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[Carbohydrate metabolic characteristics of coryneform bacteria].

The peculiarities of glucose metabolism were studied in typical representatives of coryneform bacteria, and its relation to inorganic polyphosphate metabolism was shown. The activity of the first two enzymes in the pentose pathway was found to be low. The key enzymes of glycolysis were detected, viz. fructose-1,6-diphosphate aldolase and 3-phosphoglyceraldehyde dehydrogenase. The second enzyme was more active in Nocardia sp. B-293 similar in its properties to Nocardia erythropolis as compared to the enzymes of the pentose shunt. The existence of polyphosphate glucokinase more active than ATP-dependent hexokinase is indicative of inorganic polyphosphates being involved in the metabolism of the above microorganisms.

Actinomycetales↗

Role of fatty acid synthesis in the control of insulin-stimulated glucose utilization by rat adipocytes.

A decreased capacity for fatty acid synthesis is associated with a decreased insulin effect on glucose metabolism in large fat cells and fat cells from rats fed a high-fat diet. We have investigated the relationship between these processes by specifically inhibiting fatty acid synthesis with (-)-hydroxycitrate (2.5 mM), an inhibitor of citrate cleavage enzyme, and cerulenin (0.05 mM), an inhibitor of fatty acid synthetase. (-)-Hydroxycitrate and cerulenin decreased maximally insulin-stimulated fatty acid synthesis from [6-(14)C]glucose to 10% and 25% of controls, respectively, while only (-)-hydroxycitrate decreased basal values. Oxidation of [1-(14)C]glucose in the presence of insulin was markedly depressed by each inhibitor. Thus, the percent increase over basal value was decreased from 540% in controls to 151% and 154% by (-)-hydroxycitrate and cerulenin, respectively. In contrast, oxidation of [6-(14)C]glucose was slightly enhanced by both inhibitors. Thus, oxidation of glucose via the pentose shunt was reduced, while Krebs cycle oxidation was unaffected. Basal and insulin-stimulated incorporation of [1-(14)C]glucose and [6-(14)C]glucose into glyceride-glycerol and basal lactate production was unchanged by the inhibition of fatty acid synthesis. Insulin-stimulated lactate production was halved by the inhibition of fatty acid synthesis. Total glucose utilization, as assessed by measuring the disappearance of glucose from the medium, was not detectably changed by inhibiting fatty acid synthesis under basal conditions, but insulin-stimulated values were decreased to 52% and 64% of control by (-)-hydroxycitrate and cerulenin, respectively. This occurred despite the fact that neither agent affected the initial rate of 2-deoxyglucose uptake, or glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase activities. These data therefore provide direct evidence that a limitation of the fatty acid synthetic pathway decreases the ability of insulin to stimulate both pentose shunt glucose oxidation and overall glucose utilization, but not Krebs cycle oxidation or glyceride-glycerol synthesis. The enzymatic capacity of the fat cell for fatty acid synthesis is therefore an important determinant of insulin-stimulated glucose utilization.-Fried, S. K., M. Lavau, and F. X. Pi-Sunyer. Role of fatty acid synthesis in the control of insulin-stimulated glucose utilization by rat adipocytes.

Adipose Tissue↗

[Effects of prostaglandin F 2 alpha on the activity of NADP-dependent dehydrogenases].

The effects of prostaglandin F2 alpha (0,004--0,4 mg per 100 g) on the activity of NADP-dependent dehydrogenases from rat liver and uterus were studied. After 4 hrs following F2 alpha injection the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was found to increase 4,3- and 1,2-fold in the liver and 5- and 1,6-fold in the uterus, respectively. The content of glucose in the blood remained practically unchanged. No correlation between the amount of F2 alpha and the activities of the pentose phosphate pathway dehydrogenases was observed. The effect of F2 alpha on the dehydrogenases from rat uterus (but not liver) depended on the phase of the sexual cycle and reached its maximum in the period of the secretory activity (estrus). The administration of F2 alpha in combination with alpha- and beta-adrenergic blockers (phentolamine or propranolol) did not only enhance but also potentiated their stimulating effects on the activities of the dehydrogenases. F2 alpha shifted the ratio of reduced and oxidized forms of pyridine nucleotides in the liver, increasing it by 11,2 and 33,3% for NADPH/NADP+ and NADH/NADP+, respectively. The NADP-dependent isocitrate, malate and glutamate dehydrogenases from rat liver and uterus as well as the non-oxidizing enzymes of the pentose phosphate pathway of carbohydrate metabolism were insensitive to F2 alpha.

Animals↗

[Energy metabolism of spermatozoa: present status of the subject].

The energetic metabolism of the human sperm is characterized by the predominant importance of glycolytic pathway, with the probable participation of the pentose shunt. The participation of the aerobic pathway is very small since the oxidative phosphorylation seems to be partially uncoupled at time of ejaculation. There is no correlation between the respiratory activity and the mobility of the sperm. The effects of several physicochemical conditions were studied, indicating that the spermatozoon is indeed very resistant to variations of its environment. Seminal plasma seems to be a rather unsuitable medium for investigations of the glycolytic pathway, with the probable participation of the pentose shunt.

Adenosine Triphosphate↗

[Inhibition of calcium absorption and conditions of its prolongation in the presence of carbohydrate].

Experiments were performed on adult rats. In situ ligatured ileal loops were injected with a solution 10 mM CaCl2 containing 45Ca either with or without L-xylose in concentrations of 25, 50 or 100 mM L-xylose. The rats were sacrified at either 0.5, 1.0, 1.5 or 2.0 hours after injection; the entire loop including the wall and content was removed. After ashing, the radioactivity of the loop was measured and the absorption coefficient of 45Ca was calculated. After half an hour, the absorption coefficient of the four solutions had about the same value. At this time, the 45Ca absorption was completed with the solution containing only calcium. With the solution containing pentose, the 45Ca absorption continued for a longer period of time. The maximal values of absorption coefficient were obtained after one hour with 25 and 50 mM L-xylose and after one and a half hour with 100 mM L-xylose; similarly, the maximal values increased in proportion to the concentration of pentose in the solution. The contents of the loops were fractionated successively by centrifugation, ultracentrifugation and two membrane ultrafiltration. No differences were observed between the repartition of 40Ca or 45Ca, either with or without 50 mM L-xylose. The stop or the lengthening of the absorption could be attributed to a possible influence of carbohydrate upon the mechanism of calcium transfer. This question is discussed from the point of relationship between the carbohydrate and the intestinal alkaline phosphatase.

Absorption↗

[Metabolic effects related to transketolase inhibition].

Inhibition of rat liver transketolase under conditions of hydroxythiamin or alimentary B1 avitaminosis was accompanied by an increase in intracellular concentration of 6-phosphogluconate and xylulose-5-phosphate. The alterations in their content was inversely correlated with the transketolase activity. The dehydrogenases involved in the pentose phosphate shunt exhibited various unspecific sensitivity to hydroxythiamin. The doses of the antimetabolites up to 0.4 mg per rat did not alter the activity of the pentose phosphate shunt dehydrogenases; an increase in the dose from 4 up to 20 mg led to an inhibition of the enzymes. Possible biochemical alteration, occurring in response to various degree of transketolase inhibitions, are discussed.

Animals↗

Effects of prolactin and androgens on enzymes of carbohydrate metabolism in seminal vesicles of castrated mature bonnet monkeys, Macaca radiata.

Effects of prolactin(Prl), bromocriptine(Br), testosterone propionate (TP), dihydrotestosterone (DHT) and combinations of these androgens with Prl/Br on the maximum catalytic capacities of seminal vesicular enzymes involved in the glycolytic and pentose phosphate pathways in castrated mature monkeys were studied. Castration decreased the activities of all of the enzymes studied such as hexokinase(HK), 6-phosphofructokinase(PFK), glyceraldehyde-3-phosphate dehydrogenase(G3PD), pyruvate kinase(PK), glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase(6PGD) in the seminal vesicles. Prl restored the activities of all of the enzymes to their normal values except G3PD. TP/DHT maintained all the enzyme activities at the normal tissue intact level. Prl given along with androgens further enhanced the androgen action with regard to all the enzymes activities except G3PD. Br decreased all of the enzymes but Br with androgens maintained all the enzyme activities at the normal level. Castration decreased significantly serum T/DHT titres but Prl did not alter Prl levels. Prl+TP/DHT elevated Prl levels. Br alone decreased serum Prl, T and DHT titres, but Br+TP/DHT decreased only Prl, elevated T and maintained DHT levels. These results suggest that Prl has a direct as well as a synergistic action with androgens on the activities of the enzymes of glycolysis and pentose phosphate pathways in the seminal vesicles of castrated monkeys.

Androgens↗

Effects of chlorhexidine diacetate and cetylpyridinium chloride on whole cells and protoplasts of Saccharomyces cerevisiae.

Chlorhexidine diacetate (CHA) and the quaternary ammonium compound, cetylpyridinium chloride (CPC), were fungicidal to Saccharomyces cerevisiae A364A and CHA to its mannoprotein mutant LB6-5D. Both CHA and CPC induced leakage of K+ and pentose material from both strains and both agents induced protoplast lysis as well as interacting with crude cell sap. Differences were observed between CHA and CPC in protoplast lysis and in cell sap interaction. Q25 values for A364A with CHA as test agent were 5.6 and 1.6 (fungicidal activity, depending on method of calculation), 1.46 (cell sap interaction) and 0.77 (leakage of pentoses). A sub-lytic concentration of CHA reduced considerably protoplast regeneration. Although the plasma membrane is an important target site for both agents, interaction with other cellular components also contributes to fungal death.

Cetylpyridinium↗

[The interrelationship of changes in carbohydrate-energy metabolism and potassium homeostasis in children with diffuse suppurative peritonitis].

The parameters of potassium homeostasis (potassium concentration in the plasma, red cells, and urine and potassium to sodium ratio in the urine) and of carbohydrate-energy metabolism (CEM) assessed from the activities of the key enzymes of glycolysis and pentose phosphate route of glucose oxidation were determined in 52 patients on day 2 postoperation. Relationships between potassium metabolism and CEM status were revealed in children with diffuse peritonitis. Disorders in potassium homeostasis were associated with CEM changes. In children with a favorable course of the disease the adaptation changes in the metabolic system, characterized by increased intensity of glucose oxidation and intactness of cellular energy homeostasis, permit the maintenance of potassium homeostasis at optimal potassemia of low therapeutic value. In case of a lethal outcome failure of therapeutic measures aimed at correction of potassium homeostasis is coupled with CEM disorders manifesting by reduced glucose oxidation after the pentose phosphate route, reduced concentration of adenyl nucleotides, disordered membrane permeability, and accumulation of organic acids.

Acid-Base Equilibrium↗

Deoxycytidine kinase from calf thymus. Substrate and inhibitor specificity.

Kinetic constants were determined for 34 nucleoside substrates of deoxycytidine kinase (EC 2.7.1.74) from calf thymus. Substrate efficiency was assessed by the ratio of Vmax to Km. Inhibition constants were determined for 61 nonsubstrate nucleosides or nucleoside analogues. The enzyme was relatively specific for the pentose moiety of nucleoside substrates. beta-D-2'-Deoxyribonucleosides were more efficient substrates than the corresponding beta-D-arabinonucleosides. Unexpectedly, the L isomer of the beta-arabinonucleoside of cytosine was a more efficient substrate than was the D isomer. beta-Cytidine and beta-5-azacytidine were the only beta-D-ribonucleosides studied that had detectable substrate activity. alpha-Cytidine was an inhibitor but not a substrate. Nucleosides containing a variety of sugar moieties other than those mentioned above did not have detectable substrate activity. The enzyme was relatively nonspecific for the base moiety of nucleoside substrates. 2'-Deoxyribonucleosides of a variety of pyrimidines, purines, and other heterocycles were substrates. Cytosine was the most preferred pyrimidine moiety. 5-Substitution, except with fluorine, decreased substrate efficiency with nucleosides of cytosine or uracil. 2-Fluoradenine was the most preferred purine moiety. The effects of various purine ring substituents were interdependent. Nucleosides containing bulky, hydrophobic substituents on either the base or the pentose moiety had no substrate activity but were relatively potent competitive inhibitors. This suggested the presence of a hydrophobic region on the surface of the enzyme near the active site.

Adenine↗

THE OCCURRENCE OF APIOSE IN LEMNA (DUCKWEED) AND OTHER ANGIOSPERMS.

1. The branched-chain pentose apiose reacts with the benzidine-trichloroacetic acid reagent on paper chromatograms to give a yellow spot with.an intense white fluorescence in ultraviolet light; on chromatograms developed with butanol-acetic acid-water this spot lies between fucose and rhamnose. 2. Examination of paper chromatograms of hydrolysates of whole plant material has shown the presence of a substance with these properties in a wide variety of species. 3. Among the plants examined two members of the Lemnaceae (Lemna minor and Wolffia arrhiza) were found to be especially rich sources, comparable with Posidonia australis (Bell, Hardwick, Isherwood & Cahn, 1954). 4. Measurements of the apiose content of fractions derived from Lemna have shown that the sugar is present at a concentration of about 4% in the holocellulose, and part of this is retained in the alpha-cellulose left by extraction with 24% (w/v) potassium hydroxide containing borate.

Araceae↗

[Radiological innovations in the screening and diagnosis of the inborn errors of metabolism].

New metabolic diseases are regularly identified by a genetic or biochemical approach. Indeed, the metabolic diseases result from an enzymatic block with accumulation of a metabolite upstream to the block and deficit of a metabolite downstream. The characterization of these abnormal metabolites by MRI spectroscopy permitted to identify the deficient enzyme in two new groups of diseases, creatine deficiencies and polyol anomalies. Creatine deficiency is implicated in unspecific mental retardation. A low peak of creatine at MRI spectroscopy is evocating of creatine deficiency which is treatable by creatine administration. Deficiency of synthesis of polyols, metabolites on the pentose pathway, represent new described metabolic diseases with variable symptoms including a neurological distress, liver disease, splenomegaly, cutis laxa and renal insufficiency. The deficit of ribose-5-phosphate isomerase, one of the enzymes whose diagnosis is evoked in front of the accumulation of ribitol, arabitol and xylitol leads to a leucodystrophy in adults. This new deficit was highlighted by the identification of an abnormal peak in cerebral MRI-spectroscopy corresponding to the abnormal accumulation of polyols in brain. Congenital hyperinsulinism (HI) is characterized by profound hypoglycaemia related to inappropriate insulin secretion. Focal and diffuse forms of hyperinsulinism share a similar clinical presentation but their treatment is dramatically different. Until recently, preoperative differential diagnosis was based on pancreatic venous sampling, an invasive and technically demanding technique. Positron emission tomography (PET) after injection of [18F]Fluoro-L-DOPA has been evaluated for the preoperative differentiation between focal and diffuse HI, by imaging uptake of radiotracer and the conversion of [18F]Fluoro-L-DOPA into dopamine by DOPA decarboxylase. PET with [18F]Fluoro-L-DOPA has been validated as a reliable test to differentiate diffuse and focal HI and is now a major differential diagnosis tool in infantile hyperinsulinemic hypoglycaemia.

Aldose-Ketose Isomerases↗

BACTEROIDES ORALIS, PROPOSED NEW SPECIES ISOLATED FROM THE ORAL CAVITY OF MAN.

Loesche, W. J. (Forsyth Dental Center and Harvard School of Dental Medicine, Boston, Mass.), S. S. Socransky, and R. J. Gibbons. Bacteroides oralis, proposed new species isolated from the oral cavity of man. J. Bacteriol. 88:1329-1337. 1964.-Twenty-seven strains of oral bacteroides were characterized and compared with a National Collection of Type Cultures strain of Bacteroides fragilis as well as strains of intestinal bacteroides. A combination of acid end products, gas production, pentose fermentation, and 10% bile tolerance afforded a basis for separation of oral strains from intestinal strains. The oral strains produced succinic, lactic, acetic, and formic acids but no gas. They were inhibited by 10% bile, and did not ferment xylose and arabinose. Intestinal strains consistent with the description for B. fragilis produced succinic, formic, acetic, and propionic acids and gas, with a few strains forming lactic acid. They were stimulated by 10% beef bile, and fermented xylose and arabinose. The oral strains are sufficiently different from intestinal strains of the B. fragilis type, so that they appear to constitute a separate species. It is proposed that they be recognized as B. oralis, sp. n.

Acetates↗

Effect of methionine on glycolysis in tumor cells: in vivo and in vitro NMR studies.

Inhibition of glycolysis by methionine is a phenomenon previously shown in transformed cells growing in culture. In a recent paper, [Collet V. et al., Q. Magn. Res. Biol. Med. 11, 127-134 (1995)] we investigated this effect in vivo by 13C nuclear magnetic resonance spectroscopy, but the results did not clearly support this hypothesis. In this work, in vivo 13C NMR spectroscopy has been performed on tumors developing in nude mice following the injection of two types of cells established in culture: (1) rat kidney cells transformed by Kirsten murine sarcoma virus, (NRK-K), i.e. the same tumor cell line as that used in the original paper; and (2) a well dedifferentiated human prostate adenocarcinoma cell line (PC3). Furthermore, in vitro experiments were performed with the same tumor cell lines. The effect of methionine on glycolysis was assayed by biochemical monitoring of lactate production in the supernatant of these cells grown in vitro. Lastly, 1H in vitro NMR spectroscopy of the PC3 line performed on perchloric extracts of both supernatants and cells growing in the presence of (1-13C) glucose, allowed simultaneous detection of glucose and lactate as well as estimation of the lactate-specific enrichment. The in vitro experiments confirmed the inhibiting effect of methionine on glycolysis and demonstrated the absence of a significant modification of the pentose phosphate pathway activity by this aminoacid. In contrast, none of the in vivo experimental results were compatible with this phenomenon, which is probably affected by more general physiological events.

Adenocarcinoma↗

Intracellular fluxes in a recombinant xylose-utilizing Saccharomyces cerevisiae cultivated anaerobically at different dilution rates and feed concentrations.

A metabolic flux model was constructed for the yeast Saccharomyces cerevisiae comprising the most important reactions during anaerobic metabolism of xylose and glucose. The model was used to calculate the intracellular fluxes in a recombinant, xylose-utilizing strain of S. cerevisiae (TMB 3001) grown anaerobically in a defined medium at dilution rates of 0.03, 0.06, and 0.18 h(-1). The feed concentration was varied from 0 g/L xylose and 20 g/L glucose to a mixture of 15 g/L xylose and 5 g/L glucose, so that the total concentration of carbon source was kept at 20 g/L. The specific uptake of xylose increased with the xylose concentration in the feed and with increasing dilution rate. The excreted xylitol was less than half of the xylose consumed. With increasing xylose concentration in the feed, the fluxes in the pentose phosphate pathway increased, whereas the flux through glycolysis decreased. Under all cultivation conditions, nicotinamide adenine dinucleotide (NADH) was the preferred cofactor for xylose reductase. The model showed that the flux through the reaction from ribulose 5-phosphate to xylulose 5-phosphate was very low under all cultivation conditions.

Aldehyde Reductase↗

Profiling of dynamic changes in hypermetabolic livers.

The liver plays an important role in the overall negative nitrogen balance leading to muscle wasting commonly observed in patients following many conditions, including severe injury, cancer, and diabetes. In order to study changes in liver metabolism during the establishment of such catabolic states, we used a rat skin burn injury model that induces hypermetabolism and muscle wasting. At various times during the first week following the injury, livers were isolated and perfused in a recirculating system under well-defined conditions. We applied a steady-state metabolic flux analysis model of liver metabolism and then used k-means clustering to objectively group together reaction flux time profiles. We identified six distinct groups of reactions that were differentially responsive: (1) pentose phosphate pathway (PPP); (2) amino acid oxidation reactions leading to the formation of tricarboxylic acid (TCA) cycle intermediates; (3) gluconeogenesis; (4) TCA-cycle and mitochondrial oxidation; (5) lipolysis, beta-oxidation, and ketone body formation; and (6) urea-cycle. Burn injury sequentially upregulated the urea-cycle, the PPP, and the TCA-cycle, in order, while beta-oxidation and gluconeogenesis remained unchanged. The upregulation of the PPP was transient, whereas the rise in urea- and TCA-cycle fluxes was sustained. An ATP balance predicted an increased production of ATP and energy expenditure starting on day 3 post-burn, which correlated with the induction of the oxidative phosphorylation uncoupler uncoupling protein-2. We conclude that metabolic profiling using flux analysis and clustering analysis is a useful methodology to characterize the differential activation of metabolic pathways in perfused organs and to identify specific key pathways that are sensitive to a stimulus or insult without making a priori assumptions.

Adaptation, Physiological↗

Metabolic network analysis of lysine producing Corynebacterium glutamicum at a miniaturized scale.

We present a straightforward approach comprising (13)C tracer experiments at 200-microL volume in 96-well microtiter plates with on-line measurement of dissolved oxygen for quantitative high-throughput metabolic network analysis at a miniaturized scale. This method was successfully applied for cultivation and (13)C metabolic flux analysis of two mutants of lysine producing Corynebacterium glutamicum (ATCC 13287 and ATCC 21543). Microtiter-plate cultivations showed excellent accordance in kinetics and stoichiometry of growth and product formation as well as in intracellular flux distributions as compared with parallel shake-flask experiments. These cultivations further allowed clear identification of strain-specific flux differences such as increased flux toward lysine, increased flux through the pentose phosphate pathway (PPP), decreased flux through the tricarboxylic (TCA) cycle, and increased dihydroxyacetone formation in C. glutamicum ATCC 21543 compared with ATCC 13287. The present approach has strong potential for broad quantitative screening of metabolic network activities, especially those involving high-cost tracer substrates.

Carbon Isotopes↗

Interpretation of metabolic flux maps by limitation potentials and constrained limitation sensitivities.

Two new concepts, "Limitation Potential" and "Constraint Limitation Sensitivity" are introduced that use definitions derived from metabolic flux analysis (MFA) and metabolic network analysis (MNA). They are applied to interpret a measured flux distribution in the context of all possible flux distributions and thus combine MFA with MNA. The proposed measures are used to quantify and compare the influence of intracellular fluxes on the production yield. The methods are purely based on the stoichiometry of the network and constraints that are given from irreversible fluxes. In contrast to metabolic control analysis (MCA), within this approach no information about the kinetic mechanisms are needed. A limitation potential (LP) is defined as the reduction of the reachable (theoretical) maximum by a measured flux. Measured fluxes that strongly narrow the reachable maximum are assumed to be limiting as the network has no ability to counterbalance the restriction due to the observed flux. In a second step, the sensitivity of the reduced maximum is regarded. This measure provides information about the necessitated changes to reach higher yields. The methods are applied to interpret the capabilities of a network based on measured fluxes for a L-phenylalanine producer. The strain was examined by a series of experiments and three flux maps of the production phase are analyzed. It can be shown that the reachable yield is drastically reduced by the measured efflux into the TCA cycle, while the oxidative pentose-phosphate pathway only plays a secondary role on the reachable maximum.

Carbon Isotopes↗