1H, 15N, 13C resonance assignment of folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster

SUMO, an important post-translational modifier of variety of substrate proteins, regulates different cellular functions. Here, we report the NMR resonance assignment of the folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster (dsmt3).     

Oxidant-antioxidant imbalance in the erythrocytes of sporadic amyotrophic lateral sclerosis patients correlates with the progression of disease

Free radicals are implicated in numerous disease processes including motor neuron degeneration (MND). Antioxidant defense enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the erythrocytes are capable of detoxifying reactive oxygen species produced endogenously or exogenously. In the present study, the extent of lipid peroxidation (LPO) and antioxidant defenses were evaluated in the erythrocytes of 20 sporadic amyotrophic lateral sclerosis (ALS) patients and 20 controls. We observed that lipid peroxidation in the erythrocytes of amyotrophic lateral sclerosis patients significantly increased with respect to controls (P<0.001). On the other hand, catalase activity was found to be significantly lower (P<0.001). The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione levels were also found to be significantly reduced in ALS patients compared to healthy subjects (P<0.001, P<0.01 and P<0.01, respectively). It was further observed that lipid peroxidation started to increase and catalase, glutathione reductase, glucose-6-phosphate dehydrogenase enzyme activities and glutathione levels started to decrease as amyotrophic lateral sclerosis progressed from 6 to 24 months, suggesting a correlation between these parameters and duration of amyotrophic lateral sclerosis. This study confirms the involvement of oxidative stress during the progression of amyotrophic lateral sclerosis and the need to develop specific peripheral biomarkers.     

In vitro withanolide production by Withania somnifera L. cultures

In vitro multiple shoots, root, callus and cell suspension cultures of Withania somnifera exhibited the potentiality to produce pharmacologically active withanolides. Multiple shoots cultures exhibited an increase in withanolide A accumulation compared to shoots of the mother plant. In vitro generated root cultures as well as callus and suspension cultures also produced withanolides albeit at lower levels.     

Potent Upregulation of Glutathione and NAD(P)H:Quinone Oxidoreductase 1 by Alpha-lipoic Acid in Human Neuroblastoma SH-SY5Y Cells: Protection Against Neurotoxicant-elicited Cytotoxicity

Alpha-lipoic acid (LA) has recently been reported to afford protective effects in neurodegenerative disorders. However, the mechanisms underlying LA-mediated neuroprotection remain to be investigated. This study was undertaken to determine whether LA treatment could increase endogenous antioxidants and phase 2 enzymes in cultured human neuroblastoma SH-SY5Y cells, and whether such increased cellular defenses could afford protection against cytotoxicity induced by neurotoxicants. Incubation of SH-SY5Y cells with micromolar concentrations of LA for 24 h resulted in a significant increase in the levels of reduced glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQQ1) in a concentration-dependent fashion. Treatment of the cells with LA also led to an increased mRNA expression of γ-glutamylcysteine ligase catalytic subunit (GCLC) and NQO1. To determine the protective effects of the LA-induced cellular defenses on neurotoxicant-elicitedl cell injury, SH-SY5Y cells were pretreated with LA for 24 h and then exposed to acrolein, 4-hydroxy-2-nonenal (HNE), H₂O₂ and the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1). We observed that LA pretreatment of SH-SY5Y cells led to a marked protection against acrolein, HNE, H₂O₂ and SIN-1-mediated cytotoxicity, as detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. Taken together, this study demonstrates for the first time that LA can induce GSH and NQO1 in cultured human neuroblastoma cells and LA-upregulated cellular defenses are accompanied by a markedly increased resistance to cytotoxicity induced by various neurotoxicants. The results of this study may have important implications for the neuroprotective effects of LA.     

An Improved Method for In Vitro Large Scale Propagation of Piper betle L

A protocol for large-scale propagation of Piper betle cvs Desawari and Desi Bangla was developed through axillary shoot proliferation. Due to systemic infection as well as high phenol content the crop was very difficult to establish in aseptic condition. But a mixture of 5 mg l^?1 each of chloramphenicol and oxytetracyclin and 100 mg l^?1 each of citric acid and ascorbic acid used in MS medium for 2 days helped in establishment. After 48 h, the explants were transferred to the antibiotic free medium having PVP and ascorbic acid (100 mg l^?1 each), citric acid (50 mg l^?1), and glutathione (20 mg l^?1). Regular subculturing of the explants into liquid medium, use of antioxidants and incubation of the cultures in the dark for initial 7–10 days played a crucial role for keeping them fresh and green. Maximum numbers of axillary shoots were obtained with 2 mg l^?1 BA and 0.2 mg l^?1 NAA as growth supplements. The plants were rooted in 0.25 mg l^?1 IBA and hardened in the soil. Phenolic compound analysis showed almost the same results in tissue-raised and in vivo grown plants in Desawari.     

Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

Background  

Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites.     

Isolation and characterization of Escherichia coli tolC mutants defective in secreting enzymatically active alpha-hemolysin.

This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed alpha-helical domain, while the remaining two mapped within the outer membrane-embedded beta-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.