Cell type-specific activation of the cytomegalovirus promoter by dimethylsulfoxide and 5-aza-2'-deoxycytidine

The cytomegalovirus promoter is a very potent promoter commonly used for driving the expression of transgenes, though it gradually becomes silenced in stably transfected cells. We examined the methylation status of the cytomegalovirus promoter in two different cell lines and characterized its mechanisms of activation by dimethylsulfoxide and 5-Aza-2'-deoxycytidine. The cytomegalovirus promoter stably transfected into Chinese hamster ovary cells is suppressed by DNA methylation-independent mechanisms, which is different from the rat embryonic cardiomyoblast H9c2-Fluc.3 cells in which the cytomegalovirus promoter is silenced by methylation. Dimethylsulfoxide and 5-Aza-2'-deoxycytidine can activate the cytomegalovirus promoter in both cell types by overlapping mechanisms. Dimethylsulfoxide activates the cytomegalovirus promoter in Chinese hamster ovary cells by promoting histone acetylation and the activation of p38 mitogen-activated protein kinase and nuclear factor kappaB (NFkappaB) signaling pathways, while 5-Aza-2'-deoxycytidine increases histone acetylation and activates the nuclear factor kappaB but not the p38 mitogen-activated protein kinase pathway. In H9c2-Fluc.3 cells, both agents promote demethylation of the cytomegalovirus promoter, and enhance its activity exclusively through activation of the nuclear factor kappaB pathway and to a lesser extent of the p38 mitogen-activated protein kinase pathway. Our findings suggest that suppression and activation of the cytomegalovirus promoter are cell type-specific. These results may be used for developing strategies to enhance the expression of transgenes and the production of recombinant proteins encoded by transgenes controlled by a cytomegalovirus promoter.     

On the use of meta-heuristics to increase the efficiency of online grid workflow scheduling algorithms

The competitiveness of online algorithms is measured based on the correctness of the results produced and processing time efficiency. Traditionally evolutionary algorithms are not favored in online paradigms because of the large number of iterations involved in the algorithm which translates directly into processing time overhead. In this paper we describe MARS (Management Architecture for Resource Services) online scheduling algorithm which uses Simulated Annealing and concepts from Tabu Search to drastically decrease the processing time of the algorithm. The paper outlines the concepts behind MARS, the components involved and scheduling methodology used. In addition we also identify the time consuming bottlenecks in the performance of the system and how evolutionary algorithms help us soar past them.

Seed priming with methyl jasmonate mitigates copper and cadmium toxicity by modifying biochemical attributes and antioxidants in Cajanus cajan

Contamination of agricultural soils with heavy metals (HMs) has posed major threat to the environment as well as human health. The aim of this study was to appraise the efficiency of key-antioxidant enzymes in enhancing plants’ tolerance to HMs (heavy metals) like copper (Cu) and Cadmium (Cd), under the action of methyl jasmonate (Me-JA) in Cajanus cajan L. Seeds of C. cajan treated with Me-JA (0, 1 nM) were discretely subjected to noxious concentrations of Cu and Cd (0, 1, 5 mM) and raised for 12 days under controlled conditions in plant growth chamber for biochemical analysis. In contrast to Cd, Cu triggered oxidative stress more significantly (44.54% in 5 mM Cu increase in MDA as compared to control) and prominently thereby affecting plants’ physiological and biochemical attributes. By activating the antioxidant machinery, Me-JA pre-treatment reduced HMs-induced oxidative stress, increased proline production, glutathione (41.95% under 5 mM Cu when treated with 1 nM Me-JA treatment) and ascorbic acid content by 160.4 % under aforemtioned treatments thus improving the redox status. Thus, in light of this our results put forward a firm basis of the positive role that Me-JA might play in the mitigation of oxidative stress caused due to HMs stress by stimulating antioxidant defense system leading to overall improvement of growth of C. cajan seedlings.     

Mucin biosynthesis: upregulation of core 2 beta 1,6 N-acetylglucosaminyltransferase by retinoic acid and Th2 cytokines in a human airway epithelial cell line

Vitamin A and the T helper 2 cytokines IL-4 and IL-13 play important roles in the induction of mucin gene expression and mucus hypersecretion. However, the effects of these agents on enzymes responsible for mucin glycosylation have received little attention. Here, we report the upregulation of core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) activity both by all-trans retinoic acid (RA) and by IL-4 and IL-13 in the H292 airway epithelial cell line. Northern blotting analysis showed that the M isoform of C2GnT, which is expressed in mucus-secreting tissues and can form all mucin glycan beta1,6-branched structures, including core 2, core 4, and blood group I antigen, was upregulated by both RA and IL-4/13. The L isoform, which forms only the core 2 structure, was moderately upregulated by IL-4/13 but not by RA. Enhancement of the M isoform of C2GnT by RA was abolished by an inhibitor of RA receptor alpha, implicating RA receptor alpha in the effect of RA. Likewise, an inhibitor of the Janus kinase 3 pathway blocked the enhancing effects of IL-4/13 on the L and M isoforms of C2GnT, suggesting a role of this pathway in the upregulation of these two C2GnTs by these cytokines. Taken together, the results suggest that IL-4/13 T helper 2 cytokines and RA can alter the activity of enzymes that synthesize branching mucin carbohydrate structure in airway epithelial cells, potentially leading to altered mucin carbohydrate structure and properties.     

A method of precise mRNA/DNA homology-based gene structure prediction

Background  

Accurate and automatic gene finding and structural prediction is a common problem in bioinformatics, and applications need to be capable of handling non-canonical splice sites, micro-exons and partial gene structure predictions that span across several genomic clones.     

Modulation of mitochondrial complex I activity by reversible Ca2+ and NADH mediated superoxide anion dependent inhibition

Complex I, a key component of the mitochondrial respiratory chain, exhibits diminished activity as a result of cardiac ischemia/reperfusion. Cardiac ischemia/reperfusion is associated with increases in the levels of mitochondrial Ca(2+) and pro-oxidants. In the current in vitro study, we sought evidence for a mechanistic link between Ca(2+), pro-oxidants, and inhibition of complex I utilizing mitochondria isolated from rat heart. Our results indicate that addition of Ca(2+) to solubilized mitochondria results in loss in complex I activity. Ca(2+) induced a maximum decrease in complex I activity of approximately 35% at low micromolar concentrations over a narrow physiologically relevant pH range. Loss in activity required reducing equivalents in the form of NADH and was not reversed upon addition of EGTA. The antioxidants N-acetylcysteine and superoxide dismutase, but not catalase, prevented inhibition, indicating the involvement of superoxide anion (O2(*-)) in the inactivation process. Importantly, the sulfhydryl reducing agent DTT was capable of fully restoring complex I activity implicating the formation of sulfenic acid and/or disulfide derivatives of cysteine in the inactivation process. Finally, complex I can reactivate endogenously upon Ca(2+) removal if NADH is present and the enzyme is allowed to turnover catalytically. Thus, the present study provides a mechanistic link between three alterations known to occur during cardiac ischemia/reperfusion, mitochondrial Ca(2+) accumulation, free radical production, and complex I inhibition. The reversibility of these processes suggests redox regulation of Ca(2+) handling.     

Sablacaurin A and B, two 19-nor-3,4-seco-lanostane-type triterpenoids from Sabal causiarum and Sabal blackburniana, respectively

In search for bioactive compounds from Sabal species, sablacaurin A [25-ethyl,23-methyl-19-nor-24-methylene-3,4-seco-4(28)-lanosten-10,3-olide] and sablacaurin B [24-ethyl,24-methyl-19-nor-3,4-seco-4(28),25(26)-lanostadiene-10,3-olide], the first 19-nor lanostane derivatives of the 3,4-seco type with a spiro element, have been isolated from the leaves of Sabal causiarum and Sabal blackburniana respectively, together with the known squalene (S. blackburniana) and ss-sitosterol (S. causiarum). From leaves of Sabal peregrina, the known triterpenes 3-oxo-24-methylenecycloartane and 24-methylcycloart-25(26)-en-3-one were isolated. The structures of these compounds were established from spectroscopic studies.     

Semi-industrial Scale Production of a New Yeast with Probiotic Traits, <Emphasis Type="Italic">Cryptococcus</Emphasis> sp. YMHS,Isolated from the Red Sea

A new yeast strain with promising probiotic traits was isolated from the Red Sea water samples. The isolate (YMHS) was subjected to genetic characterization and identified as Cryptococcus sp. Nucleotide sequence analysis of the rRNA gene internal transcribed spacer regions showed 95% sequence similarity between the isolate and Cryptococcus albidus. Cryptococcus sp. YMHS exhibited desirable characteristics of probiotic microorganisms; it has tolerance to low pH in simulated gastric juice, resistance to bile salts, hydrophobic characteristics, broad antimicrobial activity, and in vitro ability to degrade cholesterol. The isolate grew well in a semi-defined medium composed of yeast extract, glucose, KH₂PO₄, (NH₄)₂SO₄, and MgSO₄, yielding cell mass of 2.32 and 5.82 g/l in shake flask and in bioreactor cultures, respectively. Fed-batch cultivation, with controlled pH, increased the biomass gradually in culture, reaching 28.5 g/l after 32 h cultivation. Beside the feasible use as a probiotic, the new strain also could be beneficial in the development of functional foods or novel food preservatives. To our knowledge, this is the first report of yeast with probiotic properties isolated from the Red Sea.