RESUMO
A gene coding for a pullulanase from the obligately anaerobic, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus has been cloned in Escherichia coli. It consists of an open reading frame (pulA) of 2478 bp which codes for an enzyme of 95,732 Da and is flanked by two other open reading frames. A truncated version of the gene which lacks 381 bp of 5'-sequence also has pullulanase activity and it appears that the amino-terminal portion of the gene is not essential for either activity or thermostability. Amino acid sequence comparisons with other published amylases and pullulanases showed that it possesses homology to the four key regions common to these enzymes.
Assuntos
Bactérias Anaeróbias/genética , Glicosídeo Hidrolases/genética , Sequência de Aminoácidos , Bactérias Anaeróbias/enzimologia , Clonagem Molecular , Estabilidade Enzimática , Glicosídeo Hidrolases/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Homologia de Sequência de AminoácidosRESUMO
Fluconazole-susceptible Candida albicans strains accumulated [3H]fluconazole at a rate of approximately 2 pmol/min per 10(9) cells. Fluconazole accumulation was not affected by the pretreatment of cells with sodium azide or with 2-deoxyglucose. The rate of fluconazole accumulation became saturated at high fluconazole concentrations and was not affected by the addition of ketoconazole, and there was no fluconazole accumulation in cells incubated at 4 degrees C. A fluconazole-resistant mutant of C. albicans SGY-243 was isolated following growth enrichment in fluconazole-containing medium. Cells of the mutant strain, designated FR2, showed a reduced rate of fluconazole accumulation compared with SGY-243 and were not resistant to other azole antifungal agents. The rates of fluconazole accumulation by C. albicans FR2 and the other azole-resistant strains, B59630, AD, and KB, were increased in the presence of sodium azide, suggesting that fluconazole resistance in these strains may be associated with an energy-dependent drug efflux. Fluconazole-resistant C. albicans strains all contained elevated amounts (2- to 17-fold) of mRNA encoding Cdr1, and an ATP-binding cassette-type transporter. In addition, C. albicans FR2 also contained increased amounts of mRNA encoding Benr, a major facilitator superfamily transporter. These results suggest that fluconazole enters C. albicans cells by facilitated diffusion and that fluconazole resistance may involve energy-dependent drug efflux associated with increased expression of Benr and/or Cdr1.
Assuntos
Antifúngicos/farmacologia , Candida albicans/genética , Fluconazol/farmacologia , Antifúngicos/análise , Candida albicans/efeitos dos fármacos , Candida albicans/isolamento & purificação , Resistência Microbiana a Medicamentos/genética , Fluconazol/análise , Genes Fúngicos/genética , Testes de Sensibilidade Microbiana , RNA Fúngico/análiseRESUMO
4-Hydroxy-2-cyclopentenone is responsible for the anti-bacterial activity of an extract of leaves from Passiflora tetrandra with minimum inhibitory doses (MID) of ca. 10 micrograms/disk against Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa. 4-Hydroxy-2-cyclopentenone is also cytotoxic to P388 murine leukemia cells (IC50 of less than 1 microgram/ml).
