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Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

HUGO, Martin; TURELL, Lucia; MANTA, Bruno; BOTTI, Horacio; MONTEIRO, Gisele; NETTO, Luis E. S.; ALVAREZ, Beatriz; RADI, Rafael; TRUJILLO, Madia
Fonte: AMER CHEMICAL SOC Publicador: AMER CHEMICAL SOC
Tipo: Artigo de Revista Científica
ENG
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Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen`s antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together...

Biochemical characterization of serine acetyltransferase and cysteine desulfhydrase from Leishmania major

MARCIANO, Daniela; SANTANA, Marianela; MANTILLA, Brian Suarez; SILBER, Ariel Mariano; MARINO-BUSLJE, Cristina; NOWICKI, Cristina
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
ENG
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Cysteine metabolism exhibits atypical features in Leishmania parasites. The nucleotide sequence annotated as LmjF32.2640 encodes a cysteine desulfhydrase, which specifically catalyzes the breakdown of cysteine into pyruvate, NH(3) and H(2)S. Like in other pathogens, this capacity might be associated with regulatory mechanisms to control the intracellular level of cysteine, a highly toxic albeit essential amino acid, in addition to generate pyruvate for energy production. Besides, our results provide the first insight into the biochemical properties of Leishmania major serine acetyltransferase (SAT), which is likely involved in the two routes for de novo synthesis of cysteine in this pathogen. When compared with other members of SAT family, the N-terminal region of L. major homologue is uniquely extended, and seems to be essential for proper protein folding. Furthermore, unlike plant and bacterial enzymes, the carboxy-terminal-C(10) sequence stretch of L major SAT appears not to be implicated in forming a tight bi-enzyme complex with cysteine synthase. (C) 2010 Elsevier B.V. All rights reserved.; Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET); Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET); Universidad de Buenos Aires (UBA)...

Homocisteína e cisteína séricas como marcadores epigenéticos de prognóstico e preditivos de resposta em tumores de mama; Serum homocysteine and cysteine as epigenetic markers of prognosis and prediction of response in breast tumors

Raimundo, Luis Gustavo
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 28/02/2014 PT
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O câncer de mama é a principal causa de mortalidade por câncer entre as mulheres. Alguns biomarcadores e características clínicas são utilizados para avaliar o prognóstico e prever a resposta a uma série de abordagens terapêuticas. A Homocisteína é conhecida como um fator de risco para doença vascular aterosclerótica, mas sua participação na biologia do câncer ainda é incerta. Cisteína é o aminoácido sulfurado derivado da Homocisteína no ciclo da Metionina. Este ciclo metabólico origina as bases nitrogenadas e também determina o nível de metilação da molécula de DNA. É atualmente reconhecido que a hipometilação global do genoma é um evento chave na transformação maligna das células. O objetivo deste estudo foi avaliar os níveis séricos de homocisteína e cisteína como biomarcadores de sobrevida e de progressão da doença em câncer de mama. Também foi avaliado o efeito de um curso de curta duração (um mês) de tratamento hormonal sobre os níveis de Homocisteína, Cisteína e metilação do DNA. Amostras de sangue foram obtidos por ocasião da biópsia inicial (pré-tratamento) em todas as pacientes e, de tumor e de tecido normal adjacente, ao diagnóstico eem um mês após, para as pacientes que receberam o regime hormonal neo-adjuvante (pré-operatório). Todas as pacientes eram mulheres na pós-menopausa...

Cinética e mecanismo de redução de espécies de ferro-heme hipervalentes pelo H2S, cisteína e CO em relação à proteção do trato gastrointestinal e a qualidade da carne; Kinetic and mechanism of reduction of heme-iron species by H2S, Cysteine and CO in relation to the gastrointestinal tract protection and meat quality.

Libardi, Silvia Helena
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 16/12/2014 PT
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Estudos da reatividade de espécies oxidantes e a interação destas espécies com estruturas sensíveis a oxidação e antioxidantes em condições biológicassão de grande importância no entendimento dos processos redox em alimentos e no corpo humano. A mioglobina é a ferro heme proteína majoritária do músculo esquelético de mamíferos e a sua ativação por peróxido de hidrogênio dá origem às espécies reativas de ferro heme hipervalentes,perferrilmioglobina e ferrilmioglobina, que podem induzir a condição de estresse oxidativo. A reação das espécies de ferro heme hipervalentes com constituintes do meio biológico ou alimentos como proteínas ou membranas podem tanto afetar a qualidade de produtos cárneos quanto causar danos celulares no trato gastrointestinal durante sua digestão.Pequenas moléculas tais como o NO, H2S e CO são produzidas endogenamente em sistemas biológicos e, além de desempenharem importantes funções na manutenção dometabolismo celular,podem apresentar atividade antioxidante.A presente Tese procurou investigara cinética e o mecanismo para a redução das espécies perferrilmioglobina e ferrilmioglobina pelo monóxido de carbono, reação esta que apresentaconstante de velocidade de segunda ordem de k2 =(3...

Construção de uma plataforma funcional para detecção amperométrica de cisteína; Construction of a functional platform for amperometric detection of cysteine

Cecília de Carvalho Castro e Silva
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 01/08/2011 PT
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Este trabalho descreve o desenvolvimento de um sensor amperométrico para detecção eletrocatalítica de cisteína, através da construção de uma plataforma funcional para a complexação de íons cobre. O material base desta plataforma foi um nanocompósito obtido por meio da modificação de nanotubos de carbono de paredes múltiplas (MWCNTs) com poli(4-vinilpiridina) PVP, através uma reação de polimerização in situ. Foi realizada uma otimização multivariada, empregando um planejamento composto central de face centrada, para a modificação da superfície do eletrodo de carbono vítreo (ECV), sendo a condição ótima obtida quando se utiliza concentração da dispersão de nanocompósito de MWCNTs-PVP de 6,00 mg L, concentração da solução CuCl2 de 50 mmol L e tempo de complexação dos íons cobre de aproximadamente 83 minutos. A plataforma foi caracterizada por microscopia eletrônica de varredura (MEV), espectroscopia de energia dispersiva de raios-X (EDX), espectroscopia de impedância de eletroquímica (EIE), cronoamperometria e voltametria cíclica. Os valores da constante heterogênea de transferência de elétrons (ks) e da constante cinética da reação entre os íons Cu- cisteína (kobs) foram 5,78 s e 6...

Flow injection spectrofluorimetric determination of cystine and cysteine

Ensafi,Ali A.; Rezaei,B.; Nouroozi,S.
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2009 EN
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A relatively simple and sensitive procedure with spectrofluorimetric detection was developed for the determination of cystine and cysteine by flow injection system with sequential determination. This method is based on the reduction of Tl(III) with cysteine in acidic media, producing a fluorescence reagent, TlCl3(2-) (λex = 227 nm, λem = 419 nm). Before injection, the sample solution was divided into two streams. The first stream was treated with Cd reduction column and then joined with the carrier to react with Tl(III) at pH 5.0 and then passed through a 100 cm reaction coil to the flow cell of the spectrofluorimeter, where the fluorescence intensity was measured (λex = 227 nm, λem = 419 nm). This signal is related to cystine and cysteine concentrations. The second stream of sample solution was injected directly into the carrier stream to react with the reagents and then passed through the reaction coil and detector for measuring the fluorescence intensity. The signal in this step is related only to cysteine. Thus, the cystine content was determined directly from difference of the two signals. Cystine and cysteine can be determined in the range of 0.10 to 5.50 µmol L-1 and 0.20 to 8.0 µmol L-1, respectively, at a rate of 20 samples per hour. The limit of detection (3s/k) was 0.10 µmol L-1 for both analytes. The relative standard deviations for ten replicates determination of 4.0 and 3.5 µmol L-1 cystine or cysteine were 1.1% and 1.8%...

Electrochemical behaviour of dopamine at covalent modified glassy carbon electrode with l-cysteine: preliminary results

Martínez-Huitle,Carlos Alberto; Cerro-Lopez,Monica; Quiroz,Marco Antonio
Fonte: ABM, ABC, ABPol Publicador: ABM, ABC, ABPol
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2009 EN
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The surface of glassy carbon (GC) electrode has been modified by oxidation of L-cysteine. The covalent modified GC electrode with L-Cysteine has been studied, according the supporting electrolyte used. Favourable interactions between the L-cysteine film and DA enhance the current response compared to that at the Nafion GC and bare GC electrodes, achieving better performances than those other electrodes. This behaviour was as result of the adsorption of the cysteine layer film, compact and uniform formation; depending on L-cysteine solution (phosphate buffer or chloridric acid supporting electrolyte) used for modifying GC surface. In cyclic voltammetric measurements, modified electrodes can successfully separate the oxidation/reduction DA peaks in different buffer solutions, but an evident dependence in the response was obtained as function of pH and modified electrode. The modified electrode prepared with L-cysteine/HCl solution was used to obtain the calibration curve and it exhibited a stable and sensitive response to DA. The results are described and discussed in the light of the existing literature.

Lifespan extension and increased resistance to environmental stressors by N-Acetyl-L-Cysteine in Caenorhabditis elegans

Oh,Seung-Il; Park,Jin-Kook; Park,Sang-Kyu
Fonte: Faculdade de Medicina / USP Publicador: Faculdade de Medicina / USP
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/05/2015 EN
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OBJECTIVE: This study was performed to determine the effect of N-acetyl-L-cysteine, a modified sulfur-containing amino acid that acts as a strong cellular antioxidant, on the response to environmental stressors and on aging in C. elegans. METHOD: The survival of worms under oxidative stress conditions induced by paraquat was evaluated with and without in vivo N-acetyl-L-cysteine treatment. The effect of N-acetyl-L-cysteine on the response to other environmental stressors, including heat stress and ultraviolet irradiation (UV), was also monitored. To investigate the effect on aging, we examined changes in lifespan, fertility, and expression of age-related biomarkers in C. elegans after N-acetyl-L-cysteine treatment. RESULTS: Dietary N-acetyl-L-cysteine supplementation significantly increased resistance to oxidative stress, heat stress, and UV irradiation in C. elegans. In addition, N-acetyl-L-cysteine supplementation significantly extended both the mean and maximum lifespan of C. elegans. The mean lifespan was extended by up to 30.5% with 5 mM N-acetyl-L-cysteine treatment, and the maximum lifespan was increased by 8 days. N-acetyl-L-cysteine supplementation also increased the total number of progeny produced and extended the gravid period of C. elegans. The green fluorescent protein reporter assay revealed that expression of the stress-responsive genes...

Synthesis and characterization of a cysteine xyloglucan conjugate as mucoadhesive polymer

Bhalekar,Mangesh; Sonawane,Savita; Shimpi,Shamkant
Fonte: Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Publicador: Universidade de São Paulo, Faculdade de Ciências Farmacêuticas
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2013 EN
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The aim of this study was to improve the mucoadhesive potential of xyloglucan polymer by the covalent attachment of cysteine as thiol moiety. The parent polymer xyloglucan was chemically modified by introducing sulphydryl bearing compound L-cysteine HCl. Different batches of xyloglucan-cysteine conjugates were prepared at varying reaction pH (2-6) and evaluated for optimum thiol incorporation, disulphide group content, swelling behavior, rheological properties and mucoadhesive properties. The obtained conjugates characterized in vitro by quantification of immobilized thiol groups; showed maximum thiol incorporation on xyloglucan (7.67 ± 0.14 %) at pH 5. The disulphide group content was found maximum (2.83 ± 0.12) at pH 6. The water uptake at end of 4 h was 5.0 for xyloglucan and was found to decrease in thiolated derivatives with increase in thiolation. Mucoadhesion studies revealed that mucoadhesion of xyloglucan-cysteine conjugate increased more than twice compared to the unmodified polymer. The viscosity of thiomer was more than that of xyloglucan because of formation of disulphide bonds.

N-acetyl-L-cysteine depresses cardiac performance in patients with septic shock

Peake, S.; Moran, J.; Leppard, P.
Fonte: Universidade de Adelaide Publicador: Universidade de Adelaide
Tipo: Artigo de Revista Científica
Publicado em //1996 EN
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OBJECTIVE: To investigate the effects of adjunctive therapy with parenteral N-acetyl-L-cysteine in patients with newly diagnosed septic shock. DESIGN: Prospective, randomized, double-blind, placebo-controlled study. SETTING: Multidisciplinary intensive care unit at a university teaching hospital. PATIENTS: Twenty patients (N-acetyl-L-cysteine group [n = 10], placebo group [n = 10]), 15 male and five female, of mean age 64 +/- 15 (SD) yrs and Acute Physiology and Chronic health Evaluation (APACHE) II score 33 +/- 6, with septic shock within 24 hrs of diagnosis. INTERVENTIONS: After a 2-hr stabilization period (time-zero minus 2 hrs to time-zero), patients received either N-acetyl-L-cysteine in 5% dextrose (150 mg/kg in 100 mL over 15 mins, followed by 50 mg/kg in 250 mL over 4 hrs, and then 100 mg/kg/24 hrs in 500 mL for 44 hrs; N-acetyl-L-cysteine group) or the equivalent volume of 5% dextrose (placebo group). MEASUREMENTS AND MAIN RESULTS: Hemodynamic and oxygen transport indices were measured at time-zero minus 2 hrs and time-zero, and at multiple time points thereafter until completion of the trial infusion (time-zero plus 48 hrs). A daily Organ Failure Score was recorded for 14 days. Treatment group demographics and hemodynamic variables did not differ significantly between the two groups at time-zero. Mean (SD)...

A cysteine in the repetitive domain of a high-molecular-weight glutenin subunit interferes with the mixing properties of wheat dough

Gao, X.; Zhang, Q.; Newberry, M.; Chalmers, K.; Mather, D.
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
Publicado em //2013 EN
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The quality of wheat (Triticum aestivum L.) for making bread is largely due to the strength and extensibility of wheat dough, which in turn is due to the properties of polymeric glutenin. Polymeric glutenin consists of high- and low-molecular-weight glutenin protein subunits linked by disulphide bonds between cysteine residues. Glutenin subunits differ in their effects on dough mixing properties. The research presented here investigated the effect of a specific, recently discovered, glutenin subunit on dough mixing properties. This subunit, Bx7.1, is unusual in that it has a cysteine in its repetitive domain. With site-directed mutagenesis of the gene encoding Bx7.1, a guanine in the repetitive domain was replaced by an adenine, to provide a mutant gene encoding a subunit (MutBx7.1) in which the repetitive-domain cysteine was replaced by a tyrosine residue. Bx7.1, MutBx7.1 and other Bx-type glutenin subunits were heterologously expressed in Escherichia coli and purified. This made it possible to incorporate each individual subunit into wheat flour and evaluate the effect of the cysteine residue on dough properties. The Bx7.1 subunit affected dough mixing properties differently from the other subunits. These differences are due to the extra cysteine residue...

Beneficial effects of n-acetyl cysteine on pancreas and kidney following experimental pancreatic ischemia-reperfusion in rats

MEIRELLES JUNIOR, Roberto Ferreira; KUBRUSLY, Márcia Saldanha; BELLODI-PRIVATO, Marta; MOLAN, Nilza Aparecida Trindade; MACHADO, Marcel Cerqueira Cesar; D'ALBUQUERQUE, Luis Augusto Carneiro
Fonte: Faculdade de Medicina / USP Publicador: Faculdade de Medicina / USP
Tipo: Artigo de Revista Científica
ENG
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36.62%
OBJECTIVE: To evaluate the protective effects of N-acetyl cysteine on the pancreas and kidney after pancreatic ischemia reperfusion injury in a rat model. METHODS AND MATERIALS: Pancreatic ischemia reperfusion was performed in Wistar rats for 1 hour. Revascularization was achieved followed by 4 h of reperfusion. A total of 24 animals were divided into four groups: Group 1: sham; Group 2: pancreatic ischemia reperfusion without treatment; Group 3: pancreatic ischemia reperfusion plus N-acetyl cysteine intravenously; and Group 4: pancreatic ischemia reperfusion plus N-acetyl cysteine per os. Blood and tissue samples were collected after reperfusion. RESULTS: There were significant differences in amylase levels between Group 1 (6.11±0.55) and Group 2 (10.30±0.50) [p=0.0002] as well as between Group 2 (10.30±0.50) and Group 4 (7.82±0.38) [p=0.003]; creatinine levels between Group 1 (0.52 ± 0.07) and Group 2 (0.77±0.18) [p=0.035] as well as between Group 2 (0.77±0.18) and Group 3 (0.48±0.13) [p=0.012]; and pancreatic tissue thiobarbituric acid reactive substance levels between Group 1 (1.27±0.96) and Group 2 (2.60±3.01) [p=0.026] as well as between Group 2 (2.60±3.01) and Group 4 (0.52±0.56) [p=0.002]. A decrease in pancreatic tissue GST-α3 gene expression was observed in Group 2 in comparison to Group 1 (p =0.006)...

Cysteine addition on short-term cooled boar semen preservation and its relationship with swine field fertility

Severo,Carolina K; Pereira,Gabriel R; Pereira,Andressa M; Ilha,Gustavo F; Oliveira,João Francisco C. de; Soares,Marcelo; Arruda,Rubens P. de; Gonçalves,Paulo B.D
Fonte: Colégio Brasileiro de Patologia Animal - CBPA; Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Publicador: Colégio Brasileiro de Patologia Animal - CBPA; Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2011 EN
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Artificial insemination is routinely used in the swine industry to reduce the costs of production through to increase the efficiency of the refrigerated boar semen process. The objective of this study was to evaluate the effect of different levels of cysteine (CYS) added to the Beltsville Thawing Solution (BTS) extender semen during cooling for up to 72 hours. Ejaculated from three boars were collected with the gloved-hand technique and semen aliquots were diluted in BTS as follow: BTS only (BTS), BTS + 0.1mM cysteine (CYS0.1), BTS + 0.5mM cysteine (CYS0.5), BTS + 1.0mM cysteine (CYS1.0), BTS + 2.5mM cysteine (CYS2.5), BTS + 5.0mM cysteine (CYS5.0), BTS + 10.0mM cysteine (CYS10.0), and BTS + 20.0mM cysteine (CYS20.0). Evaluation of sperm integrity were analyzed using 0.5mg/ml propidium iodide (plasma membrane), 100µg/ml isothiocynate-conjugated Pisum sativun agglutinin (acrosomal membrane) and 153µM 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (mitochondria potential) after semen dilution at specific times (0, 24, 48 and 72 hours). Additionally, we also evaluated the effects of 5.0 mM CYS addition in the BTS extender on the maintenance of sperm quality and their influence on fertility in the swine production. After artificial insemination...

Beneficial effects of n-acetyl cysteine on pancreas and kidney following experimental pancreatic ischemia-reperfusion in rats

Meirelles Junior,Roberto Ferreira; Kubrusly,Márcia Saldanha; Bellodi-Privato,Marta; Molan,Nilza Aparecida Trindade; Machado,Marcel Cerqueira Cesar; D'Albuquerque,Luis Augusto Carneiro
Fonte: Faculdade de Medicina / USP Publicador: Faculdade de Medicina / USP
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2010 EN
Relevância na Pesquisa
36.62%
OBJECTIVE: To evaluate the protective effects of N-acetyl cysteine on the pancreas and kidney after pancreatic ischemia reperfusion injury in a rat model. METHODS AND MATERIALS: Pancreatic ischemia reperfusion was performed in Wistar rats for 1 hour. Revascularization was achieved followed by 4 h of reperfusion. A total of 24 animals were divided into four groups: Group 1: sham; Group 2: pancreatic ischemia reperfusion without treatment; Group 3: pancreatic ischemia reperfusion plus N-acetyl cysteine intravenously; and Group 4: pancreatic ischemia reperfusion plus N-acetyl cysteine per os. Blood and tissue samples were collected after reperfusion. RESULTS: There were significant differences in amylase levels between Group 1 (6.11±0.55) and Group 2 (10.30±0.50) [p=0.0002] as well as between Group 2 (10.30±0.50) and Group 4 (7.82±0.38) [p=0.003]; creatinine levels between Group 1 (0.52 ± 0.07) and Group 2 (0.77±0.18) [p=0.035] as well as between Group 2 (0.77±0.18) and Group 3 (0.48±0.13) [p=0.012]; and pancreatic tissue thiobarbituric acid reactive substance levels between Group 1 (1.27±0.96) and Group 2 (2.60±3.01) [p=0.026] as well as between Group 2 (2.60±3.01) and Group 4 (0.52±0.56) [p=0.002]. A decrease in pancreatic tissue GST-α3 gene expression was observed in Group 2 in comparison to Group 1 (p =0.006)...

A recombinant form of chagasin from Trypanosoma cruzi: inhibitory activity on insect cysteine proteinases

Monteiro, Ana Carolina dos Santos; Neto, Osmundo Brilhante de Oliveira; Sarto, Rafael Perseghini Del; Magalhães, Mariana Torquato Quezado de; Lima, Janaína Nascimento; Lacerda, Ariane Ferreira; Oliveira, Raquel Sampaio; Scharfstein, Julio; Silva, Maria
Fonte: Universidade Católica de Brasília Publicador: Universidade Católica de Brasília
Tipo: Artigo de Revista Científica Formato: Texto
EN
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BACKGROUND: The activity of the major digestive cysteine proteinase detected in the intestinal tract of larvae of the bean weevil, Acanthoscelides obtectus (Say), was efficiently inhibited by the well-characterized cysteine proteinase synthetic inhibitor E-64 and also by a recombinant form of chagasin (r-chagasin), a tight-binding cysteine proteinase inhibitor protein from Trypanosoma cruzi. RESULTS: Incorporation of r-chagasin into an artificial diet system at 0.1 g kg−1 retarded growth rate, decreased larval survival and led to complete mortality of A. obtectus at the end of the trial. The observed differences in growth rates occurred particularly in the first and second development stages. Artificial seeds containing high levels of r-chagasin (0.5–30 g kg−1) completely inhibited larval penetration. CONCLUSION: Together, the results reported in this paper support the hypothesis that the inhibitory activity of r-chagasin towards the major insect gut cysteine proteinase in vitro and in vivo is an accurate prediction of its insecticidal effects. The selectivity of this inhibitor against insect digestive proteinases supports the key role in parasite virulence by affecting the endogenous proteinase activity in its natural host.

Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases

Melo, Francislete Rodrigues; José, Marcia Ometto de Mello Alves; Franco, Octávio Luiz; Rigden, Daniel John; Rigden, Luciane Vieira de Mello; Genú, Aline Marques; Silva Filho, Marcio de Castro; Gleddie, Steve; Sá, Maria Fátima Grossi de
Fonte: Universidade Católica de Brasília Publicador: Universidade Católica de Brasília
Tipo: Artigo de Revista Científica Formato: Texto
EN
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Cysteine proteinases from larvae of the common bean weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae), were isolated by ion exchange affinity chromatography on a CM-Cellulose column and used to select mutant cystatins from a library made with the filamentous M13 phage display system. The library contained variant cystatins derived from the nematode Onchocerca volvulus cystatin through mutagenesis of loop 1, which contains the QVVAG motif that is involved in binding to proteinases. After three rounds of selection, the activity of variant cystatins against papain and cysteine proteinases from A. obtectus was assayed by ELISA. Two different variant cystatins (presenting amino acids DVVSA and NTSSA at positions 65–69) bound to A. obtectus cysteine proteinases more tightly than to papain. In contrast, the wild type had similar affinity for A. obtectus proteinases and for papain. These two selected variants cystatins have greater specificity towards A. obtectus cysteine proteinases than the original sequence and could represent good candidate genes for the production of transgenic plants resistant to this insect pest.

Molecular cloning of a cysteine proteinase cDNA from the cotton boll wevil anthonomus grandis (coleóptera: curculionidae).

Oliveira-Neto, Osmundo Brilhante de; Batista, João A. N.; Rigden, Daniel; Franco, Octávio; Fragoso, Rodrigo; Monnerat, Rose; Grossi-de-Sá, Maria F.
Fonte: Universidade Católica de Brasília Publicador: Universidade Católica de Brasília
Tipo: Artigo de Revista Científica Formato: Texto
EN
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Cotton boll weevil Anthonomus grandis causes severe cotton crop losses in North and South America. This report demonstrates the presence of cysteine proteinase activity in cotton boll weevil, an essential enzyme of protein metabolism for many crop pests. A cysteine proteinase cDNA from A. grandis larvae (Agcys1) was isolated using RT-PCR followed by 5’ and 3’ RACE techniques. The molecular mass and theoretical pI value of the encoded protein were 35.5 kDa and pH 6.0, respectively. Cysteine proteinase inhibitors from different sources were assayed against A. grandis proteinases but, unexpectedly, no inhibitor tested was highly effective. Despite none of the inhibitor tested was effective against A. grandis cysteine proteinases, the molecular and biochemical data of Agcys1 provided a characterization framework of this target enzymes. These data are mostly readily interpreted by proposing a molecular model of Agcys1, which shares the Cys-His-Asn catalytic triad characteristic of this proteinase family. The predicted model will be useful on discovery of novel compounds that could be utilized to inhibit cotton boll weevil cysteine proteinases.

Lipoic acid and N-acetyl cysteine decrease mitochondrial-related oxidative stress in Alzheimer disease patient fibroblasts

Moreira, Paula I.; Harris, Peggy L. R.; Zhu, Xiongwei; Santos, Maria S.; Oliveira, Catarina R.; Smith, Mark A.; Perry, George
Fonte: IOS Press Publicador: IOS Press
Tipo: Artigo de Revista Científica
ENG
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36.62%
In this study, we evaluated the effect of lipoic acid (LA) and N-acetyl cysteine (NAC) on oxidative [4-hydroxy-2-nonenal, N(epsilon)-(carboxymethyl)lysine and heme oxygenase-1] and apoptotic (caspase 9 and Bax) markers in fibroblasts from patients with Alzheimer disease (AD) and age-matched and young controls. AD fibroblasts showed the highest levels of oxidative stress, and the antioxidants, lipoic acid (1 mM) and/or N-acetyl cysteine (100 microM) exerted a protective effect as evidenced by decreases in oxidative stress and apoptotic markers. Furthermore, we observed that the protective effect of LA and NAC was more pronounced when both agents were present simultaneously. AD-type changes could be generated in control fibroblasts using N-methylprotoporphyrin to inhibit cytochrome oxidase assembly indicating that the the oxidative damage observed was associated with mitochondrial dysfunction. The effects of N-methylprotoporphyrine were reversed or attenuated by both lipoic acid and N-acetyl cysteine. These data suggest mitochondria are important in oxidative damage that occurs in AD. As such, antioxidant therapies based on lipoic acid and N-acetyl cysteine supplementation may be promising

The role of cysteine residues in the sulphate transporter, SHST1:Construction of a functional cysteine-less transporter

Howitt, Susan
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
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36.71%
We investigated the role of cysteine residues in the sulphate transporter, SHST1, with the aim of generating a functional cysteine-less variant. SHST1 contains five cysteine residues and none was essential for function. However, replacement of C421 resulted in a reduction in transport activity. Sulphate transport by C205 mutants was dependent on the size of the residue at this position. Alanine at position 205 resulted in a complete loss of function whereas leucine resulted in a 3-fold increase in sulphate transport relative to wild type SHST1. C205 is located in a putative intracellular loop and our results suggest that this loop may be important for sulphate transport. By replacing C205 with leucine and the other four cysteine residues with alanine, we constructed a cysteine-less variant of SHST1 that has transport characteristics indistinguishable from wild type. This construct will be useful for further structure and function studies of SHST1.

How Many Cysteine Residues Regulate Ryanodine Receptor Channel Activity

Dulhunty, Angela; Haarmann, Claudia; Green, Daniel; Hart, James
Fonte: Mary Ann Liebert Inc. Publicador: Mary Ann Liebert Inc.
Tipo: Artigo de Revista Científica
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36.69%
RyRs contain 80-100 cysteine residues per subunit, of which ~25% are free for covalent modification, while the remainder are either modified or form intraprotein disulfides. Oxidizing and nitrosylating reagents have several effects on single RyR channel activity, which depend on the type of modifying reagent, the isoform of the RyR, and ligands bound to the channel. We present evidence here for four major classes of functional cysteine residues associated with RyR channels, i.e., two classes with free -SH groups that either activate or inhibit channels when covalently modified and two classes, with endogenous modification, that either inhibit or activate. Single-channel characteristics provide evidence for four discrete responses within the first activating class, two responses within the second inhibiting class and two types of response within the third endogenously modified class. All but one of these changes in channel properties depend on residues located on the cytoplasmic or membrane-associated domains of the RyR; the remaining response is confined to the luminal domain. If it is assumed that each type of response depends on a separate subclass of cysteine residue and that each subclass contains a minimum of one cysteine per subunit...