Phytic acid dynamics during seed development and it’s composition in yellow and black Indian soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) genotypes through a modified extraction and HPLC method

Identifying or designing low phytic acid soybean demands an improved understanding of its dynamics and a need for a reliable and accurate phytate quantification method and hence an improved quantitative technique for accurate and rapid quantification of phytic acid (PA) using high-performance liquid chromatography is proposed in this paper. A rapid PA extraction method utilizing sonication of sample in 0.78 M HCl for 3 min followed by mechanical agitation and separation using strong anion exchange column in a vacuum manifold was optimized. The elution of PA was performed using a RP-C₁₈ column with an isocratic mobile phase [Acetonitrile, 35 mM formic acid and tetrabutylammonium hydroxide (4.8: 5.1: 0.1, v/v/v)]. The modified method was rapid, accurate, precise, and reproducible with relative standard deviation of 1.80 and 3.01 % (n = 10, for 1 mg ml^?1) for within and between days respectively with linearity (R² = 0.999, P < 0.05), low limit of detection (LOD = 7.8 μg ml^?1) and limit of quantification (LOQ = 31.25 μg ml^?1). PA dynamics was found increased in a linear trend from initial to later developing stages until maturity in both yellow (DS-9814) and black (DS-MM-64) genotypes. The PA content ranged from 2.38–4.72 g 100g^?1 in the 20 soybean genotypes screened and the variability in PA content was more in black genotypes (P < 0.0001). The black soybean genotypes JS 76-205 (2.38 g 100g^?1), Kalitur (2.50 g 100g^?1) and UPSL 652 (2.54 g 100g^?1), inherently rich in anthocyanin, contains the lowest PA content and hence can serve as potential genotypes with nutraceutical benefits.

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Graphical Abstract Pictorial representation of phytic acid extraction and quantification from soybean flour by the modified high performance liquid chromatography (HPLC) method.

     

Mechanisms of copper ion mediated Huntington's disease progression

Huntington's disease (HD) is caused by a dominant polyglutamine expansion within the N-terminus of huntingtin protein and results in oxidative stress, energetic insufficiency and striatal degeneration. Copper and iron are increased in the striata of HD patients, but the role of these metals in HD pathogenesis is unknown. We found, using inductively-coupled-plasma mass spectroscopy, that elevations of copper and iron found in human HD brain are reiterated in the brains of affected HD transgenic mice. Increased brain copper correlated with decreased levels of the copper export protein, amyloid precursor protein. We hypothesized that increased amounts of copper bound to low affinity sites could contribute to pro-oxidant activities and neurodegeneration. We focused on two proteins: huntingtin, because of its centrality to HD, and lactate dehydrogenase (LDH), because of its documented sensitivity to copper, necessity for normoxic brain energy metabolism and evidence for altered lactate metabolism in HD brain. The first 171 amino acids of wild-type huntingtin, and its glutamine expanded mutant form, interacted with copper, but not iron. N171 reduced Cu(2+)in vitro in a 1:1 copper:protein stoichiometry indicating that this fragment is very redox active. Further, copper promoted and metal chelation inhibited aggregation of cell-free huntingtin. We found decreased LDH activity, but not protein, and increased lactate levels in HD transgenic mouse brain. The LDH inhibitor oxamate resulted in neurodegeneration when delivered intra-striatially to healthy mice, indicating that LDH inhibition is relevant to neurodegeneration in HD. Our findings support a role of pro-oxidant copper-protein interactions in HD progression and offer a novel target for pharmacotherapeutics.     

Studies on esterase histochemistry of amphibian kidney

Summary In the hope that the histochemical picture of the kidney may help to understand its role in excretion and osmoregulation, an effort is here made to study the distribution of esterases in amphibian kidney.The kidneys of adults and tadpoles of the frog, Rana tigrina and the toad, Bufo melanostictus were used for this study. Some of these animals were subjected to dehydration for 3–4 days and to the effect of 150 mM NaCl for 8–12 days before their kidneys were used. The esterases were visualised using tweens, naphthol esters and 5-bromoindoxyl acetate as substrates. These were accompanied by activator/inhibitor studies.Very interesting results were obtained in the distribution of the esterases. Tween esterase and -naphthyl acetate esterase were found in the proximal tubules of the adult frog kidney only while 5-bromoindoxyl acetate esterase was found to be present in all the animals tested. On the other hand, naphthol AS acetate esterase was absent in the tadpole stages of the frog and toad. Further 5-bromoindoxyl acetate esterase and naphthol AS acetate esterase were demonstrated in the glomeruli of frogs and toads subjected to NaCl solution. Activator/ inhibitor studies helped in characteristically differentiating these different esterases.There seems to be a relationship between the distribution of the different esterases and the excretory and osmoregulatory adaptations of these animals which differ in the adult and tadpole stages and in the experimental conditions mentioned. The possible implications of the esterase distribution is discussed in considerable details.U.G.C. Research Scholar.     

Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.     

Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris)

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.     

Frequency of EGFR Mutations in 907 Lung Adenocarcioma Patients of Indian Ethnicity

Background

During the past decade, the incidence of EGFR mutation has been shown to vary across different ethnicities. It occurs at the rate of 10–15% in North Americans and Europeans, 19% in African-Americans, 20–30% in various East Asian series including Chinese, Koreans, and Japanese. Frequency of EGFR mutations in India however remains sparsely explored.

Methodology/Principal Findings

We report 23% incidence of Epidermal growth factor receptor (EGFR) mutations in 907 Non small cell lung cancer (NSCLC) patients of Indian ethnicity, in contrast to 10–15% known in Caucasians and 27–62% among East Asians. In this study, EGFR mutations were found to be more common in never-smokers 29.4% as compared to smokers 15.3%. Consistent with other populations, mutation rates among adenocarcinoma-males were predominantly lower than females with 32% incidence. However unlike Caucasians, EGFR mutation rate among adenocarcinoma-never-smoker females were comparable to males suggesting lack of gender bias among never smokers likely to benefit from EGFR targeted therapy.

Conclusions/Significance

This study has an overall implication for establishing relevance for routine EGFR mutation diagnostics for NSCLC patients in clinics and emphasizes effectiveness for adoption of EGFR inhibitors as the first line treatment among Indian population. The intermediate frequency of EGFR mutation among Indian population compared to Caucasians and East Asians is reminiscent of an ancestral admixture of genetic influence from Middle Easterners, Central Asians, and Europeans on modern- Indian population that may confer differential susceptibility to somatic mutations in EGFR.     

Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy.

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.     

Characterisation of lactic dehydrogenase fromLactobacillus casei

Lactic dehydrogenase fromLactobacillus casei ATCC 7469 has been purified to homogeneity by a two-step affinity chromatography procedure which gave an yield of 35%. The enzyme specifically catalysed the conversion of pyruvate to lactate. The enzyme was maximally active at pH 4.6, which was shifted to 5.4 in the presence of fructose 1,6-biphosphate. The enzyme had a molecular weight of 70,800 with two identical subunits, unlike the lactic dehydrogenase from other sources. Histidine and primary amino groups appeared to be involved in catalysis.