Characterization of <Emphasis Type="Italic">Brassica juncea</Emphasis> antioxidant potential under salinity stress

Present study characterizes the anti-oxidative defense potential of four Brassica juncea varieties, Pusa Jaikisan, Varuna, RLM-198, and CS-52, differing in their ability to withstand salinity stress. 7-day-old seedlings raised in MS medium supplemented with 0, 50, 100, and 150 mM NaCl were used to monitor changes in the growth profile, level of stress marker molecules, and activities of important antioxidant enzymes. Increasing NaCl concentration resulted in a significant (P ≤ 0.05) reduction of shoot fresh and dry mass and vigor index in all the varieties tested. Maximum reduction in growth was recorded for RLM-198 while CS-52 maintained better growth characteristics. Varuna and RLM-198 exhibited a limited increase in superoxide dismutase, ascorbate peroxidase, and total peroxidase activity under increasing salinity. These varieties also recorded maximum salt stress-induced damage in terms of increased lipid peroxidation, H₂O₂ content, and electrolyte leakage. On the other hand, CS-52 recorded maximum proline accumulation with minimum levels of H₂O₂, electrolyte leakage, and malondialdehyde contents. With increasing salinity stress, CS-52 recorded maximal increase in the activity of antioxidant enzymes. However, catalase activity did not correlate with alterations in H₂O₂ levels under stress. Interestingly, a lower superoxide dismutase:ascorbate peroxidase ratio in CS-52 correlated with stress tolerance trait, while a comparatively higher superoxide dismutase:ascorbate peroxidase ratio in RLM-198 marked the susceptible nature of the variety. Our results propose that superoxide dismutase:ascorbate peroxidase ratio is the critical factor, determining the degree of stress tolerance in Brassica juncea.     

Attenuated vasodilatation in lambs with endogenous and exogenous activation of cGMP signaling: role of protein kinase G nitration

Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B-type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO-cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO(-) ) generation and PKG-1α nitration, while PKG-1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and mimicked by the ONOO(-) donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Peripheral lung extracts from 4-week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24 h (with exogenous activation of cGMP) revealed increased ONOO(-) levels, elevated PKG-1α nitration, and decreased kinase activity without changes in PKG-1α protein levels. However, in Shunt lambs treated with L-arginine or lambs administered polyethylene glycol conjugated-SOD (PEG-SOD) during inhaled NO exposure, ONOO(-) and PKG-1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG-1α nitration and subsequent attenuation of activity.     

Osteopontin-stimulated expression of matrix metalloproteinase-9 causes cardiomyopathy in the mdx model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is a common and lethal form of muscular dystrophy. With progressive disease, most patients succumb to death from respiratory or heart failure, or both. However, the mechanisms, especially those governing cardiac inflammation and fibrosis in DMD, remain less understood. Matrix metalloproteinase (MMPs) are a group of extracellular matrix proteases involved in tissue remodeling in both physiologic and pathophysiologic conditions. Previous studies have shown that MMP-9 exacerbates myopathy in dystrophin-deficient mdx mice. However, the role and the mechanisms of action of MMP-9 in cardiac tissue and the biochemical mechanisms leading to increased levels of MMP-9 in mdx mice remain unknown. Our results demonstrate that the levels of MMP-9 are increased in the heart of mdx mice. Genetic ablation of MMP-9 attenuated cardiac injury, left ventricle dilation, and fibrosis in 1-y-old mdx mice. Echocardiography measurements showed improved heart function in Mmp9-deficient mdx mice. Deletion of the Mmp9 gene diminished the activation of ERK1/2 and Akt kinase in the heart of mdx mice. Ablation of MMP-9 also suppressed the expression of MMP-3 and MMP-12 in the heart of mdx mice. Finally, our experiments have revealed that osteopontin, an important immunomodulator, contributes to the increased amounts of MMP-9 in cardiac and skeletal muscle of mdx mice. This study provides a novel mechanism for development of cardiac dysfunction and suggests that MMP-9 and OPN are important therapeutic targets to mitigating cardiac abnormalities in patients with DMD.     

Electricity generation from carbon monoxide and syngas in a microbial fuel cell

Electricity generation in microbial fuel cells (MFCs) has been a subject of significant research efforts. MFCs employ the ability of electricigenic bacteria to oxidize organic substrates using an electrode as an electron acceptor. While MFC application for electricity production from a variety of organic sources has been demonstrated, very little research on electricity production from carbon monoxide and synthesis gas (syngas) in an MFC has been reported. Although most of the syngas today is produced from non-renewable sources, syngas production from renewable biomass or poorly degradable organic matter makes energy generation from syngas a sustainable process, which combines energy production with the reprocessing of solid wastes. An MFC-based process of syngas conversion to electricity might offer a number of advantages such as high Coulombic efficiency and biocatalytic activity in the presence of carbon monoxide and sulfur components. This paper presents a discussion on microorganisms and reactor designs that can be used for operating an MFC on syngas.     

Antioxidative and radioprotective activities of semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1

A semiquinone glucoside derivative (SQGD) was isolated from a radioresistant bacterium Bacillus sp. INM-1 and its antioxidant and radioprotective activities were evaluated using in vitro assays. Natural stable free radical properties of SQGD in solid as well as in solution form were estimated using Electron Paramagnetic Resonance (EPR) spectrometry. Results of the study were demonstrated high reducing power (1.267 ± 0.03356 U(abs)) and nitric oxide radicals scavenging activity (34.684 ± 2.132%) of SQGD. Maximum lipid peroxidation inhibitory activity of SQGD was found to be 74.09 ± 0.08% at 500 μg/ml concentration. Similarly, significant (39.54%; P < 0.05) protection to the liposomal artificial membrane against gamma radiation was observed by SQGD in terms of neutralization of gamma radiation-induced TBARS radicals in vitro. OH(-) radicals scavenging efficacy of SQGD was estimated in terms of % inhibition in deoxy D: -ribose degradation by non-site-specific and site-specific assay. The maximum (54.01 ± 1.01%) inhibition of deoxy D: -ribose degradation was observed in non-site-specific manner, whereas, site-specific inhibition was observed to be 46.36 ± 0.5% at the same concentration (250 μg/ml) of SQGD. EPR spectroscopic analysis of the SQGD indicated ~80% reduction of DPPH radicals at 6.4% concentration. EPR spectral analysis of SQGD was revealed an appearance of very strong EPR signal of 2.00485 (crystalline form) and 2.00520 (solution form) g(y) tensor value, which were an established characteristic of o-semiquinone radicals. Therefore, it can be concluded that SQGD is a natural stable o-semiquinone-type radical, possessing strong antioxidant activities and can effectively neutralize radiation induced free radicals in biological system.     

Prior exposure to oxidized low-density lipoprotein limits apoptosis in subsequent generations of endothelial cells by altering promoter methylation

Oxidized LDL (ox-LDL) plays a critical role in atherogenesis, including apoptosis. As hypercholesterolemia causes epigenetic changes resulting in long-term phenotypic consequences, we hypothesized that repeated and continuous exposure to ox-LDL may alter the pattern of apoptosis in human umbilical vein endothelial cells (HUVECs). We also analyzed global and promoter-specific methylation of apoptosis-related genes. As expected, ox-LDL evoked a dose-dependent increase in apoptosis in the first passage HUVECs that was completely abrogated by lectin-like ox-LDL receptor (LOX-1)-neutralizing antibody. Ox-LDL-induced apoptosis was associated with upregulation of proapoptotic LOX-1, ANXA5, BAX, and CASP3 and inhibition of antiapoptotic BCL2 and cIAP-1 genes accompanied with reciprocal changes in the methylation of promoter regions of these genes. Subsequent passages of cells displayed attenuated apoptotic response to repeat ox-LDL challenge with blunted gene expression and exaggerated methylation of LOX-1, BAX, ANXA5, and CASP3 genes (all P < 0.05 vs. first exposure to ox-LDL). Treatment of cells with LOX-1 antibody before initial ox-LDL treatment prevented both gene-specific promoter methylation and expression changes and reduction of apoptotic response to repeat ox-LDL challenge. Based on these data, we conclude that exposure of HUVECs to ox-LDL induces epigenetic changes leading to resistance to apoptosis in subsequent generations and that this effect may be related to the LOX-1-mediated increase in DNA methylation.     

Site-specific radical formation in DNA induced by Cu(II)-H₂O₂ oxidizing system, using ESR, immuno-spin trapping, LC-MS, and MS/MS

Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death, and aging. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS, and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. However, accurate measurement of DNA damage has been a matter of debate. In particular, there have been reports of artifactual oxidation leading to erroneously high damage estimates. Further, most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)–H₂O₂ oxidizing system using immuno-spin trapping (IST) with electron paramagnetic resonance (EPR), MS, and MS/MS. The radical generated is trapped by DMPO immediately upon formation. The DMPO adduct formed is initially EPR active but subsequently is oxidized to the stable nitrone, which can then be detected by IST and further characterized by MS and MS/MS.     

Analysis of distribution and significance of simple sequence repeats in enteric bacteria Shigella dysenteriae SD197

We have explored the possible role of SSR density in genome to generate biological information. In our study, we have checked the SSR (simple sequence repeats) status in virulent and non virulent genes of enteric bacteria to see whether the SSRs distribution contributes to virulence. The genome, plasmid and virulent genes sequences in fasta format were downloaded from NCBI GenBank and VFDB. The sequences were subjected to SSR analysis using software tool ssr.exe. The resulting data was pasted in excel sheet and further analyzed for percentage of each type of SSR. Higher nucleotide repeats have been observed in our study. Overall high density of SSRs can enhance antigenic variance of the pathogen population in a strategy that counteracts the host immune response. Frequency of A and T repeats is higher in the chromosome, plasmid and the virulence genes. However, in dinucleotide repeats the frequencies of GC/CG repeats are higher in genome, whereas plasmid has more of AT/TA repeats. Genome has trinucleotide repeats having predominantly G and C whereas plasmid has trinucleotide repeats having predominantly A and T. The repeat number obtained and percentage of repeats is higher in virulence genes as compared to other gene families. Due to the presence of this large number of SSRs, the organism has an enormous potential for generating this genomic and phenotypic diversity.     

Inducing toxicity by introducing a leucine-zipper-like motif in frog antimicrobial peptide, magainin 2

Cytotoxicity, a major obstacle in therapeutic application of antimicrobial peptides, is controlled by leucine-zipper-like sequences in melittin and other naturally occurring antimicrobial peptides. Magainin 2 shows significantly lower cytotoxicity than many naturally occurring antimicrobial peptides and lacks this structural element. To investigate the consequences of introducing a leucine zipper sequence in magainin 2, a novel analogue (Mag-mut) was designed by rearranging only the positions of its hydrophobic amino acids to include this structural element. Both magainin 2 and Mag-mut showed appreciable similarities in their secondary structures in the presence of negatively charged lipid vesicles, in localizing and permeabilizing the selected bacteria and exhibiting bactericidal activities. However, Mag-mut bound and localized strongly on to the mammalian cells tested and exhibited significantly higher cytotoxicity than magainin 2. Only Mag-mut, but not magainin 2, permeabilized human red blood cells and zwitterionic lipid vesicles. In contrast with magainin 2, Mag-mut self-assembled in an aqueous environment and bound co-operatively on to zwitterionic lipid vesicles. The peptides formed pores of different sizes on to a selected mammalian cell. The results of the present study indicate an important role of the leucine zipper sequence in the cytotoxicity of Mag-mut and demonstrate that its introduction into a non-toxic peptide, without altering the amino acid composition, can render cytotoxicity.