Posthemorrhagic antipyresis: what stage of fever genesis is affected?

This study was conducted to determine the time course of metabolic changes associated with a switch from a high-fat to a low-fat diet in rats. Adult rats, maintained on a high-fat diet (42% of energy from fat) for 4-5 weeks were switched to a low-fat diet (11% of energy from fat), and the activities of several liver enzymes were followed. Three different phases could be distinguished. The early phase, complete by 2 days after the switch in diets, included an increase in the activity of glucose 6-phosphate dehydrogenase (pentose phosphate pathway), an increase in pyruvate kinase and pyruvate dehydrogenase activities (terminal end of the glycolytic pathway) and an increase in ATP-citrate lyase and fatty acid synthetase (fatty acid synthesis pathway). The early phase also included a decrease in the activity of phosphoenolpyruvate carboxykinase (PEPCK, gluconeogenesis) and a lower branched-chain amino acid dehydrogenase activity (BCAADH, branched-chain amino acid degradation). The concentration of the allosteric phosphofructokinase regulator, fructose 2,6-bisphosphate (Fru-2,6-P2, glycolysis), decreased during the early phase. An intermediate phase could also be discerned between 3 and 10 days after the switch in diets. In this phase, the decreased Fru-2,6-P2 concentration and the decreased PEPCK and BCAADH activities observed in the early phase were reversed. The late phase occurred 10 days after the dietary switch and was characterized by an increase in the activities of glucokinase (glycolytic pathway) and glycogen phosphorylase (associated with glycogenolysis) and by a decrease in glutamate dehydrogenase, PEPCK and BCAADH activities. These measurements indicate that at least 20 days are required before metabolic changes associated with a switch in diet are complete.     

Triosephosphate metabolism by mature boar spermatozoa

Boar sperm rapidly interconverted dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, produced fructose-1,6-bisphosphate, approximately equilibrium concentrations of fructose 6-phosphate and glucose 6-phosphate but not glycerol or glycerol 3-phosphate. In the presence of 3-chloro-1-hydroxypropanone, an inhibitor of stage 2 of the glycolytic pathway, the triosephosphates were metabolized faster, produced less fructose-1,6-bisphosphate, fructose 6-phosphate and glucose 6-phosphate, but not glycerol or glycerol 3-phosphate. This suggests that these cells may have the capacity to convert glycolytic intermediates into a storage metabolite to conserve carbon atoms for the eventual synthesis of lactate.     

The contribution of "cure by dapsone monotherapy'' to the reduction of prevalence of leprosy in the State of Orissa, India, 1983-1993

The activity of phosphofructokinase-2, fructose, 1,6-bisphosphatase, glucokinase, and also the level of fructose 2,6-bisphosphate and glycogen were examined in the liver of normal, and streptozotocin-diabetic rats. It was shown that the activity of phosphofructokinase-2 was decreased in the liver of diabetic rats. Besides that the activity determined at pH 6.6 (the "active" or unphosphorylated enzyme form) was 3-fold reduced whereas the "total" enzyme activity as measured at pH 8.5 was lowered 1,7-fold. The phosphofructokinase-2 activity assay at two pH values allows to estimate a degree of phosphorylation of bifunctional enzyme which is markedly enhanced in diabetes. The fall of the bifunctional enzyme k in case activity is accompanied by the lowered fructose 2.6-bisphosphate level, increased fructose 1,6-bisphosphatase activity that in turn favours the liver tissue glycolysis inhibition and gluconeogenesis enhanced in diabetes.     

The Arg-Gly-Asp motif in the cell adhesion molecule L1 promotes neurite outgrowth via interaction with the alphavbeta3 integrin

This review focuses on the mechanisms of control of heart glycolysis under conditions of normal and reduced oxygen supply. The kinetic properties and the biochemical characteristics of control steps (glucose transporters, hexokinase, glycogen phosphorylase and phosphofructokinases) in the heart are reviewed in the light of recent findings and are considered together to explain the control of glycolysis by substrate supply and availability, energy demand, oxygen deprivation and hormones. The role of fructose 2,6-bisphosphate in the control of glycolysis is analysed in detail. This regulator participates in the stimulation of heart glycolysis in response to glucose, workload, insulin and adrenaline, and it decreases the glycolytic flux when alternative fuels are oxidized. Fructose 2,6-bisphosphate integrates information from various metabolic and signalling pathways and acts as a glycolytic signal. Moreover, a hierarchy in the control of glycolysis occurs and is evidenced in the presence of adrenaline or cyclic AMP, which relieve the inhibition of glycolysis by alternative fuels and stimulate fatty acid oxidation. Insulin and glucose also stimulate glycolysis, but inhibit fatty acid oxidation. The mechanisms of control underlying this fuel selection are discussed. Finally, the study of the metabolic adaptation of glucose metabolism to oxygen deprivation revealed the implication of nitric oxide and cyclic GMP in the control of heart glucose metabolism.     

Effect of troglitazone (Rezulin) on fructose 2,6-bisphosphate concentration and glucose metabolism in isolated rat hepatocytes

The effect of troglitazone, an orally effective thiazolidinedione, on lactate- and glucagon-stimulated gluconeogenesis (in the absence of insulin) was examined in hepatocytes isolated from rats under different nutritional states. Hepatocytes obtained from fed or 20-24 hr fasted male Sprague-Dawley rats were incubated in Krebs-Henseleit Bicarbonate buffer (KHBC) (in presence or absence of 10.0 mM glucose) containing 2.0 mM [U-14C]lactate (0.1-0.25 microCi) with or without 10.0 nM glucagon and troglitazone (30.0 microM) or the appropriate vehicle. Aliquots were removed at specified endpoints and assayed for glucose and fructose 2,6-bisphosphate (F-2,6-P2) concentrations. In 20-24 hour starved hepatocytes, troglitazone produced a 26.1% inhibition of lactate-stimulated gluconeogenesis. This inhibitory effect of troglitazone on hepatic gluconeogenesis was further potentiated by incubation of the cells with glucose in vitro. In hepatocytes obtained from fasted rats (and incubated with 10 mM glucose in vitro) troglitazone reduced lactate-and glucagon-stimulated gluconeogenesis by 53% and 56%, respectively. This reduction in hepatic glucose production was associated with 1.06 and 1.04 fold increase in the hepatocyte F-2,6-P2 content. In isolated hepatocytes from fed animals and incubated with 10 mM glucose in vitro, troglitazone (15 and 30 microM) did not have any effect on either lactate- or glucagon-stimulated gluconeogenesis. However, 30 microM troglitazone significantly enhanced (36%) F-2,6-P2 concentrations during lactate-stimulated gluconeogenesis. These findings demonstrate that troglitazone decreases hepatic glucose production through alterations in the activity of one or more gluconeogenic/glycolytic enzymes, depending upon the nutritional state of the animal and the presence or absence of hormonal modulation. All of the effects of troglitazone in the present study were observed in the absence of insulin, suggesting an "insulinomimetic" effect. However, this does not exclude the possibility that troglitazone may also function as an "insulin sensitizer" in hepatic and certain other tissues.     

Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein

We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic flux in tumor cells. The glycolytic intermediate fructose 1, 6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.     

Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway

A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem. 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.     

Expression of the CB1 and CB2 receptor messenger RNAs during embryonic development in the rat

We mapped the distribution of CB1 and CB2 receptor messenger RNAs in the developing rat to gain insight into how cannabinoids may affect embryogenesis. In situ hybridization histochemistry studies were done using riboprobes specific for CB1 or CB2 receptor messenger RNAs. We found that CB1 and CB2 receptor messenger RNAs are expressed in the placental cone and in the smooth muscle of the maternal uterus at the earliest gestational periods studied [from eight days of gestation (E8) through E12]. In the embryo, as early as E11, CB1 receptor messenger RNA is expressed in some cells of the neural tube and, at later embryological stages (from E15 to E21), in several distinct structures within the central nervous system. In addition, high levels of CB1 receptor messenger RNA were also found in areas of the peripheral nervous system such as the sympathetic and parasympathetic ganglia, in the retina and in the enteric ganglia of the gastrointestinal tract. In addition to neural structures, high levels of the CB1 receptor messenger RNA were also present in two endocrine organs, the thyroid gland and the adrenal gland. On the other hand, CB2 receptor messenger RNA is expressed exclusively in the liver of the embryo as early as E13. The region-specific expression of CB1 and CB2 receptor messenger RNAs suggests that these receptors have a functional role during embryogenesis.     

In vitro maintenance of Eperythrozoon suis

In vitro maintenance of Eperythrozoon suis was attempted using a Petri dish erythrocyte culture system. In preliminary experiments, the optimal conditions for maintaining E. suis attachment to erythrocytes during incubation were anticoagulation with heparin or citrate solution, incubation with 5 or 10% CO2 at 37 degrees C, and incubation with reduced or non-reduced Eagle's minimum essential medium. Using heparin, a CO2 incubator and reduced Eagle's medium (rEM), E. suis metabolic activity was evaluated by measuring glucose consumption, and lactate and pyruvate production. Glucose consumption and lactate production were measurable while pyruvate production was not detected. Erythrocyte integrity was improved by the addition of inosine although no effect was observed on maintenance of E. suis attachment to erythrocytes or the rate of glucose consumption. To determine whether the glucose consumption observed in culture was due to E. suis glycolytic activity or enhanced erythrocyte glycolytic activity, the effect of E. suis killing by EDTA addition to medium was evaluated using rEM containing inosine (rEMI). Glucose consumption decreased proportionally with the decline in the percentage of parasitized erythrocytes induced by EDTA, indicating that glucose consumption was due to E. suis. In a subsequent experiment, the effect of different types of serum (pig or fetal calf serum) and different gaseous environments (5% CO2 incubator or candle jar) were evaluated using rEMI. Glucose consumption by E. suis was significantly increased by the addition of fetal calf serum; however, no difference in the maintenance of E. suis attachment to erythrocytes and in E. suis glycolytic activity was observed between a 5% CO2 incubator and a candle jar. Finally, the effect of medium refreshment (rEMI containing fetal calf serum) was evaluated. Maintenance of E. suis parasitism on erythrocytes and E. suis glycolytic activity were significantly improved by frequent medium refreshment. The maintenance system developed enabled successful metabolic radiolabeling of E. suis for protein/antigen analysis.     

Preparation and evaluation of sustained release cross-linked chitosan microspheres containing phenobarbitone

A procedure was developed for overexpression of Trypanosoma brucei pyruvate kinase in Escherichia coli. The enzyme was purified to near-homogeneity from the bacterial lysate by first removing nucleic acids and contaminating proteins by protamine sulfate precipitation and subsequent passage over a phosphocellulose column. The purified protein is essentially indistinguishable in its physicochemical and kinetic properties from the enzyme purified from trypanosomes. Furthermore, experiments were undertaken to locate the binding site of the allosteric effector fructose 2,6-bisphosphate. Regulation of pyruvate kinase by this effector is unique to trypanosomes and related protozoan organisms. Therefore, a three-dimensional structure model of the enzyme was made, and a putative effector-binding site could be identified in an interdomain cleft. Four residues in this cleft were mutated, and the mutant proteins were produced and purified, using the same methodology as for the wild-type pyruvate kinase. Some mutants showed only minor changes in the activation by the effector. However, substitution of Arg22 by Gly resulted in a 9.2-fold higher S(0.5) for phosphoenolpyruvate and a significantly smaller kcat than the wild-type enzyme. Furthermore, the apparent affinity of this mutant for the allosteric effectors fructose 1,6-bisphosphate and fructose 2,6-bisphosphate was 8.2- and 5.2-fold lower than that of its wild-type counterpart. Effector binding was also affected, although to a lesser extent, in a mutant Phe463Val. These data indicate that particularly residue Arg22, but also Phe463, are somehow involved in the binding of the allosteric effectors.     

Localization of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamus

In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.     

Distinct regions of frequent loss of heterozygosity of chromosome 5p and 5q in human esophageal cancer

Twenty strains of Streptococcus bovis grew more slowly on lactose (1.21 +/- 0.12 h-1) then than on glucose (1.67 +/- 0.12 h-1), and repeated transfers or prolonged growth in continuous culture (more than 200 generations each) did not enhance the growth rate on lactose. Lactose transport activity was poorly correlated with growth rate, and slow growth could not be explained by the ATP production rate (catabolic rate). Batch cultures growing on lactose always had less intracellular fructose 1,6-bisphosphate (Frul,6P2) than cells growing on glucose (6.6 mM compared to 16.7 mM), and this difference could be explained by the pathway of carbon metabolism. Glucose and the glucose moiety of lactose were metabolized by the Embden-Meyerhoff-Parnas (EMP) pathway, but the galactose moiety of lactose was catabolized by the tagatose pathway, a scheme that by-passed Frul,6P2. A mutant capable of co-metabolizing lactose and glucose grew more rapidly when glucose was added, even though the total rate of hexose fermentation did not change. Wild-type S. bovis grew rapidly with galactose and melibiose, but these galactose-containing sugars were activated by galactokinase and catabolized via EMP. On the basis of these results, rapid glycolytic flux through the EMP pathway is needed for the rapid growth (more than 1.2 h-1) of S. bovis.     

Insulin signaling in the yeast Saccharomyces cerevisiae. 1. Stimulation of glucose metabolism and Snf1 kinase by human insulin

Effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae were studied in this report. Under two conditions of growth limitation (glucose-grown cells during transition to stationary phase or spheroplasts during incubation in synthetic glucose medium), human insulin (10 and 1 microM, respectively) enhanced glycogen accumulation and glycogen synthase activity by 40-60% compared to control cells. Glycogen phosphorylase activity was also increased under the same conditions, but this stimulation was diminished by 35-45% in insulin-treated compared to control cells. Thus, under growth limitation, insulin causes glycogen phosphorylase and glycogen synthase to become more sensitive to inactivation and activation, respectively. In glucose-induced spheroplasts, insulin (1 microM), in addition to glycogen accumulation, led to about 2-fold increases of the rates of ethanol production and glucose oxidation compared to control cells, and the maximal concentration of hexose 6-phosphate was increased by 30-40%. In contrast, glucose transport as well as the levels of the allosteric regulators, fructose 2,6-bisphosphate and cAMP, were not altered at all. Snf1 kinase is assumed to be involved in the regulation of glycogen metabolism in yeast, although it does not seem to be modulated directly by the glucose concentration. Snf1 kinase activity was elevated 5-10-fold in response to insulin both during glucose induction of yeast spheroplasts and during transition to stationary phase of glucose-grown cells. We conclude that Saccharomyces cerevisiae and insulin-sensitive mammalian cells share some parts of the signaling cascades regulating oxidative and nonoxidative glucose metabolism in response to glucose and insulin.     

Skewed oligomers and origins of replication

In pigs, induction of embryonic degeneration, by exogenous oestrogens given early in gestation, has been long recognised. However, the underlying mechanisms responsible for this degeneration remain unclear. The present study was conducted to determine whether oestrogen-induced early porcine embryonic mortality was associated with changes in the levels of tumour necrosis factor-alpha (TNF-alpha) messenger RNA in the uterine endometrium. Prepubertal gilts were induced into oestrus with PG600 and artificially inseminated at their second natural oestrus and again 24 h later. After insemination, gilts were randomly assigned to treatment and given 0.5 ml intramuscular injections of either oestradiol valerate (10 mg ml-1) or corn oil on day 9 and 10 of gestation. The gilts were slaughtered on day 12, 15 or 18 of gestation. The reproductive tract was removed from each gilt and the uterine horns were flushed to check for the presence and integrity of embryos. Samples of uterine endometrial tissues were collected, snap-frozen in liquid nitrogen and stored at -80 degrees C. Total cellular RNA was isolated from frozen tissues using a guanidine isothiocyanate-cesium chloride method. The abundance of TNF-alpha messenger RNA was determined by Northern blot hybridisation analysis. Treatment of pregnant gilts with oestrogen resulted in severe fragmentation of embryos on days 15 (2/3) and 18 (2/2), confirming the embryocidal effect of exogenous oestrogen. Uterine TNF-alpha messenger RNA level was elevated in oestrogen-treated gilts compared with controls (P < 0.05). This observation of an association between increased levels of TNF-alpha mRNA in the uterus and embryonic degeneration in oestrogen-treated gilts suggests that TNF-alpha may be involved in mediating oestrogen-induced early embryonic mortality in the pig.     

What makes a life good?

OBJECTIVE: The objective of this study was to evaluate the longitudinal changes in energy expenditure and body composition in relationship to alterations in carbohydrate metabolism in women with normal and abnormal glucose metabolism. We hypothesized that women with decreased insulin sensitivity before conception would have less fat accretion and smaller increases in energy expenditure. STUDY DESIGN: Six women with normal glucose tolerance and 10 women with abnormal glucose tolerance were evaluated before conception, and in early (12 to 14 weeks) and late (34 to 36 weeks) gestation. Body composition was estimated by hydrodensitometry, resting energy expenditure, and glucose and fat metabolism by indirect calorimetry, endogenous glucose production by infusion of [6-6 2H2] glucose, and insulin sensitivity using a hyperinsulinemic-euglycemic clamp (40 mU/m2/min). RESULTS: There was a smaller increase in fat mass (1.3 kg [P = .04]) in early pregnancy in women with abnormal glucose tolerance before pregnancy. Indirect calorimetry measured gestational age-related increases in basal oxygen utilization, with or without correction for fat-free mass (VO2, P = .002), resting energy expenditure (expressed in kilocalories, P = .0001), and carbohydrate oxidation (P = .0003). The insulin-mediated elevation in VO2 increased in later gestation VO2 (P = .005), as did resting energy expenditure (P = .0001) and fat oxidation (P = 0.0001). However, there was a decrease in respiratory quotient (P = .0001), carbohydrate oxidation (P = .002), and nonoxidative carbohydrate metabolism (P = .0001) with advancing gestation during insulin infusion. In early pregnancy, changes in fat mass correlated inversely with changes in insulin sensitivity (r= -0.52, P = .04) and changes in basal VO2 correlated inversely with decreases in basal endogenous glucose production (r = -0.74, P = .01). CONCLUSION: In early gestation, the changes in maternal fat mass and basal oxygen consumption are inversely related to the changes in insulin sensitivity. This response in lean women with decreased insulin sensitivity before conception may have survival value by providing a larger amount of available substrate to meet fetoplacental needs during gestation.     

Regulation of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Role of the NH2-terminal region

The role of the NH2-terminal region of the liver and skeletal muscle 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases was investigated, as well that of a mutant of the liver isoform lacking the first 22 amino acids, by the overexpression of these enzymes in Escherichia coli and the comparison of their kinetic properties. The muscle isoform and the deletion mutant had Km values for fructose 6-phosphate which were 50- and 20-fold higher, respectively, than that of the liver isoform, and the bisphosphatase maximal velocity of the liver deletion mutant was 4-fold higher than that of the native liver isoform. Phosphorylation of the liver isoform increased bisphosphatase activity by 2-3-fold and the Km for fructose 6-phosphate of the 6-phosphofructo-2-kinase by 10-15-fold, but these kinetic effects were greatly diminished for the deletion mutant despite equivalent phosphorylation by cAMP-dependent protein kinase. Arg-173 of the skeletal muscle isoform was found to be functionally equivalent to the residue corresponding to the essential fructose 6-phosphate binding residue of the liver kinase domain, Arg-195. The results suggest that 1) the NH2-terminal regions of the liver and skeletal muscle isoforms are important determinants of fructose 6-phosphate affinity, and 2) the initial 22 amino acids of the liver isoform exert an inhibitory influence on the bisphosphatase and mediate, at least in part, the response of both activities of the enzyme to cAMP-dependent phosphorylation.     

Prolonged exposure to halothane and associated changes in carbohydrate metabolism in rat muscles in vivo

Halothane, an anesthetic presently used in animal experimentation, is reported to stimulate glycogen breakdown in isolated preparations of rat skeletal muscles, suggesting that it may not be a suitable anesthetic for the study of glycogen metabolism in rats in vivo. The purpose of this study was to establish whether prolonged exposure to halothane in rats in vivo is associated with accelerated glycogenolysis. Exposure of rats to halothane for up to 1 h was not accompanied by either any change in the levels of glycogen or increase in activity ratios of glycogen phosphorylase in muscles, irrespective of their fiber compositions. In marked contrast, the levels of lactate, inorganic phosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-bisphosphate, and fructose 2, 6-bisphosphate changed progressively during anesthesia. Accordingly, the interpretation of muscle metabolite levels must be performed with caution in experiments involving prolonged exposure to halothane. Overall, our findings indicate that the reported halothane-mediated stimulation of glycogen breakdown in vitro is likely to be an artifact and that halothane is a suitable anesthetic for experiments concerned with glycogen metabolism in rats.     

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.