Eep Confers Lysozyme Resistance to Enterococcus faecalis via the Activation of the Extracytoplasmic Function Sigma Factor SigV

Enterococcus faecalis is a commensal bacterium found in the gastrointestinal tract of most mammals, including humans, and is one of the leading causes of nosocomial infections. One of the hallmarks of E. faecalis pathogenesis is its unusual ability to tolerate high concentrations of lysozyme, which is an important innate immune component of the host. Previous studies have shown that the presence of lysozyme leads to the activation of SigV, an extracytoplasmic function (ECF) sigma factor in E. faecalis, and that the deletion of sigV increases the susceptibility of the bacterium toward lysozyme. Here, we describe the contribution of Eep, a membrane-bound zinc metalloprotease, to the activation of SigV under lysozyme stress by its effects on the stability of the anti-sigma factor RsiV. We demonstrate that the Δeep mutant phenocopies the ΔsigV mutant in lysozyme, heat, ethanol, and acid stress susceptibility. We also show, using an immunoblot analysis, that in an eep deletion mutant, the anti-sigma factor RsiV is only partially degraded after lysozyme exposure, suggesting that RsiV is processed by unknown protease(s) prior to the action of Eep. An additional observation is that the deletion of rsiV, which results in constitutive SigV expression, leads to chaining of cells, suggesting that SigV might be involved in regulating cell wall-modifying enzymes important in cell wall turnover. We also demonstrate that, in the absence of eep or sigV, enterococci bind significantly more lysozyme, providing a plausible explanation for the increased sensitivity of these mutants toward lysozyme.     

Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation

Fermentation-derived products are in greater demand to meet the increasing global market as well as to overcome environmental problems. In this work, Escherichia coli has been metabolically engineered with acrylate pathway genes from Clostridium propionicum for the conversion of d-lactic acid to propionic acid. The introduced synthetic pathway consisted of seven genes encoding the enzymes propionate CoA-transferase (Pct), lactoyl-CoA dehydratase (Lcd) and acryloyl-CoA reductase (Acr). The engineered strain synthesised propionic acid at a concentration of 3.7?±?0.2 mM upon fermentation on glucose. This low production level could be attributed to the low activity of the recombinant enzymes in particular the rate-limiting enzyme, Acr. Interestingly, the recombinant pathway caused an increased lactate production in E. coli with a yield of 1.9 mol/mol of glucose consumed along with a decrease in other by-products. Down-regulation of the pfl (pyruvate formate lyase) genes and a possible inhibition of Pfl activity by the acrylate pathway intermediate, acryloyl-CoA, could have reduced carbon flow to the Pfl pathway with a concomitant increase in lactate production. This study reports a novel way of synthesising propionic acid by employing a non-native, user-friendly organism through metabolic engineering.     

Inhibition of 5-lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon

Previous studies have shown that human prostate cancer cells constitutively generate 5-lipoxygenase (5-LOX) metabolites from arachidonic acid, and inhibition of 5-LOX blocks production of 5-LOX metabolites and triggers apoptosis in prostate cancer cells. This apoptosis is prevented by exogenous metabolites of 5-LOX, suggesting an essential role of 5-LOX metabolites in the survival of prostate cancer cells. However, downstream signaling mechanisms which mediate the survival-promoting effects of 5-LOX metabolites in prostate cancer cells are still unknown. Recently, we reported that MK591, a specific inhibitor of 5-LOX activity, induces apoptosis in prostate cancer cells without inhibition of Akt, or ERK, two well-characterized regulators of pro-survival mechanisms, suggesting the existence of an Akt and ERK-independent survival mechanism in prostate cancer cells regulated by 5-LOX. Here, we report that 5-LOX inhibition-induced apoptosis in prostate cancer cells occurs via rapid inactivation of protein kinase C-epsilon (PKCε), and that exogenous 5-LOX metabolites prevent both 5-LOX inhibition-induced down-regulation of PKCε and induction of apoptosis. Interestingly, pre-treatment of prostate cancer cells with diazoxide (a chemical activator of PKCε), or KAE1-1 (a cell-permeable, octa-peptide specific activator of PKCε) prevents 5-LOX inhibition-induced apoptosis, which indicates that inhibition of 5-LOX triggers apoptosis in prostate cancer cells via down-regulation of PKCε. Altogether, these findings suggest that metabolism of arachidonic acid by 5-LOX activity promotes survival of prostate cancer cells via signaling through PKCε, a pro-survival serine/threonine kinase.     

Cloning, expression, purification and characterization of a single chain variable fragment specific to tumor necrosis factor alpha in Escherichia coli

Anti TNF-α molecules have been used as therapeutic agents in a variety of human diseases such as Rheumatoid arthritis, Ankylosing spondylitis, Chron's diseases, Psoriasis, etc., where high levels of TNF-α plays a destructive role. The limitations of the present TNF-α inhibitors in terms of size, tissue penetration and immunogenicity, etc., provoked the search for small anti TNF-α molecules. In the present study, a single chain variable fragment (ScFv) construct was made from a monoclonal antibody of the class IgG raised against TNF-α was used. The anti TNF-α ScFv was well expressed as soluble form in Escherichia coli BL21 (DE3), which was purified to homogeneity by commercial methacrylate monolith-convective interaction media (CIM) supports using two different chemistries, immobilized metal affinity chromatography (IMAC) with copper ions followed by anion exchange chromatography. The anti TNF-α ScFv found to be inhibiting the TNF-α mediated cytotoxicity in MCF-7 cells with an IC₅₀ of 8 μg. Data presented here are promising and encouraging to further optimize anti TNF-α ScFv production in larger scale with higher recovery at a cheaper price for therapeutic purposes.     

A database for the predicted pharmacophoric features of medicinal compounds

Pharmacophore feature is defined by a set of chemical structure patterns having the active site of drug like molecule. Pharmacophore can be used to assist in building hypothesis about desirable chemical properties in drug molecule and hence it can be used to refine and modify drug candidates. We predicted the pharmacophoric features of 150 medicinal compounds from plants for anti-cancer, anti-carcinogenic, anti-diabetic, anti-microbial, and anti-oxidant. Estimation of pharmacophoric feature is necessary to ensure the optimal supramolecular interaction with a biological target and to trigger or block its biological response. We subsequently make this data available to open access using a database at the URL: http://www.hccbif.info/index.htm AVAILABILITY: The database is available for free at http://www.hccbif.info/index.htm.     

The prolyl hydroxylase oxygen-sensing pathway is cytoprotective and allows maintenance of mitochondrial membrane potential during metabolic inhibition

The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase domain (PHD)-containing enzymes, whose reduction of activity initiate a hypoxic signal cascade. In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes. PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels in glucose-free medium containing cyanide. The addition of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) caused a decline of ATP levels that was indistinguishable between control and PHI-treated myocytes. Despite the comparable levels of ATP depletion, PHI-preconditioned myocytes remained significantly protected. As expected, mitochondrial membrane potential (_mito) collapses in control myocytes during cyanide and 2-DG treatment and it fails to completely recover upon washout. In contrast, _mito is partially maintained during metabolic inhibition and recovers completely on washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse _mito in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of _mito in PHI-treated myocytes, whereas a role for the "reverse mode" operation of the F₁F₀-ATP synthase was ruled out. Further examination of mitochondrial function revealed that PHI treatment downregulated basal oxygen consumption to only 15% that of controls. Oxygen consumption rates, although initially lower in PHI-preconditioned myocytes, recovered completely upon removal of metabolic poisons, while reaching only 22% of preinsult levels in control myocytes. We conclude that PHD oxygen-sensing mechanism directs multiple compensatory changes in the cardiomyocyte, which include a low-respiring mitochondrial phenotype that is remarkably protected against metabolic insult. fumarate; hibernation; cardioprotection; anaplerotic     

Isolation and characterization of dihydromonacolin-MV from <Emphasis Type="Italic">Monascus purpureus</Emphasis> for antioxidant properties

The methanolic extract of Monascus purpureus cultivated by solid-state fermentation on rice showed strong 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and better yield as compared to other polarity based extracted fractions. It was selected for further purification of the antioxidant. The activity-guided repeated fractionation of methanolic extract on a silica gel column chromatography yielded a compound that exhibited strong antioxidant activity. Based on the spectroscopic analysis by UV, IR, ¹H NMR, ¹³C NMR, 2D-HSQCT NMR, and MS, the antioxidant isolated was elucidated as a derivative of dihydromonacolin-K, where the ester group is 2-methyl propionate, designated as dihydromonacolin-MV. The DPPH radical was significantly scavenged by the dihydromonacolin-MV (IC₅₀ 20±1 μg ml⁻¹). The dihydromonacolin-MV showed strong inhibition of lipid peroxidation in a liposome model with an IC₅₀ value of 5.71±0.38 μg ml⁻¹ and superoxide radical scavenging activity with an IC₅₀ value of 163.97±2.68 μg ml⁻¹.     

Role of pectin from cucumber (Cucumis sativus) in modulation of protein kinase C activity and regulation of glycogen metabolism in rats

The regulatory role of protein kinase C (PKC) in glycogen metabolism in pectin fed rats was investigated. Administration of pectin (5 g/kg body wt/day) from cucumber (Cucumis sativius L.) led to inhibitory effects on PKC activity in the liver of rats. In the brain and pancreas, PKC activity was significantly higher in pectin-treated rats as compared to the control group. Level of blood glucose was significantly lowered and the level of glycogen in the liver was significantly increased in pectin-administered rats. Glycogen synthase activity was enhanced, while glycogen phosphorylase enzyme showed inhibition in pectin-treated rats. Results indicated that pectin administration might have caused an increase in the secretion of the insulin, which, in turn, had a stimulatory effect on the PKC activity in the pancreas. The decreased PKC activity in the liver and increased PKC activity in the brain and pancreas on pectin administration indicated enhanced glycogenesis and reduced glycogenolysis.