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BjussuSP-I: A new thrombin-like enzyme isolated from Bothrops jararacussu snake venom

ANA, Carolina D. Sant; TICLI, Fabio K.; OLIVEIRA, Leandro L.; GIGLIO, Jose R.; RECHIA, Carem G. V.; FULY, Andre L.; ARAUJO, Heloisa S. Selistre de; FRANCO, Joao J.; STABELI, Rodrigo G.; SOARES, Andreimar M.; SAMPAIO, Suely V.
Fonte: ELSEVIER SCIENCE INC Publicador: ELSEVIER SCIENCE INC
Tipo: Artigo de Revista Científica
Português
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A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr = 61,000 under reducing conditions and pI similar to 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated scrine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca2+ and Mg2+). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against I I venom samples of Bothrops, I of Crotalus, and I of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic...

Isoflavone genistein inhibits the angiotensin-converting enzyme and alters the vascular responses to angiotensin I and bradykinin

MONTENEGRO, Marcelo F.; PESSA, Lisandra R.; TANUS-SANTOS, Jose E.
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
Português
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Genistein produces antihypertensive and beneficial cardiovascular effects, although the mechanisms for these effects are not known. We examined whether genistein inhibits the in vivo responses to angiotensin I or enhances the responses to bradykinin in anaesthetized rats as a result of angiotensin-converting enzyme inhibition. We have also studied the in vitro effects produced by genistein on the angiotensin-converting enzyme activity. We measured the changes in systemic arterial pressure induced by angiotensin I in doses of 0.03 to 10 mu g/kg, by angiotensin II in doses of 0.01 to 3 mu g/kg, and to bradykinin in doses of 0.03 to 10 mu g/kg in anaesthetized rats pretreated with vehicle (controls), or a single i.v. dose of genistein 25 mg/kg, or daily genistein 25 mg/kg i.v for two days, or a single i.v. dose of captopril 2 mg/kg. Plasma angiotensin-converting enzyme activity was determined in controls and genistein-treated rats using a fluorometric method. The effects of genistein (3-300 mu mol/1) on in vitro angiotensin-converting enzyme activity were assessed by adding genistein to plasma samples and measuring angiotensin-converting enzyme activity. We found significant lower angiotensin-converting enzyme activity in plasma samples from rats pretreated with genistein compared with those found in the Control group (77.7 +/- 8.1 his-leu nmol/min/ml and 108.7 +/- 8.4 his-leu nmol/min/ml...

Planejamento de inibidores da enzima gliceraldeído-3-fosfato desidrogenase de Trypanosoma cruzi por biocalorimetria; Structure-based inhibitor design of the Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase enzyme using isothermal titration calorimetry

Wiggers, Hélton José
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 18/04/2007 Português
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A identificação de compostos bioativos que atuem em proteínas é imprescindível no conceito atual da Química Medicinal. Quase todos os processos biossintéticos dos organismos vivos são regulados por enzimas. Dentro deste contexto a enzima glicossomal Gliceraldeído-3-Fosfato desidrogenase (GAPDH) do Tripanosoma cruzi (agente etiológico causador da Doença de Chagas) tem-se mostrado um alvo promissor para o planejamento de inibidores contra o parasito. A Calorimetria de Titulação Isotérmica (ITC) é uma ferramenta robusta na identificação e otimização de compostos bioativos. Através da ITC podem-se determinar parâmetros cinéticos da atividade enzimática (kcat, KM e Ki) e parâmetros termodinâmicos de interação (DH, DG e DS) entre o alvo biomacromolecular e o composto bioativo. Neste trabalho desenvolveu-se um protocolo de ensaio cinético para a enzima GAPDH através da ITC, visando à identificação de novos compostos tripanossomicidas. A atividade da enzima foi avaliada na presença de diferentes concentrações (0 a 10 % v/v) de dois solventes orgânicos padrões (dimetilsulfóxido e metanol). Esses solventes são utilizados em bioensaios e também para aumentar a solubilidade de substâncias em água. A concentração de 5 % v/v de ambos solventes orgânicos foi determinada como aquela em que a GAPDH manteve-se estável. Houve um aumento significativo da atividade enzimática em relação aos experimentos sem uso de co-solventes. Através do ensaio padronizado foram testados os seguintes compostos frente à GAPDH: ácido 4-butilfenil-amino-metileno-fosfônico...

Estudos da correlação estrutura-função da enzima Clorocatecol 1,2-Dioxigenase de Pseudomonas putida; Studies of the structure-function correlation of the chlorocatechol 1,2-dioxygenase enzyme from Pseudomonas putida

Mesquita, Nathalya Cristina de Moraes Roso
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 13/02/2012 Português
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O intenso uso de compostos orgânicos em conjunto com o grande avanço industrial culminou em um enorme acúmulo de poluentes orgânicos no meio ambiente. Dentre estes poluentes têm-se destacado a presença de hidrocarbonetos aromáticos altamente tóxicos e resistentes à degradação física, química, fotolítica e biológica. Desta maneira, uma nova forma de combater a presença deste tipo de composto no meio ambiente têm sido estudada: o uso de microorganismos, naturais ou geneticamente modificados, capazes de transformá-los em substâncias inertes, como CO2 e água. Tal metodologia é denominada biorremediação. Dentres estes microorganismos destacam-se bactérias dos gêneros Pseudomonas, Aeromonas, Beijerinckia, dentre outros, que têm sido estudadas para esta finalidade. A enzima clorocatecol 1,2-dioxigenase (Pp 1,2-CCD) é uma das proteínas expressas por bactérias do gênero Pseudomonas putida, sendo responsável pela clivagem de hidrocarbonetos aromáticos através da incorporação de ambos os átomos de uma molécula de oxigênio à estrutura do anel aromático, sendo a proteína escolhida para desenvolvermos o presente trabalho. Mais especificamente, nos interessa estudar como o mecanismo de ação da referida enzima é controlado por moléculas extrínsecas...

Modelagem molecular da enzima gliceraldeído-3-fosfato desidrogenase de T. cruzi e análise de potenciais inibidores específicos; Molecular modeling of the enzyme glyceraldehyde-3-phosphate dehydrogenase from T.cruzi and analysis of potential specific inhibitors

Panepucci, Ezequiel Horácio
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 16/12/1994 Português
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Foi construído o modelo tridimensional da enzima gliceraldeído-3- fosfato desidrogenase de Trypanosoma cruzi, o causador da doença de Chagas, usando técnicas computacionais de modelagem por homologia. O modelo foi comparado a enzima muscular homóloga de humanos quanto as diferenças no sitio de ligação do cofator NAD+. Sobre o modelo da enzima de T.cruzi foram construidas moléculas anaJogas ao grupo adenosina do cofator NAD+ como tentativa de se obter um inibidor seletivo a enzima do parasita e não efetivo quanto à enzima de humanos. Alguns dos compostos haviam sido ensaiados quanto à afinidade pela enzima análoga de Trypanosoma brucei. Foi escrito um programa de visualização gráfica de modelos moleculares que permite a análise dos parâmetros esteroquímicos com rapidez e simplicidade; The three-dimensional model of the glycossomal enzyme gliceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi, the causative agent of Chagas disease, was built using homology modeling computational techniques. The model was compared against the human muscle homologous enzyme with regard to the differences in the NAD+ binding site. Adenosine analogs were designed based on the model of the T.cruzi enzyme as an attempt to build selective inhibitors of the parasite enzyme with no activity against the human enzyme. Some of the compounds were previously tested for their affinity for the homologous enzyme from Trypanosoma brucei. A graphical program was written that allows the representation and analysis of stereo chemical parameters of molecular models with ease and speed

Tratamento inovador da compressão medular com reposição enzimática intratecal nas mucopolissacaridoses tipos I e VI : relato de uma série de casos; Innovative treatment of cord compression with trathecal enzyme replacement therapy in mucopolysaccharidoses I and VI: report of a case series

Munõz Rojas, Maria Verônica
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Tese de Doutorado Formato: application/pdf
Português
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As mucopolissacaridoses apresentam uma história natural progressiva, causada por defeitos no metabolismo dos glicosaminoglicanos. Frequentemente graves, as mucopolissacaridoses encurtam de forma considerável a expectativa de vida do paciente. Apesar de que em muitos casos a função intelectual é normal, morbidade neurológica considerável pode ser causada por compressão medular secundária ao acúmulo de glicosaminoglicanos nas meninges. O tratamento deste problema pode requerer a descompressão medular através de laminectomia cervical. A terapia de reposição enzimática endovenosa, para o tratamento de mucopolissacaridose, reduz o acúmulo lisossômico e alivia muitos dos sintomas da doença, porém não oferece benefício direto para o sistema nervoso central uma vez que não atravessa a barreira hemato-encefálica. Esta limitação da reposição enzimática endovenosa levou alguns pesquisadores a trabalhar com uma nova opção de via de liberação medicamentosa de alcance direto no sistema nervoso central, aproveitando o extenso contato que existe entre o líquido cefaloraquidiano e as meninges e com as granulações aracnoideas, para o tratamento de algumas doenças de depósito lisossomal. Estudos em modelos animais têm sido conduzidos e com resultados promissores. Este trabalho se propõe a estudar uma nova via de administração da enzima recombinante...

Effect of enzyme supplementation of broiler diets based on corn and soybeans

Zanella, I.; Sakomura, N. K.; Silversides, F. G.; Fiqueirdo, A.; Pack, M.
Fonte: Poultry Science Assoc Inc Publicador: Poultry Science Assoc Inc
Tipo: Artigo de Revista Científica Formato: 561-568
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Digestibility of diets based on corn and soybean meal or soybeans treated by roasting or extrusion, with or without an enzyme supplementation, was measured by true (Sibbald) methods, by analysis of excreta, and by analysis of ileal digesta. Only analysis of ileal digesta was able to consistently measure differences between soybean and enzyme treatments in the digestibility of CP, starch, fat, and ME. The amino acid (AA) digestibility of the diets was measured by analysis of the ileal contents. Whereas enzyme supplementation improved overall CP digestibility by 2.9%, this improvement was not equal for all AA. of the AA most important for broilers fed corn-soybean diets, the digestibilities of Lys, Met, and Arg were not improved or not improved significantly by the enzyme supplementation; however, that of Val was improved by 2.3% and that of Thr was improved by 3.0%. A performance trial demonstrated that enzyme supplementation with equal diet formulation improved BW and the feed conversion ratio by 1.9 and 2.2%, respectively. A second performance trial compared standard diet formulations with formulations using enzyme supplementation and energy levels that were reduced by the amount of improvement provided by the inclusion of enzyme in the first performance trial. No difference was seen between treatments...

Determination of the enzyme reaction rate in a differential fixed-bed reactor: a case study

Baruque Filho,E.A.; Baruque,M.G.A.; Sant’Anna Jr.,G.L.
Fonte: Brazilian Society of Chemical Engineering Publicador: Brazilian Society of Chemical Engineering
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2001 Português
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The reaction rate of starch hydrolysis catalyzed by a glucoamylase covalently bound to chitin particles was measured in a Differential Fixed-Bed Reactor (DFBR). Under selected test conditions the initial reaction rate may represent biocatalyst activity. Some aspects which influence measurement of the initial reaction rate of an immobilized enzyme were studied: the amount of desorbed enzyme and its hydrolytic activity, the extent of pore blockage of the biocatalyst caused by substrate solution impurities and the internal and external diffusional mass transfer effects. The results showed that the enzyme glucoamylase was firmly bound to the support, as indicated by the very low amount of desorbed protein found in the recirculating liquid. Although this protein was very active, its contribution to the overall reaction rate was negligible. It was observed that the biocatalyst pores were susceptible to being blocked by the impurities of the starch solution. This latter effect was accumulative, increasing with the number of sequential experiments carried out. When the substrate solution was filtered before use, very reliable determinations of immobilized enzyme reaction rates could be performed in the DFBR. External and internal diffusional resistences usually play a significant role in fixed-bed reactors. However...

Crude protein equivalence value of a multi-enzyme product for 28- and 42-day-old broilers

Malakzadegan,A; Zaghari,M; Khalaji,S; Shivazad,M
Fonte: Fundação APINCO de Ciência e Tecnologia Avícolas Publicador: Fundação APINCO de Ciência e Tecnologia Avícolas
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2012 Português
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In order to estimate the crude protein (CP) equivalence value of Natuzyme-p (NP) enzyme by using regression response equations, two experiments were carried out using Ross (308) broiler chicks. Graded levels of dietary CP (while amino acids levels were kept constant) and NP enzyme were used to derive the regression equation in the first experiment. Four levels of dietary CP and NP enzyme were fed to 160 feather-sexed male broiler chicks during the starter (0-28 d of age) and grower (28-42 d of age) period. Each diet was offered to four replicates of five chicks in a completely randomized design. Results obtained in experiment one failed to fit a regression equation between BW, dietary CP levels and NP enzyme. In experiment two, graded levels of CP changed along with the levels of lysine (Lys), Met+Cys and threonine (Thr). Regression equations between BW and dietary CP and NP enzyme were derived. Nonlinear and linear equations were generated for enzyme and CP. Based on an assessment of r² and P value, nonlinear equations were used to determine enzyme equivalence. The derived regression equations of body weight for CP were set to be equal with those obtained for NP and were solved; enzyme equivalence value for CP was calculated by subtracting the obtained value from CP content of basal diet. Crude protein equivalence value of NP at 28 and 42 d of age was estimated to be 0.96 and 0.38 %...

Angiotensin-converting enzyme gene 2350 G/A polymorphism and susceptibility to atrial fibrillation in Han Chinese patients with essential hypertension

Jiang,Min-Hui; Su,Ya-Min; Tang,Jian-Zhong; Shen,Yan-Bo; Deng,Xin-Tao; Yuan,Ding-Shan; Wu,Jie; Pan,Min; Huang,Zhong-Wei
Fonte: Faculdade de Medicina / USP Publicador: Faculdade de Medicina / USP
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/11/2013 Português
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OBJECTIVE: The angiotensin-converting enzyme gene is one of the most studied candidate genes related to atrial fibrillation. Among the polymorphisms of the angiotensin-converting enzyme gene, the 2350 G/A polymorphism (rs4343) is known to have the most significant effects on the plasma angiotensin-converting enzyme concentration. The aim of the present study was to investigate the association of the angiotensin-converting enzyme 2350 G/A polymorphism with atrial fibrillation in Han Chinese patients with essential hypertension. METHODS: A total of 169 hypertensive patients were eligible for this study. Patients with atrial fibrillation (n = 75) were allocated to the atrial fibrillation group, and 94 subjects without atrial fibrillation were allocated to the control group. The PCR-based restriction fragment length polymorphism technique was used to assess the genotype frequencies. RESULTS: The distributions of the angiotensin-converting enzyme 2350 G/A genotypes (GG, GA, and AA, respectively) were 40.43%, 41.49%, and 18.08% in the controls and 18.67%, 46.67%, and 34.66% in the atrial fibrillation subjects (p = 0.037). The frequency of the A allele in the atrial fibrillation group was significantly greater than in the control group (58.00% vs. 38.83%...

Functional Roles of the Tetramer Organization of Malic Enzyme*

Hsieh, Ju-Yi; Chen, Shao-Hung; Hung, Hui-Chih
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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Malic enzyme has a dimer of dimers quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In addition, the enzyme has distinct active sites within each subunit. The mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) isoform behaves cooperatively and allosterically and exhibits a quaternary structure in dimer-tetramer equilibrium. The cytosolic NADP+-dependent malic enzyme (c-NADP-ME) isoform is noncooperative and nonallosteric and exists as a stable tetramer. In this study, we analyze the essential factors governing the quaternary structure stability for human c-NADP-ME and m-NAD(P)-ME. Site-directed mutagenesis at the dimer and tetramer interfaces was employed to generate a series of dimers of c-NADP-ME and m-NAD(P)-ME. Size distribution analysis demonstrated that human c-NADP-ME exists mainly as a tetramer, whereas human m-NAD(P)-ME exists as a mixture of dimers and tetramers. Kinetic data indicated that the enzyme activity of c-NADP-ME is not affected by disruption of the interface. There are no significant differences in the kinetic properties between AB and AD dimers, and the dimeric form of c-NADP-ME is as active as tetramers. In contrast, disrupting the interface of m-NAD(P)-ME causes the enzyme to be less active than wild type and to become less cooperative for malate binding; the kcat values of mutants decreased with increasing Kd...

Heparin/Heparan Sulfate 6-O-Sulfatase from Flavobacterium heparinum: INTEGRATED STRUCTURAL AND BIOCHEMICAL INVESTIGATION OF ENZYME ACTIVE SITE AND SUBSTRATE SPECIFICITY*

Myette, James R.; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

Intracisternal enzyme replacement therapy in lysosomal storage diseases: routes of absorption into brain

Jolly, R.; Marshall, N.; Perrott, M.; Dittmer, K.; Hemsley, K.; Beard, H.
Fonte: Blackwell Science Ltd Publicador: Blackwell Science Ltd
Tipo: Artigo de Revista Científica
Publicado em //2011 Português
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Aims:The research concerns enzyme replacement therapy in lysosomal storage diseases with central nervous system involvement. The principle aim was to understand the routes of entry of enzyme into the brain when delivered directly into the cerebrospinal fluid (CSF) via the cerebellomedullary cistern. Methods: Pathways for absorption of replacement enzymewere investigated in dogs withmucopolysaccharidosis IIIA (MPSIIIA) following intracisternal injections of human recombinant N-sulphoglucosamine sulphohydrolase (rhSGSH, EC3.10.1.1) by light and confocal microscopy using chromogenic and fluorescent immune probes. Results: Enzyme entered the brain superficially by penetration of the pia/glia limitans interface, but the main routewas perivascular along large veins, arteries and arterioles extending onto capillaries. It further dispersed into surrounding neuropil to be taken up by neurones, macrophages, astrocytes and oligodendroglia. Enzyme also entered the lateral ventricles adjacent to the choroid plexus, probably also by the tela choroidea and medullary velum, with further spread throughout the ventricular system and spinal canal. There was secondary spread back across the ependyma into nervous tissue of brain and spinal cord. Conclusions: Enzyme mainly enters the brain by a perivascular route involving both arteries and veins with subsequent spread within the neuropil from where it is taken up by a proportion of neurones and other cells. Penetration of enzyme through the pia/glia limitans is minor and superficial.; R. D. Jolly...

Intracisternal enzyme replacement therapy in lysosomal storage diseases: dispersal pathways, regional enzyme concentrations and the effect of posttreatment posture

Jolly, R.D.; Marshall, N.R.; Marshall, J.; Hartman, A.; Hemsley, K.M.; Winner, L.K.
Fonte: Wiley Publicador: Wiley
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
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AIMS: To investigate routes of dispersal of enzyme, its regional uptake and the effect of posture when replacement enzyme is administered directly into the cerebrospinal fluid (CSF). METHODS: Dispersal pathways of particles and solutes were investigated using intracisternal injections of india ink with visual assessment, and a contrast medium (Iohexol) with computer tomography (CT). Replacement enzyme was measured at 46 loci within the central nervous system (CNS) in four groups of dogs subjected to different post-injection postural changes. RESULTS: India ink and CT studies showed dispersal pathways for CSF to be mainly via cisterns and sulci. Replacement enzyme reached all areas of the CNS tested, although mean concentrations varied 49-fold over different areas of the brain. Posttreatment posture had only modest effects on enzyme uptake in limited anatomical sites. CONCLUSIONS: Dispersal of solutes after injection is rapid and initially enhanced by the injection process. Preferential pathways for CSF flow in the subarachnoid spaces of the brain involve cisterns and sulci. The splenial and suprasplenial sulci in particular appear important conduits for dispersal to more dorsal and rostral areas of the brain. Expansion and contraction of these sulci during brain pulsation is considered important to the forward flow of solutes in CSF through these compartments. Following intracisternal enzyme replacement therapy...

Increased aortic NADPH oxidase activity in rats with genetically high angiotensin-converting enzyme levels

Ocaranza, María Paz; Bargetto, Jorge; Pérez, Alfonso; Galaz, Alfonso; Lavandero González, Sergio; Jalil Milad, Jorge
Fonte: LIPPINCOTT WILLIAMS & WILKINS Publicador: LIPPINCOTT WILLIAMS & WILKINS
Tipo: Artículo de revista
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In humans and rats, angiotensin I-converting enzyme activity is significantly determined by a gene polymorphism. Homozygous Brown Norway rats have higher plasma angiotensin I-converting enzyme activity and circulating angiotensin II (Ang II) levels than Lewis rats. Because Ang II induces NAD(P) H oxidase activation, we hypothesized here that Brown Norway rats have higher vascular NAD(P) H oxidase activity and superoxide anion production than Lewis rats. Homozygous Brown Norway (n = 15) and Lewis (n = 13) male rats were used. Plasma angiotensin I-converting enzyme activity (by fluorimetry), Ang II levels (by high-performance liquid chromatography and radioimmunoassay), and aortic NAD(P) H oxidase activity, as well as superoxide anion production ( by chemiluminescence with lucigenin) were measured. Plasma angiotensin I-converting enzyme activity and Ang II levels were 100% higher in Brown Norway rats than in Lewis rats (P < 0.05). Aortic angiotensin I-converting enzyme, but not Ang II, was elevated (P < 0.05). Aortic superoxide anion production and NAD(P) H oxidase activity were 300% and 260% higher in Brown Norway than in Lewis rats, respectively (P < 0.05), which was not observed in Brown Norway rats treated with candesartan (10 mg/kg per day for 7 days). Endothelial NO synthase activity in the aorta from Brown Norway rats was significantly lower than in Lewis rats. However...

Integron-sequestered dihydrofolate reductase: a recently redeployed enzyme

Alonso, Hernan; Gready, Jill
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
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The introduction and wide use of antibacterial drugs has resulted in the emergence of resistant organisms. DfrB dihydrofolate reductase (DHFR) is a bacterial enzyme that is uniquely associated with mobile gene cassettes within integrons, and confers resistance to the drug trimethoprim. This enzyme has intrigued microbiologists since it was discovered more than thirty years ago because of its simple structure, enzymatic inefficiency and its virtual insensitivity to trimethoprim. Here, for the first time, a comprehensive discussion of genetic, evolutionary, structural and functional studies of this enzyme is presented together. This information supports the ideas that DfrB DHFR is a poorly adapted catalyst and has recently been recruited to perform a novel enzymatic activity in response to selective pressure.

Purification and properties of a phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279

Farouk, Abd-ElAziem; Greiner, Ralf; Hussin, Anis Shobirin Meor
Fonte: Journal of Biotechnology and Biodiversity Publicador: Journal of Biotechnology and Biodiversity
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; Formato: application/pdf
Publicado em 20/04/2012 Português
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An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The enzyme was purified about 66-fold with a recovery of 27%. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Michaelis constant (KM) and turnover number (kcat ) for sodium phytate at pH 5.0 and 50°C were calculated from the Lineweaver-Burk plot to be 760 µM and 4.14s-1, respectively. The enzyme showed narrow substrate specificity and not phytate, but GTP was dephosphorylated with the highest relative rate of hydrolysis. However, according to the kcat/KM values, phytate was concluded to be the in vivo substrate of the enzyme. Optimal activity was determined at pH 4.5 and 45-55°C. The enzyme was strongly inhibited by Fe3+, Cu2+, Zn2+, molybdate, vanadate, fluoride and phosphate (1 mM). Key-words: Enterobacter sakazakii; phytate-degrading enzyme; phytate, purification

Linamarase Enzyme from Lactobacillus delbrueckii NRRL B-763: Purification and Some Properties of a β-Glucosidase

Nwokoro,Ogbonnaya; Anya,Florence Onyebuchi
Fonte: Sociedad Química de México A.C. Publicador: Sociedad Química de México A.C.
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2011 Português
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Some biochemical properties and purification of linamarase enzyme from Lactobacillus delbrueckii NRRL B-763 were studied. The crude enzyme was used to detoxify cassava flour cyanide and samples of 150 μm particle size treated with the crude enzyme showed a reduction from 2.1 mg HCN/10g sample to 0.11 mg HCN/10 g sample after 20 h (95% reduction). Untreated control samples of 0.5 mm particle size showed a reduction from 2.1mg HCN/10 g sample to 1.98 mg HCN/10 g sample after 40 h (5.7% reduction). Lhe enzyme was purified 33 fold with a 40% yield through a series of four steps namely, ammonium sulphate precipitation, acetone precipitation, ion exchange chromatography and gel Alteration chromatograrphy using Sephadex G-200. Lhe purified enzyme showed maximum activity at pH 4.5. Lhe enzyme showed 100% stability at the pH range of 5.0 and 6.0. Maximum activity of the enzyme was observed at a temperature of 50 °C and maximum stability at a temperature range of 40 and 50 °C. Lhe approximate enzyme molecular weight was estimated to be 56 kDa by Sephadex G-200 gel filteration chromatography. Lhe linamarase enzyme could be adapted for improved degradation of cassava cyanide and other biotechnological applications.

Prescription patterns of enzyme-containing products in South Africa over a 2-year period

Truter,Ilse
Fonte: South African Journal of Science Publicador: South African Journal of Science
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/10/2014 Português
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Enzymes are traded in five categories, namely medical (intervention), diagnostic (detection and quantification), molecular biology, biofuel and industrial. Therapeutic enzymes have been investigated for different uses, for example, for the treatment of genetic disorders, blood clotting disorders, cancer and infectious diseases and for burn debridement. No studies on the prescription of enzyme-containing products in South Africa could be found. Enzymes are classified in the Monthly Index of Medical Specialities under digestants, enzymes and fibrinolytics. The primary aim of this study was to investigate the prescription patterns and cost of enzyme-containing products in South Africa. A private health-care medicines claims database for 2010 and 2011 of approximately 4.5 million records was analysed retrospectively. Enzyme-containing products constituted a small percentage of medical insurance claims (only 0.02% of approximately 4.5 million claims for products and procedures), yet they were relatively expensive. A total of 906 products was prescribed at a cost of almost ZAR2 million over the 2 years. Hyaluronidase was the most frequently prescribed (60.04%), followed by pancreatin-containing products (34.66%). Pancreatin (lipase/ protease/amylase) is primarily used in the management of pancreatic exocrine insufficiency. The average cost per hyaluronidase prescription paid by the medical insurance schemes was ZAR280. Other enzyme-containing products prescribed were imiglucerase...

High levels of maize in broiler diets with or without microbial enzyme supplementation

Bhuiyan,M.M.; Islam,A.F.; Iji,P.A.
Fonte: South African Journal of Animal Science Publicador: South African Journal of Animal Science
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2013 Português
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A total of 210 day-old male Cobb broiler chickens were randomly assigned to six treatments in a 3 x 2 factorial design. The treatments consisted of three levels of maize: 250 g/kg (LM), 500 g/kg (MM) and 750 g/kg diet (HM) and two levels of enzymes: plus enzyme and no enzyme. Each treatment was replicated five times, with seven birds per replicate. Chickens were reared in multi-tiered brooder cages to 21 days of age in a climate-controlled room. Feed and water were provided ad libitum. Over the feeding period (21 d), there was an increase in feed intake as maize inclusion level (MIL) increased in diets, while supplementation with microbial enzyme improved feed intake only in the MM diet. There was an improvement in live weight (LW) in chickens with increased MIL in their diets. The microbial enzyme supplement also improved LW, but only on the MM diet. The feed conversion ratio (FCR) was improved with increase in MIL in diets, but the enzyme supplements had no effect on FCR up to day 21. At day 21 there was an increase in relative weight of the small intestine with an increase in MIL, but this was not affected by enzyme supplementation. The weight of the liver increased with increase in MIL and enzyme supplementation. At day 21 the pH of the digesta in the gizzard declined with an increase in MIL in diets. In general...