Construction of Recombinant Klebsiella pneumoniae to Increase Ethanol Production on Residual Glycerol Fed-Batch Cultivations.

K. pneumoniae BLh-1 strain was genetically modified aiming at obtaining high ethanol productivity in cultivations using residual glycerol from biodiesel synthesis as substrate. The recombinant strain K. pneumoniae Kp17 was obtained by inserting the multicopy plasmid pTOPOBL17 containing the AdhE gene, and its own promoter, from K. pneumoniae BLh-1. Influence of Fe2+ supplementation and initial glycerol concentration on culture conditions were analyzed, both in rotatory shaker and in batch bioreactors. In the bioreactor cultures, K. pneumoniae Kp17 strain produced 4.5 g L-1 of ethanol (productivity of 0.50 g L-1 h-1 and yields of 0.15 g g-1) after 24-h cultivation, corresponding to an increase of approximately 40% in ethanol concentration compared to wild strain, K. pneumoniae BLh-1. Best conditions were then applied in exponential fed-batch bioreactors, with final ethanol concentration of 17.30 g L-1 (productivity of 0.59 g L-1 h-1 and yields of 0.16 g g-1) after 30 h of feeding, representing 11.5% of increment in titer of ethanol compared to the wild strain. Mutant cells kept 92.5% of the plasmids under batch in 24 h, and 71.9% under fed-batch after 27 h of exponential feeding. The findings in this work show the possibility of using a simple approach to genetically modify K. pneumoniae to be employed this versatile bacterium for the bioconversion of residual glycerol into ethanol.

Effect of antioxidant treatment on fibrogenesis in rats with carbon tetrachloride-induced cirrhosis.

Aim. This study aimed to assess the antioxidant activity of quercetin (Q) in an experimental model of cirrhosis induced by CCl(4) inhalation. Materials and Methods. We used 25 male Wistar rats (250 g) that were divided into 3 groups: control (CO), CCl(4), and CCl(4) + Q. The rats were subjected to CCl(4) inhalation (2x/week) for 16 weeks, and they received phenobarbital in their drinking water at a dose of 0.3 g/dL as a P450 enzyme inducer. Q (50 mg/Kg) was initiated intraperitoneally at 10 weeks of inhalation and lasted until the end of the experiment. Statistical analysis was by ANOVA Student Newman-Keuls (mean ± SEM), and differences were considered statistically significant when P < 0.05. Results. After treatment with quercetin, we observed an improvement in liver complications, decreased fibrosis, as analyzed by picrosirius for the quantification of collagen, and decreased levels of matrix metalloproteinase 2 (MMP-2) compared with the CCl(4) group. It also reduced oxidative stress, as confirmed by the decrease of substances reacting to thiobarbituric acid (TBARS), the increased activity of antioxidant enzymes, and the reduced glutathione ratio and glutathione disulfide (GSH/GSSG). Conclusion. We suggest that the use of quercetin might be promising as an antioxidant therapy in liver fibrosis.

Role of quercetin in preventing thioacetamide-induced liver injury in rats.

In hepatic toxicity induced in rats by two injections of thioacetamide (TAA, 350 mg/kg with an interval of 8 hr), the action of quercetin was investigated. After 96 hr, TAA administration resulted in hepatic necrosis, significant increases in serum transaminase activity, and increases in hepatic lipoperoxidation. Thioacetamide-induced hepatotoxicity also showed changes in antioxidant enzymes in the liver of rats, with alterations in p-ERK 1/2 (phosphorylated extracellular-signal related kinase 1/2) as well as an imbalance between proapototic protein Bax and anti-apoptotic protein Bcl-2 expression. With administration of the flavonoid quercetin (50 mg/Kg i.p.) for four consecutive days following TAA, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity were close to normal values in rats. Histological findings suggested that quercetin had a preventive effect on TAA-induced hepatic necrosis. Quercetin treatment caused significant decreases in lipid peroxide levels in the TAA-treated rats, with some changes in antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Quercetin also inhibited the change of the p-ERK1/2 by TAA and significantly prevented the increase in Bax/Bcl-2 ratio, thus preventing apoptosis. Findings indicate that quercetin may have a preventive effect on TAA-induced hepatotoxicity by modulating the oxidative stress parameters and apoptosis pathway.

Evaluation of chronic omega-3 fatty acids supplementation on behavioral and neurochemical alterations in 6-hydroxydopamine-lesion model of Parkinson's disease.

Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have been widely associated to beneficial effects over different neuropathologies, but only a few studies associate them to Parkinson's disease (PD). Rats were submitted to chronic supplementation (21-90 days of life) with fish oil, rich in omega-3 PUFAs, and were uni- or bilaterally lesioned with 4microg of the neurotoxin 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle. Although lipid incorporation was evidenced in neuronal membranes, it was not sufficient to compensate motor deficits induced by 6-OHDA. In contrast, omega-3 PUFAs were capable of reducing rotational behavior induced by apomorphine, suggesting neuroprotection over dyskinesia. The beneficial effects of omega-3 PUFAs were also evident in the maintenance of thiobarbituric acid reactive substances index from animals lesioned with 6-OHDA similar to levels from SHAM and intact animals. Although omega-3 PUFAs did not modify the tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta and in the ventral tegmental area, nor the depletion of dopamine (DA) and its metabolites in the striatum, DA turnover was increased after omega-3 PUFAs chronic supplementation. Therefore, it is proposed that omega-3 PUFAs action characterizes the adaptation of remaining neurons activity, altering striatal DA turnover without modifying the estimated neuronal population.