Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor), Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.

Energy Expenditure, Lipid Profile, Oxidative Stress, and Cardiac Energy Metabolism After Growth Hormone Treatment in Obese Young Rats

Obesity is rampant in modern society and growth hormone (GH) could be useful as adjunct therapy to reduce the obesity-induced cardiovascular damage. To investigate GH effects on obesity, initially 32 male Wistar rats were divided into two groups (n = 16): control (C) was fed standard-chow and water and hyper-caloric (H) was fed hypercaloric chow and 30% sucrose in its drinking water. After 45 days, both C and H groups were divided into two subgroups (n = 8): C + PL was fed standard-chow, water and received saline subcutaneously; C + GH was fed standard-chow, water, and received 2 mg/kg/day GH subcutaneously; H + PL was fed hypercaloric diet, 30% sucrose in its drinking water, and received saline subcutaneously; and H + GH was fed hypercaloric diet, 30% sucrose in its drinking water, and received GH subcutaneously. After 75 days of total experimental period, H + PL rats were considered obese, having higher body weight, body mass index, Lee-index, and atherogenic index (AI) compared to C + PL. Obesity was accompanied by enhanced myocardial lipid hydroperoxide (LH) and lactate dehydrogenase (LDH), as well of depressed energy expenditure (RMR) and oxygen consumption(VO(2))/body weight. H + GH rats had higher fasting RMR, as well as lower AI and myocardial LH than H + PL. Comparing C + GH with C + PL, despite no effects on morphometric parameters, lipid profile, myocardial LH, and LDH activity, GH enhanced fed RMR and myocardial pyruvate dehydrogenase. In conclusion, the present study brought new insights into the GH effects on obesity related cardiovascular damage demonstrating, for the first time, that GH regulated cardiac metabolic pathways, enhanced energy expenditure and improved the lipid profile in obesity condition. Growth hormone in standard fed condition also offered promising therapeutic value enhancing pyruvate-dehydrogenase activity and glucose oxidation in cardiac tissue, thus optimizing myocardial energy metabolism.

In vitro basal and metabolism-mediated cytotoxicity of flavonoids

The purpose of this study was to compare the basal cytotoxicity and metabolism-mediated cytotoxicity of kaempferol, quercetin and rutin. McCoy cells were exposed to various concentrations of the flavonols with and without the S9 system. The neutral red uptake assay was used to determine viability after 24 h at 35-37 degrees C. Dose-response curves were established for each flavonol in the presence and absence of external metabolizing systems. Kaempferol and quercetin were cytotoxic and provoked a dose-dependent decrease in cell viability, without the S9 system. The hepatic S9 microsomal fraction metabolized these compounds to less cytotoxic metabolites. In contrast, rutin at 500 mu g/ml failed to produce any overt signs of toxicity in either assay. (c) 2005 Elsevier Ltd. All rights reserved.

The effect of pyrazinamide and rifampicin on isoniazid metabolism in rats

Hepatotoxicity is the main concern during tuberculosis chemotherapy with the first-line drugs isoniazid (INH), rifampicin (RMP) and pyrazinamide (PYR). Since these hepatotoxic events have been associated with INH metabolites, the study aimed to measure the area under curve (AUC) parameter for INH and its metabolites acetylisoniazid (AcINH), hydrazine (Hz) and acetylhydrazine (AcHz), when groups of rats were pre-treated for 21 days with INH alone or in combination with RMP and/or PYR, in the following amounts per kg body weight: INH 100 mg; INH 100 mg + RMP 100 mg; INH 100 mg + PYR 350 mg; INH 100 mg + PYR 350 mg + RMP 100 mg. It was found that co-administration of RMP, PYR and RMP + PYR caused a significant decrease in the AUC for INH. Co-administration of PYR was the only treatment that caused a significant increase in the AUC for Hz and a decrease in the AUC for its acetylated product AcHz. The AUC for AcINH was not significantly altered in any experimental group. In conclusion, the increased metabolism of INH in all the drug combinations and the significantly higher production of Hz in the group INH + PYR might be linked with exacerbated hepatotoxic effects of these drug associations. Copyright (c) 2007 John Wiley & Sons, Ltd.

Reduction of enantioselectivity in the kinetic disposition and metabolism of verapamil in rats exposed to toluene

Toluene and verapamil are subject to extensive oxidative metabolism mediated by CYP enzymes, and their interaction can be stereoselective. In the present study we investigated the influence of toluene inhalation on the enantioselective kinetic disposition of verapamil and its metabolite, norverapamil, in rats. Male Wistar rats (n = 6 per group) received a single dose of racemic verapamil (10 mg/kg) orally at the fifth day of nose-only toluene or air (control group) inhalation for 6 h/day (25, 50, and 100 ppm). Serial blood samples were collected from the tail up to 6 h after verapamil administration. The plasma concentrations of verapamil and norverapamil enantiomers were analyzed by LC-MS/MS by using a Chiralpak AD column. Toluene inhalation did not influence the kinetic disposition of verapamil or norverapamil enantiomers (p > 0.05, Kruskal-Wallis test) in rats. The pharmacokinetics of verapamil was enantioselective in the control group, with a higher plasma proportion of the S-verapamil (AUC 250.8 versus 120.4 ng.h.mL(-1); p <= 0.05, Wilcoxon test) and S-norverapamil (AUC 72.3 versus 52.3 ng.h.mL(-1); p <= 0.05, Wilcoxon test). Nose-only exposure to toluene at 25, 50, or 100 ppm resulted in a lack of enantioselectivity for both verapamil and norverapamil. The study demonstrates the importance of the application of enantioselective methods in studies on the interaction between solvents and chiral drugs.