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At least 505 records · Page 28Linked to original sources
PubMed2005

Catalytic transformations of supercoiled DNA as studied by flow linear dichroism technique.

A catalytic turnover of supercoiled DNA (scDNA) transformation mediated by topoisomerases leads to changes in the linking number (Lk) of the polymeric substrate by 1 or 2 per cycle. As a substrate of the topoisomerization reaction it is chemically identical to its product; even a single catalytic event results in the quantum leap in the scDNA topology. Non-intrusive continuous assay to measure the kinetics of the scDNA topoisomerization was performed. The development of such a technique was hindered because of multiple DNA species of intermediate topology present in the reaction mixture. The interrelation of DNA topology, its hydrodynamics, and optical anisotropy enable us to use the flow linear dichroism technique (FLD) for continuous monitoring of the scDNA topoisomerization reaction. This approach permits us to study the kinetics of DNA transformation catalyzed by eukaryotic topoisomerases I and II, as well as mechanistic characteristics of these enzymes and their interactions with anticancer drugs. Moreover, FLD assay can be applied to any enzymatic reaction that involves scDNA as a substrate. It also provides a new way of screening drugs dynamically and is likely to be potent in various biomedical applications.

Antineoplastic Agents↗
PubMed2003

Syntaxin 1A inhibits GABA flux, efflux, and exchange mediated by the rat brain GABA transporter GAT1.

GABA transporters control extracellular GABA levels by coupling transmitter uptake to the sodium and chloride cotransport. The rat brain GABA transporter GAT1 and other members of this family are regulated by direct interactions with syntaxin 1A, a protein involved in vesicle docking and in the regulation of several ion channels and transporters. We have shown previously that syntaxin 1A exerts its effects on GAT1 by decreasing the net uptake of GABA and its associated ions through interactions with aspartic acid residues in the N-terminal tail of GAT1. This reduction in net uptake could be caused by many steps in the transport cycle, including substrate binding, substrate flux, substrate efflux, or reorientation of the unliganded transporter. To address this question, we performed GABA flux assays, measured flux- and efflux-associated ion currents, and assessed GABA exchange in multiple experimental systems expressing syntaxin 1A and wild-type GAT1 or GAT1 mutants. Syntaxin 1A caused similar reductions in forward and reverse transport that did not involve changes in apparent transport affinities for sodium, chloride, or GABA. The syntaxin 1A-mediated reduction in GABA flux and efflux was mimicked by mutations in GAT1 at the syntaxin 1A binding site. The binding site GAT1 mutant also caused a reduction in exchange. These data suggest that syntaxin 1A exerts its effects, directly or indirectly, on GAT1 function through interactions with GAT1's N-terminal tail and that the inhibition occurs at a step in the translocation process after substrate binding but which involves both unidirectional transport and transmitter exchange.

Animals↗
PubMed2003

Methionine regeneration and aminotransferases in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis.

The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L. C. Berger, J. Wilson, P. Wood, and B. J. Berger, J. Bacteriol. 183:4421-4434, 2001). The genome of Bacillus subtilis has been found to contain no subfamily Ia aminotransferase sequences. Instead, the analogous enzymes in B. subtilis were found to be members of the If subfamily. These putative aspartate aminotransferases, the yugH, ywfG, ykrV, aspB, and patA gene products, have been cloned, expressed, and characterized for methionine regeneration activity. Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor. In contrast, subcellular homogenates of B. subtilis and Bacillus cereus utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors. The two putative branched-chain aminotransferase genes in B. subtilis, ybgE and ywaA, were also cloned, expressed, and characterized. Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine. The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. subtilis ybgE gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa. Examination of B. cereus and Bacillus anthracis genome data found that there were no subfamily IIIa homologues in these organisms. In both B. cereus and B. anthracis, two putative branched-chain aminotransferases and two putative D-amino acid aminotransferases were discovered as members of subfamily IIIb. These four sequences were cloned from B. cereus, expressed, and characterized. Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. anthracis homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity. The aminooxy compound canaline was found to be an uncompetitive inhibitor of B. subtilis YbgE and also inhibited growth of B. subtilis and B. cereus in culture.

Amino Acid Sequence↗
PubMed1990

Defining hypoxia: a systems view of VO2, glycolysis, energetics, and intracellular PO2.

The necessity for defining hypoxia as O2-limited energy flux rather than low partial pressure is explored from a systems perspective. Oxidative phosphorylation, the Krebs cycle, glycolysis, substrate supply, and cell energetics interact as subsystems; the set point is a match between ATP demand and aerobic ATP production. To this end the transport subsystem must match the transcapillary and mitochondrial O2 fluxes. High transcapillary O2 flux requires intracellular PO2 in the range 1-10 Torr. In this range the O2 drive on electron transport must be compensated by adaptive changes in the phosphorylation and redox drives. Thus the metabolic subsystem supports diffusive O2 transport by maintaining O2 flux at intracellular partial pressures required for O2 release from blood. Since responses to stress are distributed according to the state of the entire system, several simultaneous metabolic measurements, including intracellular PO2 (or a known direction of change in intracellular PO2) and the O2 dependence of a measurable function are required to judge the adequacy of O2 supply. ATP demand and aerobic capacity must also be evaluated, because the hypoxic threshold depends on the ratio of ATP demand to aerobic capacity. The application and limitation of commonly used criteria of hypoxia are discussed, and a more precise terminology is proposed.

Adenosine Triphosphate↗
PubMed2002

Time course of performance changes and fatigue markers during intensified training in trained cyclists.

To study the cumulative effects of exercise stress and subsequent recovery on performance changes and fatigue indicators, the training of eight endurance cyclists was systematically controlled and monitored for a 6-wk period. Subjects completed 2 wk of normal (N), intensified (ITP), and recovery training. A significant decline in maximal power output (N = 338 +/- 17 W, ITP = 319 +/- 17 W) and a significant increase in time to complete a simulated time trial (N = 59.4 +/- 1.9 min, ITP = 65.3 +/- 2.6 min) occurred after ITP in conjunction with a 29% increase in global mood disturbance. The decline in performance was associated with a 9.3% reduction in maximal heart rate, a 5% reduction in maximal oxygen uptake, and an 8.6% increase in perception of effort. Despite the large reductions in performance, no changes were observed in substrate utilization, cycling efficiency, and lactate, plasma urea, ammonia, and catecholamine concentrations. These findings indicate that a state of overreaching can already be induced after 7 days of intensified training with limited recovery.

Adult↗
PubMedDate not supplied

Effect of excess xylitol on nitrogen and glucose metabolism in parenterally fed rats.

The objectives of this study were to investigate the effects of an excess of xylitol on nitrogen balance and glucose metabolism in parenterally fed rats. Female Sprague-Dawley rats (200-250 g, n = 17) were catheterized for total parenteral nutrition and then randomized into two groups based on subsequent diet. The two diets used were isonitrogenous (1.5 g of nitrogen per kilogram per day) and isocaloric, with half the calories (125 kcal/kg per day) being derived from lipid (125 kcal/kg per day) and the other half from either glucose or xylitol (125 kcal/kg per day). The rats were fed a half-strength total parenteral nutrition diet for the day after surgery and a full-strength total parenteral nutrition diet for the following 4 days. Urines were collected daily for the determination of nitrogen balance. On day 5, the rats were given a 7- to 8-hour infusion of 6.6-d2 glucose (6 mg/h and 2-d1 glucose (12 mg/h). At the conclusion of the isotope infusion period, the rats were killed and blood was collected. Urine output was increased by 22% per day in the xylitol-treated rats, and they excreted 46.5 mmol of xylitol per liter per kilogram per day (7.1 g/kg per day, approximately 22.7% of dose). The xylitol group lost weight, had poorer nitrogen balance (341 +/- 31 vs 83 +/- 29 mg/kg per day [mean +/- standard error of the mean], p < .05), and developed fatty livers. Analysis of the liver fat distribution pattern indicated that the source of the excess hepatic lipid was dietary fat.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Toxicology of quinone-thioethers.

Cytotoxicity associated with exposure to quinones has generally been attributed to either redox cycling, and the subsequent development of "oxidative stress," and/or to their interaction with cellular nucleophiles, such as protein and non-protein sulfhydryls. Glutathione (GSH) is the major non-protein sulfhydryl present in cells, and conjugation of potentially toxic electrophiles with GSH is usually associated with detoxication and excretion. However, this review discusses the biological (re)activity of quinone-thioethers. For example, quinone-thioethers are (1) capable of redox cycling (2) substrates for, and inhibitors of, a variety of enzymes (3) methemoglobinemic (4) potent nephrotoxicants (5) DNA reactive and (6) may contribute to quinone-mediated carcinogenicity and neurotoxicity. The ubiquitous nature of quinones, and the high intracellular concentrations of GSH, ensures that cells and tissues will be exposed to quinone-thioethers. The toxicological importance of quinone-thioethers in quinone-mediated toxicities therefore deserves further attention.

Animals↗
PubMed2005

Green tea polyphenol epigallocatechin-3-gallate blocks PDGF-induced proliferation and migration of rat pancreatic stellate cells.

AIM: To clarify the effects of epigallocatechin-3-gallate (EGCG) on the platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of pancreatic stellate cells (PSCs). METHODS: PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Cell migration was assessed using modified Boyden chambers. Cyclin D1, p21Waf1, and p27Kip1 expression and phosphorylation of PDGF beta-receptor, extracellular signal-regulated kinase, and Akt were examined by Western blotting. Activation of phospha-tidylinositol 3-kinase was examined by kinase assay using phosphatidylinositol as a substrate. Cell cycle was assessed by flow cytometry after staining with propidium iodide. RESULTS: EGCG at non-cytotoxic concentrations inhibited PDGF-induced proliferation and migration. This effect was associated with the inhibition of cell cycle progression beyond the G1 phase, decreased cyclin D1 and increased p27Kip1 expression. EGCG inhibited tyrosine phosphorylation of PDGF beta-receptor and downstream activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways. CONCLUSION: EGCG inhibited PDGF-BB-induced proliferation and migration of PSCs through the inhibition of PDGF-mediated signaling pathways.

Animals↗
PubMed1999

A novel apoptotic pathway in quiescent lymphocytes identified by inhibition of a post-proline cleaving aminodipeptidase: a candidate target protease, quiescent cell proline dipeptidase.

The vast majority of lymphocytes in vivo persist in a quiescent state. These resting lymphocytes are maintained through a cellular program that suppresses apoptosis. We show here that quiescent PBMC, but not activated PBMC or transformed lymphocytes, die in the presence of highly specific post-proline aminodipeptidase inhibitors. This form of death has the hallmarks of apoptosis, such as phosphatidylserine externalization and loss of mitochondrial transmembrane potential. However, it differs from apoptosis induced by gamma irradiation in the same cells or by Fas ligation in transformed lymphocytes in terms of caspase involvement. In addition, the aminodipeptidase inhibitor-induced cell death, but not gamma-irradiation-mediated apoptosis, can be prevented by inhibition of the proteasome complex. The target of these inhibitors is not CD26/DPPIV, but probably a novel serine protease, quiescent cell proline dipeptidase, that we have recently isolated and cloned. These studies will yield a better understanding of the requirements and the mechanisms that mediate quiescent lymphocyte homeostasis in vivo.

Apoptosis↗
PubMed2000

The divergent effects of tumour necrosis factor-alpha on skeletal muscle: implications in wasting.

Tumour necrosis factor-alpha (TNF) is a pleiotropic cytokine that can have effects on many cell types, including skeletal muscle, the most abundant tissue (representing almost 45% of body weight), where many effects of this cytokine have been described. Thus, TNF receptors have been described in muscle tissue, and different investigations have revealed effects of the cytokine on membrane potential, glucose uptake and metabolism, amino acid transport and protein turnover. However, the results found are relatively divergent, therefore the main aim of the present review has been to clarify and reconcile some of the most contradictory studies concerning the effects of TNF on metabolism in skeletal muscle.

Amino Acids↗
PubMed1992

Time correction for computing Michaelis--Menten kinetics.

A description of time correction applied to the integrated Michaelis equation in view of cancelling a non-correct estimation of the Michaelis constant due to a lack of coincidence between time zero of measurement and time zero of reaction is presented. An estimation of kinetic parameters is made with time correction on the same experimental set of data using a classical least squares iterative procedure.

Enzymes↗
PubMed1991

Ubiquinol prevents alpha-tocopherol consumption during liposome peroxidation.

In this study we investigated whether alpha-tocopherol can be spared by ubiquinol-3 during autoxidation of multilamellar liposome. A lipophilic azocompound, 2,2'-azobis-(2,4-dimethyl-valeronitrile), was chosen to initiate liposome autoxidation. The effect of either alpha-tocopherol, ubiquinol-3, or a mixture of them was compared. Rates of conjugated diene formation and concomitant disappearance of the two antioxidants was measured. Since the inhibition rate constant for the scavenging of peroxyl radical for alpha-tocopherol was higher than that for quinol-3, it was concluded that alpha-tocopherol is regenerated by ubiquinol-3.

Free Radicals↗
PubMed1989

Brown adipose tissue (Na+-K+)-ATPase activity and substrate uptake during the breeding cycle of rats.

Brown adipose tissue (Na+-K+)-ATPase activity, in vitro glucose and 2-aminoisobutyric acid uptake, as well as mitochondrial GDP-binding and succinate dehydrogenase activity were determined in order to study the relationship between these parameters and the thermogenic status. Analysis were carried out on control animal, pregnant rats, dams and pups during lactation, GDP-binding, (Na+-K+)-ATPase and glucose uptake were found to be decreased in brown adipose tissue from pregnant rats and dams, and increased in pups, 2-aminoisobutyric acid uptake was only increased in pups, but no changes were observed in the other experimental groups tested. GDP-binding and (Na+-K+)-ATPase activity showed a parallelism which suggests that the enzyme is a good index of thermogenic status of the animal.

Adipose Tissue, Brown↗
PubMed1979

Effects of 2,4-dihydroxybutyrate on lipogenesis in rat hepatocytes.

Hepatocytes were prepared from rats fasted 2 days and refed a high carbohydrate diet for 1 day. These cells contained very high levels of glycogen (about half the defatted dry weight) and carried out high rates of lipogenesis (up to 800 micron at tritium incorporation from 3HOH/g (defatted dry weight)/h), even in the absence of added substrates. Pentose cycle flux was estimated by a method involving the use of [1-14C]galactose (Rognstad, R. (1976) Int. J. Biochem. 7, 221-228). In hepatocytes from normal fasted refed rats, the amount of NADPH produced by the pentose cycle was sufficient for about one-half to three-fourths of that required for fatty acid synthesis. 2,4-Dihydroxybutyrate, a malic enzyme inhibitor (Schimerlik, M.I. & Cleland, W.W. (1977) Biochemistry 16, 565-570) markedly depressed the randomization of 14C in lactate from [6-14C]hexoses, indicating an inhibition of the pyruvate cycle. 2,4-Dihydroxybutyrate (10 mM) had only a slight inhibitory effect on overall lipogenesis, but increased the rate of the pentose cycle by 40 to 90%.

Animals↗
PubMedDate not supplied

[Effect of vitamin D3 and carbostimulin on the content of carbohydrate metabolism substrates and the tricarboxylic acid cycle in liver of rats with experimental rachitis].

The development of experimental rhachitis in rats is accompanied by a decrease in the dihydroxyacetonephosphate, glyceraldehyde-phosphate concentrations and by an increase in pyruvate, lactate, alpha-ketoglutarate, glutamate, oxalacetate and malate in the liver tissue. The feeding of vitamin D3 and carbostimulin normalizes, in the main, the concentrations of these metabolites.

Animals↗
PubMed1995

Effect of weight-training on energy expenditure and substrate utilization during sleep.

This study was performed to investigate the effect of weight-training (12 wk; 21 male subjects) on energy expenditure and substrate utilization during sleep. Sleeping metabolic rate (SMR) as measured in a respiration chamber was calculated according to three procedures: the lowest mean energy expenditure of a shiftable 3-h interval between 0:00 and 6:00 (SMR3) and SMR over predetermined intervals from 3:00 to 6:00 (SMR3-6) and from 0:00 to 6:00 (SMR0-6). In analogy with SMR the corresponding respiratory quotients were expressed as RQ3, RQ3-6, and RQ0-6. Changes in body composition were assessed from changes in body weight, body volume (densitometry), and total body water (deuterium dilution). Weight-training induced an increase in fat-free mass (+1.1 +/- 1.3 kg; P < 0.001) and a decrease in fat mass (-2.3 +/- 1.5 kg; P < 0.001) and body weight (-1.1 +/- 2.1 kg; P < 0.05). There was no significant change in SMR, irrespective of the way SMR was expressed. Only RQ3 decreased significantly (from 0.82 +/- 0.04 to 0.79 +/- 0.02; P < 0.05). Remarkably RQ3, RQ3-6, and RQ0-6 were highly negative correlated with the pre-training RQ (r = -0.93, -0.91, and -0.90, respectively: P < 0.001) resulting in a diminished variation in post-training RQ (P < 0.001). These results suggest that weight-training has no effect on SMR but increases relative fat utilization in low fat oxidizers and vice versa for individuals displaying high pre-training lipid oxidation.

Adult↗