RESUMO
Yeast communities and genetic polymorphism of prevalent Saccharomyces cerevisiae strains isolated from the spontaneous fermentation of the sugarcane juice during the production of aguardente in three distilleries in the state of Minas Gerais, Brazil, were studied. S. cerevisiae was the prevalent species during the process of aguardente production, but Schizosaccharomyces pombe was predominant in old fermentations in one distillery. Transient yeast species were found in a variable number, probably due to the daily addition of sugarcane juice, and they were different for each of the three distilleries studied. PFGE and PCR analysis of the predominant strains of S. cerevisiae isolated from the fermented must showed a high degree of genetic polymorphism among the three distilleries. A high molecular variability of S. cerevisae strains was also observed among strains isolated from the same vat at different fermentation ages. Our results showed that there was a succession of geneticly different strains of S. cerevisae during the process of aguardente production.
Assuntos
Bebidas Alcoólicas , Polimorfismo Genético , Saccharomyces cerevisiae/genética , Leveduras/crescimento & desenvolvimento , Brasil , Fermentação , Poaceae , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/isolamento & purificação , Leveduras/genética , Leveduras/isolamento & purificaçãoRESUMO
The effect of pentavalent meglumine antimoniate (glucantime) on the growth kinetics of promastigotes of 13 South American Leishmania strains isolated from patients, sylvatic reservoirs, and vectors was studied. Four of five L. (Viannia) braziliensis human isolates were obtained from drug-responsive patients and one was isolated from an unresponsive mucocutaneous-type infection. Studies involved the cell yield at the late log phase, the growth rate, and the growth-curve patterns of promastigotes in vitro. Restriction-fragment-length polymorphism, pulsed-field gel electrophoresis, and hybridization with the gene coding for a P-glycoprotein from L. (V.) guyanensis were used in an attempt to correlate the resistance phenotype with gene amplification. Consistent differences observed in both cell yield and growth rate among the isolates in the presence of glucantime indicated these parameters to be good criteria for the estimation of susceptibility to glucantime. Drug susceptibility varied widely between strains, indicating a great genetic heterogeneity of the isolates. Five L. (V.) braziliensis strains and three L. (V.) guyanensis strains were shown to be susceptible to glucantime. Five isolates were resistant, four of which were obtained from sylvatic vectors and one, from a patient with an unresponsive mucocutaneous infection. Molecular analyses of total DNA indicated the presence of a pgpA-related gene in all strains tested. No amplified sequence could be detected, suggesting that pgpA amplification is not involved in glucantime resistance in these wild Leishmania strains.
Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Animais , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Humanos , Leishmania/crescimento & desenvolvimento , Leishmania/isolamento & purificação , Leishmania braziliensis/efeitos dos fármacos , Leishmania guyanensis/efeitos dos fármacos , Antimoniato de Meglumina , Fases de Leitura Aberta , Especificidade da EspécieRESUMO
A glucantime sensitive Leishmania (V.) guyanensis strain was used to obtain in vitro resistant cell lines, by increments in glucantime concentrations employing both one step and stepwise protocols. Whereas the effective concentration of drug that inhibited the growth of wild type cells by 50% (EC50 value) was 0.20 mg Sb(v)/mL, the resistant cells were able to grow in glucantime concentrations greater than 8.0 mg/mL. The resistant cell lines were partially characterized by their in vitro response to glucantime, the stability of resistance phenotype, cross resistance to a range of drugs, and also by the analysis of total DNA fragments generated by restriction endonucleases and blot hybridization. Amplified DNA sequence similar to a P-glycoprotein analog from Leishmania tarentolae (ltpgpA gene) was observed in all the resistant cell lines obtained through the one-step protocol. These cell lines showed cross resistance to heavy metals but were sensitive to puromycin, vinblastine, and pentostam.
Assuntos
Antiprotozoários/farmacologia , Leishmania guyanensis/efeitos dos fármacos , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Antimônio/farmacologia , Tartarato de Antimônio e Potássio/farmacologia , Gluconato de Antimônio e Sódio/farmacologia , Arseniatos/farmacologia , DNA de Protozoário/análise , DNA de Protozoário/genética , Resistência a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Dosagem de Genes , Genes de Protozoários , Leishmania/genética , Leishmania guyanensis/genética , Antimoniato de Meglumina , Óxidos/farmacologia , Puromicina/farmacologia , Homologia de Sequência do Ácido Nucleico , Vimblastina/farmacologiaRESUMO
1. The activity of a pentavalent antimonial drug glucantime on the growth of promastigote forms of Leishmania strains involved in South American cutaneous and mucocutaneous leishmaniasis was investigated. A marked difference in susceptibility to glucantime among four different strains and cloned lines obtained from a single strain was observed. For the sensitive strains (L. braziliensis M2903 and L. guyanensis M1176), the cell growth was reduced in a dose-dependent manner for drug concentrations at a range of 0.23 to 23 mM. However, despite the relative sensitivity of the assay, no significant increase of effect was observed in the presence of higher drug concentrations. For the resistant strains (L. amazonensis M10996 and L. braziliensis LTB259) a dose-response line is obtained at a higher concentration range (20 mM to 70 mM). 2. The influence of the drug on surface properties, respiratory activity and incorporation of radiolabelled leucine by a sensitive strain -L. guyanensis M1176-was studied as an approach to its site of action. Despite the increased intensity of self-agglutination for cells growing in the presence of glucantime, no significant change was observed in electrophoretic mobility or Concanavalin A reactivity. Since the oxygen uptake of glucose-stimulated promastigotes was only slightly reduced in the presence of 23 mM glucantime at 28 degrees C, the reduction was not sufficient to explain the total growth inhibition observed. A significant decrease of 14C-leucine incorporation into the cold TCA-insoluble fraction of drug-treated cells was observed within the same concentration range that reduces promastigote growth.
Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Animais , Técnicas In Vitro , Leishmania/crescimento & desenvolvimento , Antimoniato de Meglumina , Fatores de TempoRESUMO
The activity of a pentavalent antimonial drug glucantime on the growth of promastigote forms of Leishmania strains involved in South American cutaneous and mucocutaneous leishmaniasis was investigated. A marked difference in susceptibility to glucantime among four different strains and cloned lines obtained from a single strain was observed. For the sensitive strains (L.braziliensis M2903 and L. guyanensis M1176), the cell growth was reduced in a dose-dependent manner for drug concentrations at a range of 0.23 to 23 mM. However, despite the relative sensitivity of the assay, no significant increase of effect was observed in the presence of higher drug concentrations. For the resistant strains (L. amazonensis M10996 and L. braziliensis LYB259) a dose-response line is obtained at a higher concentration range (20 mM to mM). The influence of the drug on surface properties, respiratory activity and incorporation of radiolabelled leucine by a sensitive strain - L-guyanensis M1176-was studied as an approach to its site of action. Despite the increased intensity of self-aglutination for cells growing in the presence of glucantime, no significant change was observed in electrophoretic mobility or Concanavian A reactivity. Since the oxygen uptake of glucose-stimulated promastigotes was only slightly reduced in the presence of 23 mM glucantime at 28-C, the reduction was not sufficient to explain the total growth inhibition observed. A significant decrease of 14C-leucine incorporation into the cold TCA-insoluble fraction of drug-treated cells was observed within the same concentration range that reduces promastigote growth
Assuntos
Animais , Antimônio/farmacologia , Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Análise de Variância , Leishmania/crescimento & desenvolvimento , Fatores de TempoRESUMO
A new DNA amplification is described from an isolate of the lizard parasite Leishmania tarentolae. This DNA is present in up to 50 copies in the Trager line of this species and present but not amplified in all other lines tested. This amplification has been named the T amplification (for Tarentolae/Trager). Restriction enzyme digestion and electrophoresis of total DNA reveal amplified fragments totalling 19 kb following staining with ethidium bromide, a finding confirmed by the use of specific hybridization probes. Much of the amplified T DNA occurs as extra-chromosomal circular molecules. No cross-hybridization was observed between the T region and other amplified DNA of Leishmania, or the maxicircle of L. tarentolae, nor was resistance to methotrexate, chloroquine or primaquine detected in the T-amplified line. Combined with our previous results showing H region amplification in 2 other unselected lab stocks, these data demonstrate the prevalence of apparently spontaneous gene amplifications in L. tarentolae.