Novel secopyrrolizidine alkaloids from Crotalaria verrucosa

Crotaverrine and O-acetylcrotaverrine, isolated from the seeds of C. verrucosa Linn., have been shown by spectroscopy and chemical evidence to be the macrocyclic diesters of otonecine and diastereoisomeric integerrinecic acid. Hitherto, diastereoisomeric integerrinecic acid esters were not known to occur in nature.     

A globule-like structure in leaf epidermis and its relation to stomatal regulation inCassia auriculata L.

The role of a globule-like structure in the leaf epidermal cells ofC. auriculata has been studied in relation to stomatal regulation. Water stress and direct sunlight promote the appearance of globules at the same time as the stomata close. Ample water supply and shade inhibit the appearance of globules and the stomata open. The number of stomata is the same on either leaf surface, but their behaviour is different because of different exposure to light.     

Gluconeogenesis from L-serine in rat liver.

Livers of fed and fasted rats were perfused $\text{in situ}$ in the presence and absence of 4.8 mM quinolinate, an $\text{in vivo}$ inhibitor of phosphoenolpyruvate carboxykinase. An assay of the hepatic activities of serine dehydratase and serine pyruvate transaminase and a comparison of the $\text{in vivo}$ incorporation of radioactivity from serine 3-¹⁴C and serine U-¹⁴C into blood glucose were also carried out in the above nutritional states. Our results demonstrate that gluconeogenesis from L-serine proceeds through two pathways. One, involving the reversal of the biosynthetic route of serine, bypasses conversion to pyruvate phosphoenolpyruvate and oxaloacetate and is not inhibited by quinolinate. This pathway appears to be the only one active in the fed state but produces a very insignificant amount of glucose. The other involves serine dehydratase mediated conversion of serine to pyruvate, is inhibited by quinolinate and becomes predominant during starvation.     

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg^?1. The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5–11.5) and temperature (40–90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K _m and V _max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min^?1 mg^?1 protein by analyzing Michaelis–Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.     

Construction of an amperometric TG biosensor based on AuPPy nanocomposite and poly (indole-5-carboxylic acid) modified Au electrode

A method is described for construction of an amperometric triglyceride (TG) biosensor based on covalent co-immobilization of lipase, glycerol kinase and glycerol-3-phosphate oxidase onto gold polypyrrole nanocomposite decorated poly indole-5-carboxylic acid electrodeposited on the surface of a gold electrode. The enzyme electrode was characterized by transmission electron microscopy, scanning electron microscopy, electrochemical impedance studies, Fourier transform infrared spectroscopy and cyclic voltammetry. Biosensor showed optimum response within 4 s at pH 6.5 and 35 °C, when polarized at +0.1 V against Ag/AgCl. There was a linear relationship between sensor response and triolein concentration in the range 50–700 mg/dl. Biosensor was employed for determination of TG in serum. Detection limit of the biosensor was 20 mg/dl. Biosensor was evaluated with 91–95 % recovery of added triolein in sera and 4.14 and 5.85 % within and between batch coefficients of variation, respectively. There was a good correlation (r = 0.99) between sera TG values by standard method (Enzymic colorimetric) and the present method. The biosensor was unaffected by a number of serum substances at their physiological concentration. Biosensor lost 50 % of its initial activity after its 100 uses over 7 months, when stored at 4 °C.     

An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid

Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V _max and K _m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 μmol mg^?1 min^?1 and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K _i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml ^? resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.     

Surface Plasmon Resonance Based Fiber Optic Ammonia Sensor Utilizing Bromocresol Purple

In this paper, a surface plasmon resonance (SPR) based fiber optic ammonia gas sensor has been designed and fabricated using bromocresol purple (BCP) as sensing element. The sensor works under wavelength modulation scheme. The detection of ammonia gas has been carried out at room temperature. Three different kinds of film coating configurations, namely silver + BCP, gold + BCP, and silver + silicon + BCP on the unclad portion of the fiber have been used for studying the role of each layer. Further, to optimize the performance of the sensor, the films of varying thicknesses were coated using thermal evaporation technique. Experiments have been performed for the ammonia concentrations ranging from 0 to 150 ppm around the probe. To record the SPR spectrum, light from a polychromatic source is launched in the fiber and the spectrum is recorded at the other end of the fiber. The spectrum has a peak at lower wavelength while a dip at the higher wavelength. The dip corresponds to SPR while the peak appears to be due to fluorescence properties of the dye. It has been observed that as the ammonia gas comes in contact of the BCP layer, it changes the refractive index of the BCP dye which, in turn, changes the resonance wavelength. Further, the change in refractive index increases as the concentration of ammonia gas increases up to certain concentration of ammonia after that it saturates. Silicon layer has been shown as a protection layer for silver and gold from oxidation and acts as a tuner of wavelength. The proposed ammonia sensor has small response as well as recovery time.     

Quantifying Missing Heritability at Known GWAS Loci

Recent work has shown that much of the missing heritability of complex traits can be resolved by estimates of heritability explained by all genotyped SNPs. However, it is currently unknown how much heritability is missing due to poor tagging or additional causal variants at known GWAS loci. Here, we use variance components to quantify the heritability explained by all SNPs at known GWAS loci in nine diseases from WTCCC1 and WTCCC2. After accounting for expectation, we observed all SNPs at known GWAS loci to explain more heritability than GWAS-associated SNPs on average (). For some diseases, this increase was individually significant: for Multiple Sclerosis (MS) () and for Crohn''s Disease (CD) (); all analyses of autoimmune diseases excluded the well-studied MHC region. Additionally, we found that GWAS loci from other related traits also explained significant heritability. The union of all autoimmune disease loci explained more MS heritability than known MS SNPs () and more CD heritability than known CD SNPs (), with an analogous increase for all autoimmune diseases analyzed. We also observed significant increases in an analysis of Rheumatoid Arthritis (RA) samples typed on ImmunoChip, with more heritability from all SNPs at GWAS loci () and more heritability from all autoimmune disease loci () compared to known RA SNPs (including those identified in this cohort). Our methods adjust for LD between SNPs, which can bias standard estimates of heritability from SNPs even if all causal variants are typed. By comparing adjusted estimates, we hypothesize that the genome-wide distribution of causal variants is enriched for low-frequency alleles, but that causal variants at known GWAS loci are skewed towards common alleles. These findings have important ramifications for fine-mapping study design and our understanding of complex disease architecture.     

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.