Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O(2) diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O(2) limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2-4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by gamma-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress. (+info)
Acute plasma volume expansion: effect on metabolism during submaximal exercise.
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To examine the effect of acute plasma volume expansion (PVE) on substrate selection during exercise, seven untrained men cycled for 40 min at 72 +/- 2% peak oxygen uptake (VO(2 peak)) on two occasions. On one occasion, subjects had their plasma volume expanded by 12 +/- 2% via an intravenous infusion of the plasma substitute Haemaccel, whereas on the other occasion no such infusion took place. Muscle samples were obtained before and immediately after exercise. In addition, heart rate and pulmonary gas and venous blood samples were obtained throughout exercise. No differences in oxygen uptake or heart rate during exercise were observed between trials, whereas respiratory exchange ratio, blood glucose, and lactate were unaffected by PVE. Muscle glycogen and lactate concentrations were not different either before or after exercise. In addition, there was no difference in total carbohydrate oxidation between trials (control: 108 +/- 2 g; PVE group: 105 +/- 2 g). Plasma catecholamine levels were not affected by PVE. These data indicate that substrate metabolism during submaximal exercise in untrained men is unaltered by acute hypervolemia. (+info)
Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion.
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The addition and endoplasmic reticulum (ER) glucosidase processing of N-linked glycans is essential for the secretion of rat hepatic lipase (HL). Human HL is distinct from rat HL by the presence of four as opposed to two N-linked carbohydrate side chains. We examined the role of N-linked glycosylation and calnexin interaction in human HL secretion from Chinese hamster ovary (CHO) cells stably expressing a human HL cDNA. Steady-state and pulse-chase labeling experiments established that human HL was synthesized as an ER-associated precursor containing high mannose N-linked glycans. Secreted HL had a molecular mass of approximately 65 kDa and contained mature N-linked sugars. Inhibition of N-linked glycosylation with tunicamycin (TM) prevented secretion of HL enzyme activity and protein mass. In contrast, incubation of cells with the ER glucosidase inhibitor, castanospermine (CST), decreased human HL protein secretion by 60%, but allowed 40% of fully active HL to be secreted. HL protein mass and enzyme activity were also recovered from the media of a CHO-derivative cell line genetically deficient in ER glucosidase I activity (Lec23) that was transiently transfected with a human HL cDNA. Co-immunoprecipitation experiments demonstrated that newly synthesized human HL bound to the lectin-like ER chaperone, calnexin, and that this interaction was inhibited by TM and CST. These results suggest that under normal conditions calnexin may increase the efficiency of HL export from the ER. Whereas a significant proportion of human HL can attain activity and become secreted in the absence of glucose trimming and calnexin association, these interrelated processes are nevertheless essential for the expression of full HL activity. (+info)
Cyclic organization of the carbohydrate metabolism in Sinorhizobium meliloti.
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The pathways of polysaccharide biosynthesis were investigated in cells of Sinorhizobium meliloti (strain Su47) using a stable isotope approach. The isotopic labeling of the periplasmic beta-1,2-glucans synthesized from glucose labeled at various positions evidenced the involvement of catabolic pathways, namely the pentose-phosphate and Entner-Doudoroff pathways, into the early steps of polysaccharide synthesis. The exopolysaccharides produced at the same time had a labeling pattern similar to that of the beta-glucans, indicating similar early steps for both polysaccharides. The results emphasized a cyclic organization of the carbohydrate metabolism in S. meliloti, in which the carbons of the initial hexose were allowed to re-enter the catabolic pathways many times. The metabolic incidences of such metabolic topology are discussed. (+info)
Effect of a prolonged low-dose lipopolysaccharide infusion on feed intake and metabolism in heifers.
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Prolonged infusions of bacterial lipopolysaccharides (LPS) are known to model gram-negative bacterial infections, but the basic mechanisms of the LPS effects on feed intake and metabolism and their potential interdependence are largely unknown. The aim of the present study was to distinguish and to better characterize the feeding suppressive and metabolic effects of LPS. Six heifers were infused intravenously for 100 min with either 1) LPS (2 microg/kg BW) with free access to feed, 2) saline with free access to feed, or 3) saline with feeding restricted to the amount of feed consumed after LPS infusion. Feed intake, body temperature, plasma concentrations of various metabolites and hormones, and the respiratory quotient and heat production were measured. The LPS reduced feed intake and induced pronounced changes in metabolic energy turnover and fat and carbohydrate metabolism that were largely independent of the concomitant feed intake reduction. Some of the metabolic changes were biphasic; the first phase resembled a stress response with increases in plasma glucose and cortisol, and the second phase reflected a beginning energy deficit with low plasma glucose and enhanced lipolysis. The coincidence of a short-term surge of plasma insulin with marked transient decreases in plasma FFA, glycerol, and beta-hydroxybutyrate as well as with the transition from hyper- to hypoglycemia indicates that insulin plays a role in some of the metabolic responses to LPS. The failure of LPS to clearly increase energy expenditure despite the increase in body temperature suggests that anaerobic mechanisms of heat production and, perhaps, a reduced peripheral blood flow contributed to the fever. Many of the initial metabolic responses occurred before and, therefore, independent of, an increase in circulating tumor necrosis factor-alpha. (+info)
Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression.
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In baker's yeast (Saccharomyces cerevisiae) the hexokinases PI (Hxk1) and PII (Hxk2) are required for triggering of the activation of the Ras-cAMP pathway and catabolite repression. Specifically, Hxk2 is essential for the establishment of glucose repression, whereas either Hxk1 or Hxk2 can sustain fructose repression. Previous studies have suggested that the extent of glucose repression is inversely correlated with hexokinase catalytic activity and hence with an adequate elevation of intracellular sugar phosphate levels. However, several lines of evidence indicate that glucose 6-phosphate is not the trigger of catabolite repression in yeast. In the present study we employed site-directed mutagenesis of amino acids important for the binding of sugar and ATP, for efficient phosphoryl transfer and for the closure of the substrate-binding cleft, to obtain an insight into the structural requirements of Hxk2 for sugar-induced signalling. We show that the ATP-binding Lys-111 is not essential for catalysis in vivo or for signal triggering. Substitution of the catalytic-centre Asp-211 caused loss of catalytic activity, but high-affinity sugar binding was retained. However, this was not sufficient to cause cAMP activation nor catabolite repression. Mutation of Ser-158 abrogated glucose-induced, but not fructose-induced, repression. Moreover, 2-deoxyglucose sustained repression despite an extremely low catalytic activity. We conclude that the establishment of catabolite repression is dependent on the onset of the phosphoryl transfer reaction on hexokinase and is probably related to the stable formation of a transition intermediate and concomitant conformational changes within the enzyme. In contrast, the role of Hxk2 in Ras-cAMP activation seems to be directly connected to its catalytic function. The implications of this model are discussed. (+info)
Thermodynamic studies of saccharide binding to artocarpin, a B-cell mitogen, reveals the extended nature of its interaction with mannotriose [3,6-Di-O-(alpha-D-mannopyranosyl)-D-mannose].
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The thermodynamics of binding of various saccharides to artocarpin, from Artocarpus integrifolia seeds, a homotetrameric lectin (M(r) 65, 000) with one binding site per subunit, was determined by isothermal titration calorimetry measurements at 280 and 293 K. The binding enthalpies, DeltaH(b), are the same at both temperatures, and the values range from -10.94 to -47.11 kJ mol(-1). The affinities of artocarpin as obtained from isothermal titration calorimetry are in reasonable agreement with the results obtained by enzyme-linked lectin absorbent essay, which is based on the minimum amount of ligand required to inhibit horseradish peroxidase binding to artocarpin in enzyme-linked lectin absorbent essay (Misquith, S., Rani, P. G., and Surolia, A. (1994) J. Biol. Chem. 269, 30393-30401). The interactions are mainly enthalpically driven and exhibit enthalpy-entropy compensation. The order of binding affinity of artocarpin is as follows: mannotriose>Manalpha3Man>GlcNAc(2)Man(3)>MealphaMan>Man>M analpha6Man> Manalpha2Man>MealphaGlc>Glc, i.e. 7>4>2>1.4>1>0.4>0.3>0.24>0.11. The DeltaH for the interaction of Manalpha3Man, Manalpha6Man, and MealphaMan are similar and 20 kJ mol(-1) lower than that of mannotriose. This indicates that, while Manalpha3Man and Manalpha6Man interact with the lectin exclusively through their nonreducing end monosaccharide with the subsites specific for the alpha1,3 and alpha1,6 arms, the mannotriose interacts with the lectin simultaneously through all three of its mannopyranosyl residues. This study thus underscores the distinction in the recognition of this common oligosaccharide motif in comparison with that displayed by other lectins with related specificity. (+info)
A putative regulatory element for carbon-source-dependent differentiation in Streptomyces griseus.
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To identify negative regulatory genes for cellular differentiation in Streptomyces griseus, DNA fragments repressing the normal developmental processes were cloned on a high-copy-number plasmid. One of these DNA fragments markedly repressed aerial mycelium and spore formation on solid media containing glucose or galactose, but not on media containing maltose or mannitol. The fragment contained three complete ORFs; precise subcloning revealed that a 249 bp fragment located in the promoter region between ORF1 and ORF3 was sufficient for repression. Quantification of the promoter activities by using a thermostable malate dehydrogenase gene as a reporter showed that the promoter for ORF3 (P(ORF3)) maintained high activity in mycelia grown in the presence of glucose but lost activity rapidly in maltose medium. P(ORF3) activity increased markedly when the promoter sequence was introduced on a high-copy-number plasmid. The results suggested that carbon-source-dependent deactivation of P(ORF3) mediated by a transcriptional repressor may initiate differentiation in S. griseus. (+info)