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1.
Lett Appl Microbiol ; 49(3): 293-8, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19531060

RESUMO

AIM: To identify the gene that encodes nigrescin, a bacteriocin produced by Prevotella nigrescens ATCC 25261. METHODS AND RESULTS: Each open reading frame (ORF) of the nig gene cluster (nigA, nigB, nigC and nigD) was transferred into an expression vector. The recombinant proteins encoded by nigA, nigB, nigC and nigD were purified and assayed for bacteriocin activity against Porphyromonas gingivalis. The ORFs of the nig gene cluster in Pr. nigrescens ATCC 25261 were re-analysed. It revealed that the position of nig ORFs was similar to previously designated locations, except that the start codon of nigC was reassigned. The new nigC gene started at the nucleotide base position 2454 and stopped at position 3608 (the position designated is relative to the first nucleotide base of the nig locus) and putatively encoded a protein with a predicted molecular mass of 41.9 kDa. The N-terminal 6xHistidine-tag recombinant proteins of NigA, NigB, NigC and NigD were overexpressed in Escherichia coli BL21 star (DE3) and were purified using Ni-NTA resins. Only recombinant NigC showed inhibitory activity against P. gingivalis A244 with minimal inhibition concentration (MIC) of 40 microg ml(-1). CONCLUSION: These results indicate that nigC is the gene that encodes nigrescin. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that indicates that the gene nigC codes for nigrescin, a bacteriocin produced by Pr. nigrescens ATCC 25261.


Assuntos
Bacteriocinas/genética , Genes Bacterianos , Prevotella nigrescens/genética , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Cromatografia de Afinidade , Clonagem Molecular , Códon de Iniciação/genética , Códon de Terminação/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Expressão Gênica , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Porphyromonas gingivalis/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Análise de Sequência de DNA
2.
Artigo em Inglês | MEDLINE | ID: mdl-9568356

RESUMO

Radiolabelled vitellogenin produced by juvenile grouper following injection of 3H-leucine, 32P-orthophosphate and estradiol-stimulation was purified by anion-exchange and gel filtration chromatography and characterized by electrophoresis and chemical analysis. The radiolabelled product was found to exist in two heterogeneous molecular weight forms by electrophoresis under nondenaturing conditions on polyacrylamide gel with M(r), of 260,000 and 525,000. It showed two protein monomers (M(r) 113,000 and 140,000) on electrophoresis under denaturing condition on a sodium dodecyl sulphate-polyacrylamide gel. The purified material contained 17.2% total lipid with 1.3% cholesterol and 2.1% triglycerides, 8-21 micrograms/mg protein total carbohydrate and 6.8 micrograms phosphate/mg protein; amino acid analysis showed a profile comparable to that found for vitellogenins isolated from the grouper, medaka, goldfish and rainbow trout. The study showed grouper vitellogenin to be a glycophospholipoprotein similar in composition to vitellogenins from other teleosts and demonstrated that vitellogenins can be induced in juveniles by injection with estradiol-17 beta.


Assuntos
Estradiol/administração & dosagem , Peixes/sangue , Vitelogeninas/sangue , Animais , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Feminino , Vitelogeninas/isolamento & purificação
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