Improved P2 phenylglycine-based hepatitis C virus NS3 protease inhibitors with alkenylic prime-side substituents

Phenylglycine has proved to be a useful P2 residue in HCV NS3 protease inhibitors. A novel π–π-interaction between the phenylglycine and the catalytic H57 residue of the protease is postulated. We hypothesized that the introduction of a vinyl on the phenylglycine might strengthen this π–π-interaction. Thus, herein is presented the synthesis and inhibitory potency of a series of acyclic vinylated phenylglycine-based HCV NS3 protease inhibitors. Surprisingly, inhibitors based on both d- and l-phenylglycine were found to be effective inhibitors, with a slight preference for the d-epimers. Furthermore, prime-side alkenylic extension of the C-terminal acylsulfonamide group gave significantly improved inhibitors with potencies in the nanomolar range (～35 nM), potencies which were retained on mutant variants of the protease.     

Role of calcium and ROS in cell death induced by polyunsaturated fatty acids in murine thymocytes

We investigated the mechanisms whereby omega‐3 and ‐6 polyunsaturated fatty acids (PUFAs) cause cell death of mouse thymocytes using flow cytometry, focusing on the respective roles of intracellular calcium concentration, [Ca²⁺]_i and reactive oxygen species (ROS). We applied the C‐22, 20, and 18 carbon omega‐3 (DHA, EPA, ALA) and omega‐6 (DTA, ARA, and LNA) fatty acids to isolated thymocytes and monitored cell death using the DNA‐binding dye, propidium iodide. When applied at 20 µM concentration, omega‐3 fatty acids killed thymocytes over a period of 1 h with a potency of DHA > EPA > ALA. The omega‐6 PUFAs were more potent. The C18 omega‐6 fatty acid, LNA, was the most potent, followed by DHA and ARA. Cell death was always accompanied by an increase in the levels of [Ca²⁺]_i and ROS. Both increases were in proportion to the potency of the PUFAs in inducing cell death. Removing extracellular calcium did not prevent the elevation in [Ca²⁺]_i nor cell death. However, the intracellular calcium chelator, BAPTA, almost totally reduced both the elevation in [Ca²⁺]_i and cell death, while vitamin E reduced the elevation in ROS and cell death. BAPTA also prevented the elevation in ROS, but vitamin E did not prevent the elevation in [Ca²⁺]_i. Thapsigargin, which depletes endoplasmic reticulum calcium, blocked the elevation in [Ca²⁺]_i, but CCCP, a mitochondrial calcium uptake inhibitor, did not. These results suggest that the six PUFAs we studied kill thymocytes by causing release of calcium from endoplasmic reticulum, which causes release of ROS from mitochondria which leads to cell death. J. Cell. Physiol. 225: 829–836, 2010. © 2010 Wiley‐Liss, Inc.     

Comparative genomic analysis of a neurotoxigenic Clostridium species using partial genome sequence: Phylogenetic analysis of a few conserved proteins involved in cellular processes and metabolism

Clostridial organisms produce neurotoxins, which are generally regarded as the most potent toxic substances of biological origin and potential biological warfare agents. Clostridium tetani produces tetanus neurotoxin and is responsible for the fatal tetanus disease. In spite of the extensive immunization regimen, the disease is an important cause of death especially among neonates. Strains of C. tetani have not been genetically characterized except the complete genome sequencing of strain E88. The present study reports the genetic makeup and phylogenetic affiliations of an environmental strain of this bacterium with respect to C. tetani E88 and other clostridia. A shot gun library was constructed from the genomic DNA of C. tetani drde, isolated from decaying fish sample. Unique clones were sequenced and sequences compared with its closest relative C. tetani E88.A total of 275 clones were obtained and 32,457 bases of non-redundant sequence were generated. A total of 150 base changes were observed over the entire length of sequence obtained, including, additions, deletions and base substitutions. Of the total 120 ORFs detected, 48 exhibited closest similarity to E88 proteins of which three are hypothetical proteins. Eight of the ORFs exhibited similarity with hypothetical proteins from other organisms and 10 aligned with other proteins from unrelated organisms. There is an overall conservation of protein sequences among the two strains of C. tetani and. Selected ORFs involved in cellular processes and metabolism were subjected to phylogenetic analysis.     

Bovine Serum Albumin Binding and Drug Delivery Studies with PVA-Ferrofluid

This paper describes a single-step method for the biomimetic synthesis of stably suspended magnetite nanoparticles in poly(vinyl alcohol) termed ferrofluids. The challenge is to synthesize water based stable magnetic colloids with a control over the particle size and morphology for biomedical applications. The polymer possibly plays a dual role of a surfactant and a functionalizing agent. Transmission electron microscopy, infrared spectroscopy and vibrating sample magnetometry were used to investigate the properties of the synthesized ferrofluids. It has a strong affinity towards the tryptophan residues in bovine serum albumin protein as determined from the fluorescence emission studies. For in vivo applications this could indirectly mean a resistance to immune response and thus ensure long-term circulation. The ability of the synthesized ferrofluid to bind a che-motherapeutic drug ceftriaxone and its ionic release was observed. The polymer hydroxyl group allows drug-binding and the magnetic property allows targeting to specific sites. Magnetic hybrid fluids with combined advantages of magnetism and polymer open up new perspectives for applications.     

Effect of lithium on hepatic and serum elemental status under different dietary protein regimens

Lithium carbonate at the dose level of 1.1 g/kg was administered in diet to normal (18% protein), low-protein-(LP; 8%) and highprotein (HP; 30% diet)-fed rats for a period of 1 mo. The LP diet resulted in a significant decrease in the hepatic levels of zinc, iron, copper, manganese, calcium, and serum levels of calcium and sodium. The HP diet caused a marked decrease in copper and calcium levels in liver, but an increase in potassium levels in serum was observed. Lithium treatment to normal rats led to a significant reduction in the hepatic contents of zinc, copper, potassium, calcium, and serum contents of potassium and sodium, whereas an elevation in serum contents of calcium was noticed. Administration of lithium to protein-deficient rats increased the hepatic concentration of manganese and serum concentration of calcium and the levels almost reached the normal limits. On the other hand, there was a marked depression in potassium contents in the serum of LP-as well as HP-fed rats following lithium treatment when compared to LP and HP groups, respectively.     

Micropropagation of Clerodendrum aculeatum through adventitious shoot induction and production of consistent amount of virus resistance inducing protein

Rapid micropropagation through adventitious shoot induction from in vitro raised leaf explants of Clerodendrum aculeatum (Verbenaceae), was successfully achieved for the first time. Basal portion of the leaves showed highest regeneration potential when grown on MS medium supplemented with BA (5.0 mg/l) and NAA and IBA (0.5 mg/l of each). Shoots after elongation in growth regulator-free medium, were rooted in MS medium containing 0.5 mg/l of NAA and IBA. Aqueous leaf extract of in vitro raised plants, induced high degree of resistance against viruses in susceptible healthy hosts when applied prior to virus inoculation. Upon purification from leaves of cultured plants, the resistance inducing protein, showed molecular mass of 34 kDa. Amount of resistance inducing protein obtained from leaves of cultured plants, was consistent throughout the year, as compared to the protein isolated from leaves of field grown plants, which showed marked seasonal fluctuation. The purified 34 kDa protein from in vitro raised plants, was serologically related to field grown plants and possessed similar characteristics. The micropropagated plants were successfully established in earthen pots under greenhouse conditions.     

A single Gbeta subunit locus controls cross-talk between protein kinase C and G protein regulation of N-type calcium channels

The modulation of N-type calcium channels is a key factor in the control of neurotransmitter release. Whereas N-type channels are inhibited by Gbetagamma subunits in a G protein beta-isoform-dependent manner, channel activity is typically stimulated by activation of protein kinase C (PKC). In addition, there is cross-talk among these pathways, such that PKC-dependent phosphorylation of the Gbetagamma target site on the N-type channel antagonizes subsequent G protein inhibition, albeit only for Gbeta(1)-mediated responses. The molecular mechanisms that control this G protein beta subunit subtype-specific regulation have not been described. Here, we show that G protein inhibition of N-type calcium channels is critically dependent on two separate but adjacent approximately 20-amino acid regions of the Gbeta subunit, plus a highly conserved Asn-Tyr-Val motif. These regions are distinct from those implicated previously in Gbetagamma signaling to other effectors such as G protein-coupled inward rectifier potassium channels, phospholipase beta(2), and adenylyl cyclase, thus raising the possibility that the specificity for G protein signaling to calcium channels might rely on unique G protein structural determinants. In addition, we identify a highly specific locus on the Gbeta(1) subunit that serves as a molecular detector of PKC-dependent phosphorylation of the G protein target site on the N-type channel alpha(1) subunit, thus providing for a molecular basis for G protein-PKC cross-talk. Overall, our results significantly advance our understanding of the molecular details underlying the integration of G protein and PKC signaling pathways at the level of the N-type calcium channel alpha(1) subunit.     

Nectarless flowers: ecological correlates and evolutionary stability

In animal-pollinated flowers, the pollinators cannot detect the presence of nectar before entering flowers, and therefore flowers may cheat by not producing nectar. An earlier model suggested that a mixed strategy of producing nectarful and nectarless flowers would be evolutionarily stable. Here we compare nectarless flowers as a cheating strategy with three competing hypotheses namely "visit-more-flowers", "cross-pollination enhancement" and "better contact". We collected field data on 28 species of plants to test some of the differential predictions of the hypotheses. Nectarless flowers were detected in 24 out of 28 plant species. Correlations of percent nectarless flowers with floral and ecological variables support the cheater flower hypothesis. We further model the cost-benefits of cheating and show that an evolutionary stable ratio of nectarless to nectarful flowers can be reached. The equilibrium ratio is mainly decided by factors associated with pollinator density and pollinator learning.