Identification of the bioactive core component of the insecticidal Vip3A toxin peptide of Bacillus thuringiensis

The potential of insecticidal Vip3Aa toxin peptide of Bacillus thuringiensis (Bt) as a resource for development of lepidopteran insect resistant transgenic crop plants has not yet been fully fathomed. The single mode of protection offered by the insecticidal Vip3Aa toxin against a broad spectrum of lepidopteran insect pests that invade crop field as secondary insect pests, carry definitive significance. However, lack of diversity amongst insecticidal Vip3A toxin towards toxicity for lepidopteran insects is often considered as disadvantage. In order to bring in improvement at this front, search for diversity and protein engineering of the toxin molecule for creation of diversity require to be undertaken in future. In that context, identification of the bioactive core component of Vip3BR toxin peptide of Bt an analogue of Vip3Aa toxin has been accomplished. The core component was found to contain enhanced potency of the insecticidal property 2–3 folds more than the native toxin against four major crop pests.     

Variant cry1Ab entomocidal Bacillus thuringiensis toxin gene facilitates the recovery of an increased number of lepidopteran insect resistant independent rice transformants against yellow stem borer (Scirpophaga incertulus) inflicted damage

The primary technical constraint plant scientists face in generating insect resistant transgenic crops with insecticidal Bacillus thuringiensis (Bt) crystal protein (Cry) genes remains failing to generate sufficiently large numbers of effective resistant transgenic plant lines. One possible means to overcome this challenge is through deployment of a Cry toxin gene that contains high levels of insecticidal specific activity for target insect pests. In the present study, we tested this hypothesis using a natural variant of the Cry1Ab toxin under laboratory conditions that possessed increased insecticidal potency against the yellow stem borer (YSB, Scirpophaga incertulus), one of the most damaging rice insect pests. Following adoption of a stringent selection strategy for YSB resistant transgenic rice lines under field conditions, results showed recovery of a significantly higher number of YSB resistant independent transgenic plant lines with the variant cry1Ab gene relative to transgenic plant lines harbouring cry1Ab berliner gene. Structural homology modelling of the variant toxin peptide with the Cry1Aa toxin molecule, circular dichroism spectral analysis, and hydropathy plot analysis indicated that serine substitution by phenylalanine at amino acid position 223 of the Cry1Ab toxin molecule resulted in a changed role for α-helix 7 in domain I of Cry1Ab for enhanced toxicity.     

RNAi mediated silencing of lipoxygenase gene to maintain rice grain quality and viability during storage

Lipoxygenase (LOX) is a common enzyme which catalyzes lipid peroxidation of seeds and consequently enhances seed quality deterioration and decreases seed viability. During seed storage, peroxidation of unsaturated fatty acids occur due to enhancement of LOX activity which directly leads to reduction in seed vigour and deterioration of grain nutritional quality. This study was undertaken to overcome these problem during rice seed storage by attenuating LOX activity using RNAi technology. To improve seed storage stability, we down regulated LOX gene activity by using a functional fragment of the LOX gene under the control of both constitutive (CaMV35S) and aleurone-specific (Oleosin-18) promoter separately. To understand the storage stability, RNAi–LOX seeds and non-transgenic control seeds were subjected to accelerated aging at 45 °C and 85 % relative humidity for 14 days. Our studies demonstrate that down regulation of LOX activity reduces the seed quality deterioration under storage condition. In addition GC–MS analysis revealed that reduction of fatty acid level in non-transgenic seeds during storage was higher when compared with that of transgenic rice seeds. Furthermore, the transgenic rice seeds with reduced LOX activity exhibited enhanced seed germination efficiency after storage than that of non-transgenic rice seeds. This study will have direct impact on nutritional stability of quality rice grains.     

Side chain modified sterols as probes into insect molting hormone metabolism. II: synthesis of monofluorocholesterols

The hydroxylations of the cholesterol side chain at C-20, 22, and 25 are key terminal events in ecdysone biogenesis. We have prepared the C-20, C-22, C-24, and C-25 monofluorinated cholesterols as potential inhibitors of these hydroxylation events, and preliminary bioassay results in Manduca sexta are reported. The synthesis of [26(14)C]-20-fluorocholesterol is also described. Although the 20-, 22-, and 25-monofluorocholesterols do not appear to affect larval growth and development, the 24-fluoro isomer shows a moderate retardation of growth and a modest increase in mortality.     

Requirement for 24(S),25-epoxycholesterol for the viability of cultured fibroblasts

Previous studies on a somatic cell mutant auxotrophic for mevalonate (Mev-1) have shown that these cells rapidly lose viability when deprived of mevalonic acid in culture medium supplemented with serum cholesterol. Testing of all known end products of mevalonate metabolism in cultured mammalian cells has been conducted to determine the basis for this mevalonate requirement. It has been found that the recently discovered mevalonate metabolite 24(S),25-epoxycholesterol produces a partial restoration of viability of Mev-1 cells starved for mevalonate, whereas other structurally similar oxysterols do not. It appears that 24(S),25-epoxycholesterol has a specific, vital cellular function in CHO-K1 cells.     

The solution structure of the S4–S5 linker of the hERG potassium channel

The human ether‐à‐go‐go related gene (hERG) encodes a protein that forms a voltage‐gated potassium channel and plays an important role in the heart by controlling the rapid delayed rectifier K⁺ current (I_Kr). The S4–S5 linker was shown to be important for the gating of the hERG channel. Nuclear magnetic resonance study showed that a peptide derived from the S4–S5 linker had no well‐ordered structure in aqueous solution and adopted a 3₁₀‐helix (E544‐Y545‐G546) structure in detergent micelles. The existence of an amphipathic helix was confirmed, which may be important for interaction with cell membrane. Close contact between side chains of residues R541 and E544 was observed, which may be important for its regulation of channel gating. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

The combined effect of Raspberry Ketone with Resveratrol against oxidative stress and steatohepatitis in rats: Pharmacokinetic and pharmacodynamic studies

Raspberry Ketone (RK) and Resveratrol (RSV) are natural phenolic antioxidants and anti-inflammatory agents. However, its combined pharmacokinetic and pharmacodynamics potentials are not reported. The study aims to assess the combined effect of RK with RSV to protect rats from carbon-tetrachloride (CCl4) induced oxidative stress and NASH. The toxicant CCl4 was employed at a concentration of 1 mL/kg as a 1:1 (v/v) mixture with olive oil twice weekly for 6 weeks to induce liver toxicity. Animal treatment was followed for 2 weeks. Silymarin was used as a standard control drug to compare the hepatoprotective effect of RK and RSV. Hepatic histology, oxidative stress, MMP, reduced glutathione (GSH), plasma levels of SGOT, SGPT, and lipid profile (total cholesterol and triglycerides) were measured. Anti-inflammation genes (IL-10), and fibrotic genes (TGF-β) were also examined in liver tissue. Oral administration of combined RK with RSV (50 + 50 mg/kg for 2 weeks) showed significantly more hepatoprotection by significantly decreasing elevated plasma markers and lipid profile than alone RK and RSV (100 mg/kg daily for 2 weeks). It also significantly alleviated the hepatic lipid peroxidation, restoring the activities of GSH levels in the liver. RT-PCR and Immunoblotting studies confirmed that significantly upregulation of anti-inflammation genes and protein expression (MMP-9) ameliorated the disease. Pharmacokinetic studies confirmed more synergistic stability in simulated gastric-intestinal fluids (FaSSGF, FaSSIF) and rat liver microsomes (CYP-450, NADPH oxidation & glucuronidation. Moreover, coadministration of drugs augmented the relative bioavailability, V_d/F (L/Kg), and MRT_0-∞(h), which leads to more efficacy. This pharmacokinetic and pharmacodynamic reveals a new adjuvant therapy for the treatment of steatohepatitis.     

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E

A critical point in the V₁ sector and entire V₁V_O complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G_1–59) of the V₁V_O ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of G_1–59 in the absence and presence of the N-terminal peptides E_1–18 and E_18–38 as well as the produced and purified C-terminal segment (E_39–233) shows specific interactions only with the peptide fragment E_18–38. The binding of this peptide occurs via the residues M₁, V₂, S₃, and K₅ as well for V₂₂, S₂₃, K₂₄, A₂₅ and R₂₆ of G_1–59. The specific E_18–38/G_1–59 binding has been confirmed by fluorescence correlation spectroscopy data. The E_18–38 peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E_18–38/G_1–59 complex reveals the orientation of E_18–38 relative to G_1–59 via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.