Nuclear localization of flavivirus RNA synthesis in infected cells

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.     

Differential response of antioxidative enzymes to various abiotic stresses in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> seedlings

Antioxidative enzyme activities and their isozyme patterns under water-deficit, salinity, high and low temperature stresses were studied in the seedlings of Pennisetum glaucum (L.) R.Br. It was observed that under water-deficit stress glutathione reductase (GR) was the key enzyme while in case of high temperature stress, GR along with catalase played a major role. Superoxide dismutase was found to be the main enzyme under low temperature stress. Co-ordinated higher expression of all the antioxidative enzymes was observed under salt stress. This study revealed the operation of different enzymatic antioxidative mechanisms under various abiotic stresses that will aid in understanding the metabolic basis of stress tolerance in pearl millet.     

Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes

Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8 h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47^phox phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress.     

Elevated CO2 increases water use efficiency by sustaining photosynthesis of water-limited maize and sorghum

Maize and grain sorghum seeds were sown in pots and grown for 39 days in sunlit controlled-environment chambers at 360 (ambient) and 720 (double-ambient, elevated) μmol mol⁻¹ carbon dioxide concentrations [CO₂]. Canopy net photosynthesis (PS) and evapotranspiration (TR) was measured throughout and summarized daily from 08:00 to 17:00 h Eastern Standard Time. Irrigation was withheld from matched pairs of treatments starting on 26 days after sowing (DAS). By 35 DAS, cumulative PS of drought-stress maize, compared to well-watered plants, was 41% lower under ambient [CO₂] but only 13% lower under elevated [CO₂]. In contrast, by 35 DAS, cumulative PS of drought-stress grain sorghum, compared to well-watered plants, was only 9% lower under ambient [CO₂] and 7% lower under elevated [CO₂]. During the 27-35 DAS drought period, water use efficiency (WUE, mol CO₂ Kmol⁻¹ H₂O), was 3.99, 3.88, 5.50, and 8.65 for maize and 3.75, 4.43, 5.26, and 9.94 for grain sorghum, for ambient-[CO₂] well-watered, ambient-[CO₂] stressed, elevated-[CO₂] well-watered and elevated-[CO₂] stressed plants, respectively. Young plants of maize and sorghum used water more efficiently at elevated [CO₂] than at ambient [CO₂], especially under drought. Reductions in biomass by drought for young maize and grain sorghum plants were 42 and 36% at ambient [CO₂], compared to 18 and 14% at elevated [CO₂], respectively. Results of our water stress experiment demonstrated that maintenance of relatively high canopy photosynthetic rates in the face of decreased transpiration rates enhanced WUE in plants grown at elevated [CO₂]. This confirms experimental evidence and conceptual models that suggest that an increase of intercellular [CO₂] (or a sustained intercellular [CO₂]) in the face of decreased stomatal conductance results in relative increases of growth of C₄ plants. In short, drought stress in C₄ crop plants can be ameliorated at elevated [CO₂] as a result of lower stomatal conductance and sustaining intercellular [CO₂]. Furthermore, less water might be required for C₄ crops in future higher CO₂ atmospheres, assuming weather and climate similar to present conditions.     

T Cell receptor clonotype influences epitope hierarchy in the CD8+ T cell response to respiratory syncytial virus infection

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vβ13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vβ subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vβ genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.     

Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms

We have developed a novel PCR-based assay for individual and simultaneous detection of three major pathogens (microsporidians, nucleopolyhedrovirus (NPV) and densovirus (DNV)) infecting the silkworm, Bombyx mori. Multiplex PCR, using three primer pairs, two of which were designed from the conserved regions of 16S small subunit ribosomal RNA gene of microsporidians, and polyhedrin gene of NPVs respectively, and a third primer pair designed from the internal sequences of B. mori DNVs (BmDNV), showed discrete and pathogen specific PCR products. The assay showed high specificity and sensitivity for the pathogenic DNA. Under optimized PCR conditions, the assay yielded a 794 bp DNA fragment from Nosema bombycis, 471 bp fragment from B. mori NPV (BmNPV) and 391 bp fragment from BmDNV. Further, this detection method was successfully applied to other silkworm species such as Antheraea mylitta and Samia cynthia ricini, in detecting same or similar pathogens infecting them. This method is a valuable supplement to the conventional microscopic diagnostic methods and can be used for the early detection of pathogens infecting silkworms. Furthermore it can assist research and extension centers for the safe supply of disease-free silkworms to farmers.     

Genetic association, post-translational modification, and protein-protein interactions in Type 2 diabetes mellitus

Type 2 diabetes mellitus is a complex disorder with a strong genetic component. Inherited complex disease susceptibility in humans is most commonly associated with single nucleotide polymorphisms. The mechanisms by which this occurs are still poorly understood. Here we focus on analyzing the effect of a set of disease-causing missense variations of the monogenetic form of Type 2 diabetes mellitus and a set of disease-associated nonsynonymous variations in comparison with that of nonsynonymous variations without any experimental evidence for association with any disease. Analysis of different properties such as evolutionary conservation status, solvent accessibility, secondary structure, etc. suggests that disease-causing variations are associated with extreme changes in the value of the parameters relating to evolutionary conservation and/or protein stability. Disease-associated variations are rather moderately conserved and have a milder effect on protein function and stability. The majority of the genes harboring these variations are clustered in or near the insulin signaling network. Most of these variations are identified as potential sites for post-translational modifications; certain predictions have already reported experimental evidence. Overall our results indicate that Type 2 diabetes mellitus may result from a large number of single nucleotide polymorphisms that impair modular domain function and post-translational modifications involved in signaling. Our emphasis is more on conserved corresponding residues than the variation alone. We believe that the approach of considering a stretch of peptide sequence involving a polymorphism would be a better method of defining the role of the polymorphism in the manifestation of this disease. Because most of the variations associated with the disease are rare, we hypothesize that this disease is a "mosaic model" of interaction between a large number of rare alleles and a small number of common alleles along with the environment, which is little contrary to the existing common disease common variant model.     

Synthesis of some new biologically active 1,3,4-oxadiazolyl nitroindoles and a modified Fischer indole synthesis of ethyl nitro indole-2-carboxylates

An efficient and modified synthesis of ethyl-4-nitro/5-nitro/6-nitro and 7-nitroindole-2-carboxylates is described. Carbohydrazides of corresponding ethyl nitroindole-2-carboxylates underwent smooth one-step transformation to 1,3,4-oxadiazolyl nitroindoles (4a-l) on reaction with aromatic carboxylic acids in the presence of phosphorus oxychloride. An alternate method to synthesize 1,3,4-oxadiazolyl nitroindoles is also described. Among the newly synthesized 1,3,4-oxadiazolyl nitroindoles, a few compounds are studied for anti-inflammatory activity.     

Synthesis and chemical characterization of 2-methoxy-N(10)-substituted acridones needed to reverse vinblastine resistance in multidrug resistant (MDR) cancer cells

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of 19 N(10)-substituted-2-methoxyacridone analogues has been synthesized. 2-Methoxyacridone and its derivatives (1-19) were synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-anisidine followed by cyclization using polyphosphoric acid. This compound undergoes N-alkylation in the presence of phase transfer catalyst (PTC). Stirring of 2-methoxy acridone with 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a two-phase system consisting of organic phase (tetrahydrofuran) and 6N potassium hydroxide in the presence of tetrabutylammonium bromide leads to the formation of compounds 2 and 11 in good yield. N-(omega-Chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with various secondary amines. Products were characterized by UV, IR, 1H and 13C NMR, mass-spectral data and elemental analysis. The lipophilicity expressed in log(10) P and pK(a) of compounds have been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 7, 10, 12, and 15-19 at 100 microM caused a 1.05- to 1.7-fold greater accumulation of vinblastine than did a similar concentration of the standard modulator, verapamil (VRP). However, the effects on VLB uptake were specific because these derivatives had little effect in the parental drug sensitive line KB-3-1. Steady state accumulation of VLB, a substrate for P-glycoprotein (P-gp) mediated efflux, was studied in the MDR cell line KBCh(R)-8-5 in the presence and absence of novel MDR modulators. Results of the efflux experiment showed that VRP and each of the modulators (1-19) significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. From among the compounds examined, 14 except 1, 2, 4, 8, and 11, exhibited greater efflux inhibiting activity than VRP. All the 19 compounds effectively compete with [(3)H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely site of action. Cytotoxicity has been determined and the IC(50) values lie in the range 8.00-18.50 microM for propyl and 4-15 microM for butyl derivatives against KBCh(R)-8-5 cells suggesting that the antiproliferative activity increases as chain length increases from 3 to 4 carbons at N(10)-position. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh(R)-8-5 cells and found that the modulators enhanced the cytotoxicity of VLB by 5- to 35-fold. Modulators 12, 14-16, and 19 like VRP, were able to completely reverse the 24-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-methoxyacridones.     

Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants

Inositol pyrophosphates are unique cellular signaling molecules with recently discovered roles in energy sensing and metabolism. Studies in eukaryotes have revealed that these compounds have a rapid turnover, and thus only small amounts accumulate. Inositol pyrophosphates have not been the subject of investigation in plants even though seeds produce large amounts of their precursor, myo‐inositol hexakisphosphate (InsP₆). Here, we report that Arabidopsis and maize InsP₆ transporter mutants have elevated levels of inositol pyrophosphates in their seed, providing unequivocal identification of their presence in plant tissues. We also show that plant seeds store a little over 1% of their inositol phosphate pool as InsP₇ and InsP₈. Many tissues, including, seed, seedlings, roots and leaves accumulate InsP₇ and InsP₈, thus synthesis is not confined to tissues with high InsP₆. We have identified two highly similar Arabidopsis genes, AtVip1 and AtVip2, which are orthologous to the yeast and mammalian VIP kinases. Both AtVip1 and AtVip2 encode proteins capable of restoring InsP₇ synthesis in yeast mutants, thus AtVip1 and AtVip2 can function as bonafide InsP₆ kinases. AtVip1 and AtVip2 are differentially expressed in plant tissues, suggesting non‐redundant or non‐overlapping functions in plants. These results contribute to our knowledge of inositol phosphate metabolism and will lay a foundation for understanding the role of InsP₇ and InsP₈ in plants.