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PubMed2000

Determination of low-abundant metabolites in plant extracts by NAD(P)H fluorescence with a microtiter plate reader.

This article describes a method for the enzymatic detection of low-abundant metabolic intermediates in plant extracts via NAD(P)H fluorescence using a microtiter plate reader. The detection of changes in NAD(P)H fluorescence (excitation 340 nm, emission 465 nm) exhibits a high signal-to-noise ratio and is as sensitive (> or = 20 pmol per well) as absorbance measurements with dual-wavelength photometers. Since up to 96 reactions can be initiated, monitored, and evaluated simultaneously, this method might be suitable for high-throughput screening programs on metabolite profiles. However, in contrast to absorbance measurements, fluorescence detection of NAD(P)H yields relative data, which can be impaired by the quench characteristics and the basic fluorescence of the extracts. Hence, extensive calibration is required to gain reproducible results. Calibration of the assay system was performed using leaf or root material (equivalent to 2-35 mg of fresh weight per well) extracted with perchloric acid, chloroform/water/methanol, or hot ethanol. Extraction with perchloric acid was found to be superior for metabolite quantification. Examples of the kinetics of individual metabolite determinations are presented and the contents of 3-phosphoglycerate, hexose phosphates, triose phosphates, pyruvate, and phosphoenolpyruvate in illuminated and darkened spinach leaves as well as leaf rosettes of Arabidopsis thaliana and leaf segments of the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum were measured via NAD(P)H fluorescence and, where possible, compared to reported data determined with dual-wavelength photometers.

Arabidopsis↗
PubMed1995

Pentose cycling and the distribution of 13C in trehalose during glucogenesis from 13C-labelled substrates in an insect.

Redistribution of 13C in trehalose (Tre) due to pentose cycling was observed in vivo in Manduca sexta during glucogenesis from [3-13C]alanine (Ala) and [2-13C]glycerol (Gly). The extent of cycling was affected by dietary composition. Larvae maintained on a low-carbohydrate diet (LCD) exhibited approximately 13% cycling, while those on a complete-balanced diet (CBD) or low-fat diet (LFD) displayed much higher rates of cycling. Significant incorporation of 13C via reversal of the non-oxidative phase was evident on all diets but was greatest on the CBD and LFD. In contrast to conclusions from previous studies with insects, the present results indicate that under normal conditions the pentose pathway is not the principal source of triose phosphates for oxidative catabolism during larval development.

Animals↗
PubMed1996

Rapid formation of advanced glycation end products by intermediate metabolites of glycolytic pathway and polyol pathway.

To clarify roles of intermediate metabolites of the glycolytic pathway and the polyol pathway in nonenzymatic glycation under physiological conditions, we incubated bovine serum albumin with intermediates of both pathways in the micromolar range as well as with 20 mmol/l glucose, and observed the formation of advanced glycation end products (AGEs). We found that triose phosphates, glyceraldehyde, and a novel polyol pathway-related metabolite, fructose 3-phosphate along with its breakdown product, 3-deoxyglucosone were extremely potent glycating agents that at nearly physiological concentrations on incubation with albumin produced substantial amounts of AGEs as early as 24 hours, while 20 mmol/l glucose afforded trace amounts of AGEs after two week incubation. The results along with the previous evidence of the increased level of intermediates in diabetic states may suggest that the intermediate metabolites rather than glucose contribute to enhanced glycation in diabetic tissues, inspite of the much lower concentrations compared with glucose.

Animals↗
PubMed1997

Studies on the essential role of ascorbic acid in the energy dependent release of insulin from pancreatic islets.

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with depolarizing levels of KCl (45 mM) and by 20 mM D-glyceraldehyde. Islets from normal guinea pigs released insulin in a K+ and D-glyceraldehyde dependent manner showing a rapid initial secretion phase followed by secondary waves of insulin release during a 120 min period. Islets from scorbutic guinea pigs were able to respond to elevated K+ in a manner identical to that of the control islets. In contrast, insulin release from ascorbic acid deficient islets in response to the secretagogue, D-glyceraldehyde, was significantly delayed and decreased responses were observed during the 120 min period after D-glyceraldehyde stimulation. The results are consistent with the site of action of ascorbic acid on energy-dependent insulin release lying between the triose-phosphate level of glycolysis and the generation of ATP by oxidative phosphorylation.

Animals↗
PubMed1998

The generation of superoxide anions in glycation reactions with sugars, osones, and 3-deoxyosones.

Glycoxidation is a process whereby glycated proteins chemically generate oxygen free radicals. Superoxide anion formation was measured by the superoxide dismutase-dependent reduction of ferricytochrome C in glycation reactions at pH 7.0 in the absence of transition metal ions. Assays were linear over 1 h, and most activity was seen after a 2 d incubation of 5 mM L-threose and 10 mM alpha-N-acetyl-lysine (N-Ac-Lys) or 10 mg/mL RNase A. Trioses, tetroses and their corresponding osones and 3-deoxyosones had the highest activity (12-16 nmoles O.-2/hr/ml) with N-Ac-Lys. Osones and 3-deoxyosones alone generated considerable O.-2, whereas aldose sugars largely did not. Xylosone and 3-deoxyxylosone produced 6 and 10 nmoles O.-2/hr/ml respectively with N-Ac-Lys, however, xylose was inactive, as were glucose and fructose. Glycation assays with 3-deoxyglucosone and glyoxal showed no activity, however, methyl glyoxal generated 1.7 and 2.0 nmoles O.-2/hr/ml with N-Ac-Lys and N-Ac-Arg, respectively. Therefore, Amadori compounds composed of lysine and short chain sugars can rapidly generate superoxide anion in the absence of metal ions.

Arginine↗
PubMed1998

Carnosine protects proteins against methylglyoxal-mediated modifications.

Methylglyoxal (MG) (pyruvaldehyde) is an endogenous metabolite which is present in increased concentrations in diabetics and implicated in formation of advanced glycosylation end-products (AGEs) and secondary diabetic complications. Carnosine (beta-alanyl-L-histidine) is normally present in long-lived tissues at concentrations up to 20 mM in humans. Previous studies showed that carnosine can protect proteins against aldehyde-containing cross-linking agents such as aldose and ketose hexose and triose sugars, and malon-dialdehyde, the lipid peroxidation product. Here we examine whether carnosine can protect protein exposed to MG. Our results show that carnosine readily reacts with MG thereby inhibiting MG-mediated protein modification as revealed electrophoretically. We also investigated whether carnosine could intervene when proteins were exposed to an MG-induced AGE (i.e. lysine incubated with MG). Our results show that carnosine can inhibit protein modification induced by a lysine-MG-AGE; this suggests a second intervention site for carnosine and emphasizes its potential as a possible non-toxic modulator of diabetic complications.

Carnosine↗
PubMed2000

Chitinous materials inhibit nitric oxide production by activated RAW 264.7 macrophages.

Chitinous materials have been studied in wound healing and artificial skin substitutes for many years. Nitric oxide (NO) has been shown to contribute to cytotoxicity in cell proliferation during inflammation of wound healing. In this study, we examined the effect of chitin and its derivatives on NO production by activated RAW 264.7 macrophages. Chitin and chitosan showed a significantly inhibitory effect on NO production by the activated macrophages. Hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose also inhibited NO production but with less potency. However, N-acetylchitotetraose, -triose, -biose, and monomer of chitin, N-acetylglucosamine and glucosamine had little effect on NO production by the activated cells. These results suggest that the promotive effect of chitinous material on wound healing be related, at least partly, to inhibit NO production by the activated macrophages.

Animals↗
PubMed1994

13C NMR study of C2- and C3-deuterated lactic acid production by parotid cells exposed to 13C-labeled glucose in the presence of D2O.

The generation of C2- and C3-deuterated lactic acid produced by rat parotid cells exposed to [1-13C] glucose, [2-13C]glucose, and [6-13C]glucose in the presence of D2O was assessed by 13C NMR. The results indicated that the escape from deuteration amounted to about 46% at the phosphoglucoisomerase level, 100% at the phosphomannoisomerase level, 65% in the reactions catalyzed by phosphofructoaldolase and triose phosphate isomerase, and 58% at the level of glutamate pyruvate transaminase. Such high values are considered to support a possible enzyme-to-enzyme tunneling of metabolic intermediates at selected sites in the glycolytic pathway.

Animals↗
PubMed1999

Temperature regulation of glucose metabolism in red blood cells of the freeze-tolerant wood frog.

The low-temperature metabolism of erythrocytes from the freeze-tolerant frog Rana sylvatica was investigated by (13)C and (31)P NMR spectroscopy. Erythrocytes readily took up high concentrations of the natural cryoprotectant, glucose, at both high (12 and 17 degrees C) and low (4 degrees C) temperatures but glucose was apparently not metabolized at 4 degrees C. Strong inhibition of glucose catabolism at low temperature would facilitate the maintenance of the very high concentrations of glucose (approximately 200 mM) that are accumulated to provide cryoprotection during freezing in wood frogs. Analysis of (13)C labeling of glycolytic intermediates at 4 degrees C showed mixing of label primarily in hexose (fructose) and hexose phosphate (glucose 6-phosphate, fructose 6-phosphate) pools but little label incorporation into triose phosphate intermediates. These data are consistent with a profound low-temperature-induced inhibition of phosphofructokinase (PFK). Investigations into potential PFK control mechanisms were undertaken. (31)P NMR analysis showed that the intracellular pH of erythrocytes increased from 7.0 to 7.3 as temperature decreased from 17 to 4 degrees C in a manner consistent with alphastat regulation. This change is exactly opposite to that expected if overall PFK activity was regulated by changes in cellular pH since PFK is less active at lower pH values in vitro. Other factors must, therefore, operate to regulate PFK at lower temperatures.

Animals↗
PubMed1993

Glycation mediated lens crystallin aggregation and cross-linking by various sugars and sugar phosphates in vitro.

Glycation of lens crystallins results in protein conformational changes, oxidation, browning and aggregation. Though glucose is the major sugar, other sugars and sugar phosphates generated as intermediates of metabolic pathways are present in the lens, albeit at low concentrations. In this study we incubated bovine lens soluble fraction with various sugars and sugar phosphates (5mM for 10 days). The reactivity was in the order trioses > tetroses > pentoses > hexoses. High molecular weight (HMW) aggregates were also formed at a comparable rate. Increased levels of fluorescence were associated with the HMW aggregates with fast reacting sugars. The phosphorylated derivatives were only slightly more reactive than their respective sugars. Interestingly, fructose-1,6-diphosphate was more reactive and cross-linked more readily than fructose-6-phosphate. Gel electrophoresis under reducing and nonreducing conditions showed formation of disulfide linked protein aggregates with slow reacting sugars such as glucose and non-disulfide covalent linked protein aggregates with fast reacting sugars such as erythrose. In contrast, if 0.1 m DTT was present in erythrose incubations (a fast reacting sugar), the HMW aggregate formation was significantly reduced. In order to show the reactivity among the slow reacting hexoses, we incubated lens proteins with 1 M hexoses for 30 days and the results showed that galactose was more reactive and showed higher cross-linking than fructose and glucose. These results thus indicate that relatively low levels of some sugars and sugar phosphates in the lens could be compensated by enhanced lens protein cross-linking and the combined effect could be rather significant with respect to cataractogenesis.

Animals↗
PubMed1994

Alignment of beta-barrels in (beta/alpha)8 proteins using hydrogen-bonding pattern.

A multiple alignment procedure for aligning the beta-sheet residues of the (beta/alpha)8-barrel structures is described. It uses a two-dimensional numbering scheme which is based on the covalent and hydrogen-bonding pattern of the beta-sheet. Two different scoring functions were used: one measured the sequence and topological similarity and the other the root-mean-square deviation of the coordinates of the matched residues. The procedure was applied to obtain multiple alignments of the beta-barrels of ten (beta/alpha)8-barrel proteins of known structure. Two kinds of alignments were derived: one in which the beta-strand numbering was preserved and another in which the beta-strands were allowed to be cyclically permuted. It is shown that-preservation of the beta-strand numbering corresponds to aligning only the layer structure of the beta-barrels. In order to obtain the optimal rotational alignment of the barrels as well, the beta-strands must be allowed to be renumbered. Although the 2-fold or 4-fold rotational symmetry of the beta-barrels makes it difficult to obtain unique rotational alignment of the barrels, the results of the alignment indicate that the beta-strands in the beta-barrel of enolase, xylose isomerase, taka-amylase, and possibly fructose biphosphate aldolase, must be cyclically permuted in order to be optimally aligned to those of the other proteins, which include triose phosphate isomerase, the alpha-subunit of tryptophan synthetase, flavocytochrome b2, ribulose-1, 5-biphosphate carboxylase/oxygenase, and glycolate oxidase.

Amino Acid Sequence↗
PubMed1999

In vivo 31P and multilabel 13C NMR measurements for evaluation of plant metabolic pathways.

Reliable measurements of intracellular metabolites are useful for effective plant metabolic engineering. This study explored the application of in situ 31P and 13C NMR spectroscopy for long-term measurements of intracellular pH and concentrations of several metabolites in glycolysis, glucan synthesis, and central carbon metabolic pathways in plant tissues. An NMR perfusion reactor system was designed to allow Catharanthus roseus hairy root cultures to grow for 3-6 weeks, during which time NMR spectroscopy was performed. Constant cytoplasmic pH (7.40+/-0.06), observed during the entire experiment, indicated adequate oxygenation. 13C NMR spectroscopy was performed on hairy root cultures grown in solutions containing 1-13C-, 2-13C-, and 3-13C-labeled glucose in separate experiments and the flow of label was monitored. Activities of pentose phosphate pathways, nonphotosynthetic CO2 fixation, and glucan synthesis pathways were evident from the experimental results. Scrambling of label in glucans also indicated recycling of triose phosphate and their subsequent conversion to hexose phosphates.

Carbon Isotopes↗
PubMed1999

Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity.

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.

Bioreactors↗
PubMed2001

Glucose metabolism and regulation of glycolysis in Lactococcus lactis strains with decreased lactate dehydrogenase activity.

The distribution of carbon flux at the pyruvate node was investigated in Lactococcus lactis under anaerobic conditions with mutant strains having decreased lactate dehydrogenase activity. Strains previously selected by random mutagenesis by H. Boumerdassi, C. Monnet, M. Desmazeaud, and G. Corrieu (Appl. Environ. Microbiol. 63, 2293-2299, 1997) were found to have single punctual mutations in the ldh gene and presented a high degree of instability. The strain L. lactis JIM 5711 in which lactate dehydrogenase activity was diminished to less than 30% of the wild type maintained homolactic metabolism. This was due to an increase in the intracellular pyruvate concentration, which ensures the maintained flux through the lactate dehydrogenase. Pyruvate metabolism was linked to the flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase, as previously postulated for the parent strain (C. Garrigues, P. Loubière, N. D. Lindley, and M. Cocaign-Bousquet (1997) J. Bacteriol. 179, 5282-5287, 1997). However, a strain (L. lactis JIM 5954) in which the ldh gene was interrupted reoriented pyruvate metabolism toward mixed metabolism (production of formate, acetate, and ethanol), though the glycolytic flux was not strongly diminished. Only limited production of acetoin occurred despite significant overflow of pyruvate. Intracellular metabolite profiles indicated that the in vivo glyceraldehyde-3-phosphate dehydrogenase activity was no longer flux limiting in the Deltaldh strain. The shift toward mixed acid fermentation was correlated with the lower intracellular trioses phosphate concentration and diminished allosteric inhibition of pyruvate formate lyase.

Base Sequence↗
PubMed2001

Analysis of the effect of changing environmental conditions on the expression patterns of exported surface-associated proteins of the oral pathogen Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans has been specifically implicated in the aetiology of one or more of the periodontal diseases, conditions in which inflammation of the gums is associated with destruction of the alveolar bone supporting the teeth. In these diseases there is loss of attachment of the gums (gingivae) to the teeth forming a periodontal pocket. The microenvironment of this pocket is extremely complex and it is likely that there will be substantial variation in the environmental conditions operating in this habitat. The aim of the current investigation was to study the effect of disease-relevant environmental factors on the production and release of secreted surface- associated proteins of A. actinomycetemcomitans. These secreted proteins contain many of the virulence determinants of this organism. A range of environmental conditions were investigated: growth in a CO(2)-enriched aerobic atmosphere vs anaerobic growth, presence of serum or blood, biofilm vs planktonic mode of growth and iron depletion. Differential expression of a number of the secreted surface-associated proteins was observed under different growth conditions and these included the glycolytic enzyme triose phosphate isomerase. An ability to adapt to prevailing environmental conditions may facilitate the survival of the organism in the changing microIenvironment of the periodontal pocket.

Adaptation, Biological↗
PubMed1993

A molecular perspective on the phylogenetic relationships of the barbeled minnows, historically assigned to the genus Hybopsis (Cyprinidae: Cypriniformes).

Allozyme products of 32 gene loci were compared for the North American cyprinid fishes Campostoma oligolepis, Cyprinella galactura, Cyprinella monacha, Erimystax cahni, E. dissimilis, E. insignis, E. x-punctatus, Hybopsis amblops, H. labrosa, H. rubriforns, H. zanema, Macrhybopsis aestivalis, M. gelida, M. meeki, Nocomis biguttatus, Phenacobius uranops, Platygobio gracilis, and P. storeriana. Triose-phosphate isomerase was found to be duplicated in all species examined. Parsimony analysis revealed that Hybopsis sensu lato did not form a monophyletic group. Cyprinella monacha was found to be the sister species to members of the genus Cyprinella and was excluded from the genus Erimystax. A clade of four species of Erimystax was identified although relationships among the four species could not be resolved. Phenacobius was identified as the sister group to Erimystax. The genus Macrhybopsis was expanded to include M. aestivalis. Macrhybopsis gelida was identified as the sister to M. meeki and M. aestivalis as the sister to the former two species. The genus Platygobio was expanded to include P. storeriana. Hybopsis labrosa and H. zanema were found to be more closely related to members of the genus Hybopsis sensu stricto than to members of Cyprinella.

Animals↗
PubMed1993

Estimating gluconeogenic rates in NIDDM.

To measure the rate of gluconeogenesis in humans directly, one must administer and determine the specific activity or the enrichment in an intermediate in the gluconeogenic process and in the glucose formed, thus obtaining the fraction of the glucose formed by gluconeogenesis. By a separate determination of the rate of hepatic glucose production, the rate of gluconeogenesis can then be calculated. The closer the intermediate is to glucose-6-P, the more complete will be the measurement of the rate. Thus, if the intermediate is below the level of the triose phosphates, gluconeogenesis from glycerol will not be included in the estimate. Estimates of rates of gluconeogenesis from estimates of PEP enrichment or specific activity require a measure of the extent of exchange of label at the level of oxaloacetate. By using 14C or 13C labeled CO2 as the intermediate and estimating the relative rates of the reactions of the tricarboxylic acid cycle relative to gluconeogenesis from the distribution of 14C from [3-14]lactate in glutamine from the glutamine conjugate of phenylacetate, the enrichment or specific activity of PEP has been estimated. Correction must be made for the incorporation into the glutamine of 14CO2 formed from the [3-14C]lactate. Data support the validity of this approach toward estimating gluconeogenesis in NIDDM, but the approach is complex, time consuming and with uncertainties. Estimates that have been made using [2-14C] acetate are invalid because of the extensive metabolism of [2-14C]acetate in other than liver. Other approaches have promise, but technical problems may exist in their use and other problems, such as hepatic zonation and exchange reactions, may compromise their application.

Acetates↗
PubMed1992

Prebiotic sugar synthesis: hexose and hydroxy acid synthesis from glyceraldehyde catalyzed by iron(III) hydroxide oxide.

Iron(III) hydroxide oxide [Fe(OH)O] efficiently catalyzed the condensation of 25 mM DL-glyceraldehyde to ketohexoses at 25 degrees C (pH 5-6). At 16 days the yields were sorbose (15.2%), fructose (12.9%), psicose (6.1%), tagatose (5.6%), and dendroketose (2.5%) with 19.6% of triose unreacted. Analysis at 96 days showed no decomposition of hexoses. Under these conditions Fe(OH)O also catalyzed the isomerization and rearrangement of glyceraldehyde to dihydroxyacetone and lactic acid, respectively. In these reactions, about 10% of the glyceraldehyde was oxidized to glyceric acid with concurrent reduction of the iron(III) to iron(II). The partial reduction of Fe(OH)O did not noticeably reduce its ability to catalyze hexose synthesis. The relationship of these results to prebiotic sugar synthesis is discussed.

Carbohydrates↗