Lily pollen alkaline phytase is a histidine phosphatase similar to mammalian multiple inositol polyphosphate phosphatase (MINPP)

Phytic acid is the most abundant inositol phosphate in cells; it constitutes 1-5% of the dry weight of cereal grains and legumes. Phytases are the primary enzymes responsible for the hydrolysis of phytic acid and thus play important roles in inositol phosphate metabolism. A novel alkaline phytase in lily pollen (LlALP) was recently purified in our laboratory. In this paper, we describe the cloning and characterization of LlALP cDNA from lily pollen. Two isoforms of alkaline phytase cDNAs, LlAlp1 and LlAlp2, which are 1467 and 1533 bp long and encode proteins of 487 and 511 amino acids, respectively, were identified. The deduced amino acid sequences contains the signature heptapeptide of histidine phosphatases, -RHGXRXP-, but shares < 25% identity to fungal histidine acid phytases. Phylogenetic analysis reveals that LlALP is most closely related to multiple inositol polyphosphate phosphatase (MINPP) from humans (25%) and rats (23%). mRNA corresponding to LlAlp1 and LlAlp2 were expressed in leaves, stem, petals and pollen grains. The expression profiles of LlAlp isoforms in anthers indicated that mRNA corresponding to both isoforms were present at all stages of flower development. The expression of LlAlp2 cDNA in Escherichia coli revealed the accumulation of the active enzyme in inclusion bodies and confirmed that the cDNA encodes an alkaline phytase. In summary, plant alkaline phytase is a member of the histidine phosphatase family that includes MINPP and exhibits properties distinct from bacterial and fungal phytases.     

Conformer and pharmacophore based identification of peptidomimetic inhibitors of chikungunya virus nsP2 protease

Chikungunya virus nsP2 replication protein is a cysteine protease, which cleaves the nonstructural nsP1234 polyprotein into functional replication components. The cleavage and processing of nsP1234 by nsP2 protease is essential for the replication and proliferation of the virus. Thus, ChikV nsP2 protease is a promising target for antiviral drug discovery. In this study, the crystal structure of the C-terminal domain of ChikV nsP2 protease (PDB ID: 4ZTB) was used for structure based identification and rational designing of peptidomimetic inhibitors against nsP2 protease. The interactions of the junction residues of nsP3/4 polyprotein in the active site of nsP2 protease have been mimicked to identify and design potential inhibitory molecules. Molecular docking of the nsP3/4 junction peptide in the active site of ChikV nsP2 protease provided the structural insight of the probable binding mode of nsP3/4 peptide and pigeonholed the molecular interactions critical for the substrate binding. Further, the shape and pharmacophoric properties of the viral nsP3/4 substrate peptide were taken into consideration and the mimetic molecules were identified and designed. The designed mimetic compounds were then analyzed by docking and their binding affinity was assessed by molecular dynamics simulations.     

Exposure to a Northern Contaminant Mixture (NCM) Alters Hepatic Energy and Lipid Metabolism Exacerbating Hepatic Steatosis in Obese JCR Rats

Non-alcoholic fatty liver disease (NAFLD), defined by the American Liver Society as the buildup of extra fat in liver cells that is not caused by alcohol, is the most common liver disease in North America. Obesity and type 2 diabetes are viewed as the major causes of NAFLD. Environmental contaminants have also been implicated in the development of NAFLD. Northern populations are exposed to a myriad of persistent organic pollutants including polychlorinated biphenyls, organochlorine pesticides, flame retardants, and toxic metals, while also affected by higher rates of obesity and alcohol abuse compared to the rest of Canada. In this study, we examined the impact of a mixture of 22 contaminants detected in Inuit blood on the development and progression of NAFLD in obese JCR rats with or without co-exposure to10% ethanol. Hepatosteatosis was found in obese rat liver, which was worsened by exposure to 10% ethanol. NCM treatment increased the number of macrovesicular lipid droplets, total lipid contents, portion of mono- and polyunsaturated fatty acids in the liver. This was complemented by an increase in hepatic total cholesterol and cholesterol ester levels which was associated with changes in the expression of genes and proteins involved in lipid metabolism and transport. In addition, NCM treatment increased cytochrome P450 2E1 protein expression and decreased ubiquinone pool, and mitochondrial ATP synthase subunit ATP5A and Complex IV activity. Despite the changes in mitochondrial physiology, hepatic ATP levels were maintained high in NCM-treated versus control rats. This was due to a decrease in ATP utilization and an increase in creatine kinase activity. Collectively, our results suggest that NCM treatment decreases hepatic cholesterol export, possibly also increases cholesterol uptake from circulation, and promotes lipid accumulation and alters ATP homeostasis which exacerbates the existing hepatic steatosis in genetically obese JCR rats with or without co-exposure to ethanol.     

A network map of thrombopoietin signaling

Thrombopoietin (THPO), also known as megakaryocyte growth and development factor (MGDF), is a cytokine involved in the production of platelets. THPO is a glycoprotein produced by liver and kidney. It regulates the production of platelets by stimulating the differentiation and maturation of megakaryocyte progenitors. It acts as a ligand for MPL receptor, a member of the hematopoietic cytokine receptor superfamily and is essential for megakaryocyte maturation. THPO binding induces homodimerization of the receptor which results in activation of JAKSTAT and MAPK signaling cascades that subsequently control cellular proliferation, differentiation and other signaling events. Despite the importance of THPO signaling in various diseases and biological processes, a detailed signaling network of THPO is not available in any publicly available database. Therefore, in this study, we present a resource of signaling events induced by THPO that was manually curated from published literature on THPO. Our manual curation of thrombopoietin pathway resulted in identification of 48 molecular associations, 66 catalytic reactions, 100 gene regulation events, 19 protein translocation events and 43 activation/inhibition reactions that occur upon activation of thrombopoietin receptor by THPO. THPO signaling pathway is made available on NetPath, a freely available human signaling pathway resource developed previously by our group. We believe this resource will provide a platform for scientific community to accelerate further research in this area on potential therapeutic interventions.     

Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility

The Na,K-ATPase, consisting of alpha- and beta-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase beta1-subunit (Na,K-beta) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility. However, until now, the mechanism by which Na,K-beta reduces cell motility remained elusive. Here, we demonstrate that Na,K-beta localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase alpha1-subunit (Na,K-alpha) and Na,K-beta with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-alpha associates with the regulatory subunit of PI3-kinase and Na,K-beta binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.     

Molecular insight into the genesis of ranked caste populations of western India based upon polymorphisms across non-recombinant and recombinant regions in genome

Background

Large-scale trade and cultural contacts between coastal populations of western India and Western-Eurasians paved for extensive immigration and genesis of wide spectrum of admixed gene pool. To trace admixture and genesis of caste populations of western India, we have examined polymorphisms across non-recombining 20 Y-SNPs, 20 Y-STRs, 18 mtDNA diagnostic sites, HVS-1 plus HVS-2 regions; and recombining 15 highly polymorphic autosomal STRs in four predominant caste populations- upper-ranking Desasth-brahmin and Chitpavan-brahmin; a middle-ranking Kshtriya Maratha; and a lower-rank peasant Dhangar.

Results

The generated genomic data was compared with putative parental populations- Central Asians, West Asians and Europeans using AMOVA, PC plot, and admixture estimates. Overall, disparate uniparental ancestries, and l.1% GST value for biparental markers among four studied caste populations linked well with their exchequer demographic histories. Marathi-speaking ancient Desasth-brahmin shows substantial admixture from Central Asian males but Paleolithic maternal component support their Scytho-Dravidian origin. Chitpavanbrahmin demonstrates younger maternal component and substantial paternal gene flow from West Asia, thus giving credence to their recent Irano-Scythian ancestry from Mediterranean or Turkey, which correlated well with European-looking features of this caste. This also explains their untraceable ethno-history before 1000 years, brahminization event and later amalgamation by Maratha. The widespread Palaeolithic mtDNA haplogroups in Maratha and Dhangar highlight their shared Proto-Asian ancestries. Maratha males harboured Anatolianderived J2 lineage corroborating the blending of farming communities. Dhangar heterogeneity is ascribable to predominantly South-Asian males and West-Eurasian females.

Conclusions

The genomic data-sets of this study provide ample genomic evidences of diverse origins of four ranked castes and synchronization of caste stratification with asymmetrical gene flows from Indo-European migration during Upper Paleolithic, Neolithic, and later dates. However, subsequent gene flows among these castes living in geographical proximity, have diminished significant genetic differentiation as indicated by AMOVA and structure.     

Dynamic site heterogeneity in amorphous maltose and maltitol from spectral heterogeneity in erythrosin B phosphorescence

We have used phosphorescence from erythrosin B (tetraiodofluorescein) dispersed in thin films of either maltose or maltitol to investigate the physical properties of these amorphous pure sugar matrixes. Intensity decays collected as a function of emission wavelength over the range from 640 to 720 nm were analyzed using a stretched exponential kinetic model in which the lifetime (tau) and the stretching exponent (beta) were the physically relevant parameters. The lifetimes varied systematically with emission wavelength in both matrixes. Analysis of the temperature dependence of the lifetime at each wavelength provided an estimate of the activation energy for nonradiative quenching of the triplet state; the activation energy also varied with emission wavelength. In addition, time-resolved emission spectra exhibited a blue shift with time following excitation. These data support a photophysical model in which probes are distributed among sites that vary in terms of overall molecular mobility and in which sites with lower rates of dipolar relaxation also have lower rates of collisional quenching of the erythrosin triplet state. The amorphous matrix of both maltose and maltitol in both the glass and the melt state is thus characterized by dynamic site heterogeneity in which different sites vary in terms of their overall molecular mobility.     

Alkaline phytase from lily pollen: Investigation of biochemical properties

Phytases catalyze the hydrolysis of phytic acid (InsP6, myo-inositol hexakisphosphate), the most abundant inositol phosphate in cells. In cereal grains and legumes, it constitutes 3-5% of the dry weight of seeds. The inability of humans and monogastric animals such as swine and poultry to absorb complexed InsP6 has led to nutritional and environmental problems. The efficacy of supplemental phytases to address these issues is well established; thus, there is a need for phytases with a range of biochemical and biophysical properties for numerous applications. An alkaline phytase that shows unique catalytic properties was isolated from plant tissues. In this paper, we report on the biochemical properties of an alkaline phytase from pollen grains of Lilium longiflorum. The enzyme exhibits narrow substrate specificity, it hydrolyzed InsP6 and para-nitrophenyl phosphate (pNPP). Alkaline phytase followed Michaelis-Menten kinetics with a K(m) of 81 microM and V(max) of 217 nmol Pi/min/mg with InsP6 and a K(m) of 372 microM and V(max) of 1272 nmol Pi/min/mg with pNPP. The pH optimum was 8.0 with InsP6 as the substrate and 7.0 with pNPP. Alkaline phytase was activated by calcium and inactivated by ethylenediaminetetraacetic acid; however, the enzyme retained a low level of activity even in Ca2+-free medium. Fluoride as well as myo-inositol hexasulfate did not have any inhibitory affect, whereas vanadate inhibited the enzyme. The enzyme was activated by sodium chloride and potassium chloride and inactivated by magnesium chloride; the activation by salts followed the Hofmeister series. The temperature optimum for hydrolysis is 55 degrees C; the enzyme was stable at 55 degrees C for about 30 min. The enzyme has unique properties that suggest the potential to be useful as a feed supplement.     

Bruchid (Coleoptera: Bruchidae) ovicidal phenylbutanoid from Zingiber purpureum

The larvicidal activity of the dichloromethane extract of Zingiber purpureum Roscoe (Zingiberaceae) rhizome against the second instar of Aedes aegypti (L.) (Diptera: Culicidae) is shown to be due to 4-(3',4'-dimethoxyphenyl)buta-1,3-diene. The diene also showed ovicidal activity against the bruchid Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). Most of the eggs laid by bruchids on treated cowpea seeds were transparent, and very few of them contained developing embryos. The few larvae produced from these embryos were unable to penetrate the seed coat and enter the seed. Similar effects were seen when adults were exposed to the compound and then placed on untreated cowpea seeds, suggesting that a new type of maternally mediated ovicidal effect was involved. Coated and impregnated granular formulations of the extract were evaluated for use in the control of bruchid infestation of stored cowpea seeds. Coated granules showed activity similar to that of the crude extract but were found to lose activity rapidly. Impregnated granules were found to be less active than the crude extract.