The contribution of electrostatic forces to the process of adherence of Candida albicans yeast cells to substrates

Abstract Thermoanaerobacter thermohydrosulfuricus Rt8.B1 catabolized xylose by the pentose phosphate pathway, and xylose isomerase and xylulokinase were inducible. The uptake of xylose was by two low-affinity, inducible systems. Both systems were resistant to the protonophore, tetrachlorosalicylanilide, the F₁F₀-ATPase inhibitor, N , N -dicyclohexylcarboiimide, and the sodium/proton antiporter, monensin. The high capacity system (100 nmol min⁻¹ (mg protein)⁻¹) was only expressed when the bacterium was grown with a high concentration of xylose (50 mM). It took more than 60 mM xylose to saturate the high capacity system. When T. thermohydrosulfuricus was grown with a low concentration of xylose (5 mM), xylose uptake was saturated by as little as 10 mM xylose (18 nmol min⁻¹ (mg protein)⁻¹). Cells grown with 50 mM xylose could not transport glucose, and high capacity xylose transport was not inhibited by glucose or non-metabolizable glucose analogues. Cells grown with 5 mM xylose transported glucose at a rapid rate (30 nmol min⁻¹ (mg protein)⁻¹), and low capacity xylose uptake was competitively inhibited by either glucose or 2-deoxy-glucose. Because the glucose uptake of cells grown on 5 mM xylose was competitively inhibited by xylose, it appeared that the low capacity xylose uptake system was a glucose/xylose carrier.     

Regional modulations in tegumental glucose transporter kinetics in the rat tapeworm

Comparisons of glucose transporter kinetics in 8-day (Km = 0.34 mM, Vmax = 14 nmole.min-1.g-1), 10-day (Km = 0.46 mM, Vmax = 18 nmole.min-1.g-1), the first quartile of 17-day (Km = 0.51 mM, Vmax = 21 nmole.min-1.g-1), and the first quartile of 32-day (Km = 0.33 mM, Vmax = 39 nmole.min-1.g-1) rat tapeworms (Hymenolepis diminuta) suggest maximal velocities may vary with age. A gradient in glucose transporter density is suggested in the rat tapeworm by changes in the estimated transporter Vmax in the first through fourth quartiles. Alterations in the physiological efficiency (as indicated by the Vmax/Km ratio) and permeability (indicated by the unsaturated permeability-area product) of the glucose transporter were determined to be significantly greater in the first quartile than in other quartiles of 17-day hymenolepids. A similar trend was apparent in older (32-day) worms. In tapeworms maintained for 30 min in glucose-free medium, maximal velocities were highest in the anterior (first) quartile, and reductions were seen in successive second, third, and fourth quartiles. When worms were maintained in a medium containing 11 mM glucose, maximal velocities were about twofold greater, but the Vmax increased in each successive quartile. The apparent half-saturation constants, which indicate that concentration of external glucose at which half of the glucose transporter proteins are occupied, are reduced approximately 50% in tapeworms maintained in glucose-free medium. These studies demonstrate that regional differences exist in the glucose transporter of the rat tapeworm, analogous to the intestinal glucose gradient. Furthermore, substrate-induced modulations in the transporter may also exhibit independent regional variability.     

Glycerol utilization in Fusarium oxysporum var. lini: regulation of transport and metabolism

Glycerol was transported in the fungus Fusarium oxysporum var. lini by a facilitated diffusion transport system with a half-saturation constant, Ks, of 0.5 mM and a maximum velocity, Vmax, of 0.9 mmol (g dry wt)-1 h-1 at pH 5 and 25 degrees C. 1,2-Propanediol was a competitive inhibitor of glycerol transport, but the cells did not actively accumulate 1,2-propanediol. The transport system was partially constitutive. In cells grown in the presence of glucose, glycerol was not transported, indicating that the synthesis of the system was under glucose repression. Glycerol kinase and NADP(+)-dependent glycerol dehydrogenase activities were present under all physiological conditions tested. A flavin-dependent glycerol phosphate dehydrogenase was induced only when glycerol was the sole energy source in the medium. This enzyme, together with the transport system, constitute the regulated steps in the glycerol metabolic pathway.     

Kinetics of NO3- Influx in Spruce

Influxes of 13NO3- across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Three kinetically distinct uptake systems for NO3- were identified. In seedlings not previously exposed to external NO3-, a single Michaelis-Menten-type constitutive high-affinity transport system (CHATS) operated in a 2.5 to 500 [mu]M range of external NO3- [NO3-]o. The Vmax of this system was 0.1 [mu]mol g-1 h-1, and the Km was approximately 15 [mu]M. Following exposure to NO3- for 3 d, this CHATS activity was increased approximately 3-fold, with no change of Km. In addition, a NO3--inducible high-affinity system became apparent with a Km of approximately 100[mu]M. The combined Vmax for these discrete saturable components was 0.7 [mu]mol g-1 h-1. In both uninduced and induced plants a linear low-affinity system, additive to CHATS and an NO3--inducible high-affinity system, operated at [NO3-]o [greater than or equal to] 1 mM. The time taken to achieve maximal rates of uptake (full induction) was 2 d from 1.5 mM [NO3-]o and 3 d from 200 [mu]M [NO3-]o.     

Gluconeogenesis predominates in periportal regions of the liver lobule

Rates of gluconeogenesis from lactate were calculated in periportal and pericentral regions of the liver lobule in perfused rat livers from increases in O2 uptake due to lactate. When lactate (0.1-2.0 mM) was infused into livers from fasted rats perfused in either anterograde or the retrograde direction, a good correlation (r = 0.97) between rates of glucose production and extra O2 uptake by the liver was observed as expected. Rates of oxygen uptake were determined subsequently in periportal and pericentral regions of the liver lobule by placing miniature oxygen electrodes on the liver surface and measuring the local change in oxygen concentration when the flow was stopped. Basal rates of oxygen uptake of 142 +/- 11 and 60 +/- 4 mumol X g-1 X h-1 were calculated for periportal and pericentral regions, respectively. Infusion of 2 mM lactate increased oxygen uptake by 71 mumol X g-1 X h-1 in periportal regions and by 29 mumol X g-1 X h-1 in pericentral areas of the liver lobule. Since the stoichiometry between glucose production and extra oxygen uptake is well-established, rates of glucose production in periportal and pericentral regions of the liver lobule were calculated from local changes in rates of oxygen uptake for the first time. Maximal rates of glucose production from lactate (2 mM) were 60 +/- 7 and 25 +/- 4 mumol X g-1 X h-1 in periportal and pericentral zones of the liver lobule, respectively. The lactate concentrations required for half-maximal glucose synthesis were similar (0.4-0.5 mM) in both regions of the liver lobule in the presence or absence of epinephrine (0.1 microM). In the presence of epinephrine, maximal rates of glucose production from lactate were 79 +/- 5 and 59 +/- 3 mumol X g-1 X h-1 in periportal and pericentral regions, respectively. Thus, gluconeogenesis from lactate predominates in periportal areas of the liver lobule during perfusion in the anterograde direction; however, the stimulation by added epinephrine was greatest in pericentral areas. Differences in local rates of glucose synthesis may be due to ATP availability, as a good correlation between basal rates of O2 uptake and rates of gluconeogenesis were observed in both regions of the liver lobule in the presence and absence of epinephrine. In marked contrast, when livers were perfused in the retrograde direction, glucose production was 28 +/- 5 mumol X g-1 X h-1 in periportal areas and 74 +/- 6 mumol X g-1 X h-1 in pericentral regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

皮状丝孢酵母同步利用葡萄糖/木糖的糖转运动力学简

皮状丝孢酵母（ Trichosporon cutaneum）能够同步利用葡萄糖和木糖生产油脂。以2脱氧葡萄糖（2 DOG）为底物,考察皮状丝孢酵母糖跨膜运输的转运动力学。结果表明：2 DOG转运符合米氏方程,表观米氏常数Km为0.19 mmol/L,最大转运速率Vmax为14.1 nmol/（ min＆#183;mg）。葡萄糖和木糖均竞争性抑制2 DOG转运,葡萄糖表观抑制常数Ki远低于木糖,表明存在一个共用转运体系,且该转运体系对葡萄糖亲和力更高。大量木糖与2 DOG同时转运到胞内,进一步说明木糖与葡萄糖共运输。代谢抑制剂和pH对糖转运有明显影响,说明质子/底物同向运输系统是该酵母的主要糖转运系统。     

Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum

The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties, such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4 M glucose/1.0 M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-d-glucopyranoside as substrate the values were Vmax (7614 U/mg), and K_M (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and K_M 0.877 mM (glucose), and 1.410 mM (xylose).     

A glycerol-3-phosphate dehydrogenase-deficient mutant of Saccharomyces cerevisiae expressing the heterologous XYL1 gene.

The gene XYL1, encoding a xylose reductase, from Pichia stipitis was transformed into a mutant of Saccharomyces cerevisiae incapable of glycerol production because of deletion of the genes GPD1 and GPD2. The transformed strain was capable of anaerobic glucose conversion in the presence of added xylose, indicating that the xylose reductase reaction can fulfill the role of the glycerol-3-phosphate dehydrogenase reaction as a redox sink. The specific xylitol production rate obtained was 0.38 g g-1 h-1.     

Transport and metabolism of glucose in an insulin-secreting cell line, beta TC-1.

Kinetic characteristics of glucose transport and glucose phosphorylation were studied in the islet cell line beta TC-1 to explore the roles of these processes in determining the dependence of glucose metabolism and insulin secretion on external glucose. The predominant glucose transporter present was the rat brain/erythrocyte type (Glut1), as determined by RNA and immunoblot analysis. The liver/islet glucose transporter (Glut2) RNA was not detected. The functional parameters of zero-trans glucose entry were Km = 9.5 +/- 2 mM and Vmax = 15.2 +/- 2 nmol min-1 (microL of cell water)-1. Phosphorylation kinetics of two hexokinase activities were characterized in situ. A low-Km (0.036 mM) hexokinase with a Vmax of 0.40 nmol min-1 (microL of cell water)-1 was present along with a high-Km (10 mM) hexokinase, which appeared to conform to a cooperative model with a Hill coefficient of about 1.4 and a Vmax of 0.3 nmol min-1 (microL of cell water)-1. Intracellular glucose at steady state was about 80% of the extracellular glucose from 3 to 15 mM, and transport did not limit metabolism in this range. In this static (nonperifusion) system, 2-3 times more immunoreactive insulin was secreted into the medium at 15 mM glucose than at 3 mM. The dependence of insulin secretion on external glucose roughly paralleled the dependence of glucose metabolism on external glucose. Simulations with a model demonstrated the degree to which changes in transport activity would affect intracellular glucose levels and the rate of the high-Km hexokinase (with the potential to affect insulin release).     

Creatine Transport in Cultured Cells of Rat and Mouse Brain

Astroglia-rich cultures derived from brains of newborn rats or mice use a transport system for the uptake of creatine. The uptake system is saturable, Na+-dependent, and highly specific for creatine and Na+. Kinetic studies on rat cells revealed a Km value for creatine of 45 microM, a Vmax of 17 nmol x h-1 x (mg of protein)-1, and a Km value of 55 mM for Na+. The carrier is competitively inhibited by guanidinopropionate (Ki = 15 microM). No such transport system was found in neuron-rich primary cultures from embryonic rat brain. It is hypothesized that creatine transport is an astroglial rather than a neuronal function.     

Binding-protein-dependent sugar transport by Agrobacterium radiobacter and A. tumefaciens grown in continuous culture

Binding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound D-galactose (KD 0.26 microM) and D-xylose (KD 0.04 microM) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0.045 h-1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyperproduced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A. radiobacter is unique amongst these transport systems in that the mutations that lead to its hyperproduction under carbon limitation render it least susceptible to repression by excess glucose during ammonia limitation, with the result that succinoglucan exopolysaccharide is produced from glucose at an enhanced rate.     

Inhibition of the intestinal transport of uracil by hexoses and amino acids

Various hexoses and amino acids were tested as potential inhibitors of the active mucosal to serosal transport of uracil across the everted rat jejunum. Uracil transport displayed Michaelis-Menten type kinetics with a Vmax of 10.4 +/- 0.2 mumol X g-1 X h-1 and an apparent Km of 0.047 +/- 0.002 mM (means +/- S.D.). Scilliroside, an inhibitor of the basolateral (Na+ + K+)-ATPase, dose-dependently inhibited the transport of uracil consistent with the Na+ dependency of uracil transport. Thymine was a full competitive inhibitor (Ki = 0.021 +/- 0.002 mM) of uracil transport. All actively transported substances tested including L-phenylalanine, L-leucine, D-galactose, D-glucose, and 3-O-methylglucose inhibited the transport of uracil. In contrast, L-glucose and fructose, substances which are not actively transported, were without effect on uracil transport. Further studies with D-galactose indicated that it acts as a partial noncompetitive inhibitor (Ki = 6.0 +/- 1.4 mM) of uracil transport. This Ki is in good agreement with the apparent Kt (5.8 +/- 1.1 mM) for D-galactose transport. Phlorizin (0.1 mM), an inhibitor of galactose transport, blocked the inhibitory effect of galactose on uracil transport. In the ileum D-galactose had no effect on uracil transport but thymine caused the same degree of inhibition as in the jejunum. The results demonstrate that heterologous inhibition is a more general phenomenon than had previously been realized.     

Muscle glucose uptake of obese Zucker rats trained at two different intensities

Exercise training reduces the muscle insulin resistance of the obese Zucker rat. The purpose of the present study was to determine whether the magnitude of this training response is exercise intensity specific. Obese Zucker rats were randomly divided into sedentary (SED), low-intensity (LI), and high-intensity (HI) exercise groups. For the LI rats, exercise training consisted of running on a rodent treadmill at 18 m/min up an 8% grade for 90 min. Rats in the HI group ran at 24 m/min up an 8% grade for four 17-min bouts with 3 min between bouts. Both exercise groups performed the same amount of work and trained 5 days/wk for 7 wk. To evaluate muscle insulin resistance, rat hindlimbs were perfused for 30 min with perfusate containing 6 mM glucose (0.15 mu Ci of D-[14C(U)] glucose/ml) and either a maximal (10.0 mU/ml) or a submaximal (0.50 mU/ml) insulin concentration. Perfusions were performed 48-56 h after the last exercise bout and a 12-h fast. In the presence of 0.5 mU/ml insulin, the rate of muscle glucose uptake was found to be significantly faster for the HI (9.56 +/- 0.66 mumol.h-1.g-1) than for the LI (7.72 +/- 0.65 mumol.h-1.g-1) and SED (6.64 +/- 0.44 mumol.h-1.g-1) rats. The difference in glucose uptake between the LI and SED rats was not significant. In the presence of 10.0 mU/ml insulin, the rate of glucose uptake was significantly faster for the HI (16.43 +/- 1.02 mumol.h-1.g-1) than for the LI rats (13.76 +/- 0.84 mumol.h-1.g-1) and significantly faster for the LI than for the SED rats (11.02 +/- 0.35 mumol.h-1.g-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1)

Influxes of 13NH4+ across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Two kinetically distinct uptake systems for NH4+ were identified. In N-deprived plants, a Michaelis-Menten-type high-affinity transport system (HATS) operated in a 2.5 to 350 [mu]M range of external NH4+ concentration ([NH4 +]o). The Vmax of this HATS was 1.9 to 2.4 [mu]mol g-1 h-1, and the Km was 20 to40 [mu]M. At [NH4+]o from 500 [mu]M to 50 mM, a linear low-affinity system (LATS) was apparent. Both HATS and LATS were constitutive. A time-dependence study of NH4+ influx in previously N-deprived seedlings revealed a small transient increase of NH4+ influx after 24 h of exposure to 100 [mu]M [NH4+]o. This was followed by a decline of influx to a steady-state value after 4 d. In seedlings exposed to 100 [mu]M external NO3- concentration for 3 d, the Vmax for NH4+ uptake by HATS was increased approximately 30% compared to that found in N-deprived seedlings, whereas LATS was down-regulated. The present study defines the much higher uptake capacity for NH4+ than for N03- in seedlings of this species.     

Exchange of 3-O-methylglucose in isolated fat cells. Concentration dependence and effect of insulin.

3-O-[14C]Methylglucose was used to study the insulin action on the sugar transport in white fat cells. The experiments comprised determinations of the 3-O-methylglucose space at stationary distribution, of the rate constants for 3-O-methylglucose equilibrium exchange under various conditions, and of the 3-O-methylglucose inhibition of the lipogenesis from glucose. The following was found. The intracellular distribution space for 3-O-methylglucose at equilibrium was unaffected by insulin and was identical with the intracellular 3H2O space. The half-time for the equilibrium exchange of 3-O-methylglucose at a concentration of 25 mM was about 240 s in the absence of insulin and about 15 s with insulin (0.7 muM) present. Addition of phloridzin (5 mM) decreased the rate of the exchange process about 25-fold in both cases. The self-exchange of 3-O-methylglucose (1 mM) was at least 50 times faster than the self-exchange of L-glucose (1 mM). The concentration dependence of the 3-O-methylglucose exchange rate was approximately hyperbolic both in the absence and the presence of insulin, although the saturation of the transport mechanism at high concentrations of sugar was not as complete as predicted. In the absence of insulin the estimate of the half-saturation constant (Kt) was about 5 mM; that of the maximal exchange rate (Vmax) varied from 0.07 mmol s-1/liter of intracellular water to 0.2 mmol s-1 liter-1. In the presence of insulin Kt remained about 5 mM, whereas Vmax was increased to about 1.7 mmol s-1 liter-1. The latter estimate was reproducible within about 20%. The incorporation of trace amounts of [U-14C]glucose into intracellular lipids was inhibited by unlabeled 3-O-methylglucose pre-equilibrated over the membrane. The inhibition constant estimated from such experiments was about 5 mM both in the absence and the presence of insulin, and the insulin-induced increase in the rate of glucose incorporation was similar to the increase in the rate of the 3-O-methylglucose exchange process. It is concluded that exchange of 3-O-methylglucose proceeds via a mechanism which shows stereospecificity and saturability and that insulin acts by increasing the maximal transport capacity without changing the half-saturation constant.     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp.

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversibility of decreased insulin-stimulated glucose transport capacity in diabetic muscle with in vitro incubation. Insulin is not required

The mechanisms by which insulin deficiency affects muscle glucose transport were investigated. Epitrochlearis muscles from rats with streptozotocin-induced diabetes and from controls were incubated in vitro for 0.5-14 h. The incubation was shown not to impair muscle energy stores or tissue oxygenation. Diabetes decreased basal 3-O-methylglucose transport by 40% (p less than 0.01), and insulin-stimulated (20 milli-units/ml) glucose transport capacity by 70% (p less than 0.001). In vitro incubation gradually normalized insulin responsiveness (3.77 +/- 0.38 before versus 8.97 +/- 0.65 mumol X ml-1 X h-1 after 12 h of incubation). Basal glucose transport remained significantly reduced. The reversal of the insulin responsiveness did not require the presence of rat serum and, furthermore, took place even in the absence of insulin. In fact, insulin responsiveness was higher after incubation (14 h) with no insulin than with 100 microunits/ml insulin (9.85 +/- 0.59 versus 8.06 +/- 0.59 mumol X ml-1 X h-1, p less than 0.05). Glucose at 30 mM did not affect the normalization of the insulin-stimulated glucose transport capacity, whereas incubation in serum from diabetic rats resulted in a slightly (26%) blunted reversal (7.60 +/- 0.39 versus 8.89 +/- 0.45 mumol X ml-1 X h-1 with diabetic versus control serum for 14 h, p less than 0.05; before incubation the value was 3.87 +/- 0.40). Inhibition of protein synthesis by cycloheximide blocked the normalization by 80%. These results suggest the presence in diabetic serum of some labile factor that might inhibit the glucose transport system. The results indicate that the decreased insulin-stimulated glucose transport capacity, in the insulin-deficient diabetic muscle, is not a direct consequence of the lack of insulin or of high glucose concentrations.     

酿酒酵母和粟酒裂殖酵母属间融合和融合子特性

酿酒酵母(Saccharomyces cerevisiae Hansen)PW218和粟酒裂殖酵母(Schizosaccharomyces pombe Lindn)PW232的原生质体用20mmol/L CaCl_2和30％PEG(MW6000)处理进行属间融合,获得了10多株融合子,融合率为0.65～1.96×10~(-5)。对F_2和F_(10)两株融合子进行了葡萄糖、木糖及葡萄糖和木糖混合液的摇瓶实验结果表明F_(10)融合子利用葡萄糖、木糖及两种糖混合液产乙醇的能力大大高于两亲株。F_2融合子对木糖以及葡萄糖和木糖混合液的发酵能力亦较两亲株高,其中利用木糖产乙醇的量分别比PW218和PW232提高1.38倍和2.65倍。     

Characterization and regulation of galactose transport in Neurospora crassa

Two galactose uptake systems were found in the mycelia of Neurospora crassa. In glucose-grown mycelia, galactose was transported by a low-affinity (Km = 400 mM) constitutive system which was distinct from the previously described glucose transport system I (R. P. Schneider and W. R. Wiley, J. Bacteriol. 106:479--486, 1971). In carbon-starved mycelia or mycelia incubated with galactose, a second galactose transport activity appeared which required energy, had a high affinity for galactose (Km = 0.7 mM), and was shown to be the same as glucose transport system II. System II also transported mannose, 2-deoxyglucose, xylose, and talose and is therefore a general monosaccharide transport system. System II was derepressed by carbon starvation, completely repressed by glucose, mannose, and 2-deoxyglucose, and partially repressed by fructose and xylose. Incubation with galactose yielded twice as much activity as starvation. This extra induction by galactose required protein synthesis, and represented an increase in activity of system II rather than the induction of another transport system. Glucose, mannose, and 2-deoxyglucose caused rapid degradation of preexisting system II; fructose and xylose caused a slower degradation of activity.