Uniconazole-induced thermotolerance in soybean seedling root tissue

Soybean [Glycine max(L.) Merr. cv. A2] seeds were germinated in 0 or 1 mg 1¹ (3.4 uM) uniconazole, after which seedling roots were excised and exposed to 22 or 48°C for 90 min. Prior to the temperature treatments there were few ultrastructural differences between uniconazole-treated seedling roots and the controls. Following exposure to 48°C, electron micrographs revealed near complete loss of normal ultrastructure in control epidermal root cells, whereas cellular integrity was maintained in treated roots, indicating that uniconazole conferred tolerance to high temperature. Total electrolyte, sugar and K⁺ leakage were all greater from control roots than treated roots during exposure to 48°C. Proline content in the roots was unaffected by uniconazole at 22°C but was 25–30% greater in treated tissue than in controls following exposure to 48°C. Malondialdehyde content was unaffected by uniconazole at 22°C but was nearly 20% less in treated tissue than in controls following high temperature exposure. This indicates that uniconazole decreased high-temperature-induced lipid peroxidation. Uniconazole elevated several antiox-idant systems in the roots, including water-soluble sulfhydryl concentration and catalase, peroxidase and superoxide dismutase activities. These findings are consistent with the hypothesis that uniconazole-induced stress tolerance is due, at least in part, to enhanced antioxidant activity which reduces stress-related oxidative damage to cell membranes.     

Crystal structure of nonspecific lipid transfer protein from Solanum melongena

Lipids are considered to protect protein allergens from proteolysis and are generally seen to exist in a bound form. One of the well‐known plant protein families with bound lipids is non‐specific lipid transfer proteins (nsLTPs). Structure‐function relationships in the case of the members of non‐specific lipid transfer protein family are not clearly understood. As part of exploring the seed proteome, we have analyzed the proteome of a member of Solanaceae family, Solanum melongena (eggplant) and a non‐specific lipid transfer protein from S. melongena, SM80.2 was purified, crystallized and the structure was determined at 1.87 Å resolution. Overall, the tertiary structure is a cluster of α‐helices forming an internal hydrophobic cavity. Absence of conserved Tyr79, known to govern the plasticity of hydrophobic cavity, and formation of hydrogen bond between Asn79 and Asn36 further reduced the pocket size. Structural analysis of SM80.2 thus gives insight about a new hydrogen bond mediated mechanism followed in closure of the binding pocket. Extra electron densities observed at two different places on the protein surface and not in the cavity could provide interesting physiological relevance. In light of allergenic properties, probably overlapping of epitopic region and ligand binding on surface could be a main reason. This work shows first crystal structure of A‐like nsLTP with a close binding pocket and extra density on the surface suggesting a plausible intermediate state during transfer.     

Visible Raman excitation laser induced power and exposure dependent effects in red blood cells

We present results of Raman spectroscopic studies carried out on optically trapped red blood cells with Raman excitation wavelength in Q‐band region of the hemoglobin (Hb) absorption spectrum. The results obtained suggest that when exposed to the Raman excitation laser the RBCs get deoxygenated due to photo‐dissociation of oxygen from hemoglobin. For smaller exposure durations (5 s) the level of deoxygenation increases with an increase in power. However, for longer exposure durations the deoxygenated hemoglobin in the cells gets irreversibly oxidized to form a low spin ferric derivative of hemoglobin. The rate of oxidation depends upon the initial level of deoxygenation; higher the initial level of deoxygenation, higher is the rate of oxidation. However, the RBCs deoxygenated via oxygen deprivation (i.e. N₂ purging) were found to be very stable against any laser induced effect. These observations suggests that in case of laser induced deoxygenation of RBCs the free oxygen generated by photo‐dissociation acts as the oxidizing agent and leads to oxidative damage of the RBCs.

     

Effect of chirality of small molecule organofluorine inhibitors of amyloid self-assembly on inhibitor potency

The effect of enantiomeric trifluoromethyl-indolyl-acetic acid ethyl esters on the fibrillogenesis of Alzheimer’s amyloid β (Aβ) peptide is described. These compounds have been previously identified as effective inhibitors of the Aβ self-assembly in their racemic form. Thioflavin-T Fluorescence Spectroscopy and Atomic Force Microscopy were applied to assess the potency of the chiral target compounds. Both enantiomers showed significant inhibition in the in vitro assays. The potency of the enantiomeric inhibitors appeared to be very similar to each other suggesting the lack of the stereospecific binding interactions between these small molecule inhibitors and the Aβ peptide.     

Identification of a New Exosite Involved in Catalytic Turnover by the Streptokinase-Plasmin Activator Complex during Human Plasminogen Activation

With the goal of identifying hitherto unknown surface exosites of streptokinase involved in substrate human plasminogen recognition and catalytic turnover, synthetic peptides encompassing the 170 loop (CQFTPLNPDDDFRPGLKDTKLLC) in the β-domain were tested for selective inhibition of substrate human plasminogen activation by the streptokinase-plasmin activator complex. Although a disulfide-constrained peptide exhibited strong inhibition, a linear peptide with the same sequence, or a disulfide-constrained variant with a single lysine to alanine mutation showed significantly reduced capabilities of inhibition. Alanine-scanning mutagenesis of the 170 loop of the β-domain of streptokinase was then performed to elucidate its importance in streptokinase-mediated plasminogen activation. Some of the 170 loop mutants showed a remarkable decline in k_cat without any alteration in apparent substrate affinity (K_m) as compared with wild-type streptokinase and identified the importance of Lys¹⁸⁰ as well as Pro¹⁷⁷ in the functioning of this loop. Remarkably, these mutants were able to generate amidolytic activity and non-proteolytic activation in “partner” plasminogen as wild-type streptokinase. Moreover, cofactor activities of the 170 loop mutants, pre-complexed with plasmin, against microplasminogen as the substrate showed a similar pattern of decline in k_cat as that observed in the case of full-length plasminogen, with no concomitant change in K_m. These results strongly suggest that the 170 loop of the β-domain of streptokinase is important for catalysis by the streptokinase-plasmin(ogen) activator complex, particularly in catalytic processing/turnover of substrate, although it does not seem to contribute significantly toward enzyme-substrate affinity per se.     

Kinetic studies of laccase enzyme of Coriolus versicolor MTCC 138 in an inexpensive culture medium

An attempt was made to use cyanobacterial biomass of water bloom, groundnut shell (GNS) and dye effluent as culture medium for laccase enzyme production by Coriolus versicolor. Laccase production was found to be 10.15 ± 2.21 U/ml in the medium containing groundnut shell and cyanobacterial bloom in a ratio of 9:1 (dry weight basis) in submerged fermentation at initial pH 5.0 and 28 ± 2 °C temperature. Half life of enzyme was found to be 74 min at 60 °C. Kinetic analysis of laccase when made with substrate ABTS, K_m and V_max were found to be 0.29 mM and 9.49 μmol/min respectively. Azide and hydroxylamine were found to exert significant inhibition on thermostable laccase. Inhibitor constant (k_i) for azide and hydroxylamine were 1.33 and 0.18 mM respectively. This study forms the first report on the potential application of waste water cyanobacterial bloom and dyeing effluent as a medium for laccase production by C. versicolor MTCC138.     

Risk Factors for MDR and XDR-TB in a Tertiary Referral Hospital in India

Background

India has a high burden of drug resistant TB, although there are few data on XDR-TB. Although XDR-TB has existed previously in India, the definition has not been widely applied, and surveillance using second line drug susceptibility testing has not been performed. Our objective was to analyze clinical and demographic risk factors associated with isolation of MDR and XDR TB as compared to susceptible controls, at a tertiary center.

Methodology/Findings

Retrospective chart review based on positive cultures isolated in a high volume mycobacteriology laboratory between 2002 and 2007. 47 XDR, 30 MDR and 117 susceptible controls were examined. Drug resistant cases were less likely to be extrapulmonary, and had received more previous treatment regimens. Significant risk factors for XDR-TB included residence outside the local state (OR 7.43, 3.07-18.0) and care costs subsidized (OR 0.23, 0.097-0.54) in bivariate analysis and previous use of a fluoroquinolone and injectable agent (other than streptomycin) (OR 7.00, 95% C.I. 1.14-43.03) and an initial treatment regimen which did not follow national guidelines (OR 5.68, 1.24-25.96) in multivariate analysis. Cavitation and HIV did not influence drug resistance.

Conclusions/Significance

There is significant selection bias in the sample available. Selection pressure from previous treatment and an inadequate initial regimen increases risk of drug resistance. Local patients and those requiring financial subsidies may be at lower risk of XDR-TB.