RESUMO
The expression of the high-affinity trehalose-H+ symport was investigated in various Saccharomyces cerevisiae strains and culture conditions. Previous kinetic studies of trehalose transport in yeast have revealed the existence of at least two different uptake mechanisms: a high-affinity trehalose-H+ symport activity repressed by glucose, and a constitutive low-affinity transport activity, a putative facilitated diffusion process. Exogenously added trehalose was not an inducer of the high-affinity transport activity, and a correlation between trehalose and maltose uptake by yeast cells was found. Our results indicate that the maltose-H+ symporters encoded by MAL11, MAL21, and MAL41 are not responsible for the trehalose transport activity. The analysis of both trehalose and maltose transport activities in wild-type and in laboratory strains with defined MAL genes showed that the trehalose-H+ symporter was under control of MAL regulatory genes. Our results also suggest that the recently characterized AGT1 gene of S. cerevisiae may encode the high-affinity trehalose-H+ symporter. During diauxic growth on glucose the transport activity was low during the first exponential phase of growth, increased as glucose was exhausted from the medium, and decreased again as the cells reached the late stationary phase. This pattern was coincident with that of the intracellular levels of trehalose. The strong correlation between these two parameters may be of physiological significance during adaptation of yeast cells to stress conditions.
Assuntos
Proteínas de Transporte/genética , Regulação Fúngica da Expressão Gênica/genética , Transporte de Íons/fisiologia , Proteínas de Transporte de Monossacarídeos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Simportadores , Trealose/metabolismo , Transporte Biológico/fisiologia , Proteínas de Transporte/classificação , Proteínas de Transporte/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica/genética , Genes Fúngicos/genética , Glucose/metabolismo , Glucose/farmacologia , Maltose/farmacologia , Dados de Sequência Molecular , Saccharomyces cerevisiae/metabolismoRESUMO
The effect of ethanol on stability of intact yeast cells has been investigated. Several strains with differences in trehalose metabolism were examined for their ability to survive in the presence of 10% (v/v) ethanol. A positive correlation was observed between cell viability and trehalose concentration. When leakage of electrolytes from the cells was recorded by observing changes in conductivity of the medium, we found that ethanol increases leakage, but the presence of trehalose reverses that effect. Similar studies were done with liposomes of similar composition to those seen in intact cells in log and stationary phases. In the presence of ethanol, carboxyfluorescein trapped in the liposomes leaked to the medium. When trehalose was added inside, outside or on both sides of the membrane, the ethanol-induced leakage was strongly inhibited. More leakage was observed in liposomes in gel phase state than in liquid-crystalline phase, suggesting that the thermotropic behavior of the lipids in the plasma membrane, together with trehalose, plays a role in enhancing ethanol tolerance.
Assuntos
Etanol/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Trealose/farmacologia , 1,2-Dipalmitoilfosfatidilcolina , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Etanol/toxicidade , Fluoresceínas , Lipossomos , Fosfatidilcolinas , Saccharomyces cerevisiae/genéticaRESUMO
A new method for the preparation of [14C]trehalose was developed, based on the ability of yeast cells to accumulate trehalose under stress. The method is simple and reliable. It utilizes a yeast strain in which the gene that encodes for phosphoglucoisomerase has been deleted. Thus, exogenously supplied glucose is not metabolized, but is instead converted to trehalose. The [14C]-trehalose obtained is pure, it is hydrolyzed by trehalase, and it is not susceptible to the action of alpha-glucosidase. The yield of this method is in the order of 35% of the [14C]glucose supplied.
Assuntos
Trealose/biossíntese , Radioisótopos de Carbono , Estudos de Avaliação como Assunto , Frutose/metabolismo , Deleção de Genes , Genes Fúngicos , Glucose/metabolismo , Glucose-6-Fosfato Isomerase/genética , Cinética , Métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Temperatura , Trealose/isolamento & purificaçãoRESUMO
OBJECTIVE: To investigate lipid tolerance in sick, ventilator-dependent, very low birth weight infants from the first day of life and the effects of early introduction of intravenously administered lipid (IVL) on glucose homeostasis. METHOD: Twenty-nine infants in the neonatal intensive care unit with birth weight less than 1500 gm received isocaloric, isonitrogenous parenteral feedings from day 1 with either IVL, 1 gm/kg from day 1 to 3 gm/kg from day 4 (group I; n = 16), or IVL added only from day 8 (group II; n = 13). Possible adverse clinical effects were monitored. Blood metabolites, nonesterified fatty acids, serum triglycerides, and insulin levels were determined daily. Arterial blood gases were measured and changes in partial pressures of oxygen and of carbon dioxide in arterial blood were compared between the two groups. RESULTS: Early lipid infusion did not appear to have deleterious effects on blood gas tensions or to increase respiratory morbidity. The incidence of other adverse clinical effects that may be associated with IVL was not increased by earlier introduction of lipid. Serum lipid values were comparable to those of preterm infants receiving IVL at a later postnatal age. Blood glucose concentrations were higher in group II (mean, 7.50 (SEM 0.43) mmol/L) than in group I (mean, 6.01 (SEM 0.28) mmol/L; p less than 0.05). There was no evidence of increased gluconeogenesis in infants in group I and no correlation between blood glucose concentrations and serum nonesterified fatty acid concentrations. CONCLUSION: When given infusion rates not exceeding 0.15 gm/kg/hr, sick, very low birth weight infants can tolerate IVL with stepwise dose increases from the first day of life without an increased incidence of possible adverse effects.
Assuntos
Emulsões Gordurosas Intravenosas/administração & dosagem , Recém-Nascido de Baixo Peso , Peso ao Nascer , Glicemia/efeitos dos fármacos , Emulsões Gordurosas Intravenosas/farmacologia , Idade Gestacional , Humanos , Recém-Nascido , Infusões Intravenosas , Insulina/sangue , Unidades de Terapia Intensiva Neonatal , Nutrição Parenteral , Troca Gasosa Pulmonar/efeitos dos fármacosRESUMO
We have isolated and characterized a membrane vesicle fraction from yeasts that is capable trehalose transport. The kinetics of the transport system were similar to those seen in the intact cells. The transport depends on a transmembrane pH gradient. If the gradient is collapsed, trehalose accumulated inside the vesicles is leaked into the medium. After aging for several days the ability of the vesicles to transport was lost. However, transport was partially restored by elevating internal pH in the vesicles.