Developmental changes in rat liver alcohol dehydrogenase

Hepatic alcohol dehydrogenase activity and mass content change coordinately during development in male rats. Enzyme activity and mass content increase continuously after birth to 100 and 80% of maximal values within 6 weeks (2.6 ± 0.4 μmole/min/g liver and 92 ± 20 μg/g liver), respectively. When expressed per milligram of soluble proteins, both parameters peak at 3 weeks (0.052 ± 0.002 μmole/min/mg protein and 2.0 ± 0.4 μg/mg protein) and then decrease gradually to plateau levels. These decreases probably arise from a “surge” in soluble liver protein levels that occurs after weaning. Similar developmental patterns also occur in female rats. These findings are the first quantitative measurements of this enzyme in developing animals.     

Functional assignment to JEV proteins using SVM

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).     

Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars

Banana streak virus strain OL (BSV-OL) commonly infects new Musa hybrids, and this infection is thought to arise de novo from integrated virus sequences present in the nuclear genome of the plant. Integrated DNA (Musa6+8 sequence) containing the whole genome of the virus has previously been cloned from cv. Obino l’Ewai (Musa AAB group), a parent of many of the hybrids. Using a Southern blot hybridization assay, we have examined the distribution and structure of integrated BSV-OL sequences in a range of Musa cultivars. For cv. Obino l’Ewai, almost every restriction fragment hybridizing to BSV-OL was predicted from the Musa6+8 sequence, suggesting that this is the predominant type of BSV-OL integrant in the genome. Furthermore, since only two junction fragments of Musa/BSV sequence were detected, and the Musa6+8 sequence is believed to be integrated as multiple copies in a tandem array, then the internal Musa spacer sequences must be highly conserved. Similarly sized restriction fragments were detected in four BB group cultivars, but not in six AA or AAA group cultivars, suggesting that the BSV-OL sequences are linked to the B-genome of Musa. We also provide evidence that cv. Williams (Musa AAA group) contains a distinct badnavirus integrant that is closely related to the ‘dead’ virus integrant previously characterized from Calcutta 4 (Musa acuminata ssp. burmannicoides). Our results suggest that the virus integrant from cv. Williams is linked to the A-genome, and the complexity of the hybridization patterns suggest multiple sites of integration and/or variation in sequence and structure of the integrants.     

Ligand requirements for the relaxation of adenylate cyclase from activated and inhibited states

The turkey erythrocyte adenylate cyclase system binds tightly the inhibitory nucleotide GDP, and a pretreatment step with isoproterenol and GMP is required to restore activation. Under identical pretreatment conditions, the release of labeled nucleotide is complete within 1 min whereas the restoration of activation by Gpp(NH)p requires 15 min. A study of the ligand requirements of the slow step shows the following: (a) The role of GMP is that of an obligatory allosteric regulator. (b) Cholera toxin modification of the system abolishes the requirement for GMP with a considerable enhancement in the reaction rate. (c) GMP is without effect on the relaxation process with the activator Gpp(NH)p as the resident nucleotide. In sharp contrast, ethylenediamine-tetraacetic acid (without effect in a GDP-occupied complex) markedly potentiates alterations from the Gpp(NH)p-occupied state. (d) Formation of a GDP/guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) hybrid leads to the suppression of both F- and Gpp(NH)p activation. F- activation is restored by isoproterenol alone, while GMP is still required to restore Gpp(NH)p activation. The results suggest that covalent modification or nucleotide analogue occupancy of the regulatory complex can modify the allosteric role for GMP, with consequences for the rate of the slow step.     

Effects of GTP on binding of (3H) glucagon to receptors in rat hepatic plasma membranes.

In this study, we report the preparation of [3H]glucagon and its characteristics of binding to receptors in the rat liver plasma membrane. Binding of the labeled hormone is optimal at pH 7.0. In the absence of GTP, [3H]glucagon binding to receptors is slow and the time of equilibration is inversely proportional to the hormone concentration. In the presence of GTP, equilibrium is reached within 30 s regardless of hormone levels, and the kinetics of binding are in accord with the kinetics of activation of adenylate cyclase by native glucagon in the presence of the nucleotide. Equilibrium binding measurements indicate that, in the absence of GTP, the binding isotherm is sigmoidal with an apparent Kd of 2 nM. The addition of GTP results in a complex binding isotherm with about 90% of the binding sites having a considerably lower apparent dissociation constant (greater than 10 nM) and a small population of sites having high affinity for the hormone. The binding properties of [3H]glucagon are compared with those of 125I-glucagon, and the implications of the actions of GTP on glucagon binding are discussed in relation to the overall regulation of adenylate cyclase by hormone and the nucleotide.     

Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide.

A 37,000 X g supernatant fraction prepared from fat lung homogenate demonstrated a 2- to 3-fold increase in guanylate cyclase activity after incubation at 30 degrees for 30 min (preincubation). Treatment of the supernatant fraction with Triton X-100 increased activity to approximately the same extent as preincubation, but would not increase the activity after preincubation. By chromatography on Sepharose 2B, before and after preincubation, it was demonstrated that the increase in activity was only associated with the soluble guanylate cyclase, and not the particulate enzyme. Activation by preincubation required O2. It was completely inhibited by thiols such as 2-mercaptoethanol, and by bovine serum albumin, KCN, and sodium diethyldithiocarbamate. These inhibitors suggested a copper requirement for activation, and this was confirmed by demonstrating that 20 to 60 muM CuCl2 could relieve the inhibition by 0.1 mM sodium diethyldithiocarbamate. 2-Mercaptoethanol inhibition could also be reversed by removal of the thiol on a Sephadex G-25 column, however, this treatment partially activated the enzyme. Addition of 2-mercaptoethanol to a preincubated preparation would not reverse the activation. H2O2 was found to activate guanylate cyclase, either by its generation in the lung supernatant with glucose oxidase and glucose, or by its addition to a preparation in which the catalase was inhibited with KCN. KCN or bovine serum albumin was able to partially inhibit activation by glucose oxidase plus glucose, however, larger amounts of glucose oxidase could overcome that inhibition, indicating a catalytic role for Cu2+ at low H2O2 concentrations. No direct evidence for H2O2 formation during preincubation could be found, however, indirect evidence was obtained by the spectrophotometric detection of choleglobin formation from hemoglobin present in the lung supernatant fluid. The H2O2 is believed to result from the reaction of oxyhemoglobin with ascorbate.     

Molecular Markers Reveal Only Two Mud Crab Species of Genus Scylla (Brachyura: Portunidae) in Indian Coastal Waters

The taxonomic ambiguity of the Indian mud crab (genus Scylla de Hann 1833) is still a cause of concern as several papers have been published with misleading identification. This is the first attempt to resolve the taxonomic uncertainty of the mud crab commonly available in Indian coastal waters using molecular genetic markers (ITS-1 and sequencing of COI gene) combined with traditional morphometry. Additionally, we developed a PCR method by which Indian mud crab species can be identified rapidly and effectively. The results clearly indicate that the green morph of the Indian mud crab is Scylla serrata and the brown morph is S. olivacea. The S. serrata commonly mentioned in the literature from India is S. olivacea; the S. tranquebarica noted by many Indian researchers should belong to S. serrata. Caution should be taken when interpreting or implementing the biological, molecular, and aquaculture data in the literature.     

Significance of PELP1 in ER-negative breast cancer metastasis

Breast cancer metastasis is a major clinical problem. The molecular basis of breast cancer progression to metastasis remains poorly understood. PELP1 is an estrogen receptor (ER) coregulator that has been implicated as a proto-oncogene whose expression is deregulated in metastatic breast tumors and whose expression is retained in ER-negative tumors. We examined the mechanism and significance of PELP1-mediated signaling in ER-negative breast cancer progression using two ER-negative model cells (MDA-MB-231 and 4T1 cells) that stably express PELP1-shRNA. These model cells had reduced PELP1 expression (75% of endogenous levels) and exhibited less propensity to proliferate in growth assays in vitro. PELP1 downregulation substantially affected migration of ER-negative cells in Boyden chamber and invasion assays. Using mechanistic studies, we found that PELP1 modulated expression of several genes involved in the epithelial mesenchymal transition (EMT), including MMPs, SNAIL, TWIST, and ZEB. In addition, PELP1 knockdown reduced the in vivo metastatic potential of ER-negative breast cancer cells and significantly reduced lung metastatic nodules in a xenograft assay. These results implicate PELP1 as having a role in ER-negative breast cancer metastasis, reveal novel mechanism of coregulator regulation of metastasis via promoting cell motility/EMT by modulating expression of genes, and suggest PELP1 may be a potential therapeutic target for metastatic ER-negative breast cancer.     

Individual Differences in Scotopic Visual Acuity and Contrast Sensitivity: Genetic and Non-Genetic Influences

Despite the large amount of variation found in the night (scotopic) vision capabilities of healthy volunteers, little effort has been made to characterize this variation and factors, genetic and non-genetic, that influence it. In the largest population of healthy observers measured for scotopic visual acuity (VA) and contrast sensitivity (CS) to date, we quantified the effect of a range of variables on visual performance. We found that young volunteers with excellent photopic vision exhibit great variation in their scotopic VA and CS, and this variation is reliable from one testing session to the next. We additionally identified that factors such as Circadian preference, iris color, astigmatism, depression, sex and education have no significant impact on scotopic visual function. We confirmed previous work showing that the amount of time spent on the vision test influences performance and that laser eye surgery results in worse scotopic vision. We also showed a significant effect of intelligence and photopic visual performance on scotopic VA and CS, but all of these variables collectively explain <30% of the variation in scotopic vision. The wide variation seen in young healthy volunteers with excellent photopic vision, the high test-retest agreement, and the vast majority of the variation in scotopic vision remaining unexplained by obvious non-genetic factors suggests a strong genetic component. Our preliminary genome-wide association study (GWAS) of 106 participants ruled out any common genetic variants of very large effect and paves the way for future, larger genetic studies of scotopic vision.