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.
Antalis, Toni M.; Buzza, Marguerite S.; Hodge, Kathryn M.; Hooper, John D.; Netzel-Arnett, Sarah
d-Serine enhances fear extinction by increasing GluA2-containing AMPA receptor endocytosis.
Activation of the N-methyl-d-aspartate receptor (NMDAR) glycine site has been shown to enhance memory extinction in physiological and pathological conditions. In the current study, we examined the effects of d-serine, an endogenous NMDAR glycine site agonist, on fear extinction and ?-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) endocytosis in the hippocampus during the process of fear extinction. In inhibitory avoidance task, systemic administration of d-serine (800mg/kg, i.p.) significantly accelerated memory extinction. The Western blot analyses showed that the acceleration of memory extinction was accompanied by an increase in postsynaptic AMPAR endocytosis in the hippocampus. Furthermore, the application of a synthetic peptide Tat-GluA23Y (3.0?mol/kg, i.p.) that interferes with the endocytosis of AMPARs succeeded in preventing the enhancement of fear extinction and AMPAR endocytosis induced by d-serine. These results suggest that d-serine might enhance fear extinction through increasing GluA2-containing AMPA receptor endocytosis, and that d-serine may be a potential therapeutic agent against learning and memory disorders. PMID:24861709
Bai, Yanrui; Zhou, Limin; Wu, Xiaoyan; Dong, Zhifang
Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase
Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D-serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca2+ produced dilation of penetrating arterioles in a manner attenuated by scavenging D-serine with D-amino acid oxidase, deleting the enzyme responsible for D-serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D-serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling.
Stobart, Jillian L. LeMaistre; Lu, Lingling; Mori, Hisashi; Anderson, Christopher M.
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.
Martineau, Magalie; Parpura, Vladimir; Mothet, Jean-Pierre
Serine protease activity contributes to control of Mycobacterium tuberculosis in hypoxic lung granulomas in mice.
The hallmark of human Mycobacterium tuberculosis infection is the presence of lung granulomas. Lung granulomas can have different phenotypes, with caseous necrosis and hypoxia present within these structures during active tuberculosis. Production of NO by the inducible host enzyme NOS2 is a key antimycobacterial defense mechanism that requires oxygen as a substrate; it is therefore likely to perform inefficiently in hypoxic regions of granulomas in which M. tuberculosis persists. Here we have used Nos2-/- mice to investigate host-protective mechanisms within hypoxic granulomas and identified a role for host serine proteases in hypoxic granulomas in determining outcome of disease. Nos2-/- mice reproduced human-like granulomas in the lung when infected with M. tuberculosis in the ear dermis. The granulomas were hypoxic and contained large amounts of the serine protease cathepsin G and clade B serine protease inhibitors (serpins). Extrinsic inhibition of serine protease activity in vivo resulted in distorted granuloma structure, extensive hypoxia, and increased bacterial growth in this model. These data suggest that serine protease activity acts as a protective mechanism within hypoxic regions of lung granulomas and present a potential new strategy for the treatment of tuberculosis. PMID:20679732
Reece, Stephen T; Loddenkemper, Christoph; Askew, David J; Zedler, Ulrike; Schommer-Leitner, Sandra; Stein, Maik; Mir, Fayaz Ahmad; Dorhoi, Anca; Mollenkopf, Hans-Joachim; Silverman, Gary A; Kaufmann, Stefan H E
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.
Schell, Michael J
Investigation of charging behavior of PS particles in nonpolar solvents
NASA Astrophysics Data System (ADS)
The charging behavior of PS (polystyrene) particles dispersed in nonpolar solvent containing surfactant AOT (sodium di-2-ethylhexylsulfosuccinate) was researched by phase angle light scattering (PALS). The effects of the AOT concentration, the particle concentration and the particle size on the zeta potential of the particles were analyzed systemically. The results showed, at different particle concentrations (expressed in the volume fraction of 10 - 5-10 - 3), that the zeta potential could be adjusted by surfactant AOT over a wide concentration range of 0.001-100 mM. An obvious difference of zeta potential on particle concentration was revealed between the high AOT concentrations (beyond 10 mM) and the low ones (below 1 mM). Meantime, it was found that the relationship of particle size to zeta potential showed a great discrepancy between the dilute particle concentrations (below 10 - 4) and the concentrated ones (beyond 6 � 10 - 4). These findings were consistent with the mechanism of preferential adsorption of the charged micelles in nonpolar solvent.
Cao, Huiying; Cheng, Yongjian; Huang, Pinwen; Qi, Ming
Dynamics of ps-pulse induced gratings in LC panels
NASA Astrophysics Data System (ADS)
In the present work we focused our attention on studies of PVK:TNF hybrid polymer liquid crystal panels under short pulse laser illumination conditions. The diffraction gratings in a LC panel were induced by crossed beams generated by doubled in frequency Nd:YAG laser ((lambda) equals 532 nm) delivering pulses of 20 ps duration. So induced gratings were read by a cw laser radiation coming from a weak power He-Ne laser working at (lambda) equals 632.8 nm. The temporal evolution of intensity of first order diffraction measured in PVK:TNF hybrid liquid crystal panels shows many interesting features and complexity dependent on various experimental conditions. The substantial diffraction is observed already in time less than 1 ms after the pulse and the grating decay is completed within hundreds of milliseconds. At least three different steps of grating build-up can be distinguished which depend in various ways on the experimental conditions. A tentative mechanism of the observed responses is discussed in connection with the photoconductive properties of polymeric layers and the optical and electrical properties of the used liquid crystal E-7 (Merck).
Bartkiewicz, Stanislaw; Miniewicz, Andrzej; Sahraoui, Bouchta; Kajzar, Francois
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.
Processing of metals and dielectric materials with ps-laserpulses: results, strategies, limitations and needs
Ultra short (ps, fs) laser pulses are used, when high requirements concerning accuracy, surface roughness, heat affected zone etc. are demanded for surface structuring. Ps-laser systems that are suited to be operated in industrial environments are of great interest for many practical applications. Here results in the field of 3-d structuring (metals and transparent materials), induced processes and structuring of
Beat Neuenschwander; Guido F. Bucher; Christian Nussbaum; Benjamin Joss; Martin Muralt; Urs W. Hunziker; Peter Schuetz
Toughening mechanisms in melt manipulated thermoplastics (PS and PC) and in novel modified epoxies
This study examined the deformation mechanisms associated with the fatigue and fracture of polystyrene (PS) and polycarbonate (PC) processed using a novel polymer melt manipulation technique, vibration-assisted injection molding (VAIM), as well as the toughening mechanisms of several epoxy resins modified with several novel block co-polymers. VAIM processed PS was found to possess a tensile strength 28% greater than conventionally
Ryan Michael Hydro
1000-V, 300-ps pulse-generation circuit using silicon avalanche devices
A Marx configured avalanche transistor string and a pulse rise-time peaking diode are used to generate pulses of >1000 V into a 50-? load with rise times of less than 300 ps. The trigger delay of this circuit is about 7�10 ns, with jitter <100 ps. This circuit has been used to generate pulses at a repetition rate up to
D. M. Benzel; M. D. Pocha
Immortalization eliminates a roadblock during cellular reprogramming into iPS cells
The overexpression of defined transcription factors in somatic cells results in their reprogramming into induced pluripotent stem (iPS) cells. The extremely low efficiency and slow kinetics of in vitro reprogramming suggest that further rare events are required to generate iPS cells. The nature and identity of these events, however, remain elusive. We noticed that the reprogramming potential of primary murine
Jochen Utikal; Jose M. Polo; Matthias Stadtfeld; Nimet Maherali; Warakorn Kulalert; Ryan M. Walsh; Adam Khalil; James G. Rheinwald; Konrad Hochedlinger
Phytophthora sojae Effector PsCRN70 Suppresses Plant Defenses in Nicotiana benthamiana
Phytophthora sojae, an oomycete pathogen, produces a large number of effector proteins that enter into host cells. The Crinklers (Crinkling and Necrosis, CRN) are cytoplasmic effectors that are conserved in oomycete pathogens and their encoding genes are highly expressed at the infective stages in P. sojae. However, their roles in pathogenesis are largely unknown. Here, we functionally characterized an effector PsCRN70 by transiently and stably overexpressing it in Nicotiana benthamiana. We demonstrated that PsCRN70 was localized to the plant cell nucleus and suppressed cell death elicited by all the tested cell death-inducing proteins, including BAX, PsAvh241, PsCRN63, PsojNIP and R3a/Avr3a. Overexpression of the PsCRN70 gene in N. benthamiana enhanced susceptibility to P. parasitica. The H2O2 accumulation in the PsCRN70-transgenic plants was reduced compared to the GFP-lines. The transcriptional levels of the defense-associated genes, including PR1b, PR2b, ERF1 and LOX, were also down-regulated in the PsCRN70-transgenic lines. Our results suggest that PsCRN70 may function as a universal suppressor of the cell death induced by many elicitors, the host H2O2 accumulation and the expression of defense-associated genes, and therefore promotes pathogen infection.
Ru, Yanyan; Liu, Tingli; Xu, Jing; Liu, Li; Mafurah, Joseph Juma; Dou, Daolong
Genome Sequence of Non-O1 Vibrio cholerae PS15.
The draft genome sequence of a non-O1 Vibrio cholerae strain, PS15, organized into 3,512 open reading frames within a 3.9-Mb genome, was determined. The PS15 genome sequence will allow for the study of the evolution of virulence and environmental adaptation in V.�cholerae. PMID:23409261
Kumar, Sanath; Lindquist, Ingrid E; Sundararajan, Anitha; Rajanna, Chythanya; Floyd, Jared T; Smith, Kenneth P; Andersen, Jody L; He, Guixin; Ayers, Ryan M; Johnson, Judith A; Werdann, James J; Sandoval, Ava A; Mojica, Nadia M; Schilkey, Faye D; Mudge, Joann; Varela, Manuel F
Genome Sequence of Non-O1 Vibrio cholerae PS15
The draft genome sequence of a non-O1 Vibrio cholerae strain, PS15, organized into 3,512 open reading frames within a 3.9-Mb genome, was determined. The PS15 genome sequence will allow for the study of the evolution of virulence and environmental adaptation in V.�cholerae.
Kumar, Sanath; Lindquist, Ingrid E.; Sundararajan, Anitha; Rajanna, Chythanya; Floyd, Jared T.; Smith, Kenneth P.; Andersen, Jody L.; He, Guixin; Ayers, Ryan M.; Johnson, Judith A.; Werdann, James J.; Sandoval, Ava A.; Mojica, Nadia M.; Schilkey, Faye D.; Mudge, Joann
The directed differentiation of human iPS cells into kidney podocytes.
The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm's tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine. PMID:23029522
Song, Bi; Smink, Alexandra M; Jones, Christina V; Callaghan, Judy M; Firth, Stephen D; Bernard, Claude A; Laslett, Andrew L; Kerr, Peter G; Ricardo, Sharon D
The Directed Differentiation of Human iPS Cells into Kidney Podocytes
The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm�s tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.
Song, Bi; Smink, Alexandra M.; Jones, Christina V.; Callaghan, Judy M.; Firth, Stephen D.; Bernard, Claude A.; Laslett, Andrew L.; Kerr, Peter G.; Ricardo, Sharon D.
Mechanical Design Philosophy and Construction of the Amsterdam Pulse Stretcher Ring AmPS.
National Technical Information Service (NTIS)
The design philosophy and the construction of the Amsterdam Pulse Stretcher (AmPS) are described with emphasis on the vacuum components as well as on the alignment system and on the supports for the magnets and the monitors. The AmPS ring is a 900 MeV ele...
A. P. Kaan J. A. Heemskerk J. Touw J. Bijleveld H. B. Rookhuizen
Optical Design of AmPS: Elements of the Design of a Pulse Stretcher.
National Technical Information Service (NTIS)
The optical design of the AmPS (Amsterdam Pulse Stretcher) is addressed. The basic concepts of circular machine theory are presented and the structure of the AmPS is described. The considerations which led to this design are given; the resulting parameter...
Y. Wu
80 kV Electrostatic Wire Septum for AmPS.
National Technical Information Service (NTIS)
The characteristics of the wire septum for the Amsterdam Pulse Stretcher (AmPS) are summarized. In the extraction process of the AmPS the extracted beam is intercepted from the circulating beam by the 1 m long electrostatic wire septum. For a bending angl...
A. Vanderlinden J. H. M. Bijleveld H. B. Rookhuizen P. J. T. Bruinsma E. Heine
NASA PS400: A New Temperature Solid Lubricant Coating for High Temperature Wear Applications
NASA Technical Reports Server (NTRS)
A new solid lubricant coating, NASA PS400, has been developed for high temperature tribological applications. This plasma sprayed coating is a variant of the patented PS304 coating and has been formulated to provide higher density, smoother surface finish and better dimensional stability than PS304. PS400 is comprised of a nickel-molybdenum binder that provides strength, creep resistance and extreme oxidative and dimensional stability. Chromium oxide, silver and barium-calcium fluoride eutectic are added to the binder to form PS400.Tribological properties were evaluated with a pin-on-disk test rig in sliding contact to 650 C. Coating material samples were exposed to air, argon and vacuum at 760 C followed by cross section microscopic analysis to assess microstructure stability. Oil-Free microturbine engine hot section foil bearing tests were undertaken to assess PS400 s suitability for hot foil gas bearing applications. The preliminary results indicate that PS400 exhibits tribological characteristics comparable to the PS304 coating but with enhanced creep resistance and dimensional stability suitable for demanding, dynamic applications.
DellaCorte, C.; Edmonds, B. J.
Immunolocalization of PsNLEC-1, a lectin-like glycoprotein expressed in developing pea nodules.
The pea (Pisum sativum) nodule lectin gene PsNlec1 is a member of the legume lectin gene family that is strongly expressed in infected pea nodule tissue. A full-length cDNA sequence of PsNlec1 was expressed in Escherichia coli and a specific antiserum was generated from the purified protein. Immunoblotting of material from isolated symbiosomes revealed that the glycoprotein was present in two antigenic isoforms, PsNLEC-1A and PsNLEC-1B. The N-terminal sequence of isoform A showed homology to an eight-amino acid propeptide sequence previously identified from the cDNA sequence of isoform B. In nodule homogenates the antiserum recognized an additional fast-migrating band, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was associated with a 100,000 g nodule membrane fraction, suggesting an association with cytoplasmic membrane or vesicles. Immunogold localization in pea nodule tissue sections demonstrated that the PsNLEC-1 antigen was present in the symbiosome compartment and also in the vacuole but revealed differences in distribution between infected host cells in different parts of the nodule. These data suggest that PsNLEC-1 is subject to posttranslational modification and that the various antigenic isoforms can be used to monitor membrane and vesicle targeting during symbiosome development.
Dahiya, P; Kardailsky, I V; Brewin, N J
Initial point selection and validation in PS-InSAR using integrated amplitude calibration
SAR amplitude calibration is performed prior to the selection of potential Persistent Scatterers (PS) to avoid amplitude variations due to sensor characteristics and viewing geometry. As only the interferometric phases of a small percentage of the radar pixels in an image is used in the PS-InSAR analysis, it is investigated if this time and storage space consuming step can be
Gini Ketelaar; Freek van Leijen; Petar Marinkovic; Ramon Hanssen
Elastomeric Capture Microparticles (ECmuPs) and Their use with Acoustophoresis to Perform Affinity Capture Assays
NASA Astrophysics Data System (ADS)
This dissertation describes the development of elastomeric capture microparticles (ECmicroPs) and their use with acoustophoresis to perform affinity capture assays. EC?Ps that function as negative acoustic contrast particles were developed by crosslinking emulsion-based droplets composed of commercially available silicone precursors followed by functionalization with avidin/biotin reagents. The size distribution of the EC?Ps was very broad or narrow depending on the emulsion system that was used during the synthesis process. Elastomeric particles exhibited a very broad size distribution when a bulk-emulsion process was used; however, when microfluidic systems were utilized, their size distribution became comparatively narrow. The functionalization of elastomeric particles was accomplished by the non-specific adsorption of avidin protein followed by bovine serum albumin (BSA) blocking and bio-specific adsorption of a biotinylated-capture antibody. Polydisperse EC?Ps were functionalized to bind prostate specific antigen (PSA) or IgG-phycoerythrin (PE) in aqueous media (buffer, plasma, blood); whereas monodisperse EC?Ps were functionalized to bind a high density lipoprotein in the aqueous media. Polydisperse EC?Ps functionalized to bind PSA in a physiological buffer (PBS pH 7.4) demonstrated nanomolar detection using flow cytometry analysis; whereas EC?Ps functionalized to bind IgG-PE demonstrated picomolar detection in 10% porcine plasma. EC?Ps have a specific density of ~1.03 and are more compressible than their surrounding aqueous media; which allowed the EC?Ps to exhibit negative acoustic contrast properties under an ultrasonic acoustic standing wave field. The negative acoustic contrast property of EC?Ps was advantageously utilized in an IgG-PE assay conducted in 0.1% whole porcine blood. The ligand-bound EC?Ps suspended in the diluted blood sample were flowed through an acoustofluidic device where the application of an ultrasonic acoustic standing wave field focused the ligand-bound EC?Ps to pressure antinodes and the positive acoustic contrast blood cells to the central pressure node of the microchannel. As a result of laminar flow, focused ligand-bound EC?Ps and blood cells were flowed into properly aligned outlet channels at the downstream trifurcation, where they where collected separately off-chip. The cell-free fraction containing ligand-bound EC?Ps was analyzed using flow cytometry; where the detection of IgG-PE was in the picomolar range. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.
Cushing, Kevin Wallace
iPS cell technologies: significance and applications to CNS regeneration and disease.
In 2006, we demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling. In this review article, we outline the current status of applications of iPS technology to cell therapies (particularly for spinal cord injury), as well as neurological disease-specific iPS cell research (particularly for Parkinson's disease and Alzheimer's disease). Finally, future directions of iPS cell research are discussed including a) development of an accurate assay system for disease-associated phenotypes, b) demonstration of causative relationships between genotypes and phenotypes by genome editing, c) application to sporadic and common diseases, and d) application to preemptive medicine. PMID:24685317
Okano, Hideyuki; Yamanaka, Shinya
Study of mechanical properties of polyvinyl chloride (PVC) and polystyrene (PS) polymers and their blends
NASA Astrophysics Data System (ADS)
Presented work is an effort to observe the variation in mechanical properties of two thermoplastic materials PVC, PS and their blends. PVC and PS are taken in the ratio of 100:0, 70:30, 50:50, and 0:100. Mixing of PVC and PS is carried out by solution casting method using tetra hydro furan as solvent. Dynamical mechanical analyzer (DMA) is used to study mechanical properties. The storage modulus, loss modulus and mechanical loss factor (tan ?) are determined with temperature. The pallets of pure PS, PVC and their blends are scanned over a temperature range from room to 140 �C. The variation of modulus, tan ? of pure PVC & pure PS and their blends with temperature were studied. The observed variation in modulus and tan ? could be accounted for their thermal behavior and compositions.
Agarwal, Shalini; Saxena, N. S.; Agrawal, R.; Saraswat, Vibhav K.
TG/FTIR analysis on co-pyrolysis behavior of PE, PVC and PS.
The pyrolysis and co-pyrolysis behaviors of polyethylene (PE), polystyrene (PS) and polyvinyl chloride (PVC) under N2 atmosphere were analyzed by Thermal gravimetric/Fourier transform infrared (TG/FTIR). The volatile products were analyzed to investigate the interaction of the plastic blends during the thermal decomposition process. The TGA results showed that the thermal stability increased followed by PVC, PS and PE. The pyrolysis process of PE was enhanced when mixed with PS. However, PS was postponed when mixed with PVC. As for PE and PVC, mutual block was happened when mixed together. The FTIR results showed that the free radical of the decomposition could combine into a stable compound. When PE mixed with PVC or PS, large amount of unsaturated hydrocarbon groups existed in products while the content of alkynes was decreased. The methyl (-CH3) and methylene (-CH2-) bonds were disappeared while PVC mixed with PE. PMID:24411064
Wu, Jingli; Chen, Tianju; Luo, Xitao; Han, Dezhi; Wang, Zhiqi; Wu, Jinhu
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.
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.
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; Fikrig, Erol
GIT1 Phosphorylation on Serine 46 by PKD3 Regulates Paxillin Trafficking and Cellular Protrusive Activity*
The continuous assembly and disassembly of focal adhesions is required for efficient cell spreading and migration. The G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein whose dynamic localization to sites of cytoskeletal remodeling is critically involved in the regulation of these processes. Here we provide evidence that the subcellular localization of GIT1 is regulated by protein kinase D3 (PKD3) through direct phosphorylation on serine 46. GIT1 phosphorylation on serine 46 was abrograted by PKD3 depletion, thereby identifying GIT1 as the first specific substrate for this kinase. A GIT1 S46D phosphomimetic mutant localized to motile, paxillin-positive cytoplasmic complexes, whereas the phosphorylation-deficient GIT1 S46A was enriched in focal adhesions. We propose that phosphorylation of GIT1 on serine 46 by PKD3 represents a molecular switch by which GIT1 localization, paxillin trafficking, and cellular protrusive activity are regulated.
Huck, Bettina; Kemkemer, Ralf; Franz-Wachtel, Mirita; Macek, Boris; Hausser, Angelika; Olayioye, Monilola A.
GIT1 phosphorylation on serine 46 by PKD3 regulates paxillin trafficking and cellular protrusive activity.
The continuous assembly and disassembly of focal adhesions is required for efficient cell spreading and migration. The G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein whose dynamic localization to sites of cytoskeletal remodeling is critically involved in the regulation of these processes. Here we provide evidence that the subcellular localization of GIT1 is regulated by protein kinase D3 (PKD3) through direct phosphorylation on serine 46. GIT1 phosphorylation on serine 46 was abrograted by PKD3 depletion, thereby identifying GIT1 as the first specific substrate for this kinase. A GIT1 S46D phosphomimetic mutant localized to motile, paxillin-positive cytoplasmic complexes, whereas the phosphorylation-deficient GIT1 S46A was enriched in focal adhesions. We propose that phosphorylation of GIT1 on serine 46 by PKD3 represents a molecular switch by which GIT1 localization, paxillin trafficking, and cellular protrusive activity are regulated. PMID:22893698
Huck, Bettina; Kemkemer, Ralf; Franz-Wachtel, Mirita; Macek, Boris; Hausser, Angelika; Olayioye, Monilola A
Serine Proteases of Malaria Parasite Plasmodium falciparum: Potential as Antimalarial Drug Targets
Malaria is a major global parasitic disease and a cause of enormous mortality and morbidity. Widespread drug resistance against currently available antimalarials warrants the identification of novel drug targets and development of new drugs. Malarial proteases are a group of molecules that serve as potential drug targets because of their essentiality for parasite life cycle stages and feasibility of designing specific inhibitors against them. Proteases belonging to various mechanistic classes are found in P. falciparum, of which serine proteases are of particular interest due to their involvement in parasite-specific processes of egress and invasion. In P. falciparum, a number of serine proteases belonging to chymotrypsin, subtilisin, and rhomboid clans are found. This review focuses on the potential of P. falciparum serine proteases as antimalarial drug targets.
Converted-wave time domain registration using the inverted pseudo-PS-wave attribute section
NASA Astrophysics Data System (ADS)
The main aim of the event registration of PP- and PS-waves in multicomponent seismic exploration is to provide the same time/depth domain data for further interpretation and application to reservoir characterization and hydrocarbon detection. Most registration methods are based on waveform similarity and phase consistency by correlating the stacked records, which may cause serious errors due to differences in PP- and PS-wave reflectivity. Our motivation was to develop a time matching workflow to improve the event registration of multicomponent seismic data and enhance joint interpretation techniques. In this paper, we present a prestack inversion method to obtain a pseudo-PS-wave (PPS) section based on prestack PP-wave gathers, and establish an attributes matching workflow based on the waveform similarity comparison of PP-, PS- and PPS-wave sections. We investigate the PP-wave prestack inversion method and the waveform similarity comparison of PP-, PS- and PPS-wave sections using synthetic data. The test on synthetic data clearly shows that the PPS-wave section has much better waveform similarity to the PS-wave section than the PP-wave due to the phase consistency of the reflectivity, increasing the correlation coefficient of waveform similarity to over 90% for PPS- and PS-waves from about 50% for PP- and PS-waves. The application to field data shows a very good improvement in the event registration, especially due to the partial nonexistence of events in the PP-wave section. The benefits for the time matching are also made evident by comparing the correlation sections of PPS- and PS-waves with PP- and PS-waves. The improved accuracy in event registration leads to better joint inversion and interpretation for the stratigraphic features and hydrocarbon detection in the subsequent applications.
Chen, Shuangquan; Li, Xiang-Yang; Tang, Jianming
Intron-exon organization of the active human protein S gene PS. alpha. and its pseudogene PS. beta. : Duplication and silencing during primate evolution
The human protein S locus on chromosome 3 consists of two protein S genes, PS{alpha} and PS{beta}. Here the authors report the cloning and characterization of both genes. Fifteen exons of the PS{alpha} gene were identified that together code for protein S mRNA as derived from the reported protein S cDNAs. Analysis by primer extension of liver protein S mRNA, however, reveals the presence of two mRNA forms that differ in the length of their 5{prime}-noncoding region. Both transcripts contain a 5{prime}-noncoding region longer than found in the protein S cDNAs. The two products may arise from alternative splicing of an additional intron in this region or from the usage of two start sites for transcription. The intron-exon organization of the PS{alpha} gene fully supports the hypothesis that the protein S gene is the product of an evolutional assembling process in which gene modules coding for structural/functional protein units also found in other coagulation proteins have been put upstream of the ancestral gene of a steroid hormone binding protein. The PS{beta} gene is identified as a pseudogene. It contains a large variety of detrimental aberrations, viz., the absence of exon I, a splice site mutation, three stop codons, and a frame shift mutation. Overall the two genes PS{alpha} and PS{beta} show between their exonic sequences 96.5% homology. Southern analysis of primate DNA showed that the duplication of the ancestral protein S gene has occurred after the branching of the orangutan from the African apes. A nonsense mutation that is present in the pseudogene of man also could be identified in one of the two protein S genes of both chimpanzee and gorilla. This implicates that silencing of one of the two protein S genes must have taken place before the divergence of the three African apes.
Ploos van Amstel, H.; Reitsma, P.H.; van der Logt, C.P.; Bertina, R.M. (University Hospital, Leiden (Netherlands))
Long term potentiation depends on release of D-serine from astrocytes
Long-term potentiation (LTP) of synaptic transmission provides an experimental model for studying mechanisms of memory1. The classical form of LTP relies on N-methyl-D-aspartate receptors (NMDARs), and it has emerged that astroglia can regulate their activation through Ca2+-dependent release of the NMDAR co-agonist D-serine2-4. Release of D-serine from glia enables LTP in cultures5 and explains a correlation between glial coverage of synapses and LTP in the supraoptic nucleus4. However, Ca2+ elevations in astroglia can also release other signalling molecules, most prominently glutamate6-8, Adenosine-5?-triphosphate9, and Tumor-Necrosis-Factor-?10,11 whereas neurons themselves can synthesise and supply D-serine12,13. Furthermore, loading an astrocyte with exogenous Ca2+ buffers does not suppress LTP in hippocampal area CA114-16, and the physiological relevance of experiments in cultures or strong exogenous stimuli applied to astrocytes has been questioned17,18. The involvement of glia in LTP induction thus remains controversial. Here we show that clamping internal Ca2+ in individual CA1 astrocytes blocks LTP induction at nearby excitatory synapses by reducing the occupancy of the NMDAR co-agonist sites. This LTP blockade can be reversed by exogenous D-serine or glycine whereas depletion of D-serine or disruption of exocytosis in an individual astrocyte blocks local LTP. We thus demonstrate that Ca2+-dependent release of D-serine from an astrocyte controls NMDAR-dependent plasticity in many thousands of excitatory synapses occurring nearby.
Henneberger, Christian; Papouin, Thomas; Oliet, Stephane H. R.; Rusakov, Dmitri A.
d-Amino acid oxidase controls motoneuron degeneration through d-serine
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving an extensive loss of motoneurons. Aberrant excitability of motoneurons has been implicated in the pathogenesis of selective motoneuronal death in ALS. d-Serine, an endogenous coagonist of N-methyl-d-aspartate receptors, exacerbates motoneuronal death and is increased both in patients with sporadic/familial ALS and in a G93A-SOD1 mouse model of ALS (mSOD1 mouse). More recently, a unique mutation in the d-amino acid oxidase (DAO) gene, encoding a d-serine degrading enzyme, was reported to be associated with classical familial ALS. However, whether DAO affects the motoneuronal phenotype and d-serine increase in ALS remains uncertain. Here, we show that genetic inactivation of DAO in mice reduces the number and size of lower motoneurons with axonal degeneration, and that suppressed DAO activity in reactive astrocytes in the reticulospinal tract, one of the major inputs to the lower motoneurons, predominantly contributes to the d-serine increase in the mSOD1 mouse. The DAO inactivity resulted from expressional down-regulation, which was reversed by inhibitors of a glutamate receptor and MEK, but not by those of inflammatory stimuli. Our findings provide evidence that DAO has a pivotal role in motoneuron degeneration through d-serine regulation and that inactivity of DAO is a common feature between the mSOD1 ALS mouse model and the mutant DAO-associated familial ALS. The therapeutic benefit of reducing d-serine or controlling DAO activity in ALS should be tested in future studies.
Sasabe, Jumpei; Miyoshi, Yurika; Suzuki, Masataka; Mita, Masashi; Konno, Ryuichi; Matsuoka, Masaaki; Hamase, Kenji; Aiso, Sadakazu
Role of zinc ion for catalytic activity in d-serine dehydratase from Saccharomyces cerevisiae.
d-Serine dehydratase from Saccharomyces cerevisiae (DsdSC) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme catalyzing d-serine dehydration. The enzyme contains 1 mol Zn(2+) in its active site and shows a unique zinc dependence. The Zn(2+) is essential for the d-serine dehydration, but not for the ?,?-elimination of ?-Cl-d-alanine catalyzed as a side-reaction. The fact that dehydration of d-threonine and d-allo-threonine, also catalyzed by DsdSC, is likewise Zn(2+) dependent indicates that Zn(2+) is indispensable for the elimination of hydroxyl group, regardless of the stereochemistry of C(?) . Removal of Zn(2+) results in a less polar active site without changing the gross conformation of DsdSC. (1) H NMR determined the rates of ?-hydrogen abstraction and hydroxyl group elimination of d-serine in (2) H(2) O to be 9.7 and 8.5 s(-1) , respectively, while the removal of Zn(2+) abolished both reactions. Mutation of Cys400 or His398 within the Zn(2+) binding sites to Ala endowed DsdSC with similar properties to those of the Zn(2+) -depleted wild-type enzyme: the mutants lost the reactivity toward d-serine and d-threonine but retained that toward ?-Cl-d-alanine. (1) H NMR analysis also revealed that both ?-hydrogen abstraction and hydroxyl group elimination from d-serine were severely hampered in the C400A mutant. Our data suggest that DsdSC catalyzes the ?-hydrogen abstraction and hydroxyl group elimination in a concerted fashion. PMID:22176976
Ito, Tomokazu; Koga, Kazushi; Hemmi, Hisashi; Yoshimura, Tohru
Dual cross-talk between nitric oxide and D-serine in astrocytes and neurons in the brain.
The present review describes the role of the putative cross-talk between two neurotransmitters, nitric oxide (NO) and D-serine, in the brain. Under physiological conditions NO homeostasis guarantees the correct function of NO in a number of events in the brain such as neurotransmission and vascular tone regulation. D-serine, produced in astrocytes, acts synergistically with glutamate at NMDA receptors on postsynaptic neurons. Neuronal and endothelial NO synthase (nNOS and eNOS) in astrocytes cross-talk with serine racemase (SR) and D-amino acid oxydase (DAAO), catalyzing the synthesis and degradation of D-serine, respectively. SR is inhibited by NO which activates DAAO. D-serine inhibits nNOS but not eNOS and activates SR. Astrocytes and neurons also cross-talk through NO/D-serine system. D-serine released from astrocytes induces a rapid increase in NO contents in postsynaptic neurons. Overall, D-serine production in astrocytes is negatively regulated by NO. Under inflammatory conditions, pro-inflammatory cytokines or Abeta induce, first, a drop in NO contents and an increase in the amounts of D-serine in astrocytes. Together with enhanced glutamate release from presynaptic neurons, D-serine induces an increase in Ca(2+) up-take into presynaptic neurons. In astrocytes an initial drop in NO contents triggers NF-kappaB activation followed by inducible NOS (iNOS) expression. iNOS-derived massive amounts of NO may potentially be toxic. Under schizophrenic conditions, D-serine production is down-regulated. Together with reduced glutamate release, this situation leads to the decreased NO production in postsynaptic neurons. In astrocytes induction of iNOS expression becomes predominant. Initial drop in nNOS-derived NO is potentially toxic in this scenario. PMID:20021361
Darra, Elena; Ebner, Florian Heinrich; Shoji, Kazuo; Suzuki, Hisanori; Mariotto, Sofia
Mapping of Stat3 serine phosphorylation to a single residue (727) and evidence that serine phosphorylation has no influence on DNA binding of Stat1 and Stat3
During their polypeptide ligand-induced activation Stats (signal transducers and activators of tran- scription) 1 and 3 acquire, in addition to an obligatory tyrosine phosphorylation, phosphorylation on serine which boosts their transactivating potential (Wen, Z., Zhong, Z. and Darnell, J. E. Jr. (1995) Cell 82, 241-250). By examining phosphopeptide maps of wild-type and mutant protein we show here that the Stat3
Zilong Wen; James E. Darnell
Role of Saccharomyces cerevisiae serine O-acetyltransferase in cysteine biosynthesis
Some strains of Saccharomyces cerevisiae have detectable activities of L-serine O-acetyltransferase (SATase) and O-acetyl-L-serine\\/O-acetyl-L-homoserine sulfhydrylase (OAS\\/OAH-SHLase), but synthesize L-cysteine exclusively via cystathionine by cystathionine ?-synthase and cystathionine ?-lyase. To untangle this peculiar feature in sulfur metabolism, we introduced Escherichia coli genes encoding SATase and OAS-SHLase into S. cerevisiaeL-cysteine auxotrophs. While the cells expressing SATase grew on medium lacking L-cysteine, those
Hiroshi Takagi; Kenji Yoshioka; Naoki Awano; Shigeru Nakamori; Bun-ichiro Ono
Theoretical simulation of the ROA spectra of neutral cysteine and serine
NASA Astrophysics Data System (ADS)
Vibrational Raman Optical Activity (ROA) spectra have been calculated for neutral conformations of amino acids cysteine and serine. The purpose of the study was to investigate the conformational dependence of the ROA differential intensity, paying particular attention to the conformation of the polar side chains. In cysteine, the ROA intensities of side chain vibrations tend to vary fairly regularly with the value of the ?(NCCS) dihedral angle, while for serine the formation of hydrogen bonds by the side chain hydroxyl group seems to be the deciding factor determining the ROA intensities of the side chain vibrations.
Pecul, Magdalena
Transfer of serine into polypeptides and myosin by chromatographic species of seryl-transfer ribonucleic acid.
The efficiencies of two chromatographic species of [3-H]seryl-tRNA, namely peaks I and II, in cell-free amino acid incorporation were investigated. The maximum yield of polypeptide seems to be the same for the reaction mixtures containing either peak I or peak II, suggesting that the efficiency of both peaks in total protein synthesis is the same. The efficiency of transfer of serine into myosin heavy subunit (myosin H) by peaks I and II was also investigated. Peak II of [3-H]seryl-tRNA transfers three times as much serine into myosin H as peak I.
Nwagwu, M
A para-nitrophenol phosphonate probe labels distinct serine hydrolases of Arabidopsis.
Activity-based protein profiling represents a powerful methodology to probe the activity state of enzymes under various physiological conditions. Here we present the development of a para-nitrophenol phosphonate activity-based probe with structural similarities to the potent agrochemical paraoxon. We demonstrate that this probes labels distinct serine hydrolases with the carboxylesterase CXE12 as the predominant target in Arabidopsis thaliana. The designed probe features a distinct labeling pattern and therefore represents a promising chemical tool to investigate physiological roles of selected serine hydrolases such as CXE12 in plant biology. PMID:21763150
Nickel, Sabrina; Kaschani, Farnusch; Colby, Tom; van der Hoorn, Renier A L; Kaiser, Markus
Genital warts in Burmeister's porpoises: characterization of Phocoena spinipinnis papillomavirus type 1 (PsPV-1) and evidence for a second, distantly related PsPV.
We identified sequences from two distantly related papillomaviruses in genital warts from two Burmeister's porpoises, including a PV antigen-positive specimen, and characterized Phocoena spinipinnis papillomavirus type 1 (PsPV-1). The PsPV-1 genome comprises 7879 nt and presents unusual features. It lacks an E7, an E8 and a bona fide E5 open reading frame (ORF) and has a large E6 ORF. PsPV-1 L1 ORF showed the highest percentage of nucleotide identity (54-55 %) with human papillomavirus type 5, bovine papillomavirus type 3 (BPV-3) and Tursiops truncatus papillomavirus type 2 (TtPV-2). This warrants the classification of PsPV-1 as the prototype of the genus Omikronpapillomavirus. PsPV-1 clustered with TtPV-2 in the E6 and E1E2 phylogenetic trees and with TtPV-2 and BPV-3 in the L2L1 tree. This supports the hypothesis that PV evolution may not be monophyletic across all genes. PMID:17554024
Van Bressem, Marie-Fran�oise; Cassonnet, Patricia; Rector, Annabel; Desaintes, Christian; Van Waerebeek, Koen; Alfaro-Shigueto, Joanna; Van Ranst, Marc; Orth, G�rard
Competitive Activity-Based Protein Profiling Identifies Aza-?-Lactams as a Versatile Chemotype for Serine Hydrolase Inhibition
Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are needed for assigning functions to these enzymes. We recently discovered a set of aza-?-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme�s serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme alpha, beta-hydrolase-10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC50 value ~ 30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, sequence-unrelated serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors.
Zuhl, Andrea M.; Mohr, Justin T.; Bachovchin, Daniel A.; Niessen, Sherry; Hsu, Ku-Lung; Berlin, Jacob M.; Dochnahl, Maximilian; Lopez-Alberca, Maria P.; Fu, Gregory C.; Cravatt, Benjamin F.
Accurate theoretical study of PS(q) (q = 0,+1,-1) in the gas phase.
Highly correlated ab initio methods were used in order to generate the potential energy curves and spin-orbit couplings of electronic ground and excited states of PS and PS(+). We also computed those of the bound parts of the electronic states of the PS(-) anion. We used standard coupled cluster CCSD(T) level with augmented correlation-consistent basis sets, internally contacted multi-reference configuration interaction, and the newly developed CCSD(T)-F12 methods in connection with the explicitly correlated basis sets. Core-valence correction and scalar relativistic effects were examined. Our data consist of a set of spectroscopic parameters (equilibrium geometries, harmonic vibrational frequencies, rotational constants, spin-orbit, and spin-spin constants), adiabatic ionization energies, and electron affinities. For the low laying electronic states, our calculations are consistent with previous works whereas the high excited states present rather different shapes. Based on these new computations, the earlier ultraviolet bands of PS and PS(+) were reassigned. For PS(-) and in addition to the already known anionic three bound electronic states (i.e., X(3)?(-), (1)?, and 1(1)?(+)), our calculations show that the (1)?(-), (3)?(+), and the (3)? states are energetically below their quartet parent neutral state (a(4)?). The depletion of the J = 3 component of PS(-)((3)?) will mainly occur via weak interactions with the electron continuum wave. PMID:22755576
Ben Yaghlane, Saida; Francisco, Joseph S; Hochlaf, Majdi
Phase Transition and Mechanical Properties of PS/PVC/CdS Polymeric Nanocomposites
NASA Astrophysics Data System (ADS)
The present study reports the phase transition temperature and mechanical properties of CdS dispersed PS-PVC nanocomposite through Dynamic Mechanical Analyzer (DMA). Thick films of polymeric nanocomposites have been synthesized by dispersing nano-filler particles of CdS in PS/PVC binary blend matrix. The surface morphology of PS/PVC blend samples has been characterized by Scanning Electron Microscopy (SEM) while the nanostructure of the CdS filler in PS/PVC/CdS composite has been ascertained through small angle X-ray Diffraction (XRD) technique. The phase transition temperature study of PS/PVC polymeric blends reveals that glass transition temperature, Tg, of the PS phase shifts towards lower temperature with the increase in PVC content in the blend whereas for CdS embedded polymeric phases of blends i.e. for PS/PVC/CdS samples, an increase in respective Tg values have been observed. This is suggestive to the fact that phase transition temperature and mechanical properties have been significantly influenced through the dispersion of CdS nano-filler particles in the studied polymeric blend series.
Mathur, Vishal; Dixit, Manasvi; Saxena, N. S.; Sharma, Kananbala
Detection of the H_2PS Free Radical by Laser Spectroscopy
NASA Astrophysics Data System (ADS)
The previously unobserved H_2PS free radical has been detected by laser-induced fluorescence (LIF) techniques. H_2PS (and D_2PS) were produced in a pulsed discharge jet using a precursor gas mixture of Cl_3PS vapor and hydrogen (or deuterium) in high pressure argon. Our ab initio predictions of the ground and excited state frequencies and excitation energy are in good agreement with the results obtained by vibrational analysis of the LIF and single vibronic level (SVL) emission spectra. High-resolution spectra of the hybrid 0^0_0 bands of H_2PS and D_2PS were analyzed by band contour methods to obtain approximate ground and excited state rotational constants and molecular structures. The electronic transition involves promotion of an electron from the ? to the ?^* orbital and is assigned as tilde{B} ^2A'-tilde{X} ^2A'. The results will be discussed in comparison to ab initio predictions and the spectra of other X_2PS radicals recently studied in our laboratory.
Grimminger, Robert A.; Clouthier, Dennis J.; Tarroni, Riccardo
Reprogramming in vivo produces teratomas and iPS cells with totipotency features.
Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine. PMID:24025773
Abad, Mar�a; Mosteiro, Lluc; Pantoja, Cristina; Ca�amero, Marta; Rayon, Teresa; Ors, Inmaculada; Gra�a, Osvaldo; Meg�as, Diego; Dom�nguez, Orlando; Mart�nez, Dolores; Manzanares, Miguel; Ortega, Sagrario; Serrano, Manuel
Dynamic dipole polarizabilities of H{sup ?} and Ps{sup ?} in weakly coupled plasmas
The effects of weakly coupled plasmas on the dynamic dipole polarizabilities of the H{sup ?} and Ps{sup ?} ions are investigated using highly correlated exponential wave functions. The Debye-H�ckel shielding approach of plasma modeling is used to represent weakly coupled plasma environments. In free-atomic cases, results obtained from the present study for H{sup ?} are in agreement with the available calculations and results for Ps{sup ?} are reported for the first time. Frequency-dependent polarizabilities of H{sup ?} and Ps{sup ?} as functions of screening parameter are also presented for the first time.
Kar, Sabyasachi [Center for Theoretical Atomic and Molecular Physics, The Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150080, Heilongjiang (China)] [Center for Theoretical Atomic and Molecular Physics, The Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150080, Heilongjiang (China); Li, H. W. [Center for Theoretical Atomic and Molecular Physics, The Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150080, Heilongjiang (China) [Center for Theoretical Atomic and Molecular Physics, The Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150080, Heilongjiang (China); Gengdan Institute of Beijing University of Technology, Beijing 101301 (China); Jiang, Pinghui [Center for Theoretical Atomic and Molecular Physics, The Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150080, Heilongjiang (China) [Center for Theoretical Atomic and Molecular Physics, The Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150080, Heilongjiang (China); College of Electrical and Information Engineering, Heilongjiang Institute of Technology, Harbin 150050 (China)
Synthesis and crystal structure of the new quaternary thiophosphate KPbPS 4
The new lead potassium thiophosphate KPbPS4 was synthesized by reacting Pb with an in situ formed melt of K2S3, P2S5 and S. Its structure was determined by single crystal X-ray diffraction. KPbPS4 crystallizes as yellow crystals in the orthorhombic system, space group Pnma (no. 62), a=17.045(3)��, b=6.660(1)��, c=6.473(1)��, V=734.9(3)��3, Z=4. The structure is isotypic with that of KEuPS4 and consists
I. Belkyal; M. El Azhari; Y.-D. Wu; W. Bensch; K.-F. Hesse; W. Depmeier
Preparation and characterization of polystyrene (PS)\\/layered double hydroxides (LDHs) composite by a heterocoagulation method
Polystyrene (PS)\\/layered double hydroxides (LDHs) composites were prepared for the first time from LDHs aqueous suspension\\u000a and PS emulsion by electrostatic assembly. The morphology characterization based on XRD and TEM showed that the LDHs were\\u000a partially exfoliated in PS matrix. By using a reactive emulsifier, i.e., sodium 2-hydroxyl-3-(methacryloxy)propane-1-sulfonate\\u000a (HMPS), which was able to copolymerize with styrene, a strong polymer-LDHs platelets
Jintao Yang; Feng Chen; Yongchang Ye; Zhengdong Fei; Mingqiang Zhong
The encapsulation of nucleoside monophosphates into Cd 0.8PS 3
The biological molecules cytidine monophosphate (CMP) monohydrate, and adenosine monophosphate (AMP) monohydrate, have been encapsulated using exfoliated single layer Cd0.8PS3, to form restacked Cd0.8PS3(NMP)x(H2O)y, where N stands for cytidine or adenosine. The layer spacings of these inorganic\\/organic hybrid materials depend strongly on x and y. We have found a range of Cd0.8PS3 layer expansions up to 21 � for CMP,
P. Westreich; D. Yang; R. F. Frindt
PS2: managing the next step in the Pan-STARRS wide field survey system
NASA Astrophysics Data System (ADS)
The Panoramic Survey Telescope and Rapid Response System (Pan-STARRS) is unique among the existing or planned major ground-based optical survey systems as the only "distributed aperture" system. The concept of increasing system �tendue by replicating small telescopes and digital cameras presents both management opportunities and challenges. The focus in this paper is on management lessons learned from PS1, and how those have been used to form the management plan for PS2. The management plan components emphasized here include technical development, financial and schedule planning, and critical path and risk management. Finally, the status and schedule for PS2 are presented.
Burgett, William S.
Analysis of the biomacromolecular architecture of eukaryotic and prokaryotic serine proteases
Summary We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered on the serine proteases and their natural inhibitors and substrates. It is essential that these approaches emphasize the comparison of these macromolecules at the separate levels of secondary, tertiary and quaternary structure. We assume in our analysis
Michael N. Liebman
Recombinant human serine racemase: enzymologic characterization and comparison with its mouse ortholog.
D-serine plays a key role in glutamatergic neurotransmission in mammalian brain as a co-agonist of N-methyl-D-aspartate receptors. The enzyme responsible for D-serine biosynthesis, serine racemase (SR), is therefore a promising target for treatment of neuropathologies related to glutamate receptor excitotoxicity, such as stroke or Alzheimer's disease. Much of the experimental work to date has been performed on mouse serine racemase, which shares a high level of sequence identity with its human ortholog. In this work, we report the synthesis of a human SR gene variant optimized for heterologous expression in Escherichia coli and describe the expression and purification of active recombinant human SR. This strategy may be of general interest to researchers wishing to express mammalian proteins in a bacterial system. Furthermore, we conduct a thorough analysis of the kinetics and inhibitor-sensitivity of the recombinant enzyme, and we provide the first direct comparison of human and mouse SR based on our kinetic data. The orthologs behave similarly overall and exhibit identical inhibition profiles, validating the use of mouse models in SR research. PMID:18812225
Hoffman, Hillary E; Jir�skov�, Jana; Ingr, Marek; Zvelebil, Marketa; Konvalinka, Jan
Cadmium-tolerance of transgenic Ipomoea aquatica expressing serine acetyltransferase and cysteine synthase
Ipomoea aquatica (water spinach) is a common aquatic plant growing in lakes and wetlands in Southeast Asia. Due to its vigorous growth, they were considered to be potentially useful for remediation of polluted water with, for example, high sulfate and heavy metals. In previous studies, we successfully constructed transgenic I. aquatica plants, which simultaneously expressed two genes encoding serine acetyltransferase
Parichart Moontongchoon; Supachitra Chadchawan; Natchanun Leepipatpiboon; Ancharida Akaracharanya; Atsuhiko Shinmyo; Hiroshi Sano
Membrane-bound serine protease matriptase-2 (Tmprss6) is an essential regulator of iron homeostasis
Proteolytic events at the cell surface are essential in the regulation of signal trans- duction pathways. During the past years, the family of type II transmembrane serine proteases (TTSPs) has acquired an in- creasing relevance because of their privi- leged localization at the cell surface, al- though our current understanding of the biologic function of most TTSPs is lim- ited.
Alicia R. Folgueras; Fernando Martin de Lara; Alberto M. Pendas; Cecilia Garabaya; Francisco Rodriguez; Aurora Astudillo; Teresa Bernal; Ruben Cabanillas; Carlos Lopez-Otin; Gloria Velasco
Serines in the Intracellular Tail of Podoplanin (PDPN) Regulate Cell Motility*
Podoplanin (PDPN) is a transmembrane receptor that affects the activities of Rho, ezrin, and other proteins to promote tumor cell motility, invasion, and metastasis. PDPN is found in many types of cancer and may serve as a tumor biomarker and chemotherapeutic target. The intracellular region of PDPN contains only two serines, and these are conserved in mammals including mice and humans. We generated cells from the embryos of homozygous null Pdpn knock-out mice to investigate the relevance of these serines to cell growth and migration on a clear (PDPN-free) background. We report here that one or both of these serines can be phosphorylated by PKA (protein kinase A). We also report that conversion of these serines to nonphosphorylatable alanine residues enhances cell migration, whereas their conversion to phosphomimetic aspartate residues decreases cell migration. These results indicate that PKA can phosphorylate PDPN to decrease cell migration. In addition, we report that PDPN expression in fibroblasts causes them to facilitate the motility and viability of neighboring melanoma cells in coculture. These findings shed new light on how PDPN promotes cell motility, its role in tumorigenesis, and its utility as a functionally relevant biomarker and chemotherapeutic target.
Krishnan, Harini; Ochoa-Alvarez, Jhon A.; Shen, Yongquan; Nevel, Evan; Lakshminarayanan, Meenakshi; Williams, Mary C.; Ramirez, Maria I.; Miller, W. Todd; Goldberg, Gary S.
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...
Contribution of Alanine-76 and Serine Phosphorylation in ?-Synuclein Membrane Association and Aggregation in Yeasts
In Parkinson's disease (PD), misfolded and aggregated ?-synuclein protein accumulates in degenerating midbrain dopaminergic neurons. The amino acid alanine-76 in ?-synuclein and phosphorylation at serine-87 and serine-129 are thought to regulate its aggregation and toxicity. However, their exact contributions to ?-synuclein membrane association are less clear. We found that ?-synuclein is indeed phosphorylated in fission yeast and budding yeast, the two models that we employed for assessing ?-synuclein aggregation and membrane association properties, respectively. Surprisingly, blocking serine phosphorylation (S87A, S129A, and S87A/S129A) or mimicking it (S87D, S129D) altered ?-synuclein aggregation in fission yeast. Either blocking or mimicking this phosphorylation increased endomembrane association in fission yeast, but only mimicking it decreased plasma membrane association in budding yeast. Polar substitution mutations of alanine-76 (A76E and A76R) decreased ?-synuclein membrane association in budding yeast and decreased aggregation in fission yeast. These yeast studies extend our understanding of serine phosphorylation and alanine-76 contributions to ?-synuclein aggregation and are the first to detail their impact on ?-synuclein's plasma membrane and endomembrane association.
Fiske, Michael; Valtierra, Stephanie; Solvang, Keith; Zorniak, Michael; White, Michael; Herrera, Sara; Konnikova, Alina; Brezinsky, Rebecca; DebBurman, Shubhik
The natural killer cell serine protease gene Lmet1 maps to mouse chromosome 10
Cytotoxic lymphocytes play a key role in immune responses against viruses and tumors. Lymphocyte-mediated cytolysis by both cytotoxic T lymphocytes (CTL) and natural killer (NK) cells is often associated with the formation of membrane lesions on target cells caused by exocytosis of cytoplasmic granule serine proteases and a pore-forming protein, perforin. A variety of granzymes have been found to reside within the cytoplasmic granules of cytotoxic lymphocytes, but unlike perforin, isolated serine proteases are not intrinsically lytic. However, a role for serine proteases in cellular cytotoxicity has been supported by the ability of protease inhibitors to completely abrogate lymphocyte cytotoxicity, and the demonstration that serine proteases can initiate DNA fragmentation in target cells transfected or pretreated with a sublytic concentration of perforin. Granzymes cloned in human, mouse, and rat encode four granzyme activities and all are expressed in either T cells, their thymic precursors, and/or NK cells. In particular, a rat granzyme that cleaves after methionine residues, but not phenylalanine residues and its human equivalent, human Met-ase 1, are unique granzymes with restricted expression in CD3-NK cells. 24 refs., 2 figs.
Thia, K.Y.T.; Smyth, M.J. [Austin Hospital, Heidelberg (Australia); Jenkins, N.A.; Gilbert, D.J.; Copeland, N.G. [NCI-Frederick Cancer Research and Development Center, Frederick, MD (United States)
Validation of serine�threonine protein phosphatase as the herbicide target site of endothall
Endothall, an older commercial herbicide, and cantharidin, a natural product from the blister beetle (Epicauta spp.), are close chemical analogues. A comparison of the effect of endothall and cantharidin on plants revealed a similarity in their level of phytotoxicity on both Arabidopsis thaliana and Lemna paucicostata. Cantharidin is a potent inhibitor of animal serine\\/threonine protein phosphatases. Protein phosphatases and kinases
Joanna Bajsa; Zhiqiang Pan; Franck E. Dayan; Daniel K. Owens; Stephen O. Duke
Serine palmitoyltransferase: role in apoptotic de novo ceramide synthesis and other stress responses
Serine palmitoyltransferase is the first and rate-limiting enzyme of sphingolipid synthesis. As such, it is a central control point in the synthesis of bioactivate sphingolipids, and it plays an important role in mediating cellular stress responses. In this review, its role in mediating these responses is discussed within the context of de novo ceramide synthesis. Furthermore, a discussion is provided
David K. Perry
Fungal Metabolite Sulfamisterin Suppresses Sphingolipid Synthesis through Inhibition of Serine Palmitoyltransferase �
Sphingolipids and their metabolites are known to modulate various cellular events including proliferation, differentiation, and apoptosis. Serine palmitoyltransferase (SPT) is the enzyme that catalyzes the first step of the biosynthesis of all sphingolipids. Here, we report that a newly identified antibiotic, sulfamisterin, derived from the fungus Pycnidiella sp., is a specific inhibitor of SPT. The chemical structure of sulfamisterin resembles
Akiko Yamaji-Hasegawa; Atsushi Takahashi; Yasuyuki Tetsuka; Yukiko Senoh; Toshihide Kobayashi
Permeability Barrier Disruption Increases the Level of Serine Palmitoyltransferase in Human Epidermis
Sphingolipids play an important role in the homeostasis and barrier function of human stratum corneum. A disturbance of sphingolipid formation is supposed to be a crucial factor for the increased transepidermal water loss in common skin diseases like atopic eczema or psoriasis. The key enzyme for de novo sphingolipid synthesis is serine palmitoyltransferase, which consists of two different subunits, named
Silke Stachowitz; Francesca Alessandrini; Dietrich Abeck; Johannes Ring; Heidrun Behrendt
Long Distance Translocation of Sucrose, Serine, Leucine, Lysine, and Carbon Dioxide Assimilates
To determine the selectivity of movement of amino acids from source leaves to sink tissues in soybeans (Glycine max [L.] Merr. `Wells'), 14C-labeled serine, leucine, or lysine was applied to an abraded spot on a fully expanded trifoliolate leaflet, and an immature sink leaf three nodes above was monitored with a GM tube for arrival of radioactivity. Comparisons were made with 14C-sucrose and 14CO2 assimilates. Radioactivity was detected in the sink leaf for all compounds applied to the source leaflet. A heat girdle at the source leaf petiole essentially blocked movement of applied compounds, suggesting phloem transport. Transport velocities were similar (ranged from 0.75 to 1.06 cm/min), but mass transfer rates for sucrose were much higher than those for amino acids. Hence, the quantity of amino acids entering the phloem was much smaller than that of sucrose. Extraction of source, path, and sink tissues at the conclusion of the experiments revealed that 80 to 90% of the radioactivity remained in the source leaflet. Serine was partially metabolized in the transport path, whereas lysine and leucine were not. Although serine is found in greater quantities than leucine and lysine in the source leaf and path of soybeans, applied leucine and lysine were transported at comparable velocities and in only slightly lower quantities than was applied serine. Thus, no selective barrier against entry of these amino acids into the phloem exists.
Housley, Thomas L.; Peterson, David M.; Schrader, Larry E.
Functional Activity of Eukaryotic Signal Sequences in Escherichia coli: the Ovalbumin Family of Serine Protease Inhibitors
It is widely assumed that the functional activity of signal sequences has been conserved throughout evolution, at least between Gram-negative bacteria and eukaryotes. The ovalbumin family of serine protease inhibitors (serpins) provides a unique tool to test this assumption, since individual members can be secreted (ovalbumin), cytosolic (leukocyte elastase inhibitor, LEI), or targeted to both compartments (plasminogen activator inhibitor 2,
D. Belin; L.-M. Guzman; S. Bost; M. Konakova; F. Silva; J. Beckwith
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.
Genome-wide survey of putative Serine\\/Threonine protein kinases in cyanobacteria
BACKGROUND: Serine\\/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes
Xiaowen Zhang; Fangqing Zhao; Xiangyu Guan; Yu Yang; Chengwei Liang; Song Qin
Divergent Behavior of Glycosylated Threonine and Serine Derivatives in Solid Phase Peptide Synthesis
Solid phase peptide coupling of glycosylated threonine derivatives was systematically evaluated. In contrast to glycosylated serine derivatives which are highly prone to epimerization, glycosylated threonine derivatives produce only negligible amounts of epimerization. Under forcing conditions, glycosylated threonine analogs undergo ?-elimination, rather than epimerization. Mechanistic studies and molecular modeling were used to understand the origin of the differences in reactivity.
Zhang, Yalong; Muthana, Saddam M.; Barchi, Joseph J.
Purification and cloning of an extracellular serine protease from the nematode-trapping fungus Monacrosporium cystosporium.
An extracellular protease (Mc1) was isolated from the nematode-trapping fungus Monacrosporium cystosporium by gel filtration, anion-exchange, and hydrophobic interaction chromatographies. This protease had a molecular mass of approximately 38 kDa and displayed an optimal activity at pH 7-9 and 56 degrees (over 30 min). Its proteolytic activity was highly sensitive to the serine protease inhibitor PMSF (phenylmethylsulfonylfluoride, 0.1 mM), indicating that it belonged to the serine-type peptidase group. The Michaelis constant (Km) and Vmax for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were 1.67x10-4 M and 0.6071 OD410 per 30 s, respectively. This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. Moreover, the enzyme could immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, suggesting that it might play a role in infection against nematodes. The encoding gene of Mc1 was composed of one intron and two exons, coding for a polypeptide of 405 amino acid residues. The deduced amino acid sequence of Mc1 showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mc1 possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases. PMID:18633281
Yang, Jin-Kui; Ye, Feng-Ping; Mi, Qi-Li; Tang, Song-Qing; Li, Juan; Zhang, Ke-Qin
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, UASCHA. We report isolation of a mutation, cha4-1, that impairs serine/threonine induction of CHA1 transcription. The cha4-1 allele causes noninducibility of a CHA1 p-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 Cys6 zinc cluster class. Gel retardation employing a recombinant peptide, Cha4p1-174, demonstrated that the peptide in vitro specifically binds UASCHA. Binding is abolished by a G-C to T-A mutation in the middle bases of the two CEZ-elements in UASCHA. The transcriptional activating ability of UASCHA derivatives in vivo correlates with their ability to bind Cha4p1-174 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 UASCHA. PMID:8889513
Holmberg, S; Schjerling, P
Nucleic acid molecules encoding a transmembrane serine protease 10, the encoded polypeptides and methods based thereon
US Patent & Trademark Office Database
Provided herein are type II transmembrane serine protease 10 (MTSP10) polypeptides. Zymogen and activated forms of these polypeptides as well as single and two chain forms of the protease domain are also provided. Methods using the polypeptides to identify compounds that modulate the activity of an MTSP10 are provided.