RESUMO
AIMS: In order to improve the quality and to create a biological basis for obtainment of the protected denomination of origin (PDO), indigenous yeast were isolated and characterized for use in Salinas city (the Brazilian region of quality cachaça production). MATERIAL AND METHODS: Seven thousand and two hundred yeast colonies from 15 Salinas city distilleries were screened based on their fermentative behaviour and the physicochemical composition of cachaça. Molecular polymorphic analyses were performed to characterize these isolates. RESULTS: Two Saccharomyces cerevisiae strains (nos. 678 and 680) showed appropriate characteristics to use in the cachaça production: low levels of acetaldehyde and methanol, and high ethyl lactate/ethyl acetate ratio respectively. They also presented polymorphic characteristics more closely related between themselves even when compared to other strains from Salinas. CONCLUSIONS: The application of selected yeast to cachaça production can contribute for the improvement of the quality product as well as be used as a natural marker for PDO. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the use of selected yeast strains could contribute to obtain a cachaça similar to those produced traditionally, while getting wide acceptation in the market, yet presenting more homogeneous organoleptic characteristics, and thus contributing to the PDO implementation.
Assuntos
Bebidas Alcoólicas/microbiologia , Saccharomyces cerevisiae/metabolismo , Acetaldeído/análise , Acetaldeído/metabolismo , Bebidas Alcoólicas/análise , Brasil , Fermentação , Metanol/análise , Metanol/metabolismo , Melhoria de Qualidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Kluyveromyces lactis strains are able to assimilate lactose. They have been used industrially to eliminate this sugar from cheese whey and in other industrial products. In this study, we investigated specific features and the kinetic parameters of the lactose transport system in K. lactis JA6. In lactose grown cells, lactose was transported by a system transport with a half-saturation constant (K s) of 1.49 ± 0.38 mM and a maximum velocity (V max) of 0.96 ± 0.12 mmol. (g dry weight)(-1) h(-1) for lactose. The transport system was constitutive and energy-dependent. Results obtained by different approaches showed that the lactose transport system was regulated by glucose at the transcriptional level and by glucose and other sugars at a post-translational level. In K. lactis JA6, galactose metabolization was under glucose control. These findings indicated that the regulation of lactose-galactose regulon in K. lactis was similar to the regulation of galactose regulon in Saccharomyces cerevisiae.
Assuntos
Kluyveromyces/metabolismo , Lactose/metabolismo , Transporte Biológico/fisiologia , Regulação Fúngica da Expressão Gênica , CinéticaRESUMO
Recently, much attention has been given to the use of probiotics as an adjuvant for the prevention or treatment of gastrointestinal pathology. The great advantage of therapy with probiotics is that they have few side effects such as selection of resistant bacteria or disturbance of the intestinal microbiota, which occur when antibiotics are used. Adhesion of pathogenic bacteria onto the surface of probiotics instead of onto intestinal receptors could explain part of the probiotic effect. Thus, this study evaluated the adhesion of pathogenic bacteria onto the cell wall of Saccharomyces boulardii and Saccharomyces cerevisiae strains UFMG 905, W303 and BY4741. To understand the mechanism of adhesion of pathogens to yeast, cell-wall mutants of the parental strain of Saccharomyces cerevisiae BY4741 were used because of the difficulty of mutating polyploid yeast, as is the case for Saccharomyces cerevisiae and Saccharomyces boulardii. The tests of adhesion showed that, among 11 enteropathogenic bacteria tested, only Escherichia coli, Salmonella Typhimurium and Salmonella Typhi adhered to the surface of Saccharomyces boulardii, Saccharomyces cerevisiae UFMG 905 and Saccharomyces cerevisiae BY4741. The presence of mannose, and to some extent bile salts, inhibited this adhesion, which was not dependent on yeast viability. Among 44 cell-wall mutants of Saccharomyces cerevisiae BY4741, five lost the ability to fix the bacteria. Electron microscopy showed that the phenomenon of yeast-bacteria adhesion occurred both in vitro and in vivo (in the digestive tract of dixenic mice). In conclusion, some pathogenic bacteria were captured on the surface of Saccharomyces boulardii, Saccharomyces cerevisiae UFMG 905 and Saccharomyces cerevisiae BY4741, thus preventing their adhesion to specific receptors on the intestinal epithelium and their subsequent invasion of the host.
Assuntos
Aderência Bacteriana/fisiologia , Parede Celular/microbiologia , Escherichia coli/fisiologia , Probióticos/metabolismo , Saccharomyces/fisiologia , Salmonella typhimurium/fisiologia , Animais , Humanos , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos NOD , Saccharomyces/classificaçãoRESUMO
Dimorphandra mollis (Leguminosae), known as faveiro and fava d'anta, is a tree that is widely distributed throughout the Brazilian Cerrado (a savanna-like biome). This species is economically valuable and has been extensively exploited because its fruits contain the flavonoid rutin, which is used to produce medications for human circulatory diseases. Knowledge about its genetic diversity is needed to guide decisions about the conservation and rational use of this species in order to maintain its diversity. DNA extraction is an essential step for obtaining good results in a molecular analysis. However, DNA isolation from plants is usually compromised by excessive contamination by secondary metabolites. DNA extraction of D. mollis, mainly from mature leaves, results in a highly viscous mass that is difficult to handle and use in techniques that require pure DNA. We tested four protocols for plant DNA extraction that can be used to minimize problems such as contamination by polysaccharides, which is more pronounced in material from mature leaves. The protocol that produced the best DNA quality initially utilizes a sorbitol buffer to remove mucilaginous polysaccharides. The macerated leaf material is washed with this buffer until there is no visible mucilage in the sample. This protocol is adequate for DNA extraction both from young and mature leaves, and could be useful not only for D. mollis but also for other species that have high levels of polysaccharide contamination during the extraction process.
Assuntos
DNA de Plantas/isolamento & purificação , Fabaceae/genética , Folhas de Planta/genética , BrasilRESUMO
Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H(+)-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H(+)-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca(2+)-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H(+)-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H(+)-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP(3). Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H(+)-ATPase activation.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/enzimologia , Glucose/farmacologia , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Vacúolos/metabolismo , Compostos de Boro/farmacologia , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Ácido Egtázico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Modelos Biológicos , Mutação/genética , Nifedipino/farmacologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Canais de Cátion TRPC/metabolismo , Vacúolos/efeitos dos fármacosRESUMO
The cursor complex is a group within the Akodon genus of South American rodents, formed by Akodon cursor and A. montensis. Correct distinction between these two species is of great importance since they can harbor different Hantavirus strains. These species are only distinguishable by means of karyotypic or internal anatomic features, requiring dissection; recently, some other genetic methods have become available. We developed RAPD markers capable of distinguishing between A. cursor and A. montensis. Samples included 42 individuals of A. cursor from four localities and 16 individuals of A. montensis from two localities. Fifty-five bands, 41 of which were polymorphic, were analyzed. A principal component analysis showed that this set of markers could successfully distinguish between the two species, mainly based on three RAPD bands. The number of bands in each population was compared within a 95% confidence interval as a measure of intraspecific variability. The A. cursor populations were found to have marked genetic structure across the study area (AMOVA; F(ST )= 0.21), which in part might be because of the relatively limited dispersal capabilities of this species. Species-specific bands, with potential for species identification, were identified.
Assuntos
Arvicolinae/genética , Reservatórios de Doenças/veterinária , Infecções por Hantavirus/veterinária , Polimorfismo Genético , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Animais , Arvicolinae/anatomia & histologia , Arvicolinae/classificação , Brasil , Reservatórios de Doenças/virologia , Feminino , Marcadores Genéticos , Orthohantavírus/patogenicidade , Infecções por Hantavirus/prevenção & controle , Infecções por Hantavirus/transmissão , Infecções por Hantavirus/virologia , Cariotipagem , Masculino , Filogenia , Análise de Componente Principal , Especificidade da EspécieRESUMO
Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels.
Assuntos
Neurotoxinas/biossíntese , Neurotoxinas/farmacologia , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Animais , Cálcio/fisiologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Linhagem Celular , Clonagem Molecular , Receptores de Inositol 1,4,5-Trifosfato/biossíntese , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Canal de Liberação de Cálcio do Receptor de Rianodina/biossíntese , Venenos de Aranha/biossíntese , Aranhas/químicaRESUMO
The PKC1 gene in the yeast Saccharomyces cerevisiae encodes for protein kinase C which is known to control a MAP kinase cascade consisting of different kinases: Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of pkc1Delta mutants suggests additional targets that have not yet been identified [Heinisch et al., Mol. Microbiol. 32 (1999) 671-680]. The pkc1Delta mutant, as opposed to other mutants in the MAP kinase cascade, displays defects in the control of carbon metabolism. One of them occurs in the derepression of SUC2 gene after exhaustion of glucose from the medium, suggesting an involvement of Pkc1p in the derepression process that is not shared by the downstream MAP kinase cascade. In this work, we demonstrate that Pkc1p is required for the increase of the activity of enzymatic systems during the derepression process. We observed that Pkc1p is involved in the derepression of invertase and alcohol dehydrogenase activities. On the other hand, it seems not to be necessary for the derepression of the enzymes of the GAL system. Our results suggest that Pkc1p is acting through the main glucose repression pathway, since introduction of an additional mutation in the PKC1 gene in yeast strains already presenting mutations in the HXKII or MIG1 genes does not interfere with the typical derepressed phenotype observed in these single mutants. Moreover, our data indicate that Pkc1p participates in this process through the control of the cellular localization of the Mig1 transcriptional factor.
Assuntos
Proteína Quinase C/metabolismo , Saccharomyces cerevisiae/enzimologia , Ágar/farmacologia , Álcool Desidrogenase/metabolismo , Northern Blotting , Western Blotting , Divisão Celular , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Epitopos , Glucose/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Mutação , Fenótipo , Ligação Proteica , Proteína Quinase C/genética , RNA/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Fatores de Tempo , Transcrição Gênica , beta-FrutofuranosidaseRESUMO
The PKC1 gene in the yeast Saccharomyces cerevisiae encodes protein kinase C that is known to control a mitogen-activated protein (MAP) kinase cascade consisting of Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of Pkc1 mutants suggests additional targets which have not yet been identified. We show that a pkc1Delta mutant, as opposed to mutants in the MAP kinase cascade, displays two major defects in the control of carbon metabolism. It shows a delay in the initiation of fermentation upon addition of glucose and a defect in derepression of SUC2 gene after exhaustion of glucose from the medium. After addition of glucose the production of both ethanol and glycerol started very slowly. The V(max) of glucose transport dropped considerably and Northern blot analysis showed that induction of the HXT1, HXT2 and HXT4 genes was strongly reduced. Growth of the pkc1Delta mutant was absent on glycerol and poor on galactose and raffinose. Oxygen uptake was barely present. Derepression of invertase activity and SUC2 transcription upon transfer of cells from glucose to raffinose was deficient in the pkc1Delta mutant as opposed to the wild-type. Our results suggest an involvement of Pkc1p in the control of carbon metabolism which is not shared by the downstream MAP kinase cascade.
Assuntos
Glucose/metabolismo , Glicosídeo Hidrolases/genética , Proteína Quinase C/fisiologia , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Northern Blotting , Regulação Fúngica da Expressão Gênica , Mutação , Proteína Quinase C/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Transcrição Gênica , beta-FrutofuranosidaseRESUMO
As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S.cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.