Effect of cholesterol and 7-beta hydroxycholesterol on glutathione status and nitric oxide production in murine peritoneal macrophages

Present study was conducted to observe the effect of cholesterol and oxidized cholesterol (7beta-hydroxycholesterol,7beta-OH) on the nitric oxide (NO) production and the redox ratio by lipopolysaccharide-stimulated macrophages. Dose-dependent decrease in NO levels was seen with both cholesterol and 7beta-OH at different incubation intervals (6,12,18,24 hr) and concentrations (2.5,5,7.5microg/ml). On comparison, a significant decrease in the NO was observed at 24 hr interval in 7beta-OH exposed cells with all respective concentrations of cholesterol. Incubation with 7beta-OH also resulted in significant increase in levels of oxidized glutathione (GSSG) and decrease in reduced glutathione (GSH), while cholesterol showed no effect on GSSG levels. Moreover, GSH levels were lowered only at highest concentration (7.5microg/ml), and at longer incubation intervals (18,24 hr) with cholesterol exposure. This altered the redox status in both cholesterol/7beta-OH treated macrophages. Increased redox ratio and decreased NO levels indicated increased oxidative stress and decreased vasodilation by 7beta-OH compared to cholesterol.     

Revisiting cobalt chloride preconditioning to prevent hypobaric hypoxia-induced damage: identification of global proteomic alteration and key networks

Several studies have supported the hypoxia mimetic roles and cytoprotective properties of cobalt chloride in vitro and in vivo. However, a clear understanding of biological process-based mechanism that integrates the available information remains unknown. This study was aimed to explore the potential mechanism of cobalt chloride deciphering its benefits and well-known physiological challenge caused by hypobaric hypoxia that reportedly affects nearly 24 % of the global population. In order to explore the mechanism of CoCl₂, we used global proteomic and systems biology approach in rat model to provide a deeper insight into molecular mechanisms of preconditioning. Furthermore, key conclusions were drawn based on biological network analysis and their enrichment with ontological overlaps. The study was further strengthened by consistent identification of validation of proteins using immunoblotting. CoCl₂-pretreated animals exposed to hypoxia showed two significant networks, one lipid metabolism and other cell cycle associated, with a total score of 23 and eight focus molecules. In this study, we delineated two primary routes: one, by direct modulation of reactive oxygen species metabolism and, second, by regulation of lipid metabolism which was not known until now. The previously known benefits of cobalt chloride during physiological challenge by hypobaric hypoxia are convincing and could be explained by some basic set of metabolic and molecular reorganization within the hypoxia model. Interestingly, we also observed some of the completely unknown roles of cobalt chloride such as regulation of lipid that could undulate the translational roles of cobalt chloride supplementation beyond hypoxia preconditioning.     

Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica)

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic proteins found primarily in plants. The barley hva1 gene encodes a group 3 LEA protein and is induced by ABA and water deficit conditions. We report here the over expression of hva1 in mulberry under a constitutive promoter via Agrobacterium-mediated transformation. Molecular analysis of the transgenic plants revealed the stable integration and expression of the transgene in the transformants. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of hva1 in conferring tolerance. The transgenic plants showed better cellular membrane stability (CMS), photosynthetic yield, less photo-oxidative damage and better water use efficiency as compared to the non-transgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants show many fold increase in proline concentration than the non-transgenic plants and under water deficit conditions proline is accumulated only in the non-transgenic plants. Results also indicate that the production of HVA1 proteins helps in better performance of transgenic mulberry by protecting membrane stability of plasma membrane as well as chloroplastic membranes from injury under abiotic stress. Interestingly, it was observed that hva1 conferred different degrees of tolerance to the transgenic plants towards various stress conditions. Amongst the lines analysed for stress tolerance transgenic line ST8 was relatively more salt tolerant, ST30, ST31 more drought tolerant, and lines ST11 and ST6 responded well under both salinity and drought stress conditions as compared to the non-transgenic plants. Thus hva1 appears to confer a broad spectrum of tolerance under abiotic stress in mulberry.     

Importance of intracellular pH in determining the uptake and efficacy of the weakly basic chemotherapeutic drug, doxorubicin

Low extracellular pH (pH(e)), that is characteristic of many tumours, tends to reduce the uptake of weakly basic drugs, such as doxorubicin, thereby conferring a degree of physiological resistance to chemotherapy. It has been assumed, from pH-partition theory, that the effect of intracellular pH (pH(i)) is symmetrically opposite, although this has not been tested experimentally. Doxorubicin uptake into colon HCT116 cells was measured using the drug's intrinsic fluorescence under conditions that alter pH(i) and pH(e) or pH(i) alone. Acutely, doxorubicin influx across the cell-membrane correlates with the trans-membrane pH-gradient (facilitated at alkaline pH(e) and acidic pH(i)). However, the protonated molecule is not completely membrane-impermeant and, therefore, overall drug uptake is less pH(e)-sensitive than expected from pH-partitioning. Once inside cells, doxorubicin associates with slowly-releasing nuclear binding sites. The occupancy of these sites increases with pH(i), such that steady-state drug uptake can be greater with alkaline cytoplasm, in contradiction to pH-partition theory. Measurements of cell proliferation demonstrate that doxorubicin efficacy is enhanced at alkaline pH(i) and that pH-partition theory is inadequate to account for this. The limitations in the predictive power of pH-partition theory arise because it only accounts for the pH(i)/pH(e)-sensitivity of drug entry into cells but not the drug's subsequent interactions that, independently, show pH(i)-dependence. In summary, doxorubicin uptake into cells is favoured by high pH(e) and high pH(i). This modified formalism should be taken into account when designing manoeuvres aimed at increasing doxorubicin efficacy.     

Characterization of Mycolytic Enzymes of Bacillus Strains and Their Bio-Protection Role Against Rhizoctonia solani in Tomato

Four antagonists bacteria namely, Bacillus megaterium MB3, B. subtilis MB14, B. subtilis MB99 and B. amyloliquefaciens MB101 were able to produce chitinase, β-1,3-glucanase and protease in different range with the presence of Rhizoctonia solani cell wall as a carbon source. Amplification of chitinase (chiA) gene of 270 bp and β-1, 3-glucanase gene of 415 bp was given supportive evidence at molecular level of antibiosis. After in vitro screening, all antagonists were tested against R. solani under greenhouse conditions. Root treatment of Bacillus strains showed superior defense during pathogen suppression in terms of chitinase, glucanase, peroxidase, poly phenol oxidase, phenylalanine ammonia-lyase activity and total phenolic content in leaves of tomato. All these enzymes accumulated high in tomato leaves as compared to roots. Pathogenesis-related proteins and defense-related enzymes accumulation was directly correlated with plant protection and greenhouse results indicated that B. amyloliquefaciens MB101- and B. subtilis MB14-treated plants offered 69.76 and 61.51 % disease reductions, respectively, over the infected control. These results established that these organisms have the potential to act as biocontrol agents. This study could be highlighted a mutual importance of liquid formulation of antagonistic Bacillus spp. against root associated sclerotia former pathogen R. solani.     

Preparation of (+)-Trans-Isoalliin and Its Isomers by Chemical Synthesis and RP-HPLC Resolution

Naturally occurring (+)-trans-isoalliin, (R_CR_S)-(+)-trans-S-1-propenyl-L-cysteine sulfoxide, is a major cysteine sulfoxide in onion. The importance of producing it synthetically to support further research is very well recognized. The (+)-trans-isoalliin is prepared by chemical synthesis and reversed-phase (RP)-HPLC. First, S-2-propenyl-L-cysteine (deoxyalliin) is formed from L-cysteine and allyl bromide, which is then isomerized to S-1-propenyl-L-cysteine (deoxyisoalliin) by a base-catalyzed reaction. A mixture of cis and trans forms of deoxyisoalliin is formed and separated by RP-HPLC. Oxidation of the trans form of deoxyisoalliin by H₂O₂ produces a mixture of (−)- and (+)-trans-isoalliin. Finally, RP-HPLC is used successfully in separating (−)- and (+)-trans-isoalliin, and hence, (+)-trans-isoalliin is synthesized for the first time in this study. In addition, the (±) diastereomers of cis-isoalliin are also separated and purified by RP-HPLC.     

Obesity is an independent determinant of elevated C-reactive protein in healthy women but not men

Background and aims: High-sensitivity C-reactive protein (hs CRP) has emerged as an inflammatory biomarker to predict metabolic syndrome. Here, we investigate the association of hs CRP with metabolic variables and determine the risks for elevated hs CRP levels in healthy Singaporean adults.

Methods: We conducted a cross-sectional study of 225 participants (104 men). The levels of hs CRP and fasting lipid parameters were analyzed by COBAS. Body composition was determined with dual-energy X-ray absorptiometry.

Results: Twenty-one (9?%) participants had elevated hs CRP levels (>3?mg/mL). The levels of hs CRP had significant correlations (p?<0.05) with obesity and metabolic variables among women. Stepwise multivariate regression analysis identified FM (%) (accounted for 22.5% of the variability in hs CRP levels) as a major determinant of hs CRP levels. On multivariate regression, FM (%) was the independent determinant of intermediate and elevated hs CRP in women after adjustment for the potential confounders.

Conclusions: Obesity may play a direct role in the elevated hs CRP levels in women, but not men living in Singapore. This is probably due to different body composition or different effects of sex hormones on adipose tissue between men and women.     

Molecular recognition of human eosinophil-derived neurotoxin (RNase 2) by placental ribonuclease inhibitor

Placental ribonuclease inhibitor (RI) binds diverse mammalian RNases with dissociation constants that are in the femtomolar range. Previous studies on the complexes of RI with RNase A and angiogenin revealed that RI utilises largely distinctive interactions to achieve high affinity for these two ligands. Here we report a 2.0 angstroms resolution crystal structure of RI in complex with a third ligand, eosinophil-derived neurotoxin (EDN), and a mutational analysis based on this structure. The RI-EDN interface is more extensive than those of the other two complexes and contains a considerably larger set of interactions. Few of the contacts present in the RI-angiogenin complex are replicated; the correspondence to the RI-RNase A complex is somewhat greater, but still modest. The energetic contributions of various interface regions differ strikingly from those in the earlier complexes. These findings provide insight into the structural basis for the unusual combination of high avidity and relaxed stringency that RI displays.