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1.
J Microbiol Biotechnol ; 25(1): 66-73, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25152061

RESUMO

The pabS gene of Agaricus bisporus 02 encoding a putative PABA synthase was cloned, and then the recombinant protein was expressed in Escherichia coli BL21 under the control of the T7 promoter. The enzyme with an N-terminal GST tag or His tag, designated GST-AbADCS or His-AbADCS, was purified with glutathione Sepharose 4B or Ni Sepharose 6 Fast Flow. The enzyme was an aminodeoxychorismate synthase, and it was necessary to add with an aminodeoxychorismate lyase for synthesizing PABA. AbADCS has maximum activity at a temperature of approximately 25°C and pH 8.0. Magnesium or manganese ions were necessary for the enzymatic activity. The Michaelis-Menten constant for chorismate was 0.12 mM, and 2.55 mM for glutamine. H2O2 did distinct damage on the activity of the enzyme, which could be slightly recovered by Hsp20. Sulfydryl reagents could remarkably promote its activity, suggesting that cysteine residues are essential for catalytic function.


Assuntos
Agaricus/enzimologia , Agaricus/genética , Clonagem Molecular , Transaminases/genética , Transaminases/metabolismo , Escherichia coli/genética , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Oxo-Ácido-Liases/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Temperatura , Transaminases/química
2.
Molecules ; 18(5): 5723-35, 2013 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-23681057

RESUMO

Four new citrinin derivatives, including two citrinin dimers and two citrinin monomer derivatives, were isolated and identified from a marine-derived fungal strain Penicillium sp. ML226 along with six known related compounds. Their structures were elucidated by spectroscopic and chemical methods. The new compounds showed modest cytotoxic activity against HepG-2 cell line and weak antimicrobial activity against Staphylococcus aureus.


Assuntos
Antibacterianos , Organismos Aquáticos/química , Citrinina , Citotoxinas , Penicillium/química , Staphylococcus aureus/crescimento & desenvolvimento , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Citrinina/análogos & derivados , Citrinina/química , Citrinina/isolamento & purificação , Citrinina/farmacologia , Citotoxinas/química , Citotoxinas/isolamento & purificação , Citotoxinas/farmacologia , Células Hep G2 , Humanos , Estrutura Molecular
3.
World J Gastroenterol ; 8(6): 1014-8, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12439916

RESUMO

AIM: JNK cascade plays an important role in cell proliferation, differentiation and apoptosis. However, the exact function of JNK cascade for apoptosis induction remains largely unknown. In this study, the role of JNK activation stimulated by TPA in the process of apoptosis induction and its signaling transduction pathway in gastric cancer cells were investigated and determined. METHODS: Expressions of mRNA and protein were detected by Northern blot and Western blot. Transcription activity was measured by transient transfection and CAT assay. Apoptotic cells were displayed through staining the nucleus with DAPI and were observed under fluorescence microscope. The apoptotic index was determined by counting 1000 cells randomly. RESULTS: JNK protein was stimulated rapidly by TPA, and reached its highest peak within 3 hr, then decreased in a time-dependent manner, but the expression level of JNK protein induced by TPA was always keeping higher than that in untreated cells. Similar pattern was seen in c-jun mRNA level induced by TPA. TPA significantly activated the transcriptional activity of activator protein-1 with a TPA-dose-dependent manner. Furthermore, activation of JNK was mediated through PKC pathway. Treatment of cells with PKC specific inhibitor, Wortmannin, led to repression of JNK even in the presence of TPA. More importantly, all these effects were associated with induction of apoptosis in gastric cancer cells. TPA inducted apoptosis obviously in gastric cancer cells. The apoptotic cells became smaller and rounded, and their nuclei became condensation and fragmentation with brightly stained chromatin. However, suppression of JNK by PKC specific inhibitor, Wortmannin, resulted in the decrease of apoptosis induced by TPA in a time-dependent manner, apoptotic index dramatically decreased from 32.56 % to 8.71 %. CONCLUSION: TPA stimulates JNK cascade, including up-regulation of JNK protein expression level and c-jun mRNA expression level, and activation of activator protein-1 transcriptional activity. Activation of JNK is mediated through PKC pathway, which has an association with induction of apoptosis by TPA. Thus, activation of JNK via PKC pathway may represent one of important mechanisms for TPA to induce apoptosis in gastric cancer cells.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Acetato de Tetradecanoilforbol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Genes jun/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/genética , Fator de Transcrição AP-1/metabolismo , Células Tumorais Cultivadas
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