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1.
Biosci Biotechnol Biochem ; 75(6): 1113-8, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21670521

RESUMO

Schizosaccharomyces pombe treated with valproic acid died with apoptotic markers such as DNA fragmentation, loss of a mitochondrial electrochemical gradient and chromatin condensation, independently of metacaspase, a yeast homolog of metazoan caspase. Sensitivity to valproic acid was strongly dependent on growth phase. Cells in a later growth phase were much more sensitive to valproic acid than those in an earlier one. Altering the pH of the medium with HCl and with NaOH also caused remarkable changes in sensitivity. Cells in an acidic medium were more sensitive to valproic acid. This pH-dependent change in sensitivity did not require de novo protein synthesis, and a change in pH 60 min after the administration of valproic acid affected sensitivity. These results suggest that the intracellular cell death process was susceptible to extracellular pH. Although a sir2 mutant of Saccharomyces cerevisiae has been reported to be resistant to valproic acid, mutations in sir2 did not affect the sensitivity to valproic acid of S. pombe.


Assuntos
Caspases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Ácido Valproico/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores/análise , Caspases/genética , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Contagem de Colônia Microbiana , Fragmentação do DNA/efeitos dos fármacos , Deleção de Genes , Concentração de Íons de Hidrogênio , Marcação In Situ das Extremidades Cortadas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética
2.
Biochem Biophys Res Commun ; 366(1): 92-7, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18053802

RESUMO

Cadherin 23 (Cdh23), an essential factor in inner ear mechano-electric transduction, exists in two alternatively spliced forms, Cdh23(+68) and Cdh23(-68), depending on the presence and absence of exon 68. Cdh23(+68) is inner ear-specific. The exon 68-corresponding region confers an alpha-helical configuration upon the cytoplasmic domain (Cy) and includes a cysteine residue, Cys(3240). We demonstrate here that Cy(+68) as well as the transmembrane (TM) plus Cy(+68) region is present in two different forms in transfected cells, reduced and non-reduced, the latter existing in more compact configuration than the former. The observed characteristic of Cy(+68) was completely abolished by Cys(3240)Ala substitution. Treatment of TMCy(+68)-transfected cells with diethyl maleate, a glutathione depleting reagent, resulted in conversion of the non-reduced to the reduced form of TMCy(+68), suggesting glutathione to be a Cys(3240)-binding partner. Multiple alignment of mammalian Cdh23Cy sequences indicated the occurrence of conformation-inducible Cys in Cdh23Cy of mammals, but not lower vertebrates. The implications of Cys-dependent structural ambivalence of Cdh23 in inner ear mechanosensation are discussed.


Assuntos
Caderinas/química , Caderinas/metabolismo , Orelha Interna/química , Orelha Interna/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Relacionadas a Caderinas , Caderinas/ultraestrutura , Citoplasma/química , Citoplasma/metabolismo , Humanos , Dados de Sequência Molecular , Oxirredução , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/ultraestrutura , Estrutura Terciária de Proteína , Especificidade da Espécie , Relação Estrutura-Atividade
3.
Biosci Biotechnol Biochem ; 71(11): 2841-4, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17986764

RESUMO

A mutant of Schizosaccharomyces pombe deficient in both superoxide dismutase with copper and zinc as cofactors and glutathione was hypersensitive to menadione, which intracellularly generates superoxide radicals, and showed short chronological lifespan with more oxidation of proteins. Disruption of the sir2 gene in the double mutant enhanced the short chronological lifespan without more enhanced protein oxidation.


Assuntos
Glutationa/deficiência , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/crescimento & desenvolvimento , Superóxido Dismutase/genética , Coenzimas/metabolismo , Cobre/metabolismo , Farmacorresistência Fúngica/genética , Mutação , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Vitamina K 3/farmacologia , Vitaminas/farmacologia , Zinco/metabolismo
4.
J Biochem ; 138(6): 797-804, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16428309

RESUMO

Several chemical agents have been used to exert oxidative stress in the study of stress response, but differences in the effects of different reagents have received little attention. To elucidate whether such differences exist, the response of Schizosaccharomyces pombe to menadione (MD), 1-chloro-2,4-dinitrobenzene (CDNB), hydrogen peroxide and cumene hydroperoxide (CHP), which are frequently used to exert oxidative stress, was investigated. Sensitivity to these reagents differed among mutants deficient in genes involved in oxidative stress resistance. N-Acetylcysteine restored resistance to MD, CHP and hydrogen peroxide but did not change sensitivity to CDNB. The induction kinetics of genes induced by oxidative stress differed for each reagent. MD, CDNB and hydrogen peroxide caused a transient induction of genes, but the peak times of induction differed among the reagents. CHP gave quite different kinetics in that the induction continued for up to 2 h. The ctt1(+) gene was not induced by CHP. GSH rapidly decreased in the cells treated with high concentrations of these reagents, but at a low concentration only CDNB decreased GSH. These results indicated that S. pombe responded differently to the oxidative stress exerted by these different reagents.


Assuntos
Oxidantes/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Derivados de Benzeno/farmacologia , Dinitroclorobenzeno/farmacologia , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo , Vitamina K 3/farmacologia
5.
Yeast ; 22(2): 91-7, 2005 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-15645504

RESUMO

A Schizosaccharomyces pombe mutant deficient in Cu,Zn-superoxide dismutase (sod1 mutant) was hypersensitive to phloxine B, which is used as a food-colouring agent and also to distinguish diploid strains of Sz. pombe from haploid strains, under illumination with light. The pro-oxidant nature of phloxine B was confirmed biochemically. The carbonyl content of proteins (which represents protein oxidation) increased, and the reduced form of glutathione was transiently decreased by phloxine B treatment under illumination with light. When cells were treated with phloxine B under light, carbonyl content of proteins in the sod1 mutant was greater than that in the wild-type and amount of glutathione was much decreased in the sod1 mutant compared with the wild-type. Genes induced by oxidative stress were induced by phloxine B under illumination with light and some were induced by phloxine B without light.


Assuntos
Azul de Eosina I/farmacologia , Corantes de Alimentos/farmacologia , Oxidantes/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Superóxido Dismutase/deficiência , Northern Blotting , Teste de Complementação Genética , Glutationa/metabolismo , Luz , Mutação , Oxirredução , RNA Fúngico/química , RNA Fúngico/genética , Espécies Reativas de Oxigênio/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Superóxido Dismutase/metabolismo
6.
Curr Genet ; 41(2): 82-8, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12073089

RESUMO

A Cu, Zn-superoxide dismutase gene ( sod1+) deletion mutant of fission yeast Schizosaccharomyces pombe was constructed and its properties were investigated. Superoxide dismutase activity was not detected in the mutant on activity staining of polyacrylamide gels. The mutant showed cysteine or methionine and lysine auxotrophy, slow growth and sensitivity to menadione. While expression of the apt1+ gene, induction of which depends on the Pap1 transcription factor, was induced at the same concentration of menadione in both the wild-type cell and the sod1 mutant, expression of the gpx1+ gene, induction of which depends on the Atf1 transcription factor, was induced at a lower concentration of menadione in the mutant compared with the wild-type control. Expression of the sod1+ gene was induced by oxidative stress and no induction was observed in pap1, prr1 and spc1 mutants.


Assuntos
Mutação/genética , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Superóxido Dismutase/metabolismo , Aminoácidos/metabolismo , Divisão Celular , Indução Enzimática/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Mutagênese , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Proteínas Associadas a Pancreatite , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Superóxido Dismutase/deficiência , Superóxido Dismutase/genética , Vitamina K 3/farmacologia
7.
Biotechnol Bioeng ; 80(1): 22-32, 2002 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-12209783

RESUMO

The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme.


Assuntos
D-Aminoácido Oxidase/biossíntese , D-Aminoácido Oxidase/genética , Regulação Fúngica da Expressão Gênica , Saccharomycetales/enzimologia , Saccharomycetales/genética , Schizosaccharomyces/enzimologia , Catálise , Células Cultivadas , Cefalosporinas/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Vetores Genéticos , Projetos Piloto , Plasmídeos , Schizosaccharomyces/genética , Especificidade da Espécie , Transformação Genética
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