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Infrared and Raman spectroscopic characterization of the hydrogen-bonding network in l-serine crystal

Jarmelo, S.; Reva, I.; Carey, P. R.; Fausto, R.
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Artigo de Revista Científica Formato: aplication/PDF
ENG
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The IR spectra (4000-400 cm-1) of neat and isotopically substituted (ND/OD <= 10% D and [congruent with]30% D) polycrystalline l-serine ([alpha]-amino-[beta]-hydroxypropionic acid; HO-CH2-CH(NH3)+-COO-) were recorded in the temperature range 300-10 K and assigned. The isotopic-doping/low-temperature methodology, which allows for decoupling of individual proton vibrational modes from the crystal bulk vibrations, was used for estimating the lengths and energies of the different H-bonds present in l-serine crystal. To this end, the frequency shifts observed in both the NH/OH stretching and out-of-plane bending spectral regions (relatively to reference values for these vibrations in non-hydrogen-bonded l-serine molecules) were used, together with previously developed empirical correlations between these spectral parameters and the H-bond properties. In addition, the room-temperature Raman spectrum (4000-150 cm-1) of a single crystal of neat l-serine was also recorded and interpreted. A systematic comparison was made between the spectroscopic data obtained currently for l-serine and previously for dl-serine, revealing that the vibrational spectra of the two crystals reflect well the different characteristics of their hydrogen-bond networks...

The Raman spectra of serine and 3,3-dideutero-serine in aqueous solution

Jarmelo, S.; Carey, P. R.; Fausto, R.
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Artigo de Revista Científica Formato: aplication/PDF
ENG
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The Raman spectra of serine [[alpha]-amino-[beta]-hydroxypropionic acid; HOCH2CH(NH3)+COO-] and 3,3-dideutero-serine [HOCD2CH(NH3)+COO-] in aqueous solution were studied in the range 4000-300 cm-1. The data obtained for the deuterated compound are novel and provide compelling evidence that previously reported assignments for the undeuterated amino acid should be revised.; http://www.sciencedirect.com/science/article/B6THW-4KNMB16-1/1/19da7b30f32b5efeacb053228a66c4a4

Low-temperature infrared spectra and hydrogen bonding in polycrystalline dl-serine and deuterated derivatives

Jarmelo, S.; Reva, I.; Rozenberg, M.; Carey, P. R.; Fausto, R.
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Artigo de Revista Científica Formato: aplication/PDF
ENG
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The FT-IR spectra of polycrystalline dl-serine [[alpha]-amino-[beta]-hydroxypropionic acid; HO-CH2-CH(NH3)+-COO-] and isotopically substituted [ND/ODAlcohol (<10% and >90% D); CD2 (>98% D)] dl-serine were recorded in the range 4000-500 cm-1 in the temperature range 300-10 K, and fully assigned. The isotopic-doping/low-temperature methodology, which allows for decoupling of individual proton vibrational modes from the crystal bulk vibrations, was used to estimate the energies of the different H-bonds present in dl-serine crystal. To this end, the frequency shifts observed in both the NH/OH stretching and out-of-plane bending spectral regions (relatively to reference values for these vibrations in non-hydrogen-bonded dl-serine molecules) were used, together with previously developed empirical correlations. The results are compared with available structural data on this amino acid.; http://www.sciencedirect.com/science/article/B6THW-4J91R39-2/1/f8f35f435466c8a5204879d330cbadec

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

Menaldo, Danilo Luccas; Bernardes, Carolina Petri; Santos-Filho, Norival Alves; Moura, Laura de Andrade; Fuly, Andre Lopes; Arantes, Eliane Candiani; Sampaio, Suely Vilela
Fonte: ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER; PARIS Publicador: ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER; PARIS
Tipo: Artigo de Revista Científica
ENG
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This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the B beta chain and BpirSP41 on both A alpha and B beta chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner...

Serina endopeptidases de insetos e a interação inseto-planta; Insect serine-endopeptidases and plant-insect interactions

Lopes, Adriana Rios
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 03/05/2004 PT
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Serina endopeptidases de insetos, principalmente tripsinas e quimotripsinas, estão envolvidas na digestão inicial de proteínas. Genes codificadores para estas enzimas estão organizados em famílias multigênicas tendo expressão diferencial de acordo com a dieta do inseto, estando envolvidos no desenvolvimento de resistência a diferentes metabólitos secundários vegetais. Para uma melhor compreensão desta interação, fez-se necessário o isolamento destas enzimas para insetos de diferentes ordens, bem como a caracterização de suas especificidades por duas abordagens: (a) caracterização cinética dos subsítios componentes do sítio de ligação de tripsinas e quimotripsinas, utilizando diferentes substratos, modificadores químicos e inibidores e (b) estudos estruturais por modelagem molecular, clonagem, expressão e cristalização destas enzimas de insetos. Além disso, estudos evolutivos por análise de distância possibilitaram uma caracterização inicial da interação insetoplanta. Estas determinações permitiram verificar que tripsinas de insetos apresentam diferenças de especificidade tanto dentre as diferentes ordens de insetos quanto em relação às tripsinas de vertebrados, sendo que as tripsinas da ordem Lepidóptera apresentam troca de especificidade primária hidrolisando preferencialmente substratos P1 Lys. Foram também observadas diferenças de hidrofobicidade para os subsítios caracterizados sendo que estes apresentam hidrofobicidades crescentes segundo o grau de complexidade dos insetos na sua escala evolutiva. A troca de especificidade e o aumento da hidrofobicidade podem permitir a hidrólise dos inibidores vegetais protéicos. A análise das sequências de tripsinas de insetos por Neighbor Joining (NJ) compõe uma árvore de distâncias topologicamente semelhante à árvore de relações filogenéticas determinadas por morfologia. A sobreposição de estruturas pré -determinadas de tripsina complexada a diferentes inibidores permite a identificação de posições de interação enzima-inibidor que justificam a classificação em grupos distintos de enzimas sensíveis ou resistentes a presença de inibidores na dieta de insetos. Da mesma forma: a caracterização da especificidade das quimotripsinas de insetos permitiu a separação de grupos distintos de quimotripsinas. Estes grupos são sustentados pela substituição do resíduo 59 em insetos polífagos que alimentam-se de plantas que contêm cetonas naturais reativas. Estas caracterizações demonstram a importância de um estudo detalhado da especificidade de serina endopeptidases possibilitando o desenho de moléculas apropriadas para inibição destas e desenvolvimento de estratégias de controle de insetos.; Insect serine endopeptidases...

Estudo dos efeitos das serinoproteinases PA-BJ e Giroxinas isoladas de venenos de serpentes em cultura de células endoteliais; Studies on the effects of the serine proteinases PA-BJ and gyroxin, isolated from snake venoms, on endothelial cells in culture

Lima, Sergio Augusto de
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 10/05/2010 PT
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Neste estudo foram avaliados os efeitos das serinoproteinases PA-BJ e giroxina, isoladas dos venenos das serpentes Bothrops jararaca e Crotalus durissus terríficus, respectivamente, sobre células endoteliais (CEs) em cultura. Os resultados obtidos demonstraram que essas toxinas, nas concentrações utilizadas, não afetaram a viabilidade e a integridade das CEs. Por outro lado, induziram a liberação de PGI2, que foi significativamente reduzida por inibidores não seletivos e seletivos das ciclooxigenases -1 e -2 (COX-1 e -2), mas não afetaram a expressão protéica constitutiva das mesmas. Adicionalmente, foi demonstrado que o antagonista de receptores PAR-1, o SCH 79797, não alterou a liberação de PGI2, induzida pelas toxinas. Em conclusão, essas toxinas, em concentrações não citotóxicas, induziram a liberação de PGI2 a partir de CEs, de modo dependente da ativação das COX-1 e -2. Por outro lado, o receptor PAR-1 não parece ser importante para este efeito, nessas células.; In this study, the effects of PA-BJ and gyroxin, isolated from Bothrops jararaca and Crotalus durissus terrificus snake venoms, respectively, on endothelial cells in culture were investigated. Results showed that neither PA-BJ nor gyroxin affected the integrity of monolayers nor modified ECs viability in the periods of incubation tested. In contrast...

Clonagem de serino proteases do veneno da cascavel Crotalus durissus terrificus e expressão da giroxina em célula de mamífero; Cloning of serine proteases from the venom of rattlesnake Crotalus durissus terrificus and expression of a gyroxin in mammalian cells

Yonamine, Camila Miyagui
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 05/12/2007 PT
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As serino proteases participam de diversos processos fisiológicos (tal como o de coagulação) e patológicos. Essas enzimas estão amplamente distribuídas entre as espécies, são também toxinas dos venenos de serpentes, sendo denominadas SVSPs (snake venom serine proteases). Essas SVSPs são multifuncionais e contêm uma tríade catalítica formada pelos aminoácidos HDS. Algumas SVSPs são comercialmente disponíveis, sendo indicadas para o tratamento de infarto do miocárdio, tromboses e embolia pulmonar. No veneno de Crotalus durissus terrificus estão descritas até o momento, apenas duas SVSPs sendo que a mais estudada é a giroxina que representa cerca de 2,5% do veneno total. No presente estudo foi reportado a clonagem de sete serino proteases amplificadas a partir de uma biblioteca de cDNA de glândula de veneno de um único espécime adulto de Crotalus durissus terrificus. Estes clones foram analisados com relação à organização do cDNA, estrutura e prováveis funções. A construção do modelo tridimensional da giroxina permitiu verificar as similaridades com tripsina, trombina e outras SVSPs. A glicosilação e a presença de muitas pontes dissulfetos dificultam a obtenção das SVSP recombinantes na forma solúvel e com atividade...

Purification, characterization, and specificity determination of a new serine protease secreted by penicillium waksmanii

Graminho, Eduardo Rezende; Da Silva, Ronivaldo Rodrigues; De Freitas Cabral, Tatiana Pereira; Arantes, Eliane Candiani; Da Rosa, Nathalia Gonsales; Juliano, Luiz; Okamoto, Debora Noma; De Oliveira, Lilian Caroline Gonçalves; Kondo, Marcia Yuri; Juliano,
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 201-214
ENG
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The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl2, KCl, and BaCl, and partially inhibited by CuCl2, CoCl2 and totally inhibited by AlCl3 and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k cat/K m of 10,666 mM-1 s-1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k cat/K m of 7,500 mM -1 s-1. Basic and acidic side chain-containing amino acids performed best at subsite S1. Subsites S2, S3, S′ 2, and S′ 1, S ′ 3 showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain...

Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms

Fernandes de Oliveira, Liliane Maria; Ullah, Anwar; Masood, Rehana; Zelanis, Andre; Spencer, Patrick J.; Serrano, Solange M. T.; Arni, Raghuvir K.
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 282-290
ENG
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); 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 similar to 32 kDa whereas the two closely related serine proteinases from B. moojeni display apparent molecular masses of similar to 32 kDa and similar to 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. (C) 2013 Elsevier Ltd. All rights reserved.

Estudos comparativos da atividade cinética e trombina-símile de serinoproteases isoladas a partir dos venenos de Bothrops brazili e Bothrops roedingeri; Comparative studies of the kinetics and thrombin-like activity of serine proteases isolated from the venom of Bothrops brazili and Bothrops roedingeri

Augusto Vilca Quispe
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 19/03/2013 PT
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No presente trabalho duas novas serinoproteases com atividade trombina-simile, nomeadas TLBbz e TLBro isoladas a partir de Bothrops brazili e Bothrops roedingeri respectivamente, foram purificadas em um único passo cromatográfico por HPLC de fase reversa com um alto grau de pureza e homogeneidade molecular, sem perda da atividade biológica. Ambas serinoproteases foram caracterizadas fisico-quimicamente, revelando uma massa molecular relativa de TLBbz = 35,13 KDa e TLBro = 20,24 KDa por SDS-PAGE. Ambas apresentaram atividade proteolítica perante o substrato cromogenico DL-BaρNA. Os estudos da atividade cinética mostraram que as serinoproteases tiveram um comportamento michaeliano frente ao substrato DL-BaρNA, apresentando uma Vmax = 1,89 nmoles ρ-NA/Lt/min e KM = 0,853 mM para TLBbz; e Vmax = 0,0432 nmoles ρ-NA/Lt/min e KM = 0,039 mM para TLBro. Ambas serinoproteases apresentam uma atividade ótima em torno de 38 oC e pH 8,0; sendo inibidas pela ação do fluoreto de fenilmetilsulfonila (PMSF) e outros inibidores, através dos quais a atividade trombina-simile foi reduzida em mais dos 50 % (TLBbz = 86.2 % and TLBro = 42.6 %). Ambas TLBbz e TLBro possuem caráter acido ao apresentar um elevado numero de aminoácidos ácidos...

Functional and structural characterization of a new serine protease with thrombin-like activity TLBan from Bothrops andianus (Andean Lancehead) snake venom

Valeriano-Zapana, Jose Antonio; Steve Segovia-Cruz, Fernando; Miguel Rojas-Hualpa, Jose; Martins-de-Souza, Daniel; Ponce-Soto, Luis Alberto; Marangoni, Sergio
Fonte: Pergamon-Elsevier Science Ltd; Oxford Publicador: Pergamon-Elsevier Science Ltd; Oxford
Tipo: Artigo de Revista Científica
ENG
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A new serine protease with thrombin-like activity (TLBan) from Bothrops andianus (Andean Lancehead) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. TLBan is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with Mr similar to 29 kDa under reducing conditions and non-reducing similar to 25 kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65 Da) and exhibited high specificity for BA rho NA, Michaelis-Menten behavior with Km 5.4 x 10(-1) M and the V-max 7.9 x 10(-1) nmoles rho-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60 degrees C), pHs (4.0 to 8.0), was inhibited by soybean trypsin inhibitor, EDTA and phenylmethylsulfonyl fluoride (PMSF). The total amino acid sequence was obtained through sequencing of selected tryptic peptides and by inference obtained using SwissProt database http://br.expasy.org/ with the search restricted to serine proteases from Crotalinae snakes and show high amino acid sequence identity with other serine proteases from snake venom. TLBan showed the presence of His(44), Asp(91) residues and Ser was deduced (187) position, in the corresponding positions to the catalytic triad established in the serine proteases and Ser(187) are inhibited by phenylmethylsulfonyl fluoride (PMSF). In this work...

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Morgado-Díaz,José Andrés; Silva-Lopez,Raquel Elisa da; Alves,Carlos Roberto; Soares,Maurilio José; Corte-Real,Suzana; De Simone,Salvatore Giovanni
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/07/2005 EN
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Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatus larvae

Nabian,Sedigheh; Taheri,Mohammad; Ranjbar,Mohammad Mehdi; Sazmand,Alireza; Youssefy,Parastou; Nazaralipour,Gholam Reza
Fonte: Colégio Brasileiro de Parasitologia Veterinária Publicador: Colégio Brasileiro de Parasitologia Veterinária
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2014 EN
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Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets

Silva, Fatima C. B. L.; Alcazar, Alonso; Macedo, Leonardo L. P.; Oliveira, Adeliana S.; Macedo, Francisco P.; Abreu, Luiz R. D.; Santos, Elizeu A.; Sales, Mauricio P.
Fonte: Insect Biochemistry and Molecular Biology Publicador: Insect Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
POR
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SILVA, Fatima C. B. L. et al. Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets. Insect Biochemistry and Molecular Biology, v. 36, p. 561-569, 2006.ISSN: 0965-1748.DOI: 10.1016/j.ibmb.2006.04.004.; The digestive system of Ceratitis capitata was characterized during its larval development and in the insect stage. Disaccharidases against maltose and sucrose were more evident in the 2nd and 3rd day of larval development and in the adult stage, respectively. Glycosil-hydrolyses with higher specific a-galactosidasic and b-galactosidasic activities were detected in the 2nd and 3rd day of the larval stage, respectively. Specific proteolytic activities against azocasein showed an increase in the 4th and 5th day of the larval stage and in the adult stage. Specific hemoglobin activities were constant between 2nd and 6th day of the larval stage. The larvae used mainly serine proteinases, such as trypsin/chymotrypsin, and the adult insects only chymotrypsin-like enzymes in their digestive process. Two serine proteinases were separated from zymogram between the 4th and 5th day of larval development and in the adult stage. Effect of soybean trypsin inhibitor (SBTI...

Untersuchungen zur Regulation der Phosphorylierung des Insulin-Rezeptor-Substrates-1 an Serin 318; Exploring regulation of phosphorylation in insulin-receptor-substrate-1 at serine 318

Fiedler, Hendrik
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
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Die genauen molekularen Mechanismen der Insulinresistenz, welcher eine bedeutende Rolle bei der Pathogenese des Diabetes mellitus Typ 2 zukommt, sind bis heute nicht vollständig aufgeklärt. Bekannt ist jedoch, dass die zelluläre Insulinresistenz u. a. mit einer vermehrten Phosphorylierung des Insulinrezeptorsubstrates-1 (IRS-1) an mehreren Serinen und Threoninen einhergeht. IRS-1 wird nach Stimulation mit dem Phorbolester 12-OTetradecanoylphorbol-13-acetat (TPA) bzw. Insulin an der kürzlich identifizierten Serinstelle 318 phosphoryliert. Der genaue Signalweg, der zu dieser Phosphorylierung führt, ist bislang nicht bekannt. In der vorliegenden Arbeit galt es daher zu untersuchen, ob Kinasen, für die beschrieben ist, dass sie IRS-1 phosphorylieren können, an dieser TPA- bzw. Insulin-induzierten Phosphorylierung beteiligt sind. Die Phosphorylierung von IRS-1 an Serin 318 wurde in C2C12-Myotuben mittels Western-Blotting nachgewiesen. Die 30-minütige Stimulation mit TPA führte zu einer starken Phosphorylierung von IRS-1 an Serin 318. Durch 30-minütige Vorinkubation mit dem PKCInhibitor Bisindolylmaleimid I (500 nM), konnte die Phosphorylierung nach 30-minütiger Stimulation mit 100 nM TPA deutlich abgeschwächt und die Phosphorylierung nach 30-minütiger Stimulation mit 1 und 10 nM TPA auf das basale Niveau reduziert werden. Die Stimulation mit 100 nM Insulin (30 min) führte ebenfalls zu einer starken Phosphorylierung von IRS-1 an Serin 318. Die Vorinkubation mit dem Inhibitor der c-Jun-N-terminalen Kinase (JNK) SP600125 (50 mikroM...

Effects of D-Serine on Visual Working Memory in Macaque Monkeys

Manjunath, Jaishri
Fonte: Quens University Publicador: Quens University
Tipo: Tese de Doutorado
EN; EN
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Schizophrenia is characterized by positive and negative symptoms along with cognitive symptoms that include impairment in working memory (WM). WM is the storage of relevant information for short intervals of time to guide thoughts and actions. The neural correlate of WM is thought to be the persistent activity exhibited during the retention interval of WM tasks. Persistent activity is hypothesized to be mediated by the activation of NMDA receptors (NMDAR) within recurrent neuronal circuits. Consistent with this hypothesis, studies with healthy humans and monkeys have shown that the administration of the NMDAR antagonist ketamine induces memory-load dependent deficits in WM, along with increasing response time. In parallel to this, the pathophysiology of schizophrenia has been hypothesized to rest on the hypofunction of NMDAR. Previous studies in humans indicate that blockade of NMDAR induces schizophrenia-like symptoms. In addition, symptoms of schizophrenia patients are alleviated with sub-chronic treatments focusing on the activation of the NMDAR co-agonist site. Based on these observations, I tested the hypothesis that increasing the activation of NMDAR with co-agonist stimulation has beneficial effects on WM. D-serine (100mg/kg/day-6 weeks) was orally administered to two female macaque monkeys performing a visual sequential comparison task (VCST)...

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

Pérez,A.V; Rucavado,A; Sanz,L; Calvete,J.J; Gutiérrez,J.M
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2008 EN
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A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pestresistant transgenic cotton plants

Oliveira Neto, Osmundo Brilhante de; Batista, João Aguiar Nogueira; Rigden, Daniel John; Fragoso, Rodrigo da Rocha; Silva, Rodrigo Osório da; Gomes, Eliane Aparecida; Franco, Octávio Luiz; Dias, Simoni Campos; Cordeiro, Célia Maria Torres; Pontes, Ros
Fonte: Universidade Católica de Brasília Publicador: Universidade Católica de Brasília
Tipo: Artigo de Revista Científica Formato: Texto
EN
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Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin- like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin- like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor...

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode meloidogyne incognita

Fragoso, Rodrigo da Rocha; Batista, João Aguiar Nogueira; Oliveira Neto, Osmundo Brilhante; Grossi- de-Sá, Maria Fátima
Fonte: Universidade Católica de Brasília Publicador: Universidade Católica de Brasília
Tipo: Artigo de Revista Científica Formato: Texto
EN
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This report describes the Wrst serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372 bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was ampliWed from total RNA of adult females by RT-PCR and 5_ and 3_ rapid ampliWcation of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of evelopmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.

Preferred stereoselective brain uptake of D-serine-a modulator of glutamatergic neurotransmission

Bauer, Dagmar; Hamacher, Kurt; Broer, Stefan; Pauleit, Dirk; Palm, Christoph; Zilles, Karl; Coenen, Heinz; Langen, Karl-Josef
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
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Although it has long been presumed that d-amino acids are uncommon in mammalians, substantial amounts of free d-serine have been detected in the mammalian brain. d-Serine has been demonstrated to be an important modulator of glutamatergic neurotransmission and acts as an agonist at the strychnine-insensitive glycine site of N-methyl-d-aspartate receptors. The blood-to-brain transfer of d-serine is thought to be extremely low, and it is assumed that d-serine is generated by isomerization of l-serine in the brain. Stimulated by the observation of a preferred transport of the d-isomer of proline at the blood-brain barrier, we investigated the differential uptake of [3H]-d-serine and [3H]-l-serine in the rat brain 1 h after intravenous injection using quantitative autoradiography. Surprisingly, brain uptake of [3H]-d-serine was significantly higher than that of [ 3H]-l-serine, indicating a preferred transport of the d-enantiomer of serine at the blood-brain barrier. This finding indicates that exogenous d-serine may have a direct influence on glutamatergic neurotransmission and associated diseases.