CUL-2 and ZYG-11 promote meiotic anaphase II and the proper placement of the anterior-posterior axis in C. elegans

The faithful segregation of chromosomes during meiosis is vital for sexual reproduction. Currently, little is known about the molecular mechanisms regulating the initiation and completion of meiotic anaphase. We show that inactivation of CUL-2, a member of the cullin family of ubiquitin ligases, delays or abolishes meiotic anaphase II with no effect on anaphase I, indicating differential regulation during the two meiotic stages. In cul-2 mutants, the cohesin REC-8 is removed from chromosomes normally during meiosis II and sister chromatids separate, suggesting that the failure to complete anaphase results from a defect in chromosome movement rather than from a failure to sever chromosome attachments. CUL-2 is required for the degradation of cyclin B1 in meiosis and inactivation of cyclin B1 partially rescued the meiotic delay in cul-2 mutants. In cul-2 mutants, the failure to degrade cyclin B1 precedes the metaphase II arrest. CUL-2 is also required for at least two aspects of embryonic polarity. The extended meiosis II in cul-2 mutants induces polarity reversals that include reversed orientation of polarity proteins, P granules, pronuclei migration and asymmetric cell division. Independently of its role in meiotic progression, CUL-2 is required to limit the initiation/spread of the polarity protein PAR-2 in regions distant from microtubule organizing centers. Finally, we show that inactivation of the leucine-rich repeat protein ZYG-11 produces meiotic and polarity reversal defects similar to those observed in cul-2 mutants, suggesting that the two proteins function in the same pathways.     

Synthesis and evaluation of 2-amino-8-alkoxy quinolines as MCHr1 antagonists. Part 2

The continued SAR investigation of 2-amino-8-alkoxy quinolines as melanin concentrating hormone receptor-1 (MCHr1) antagonists is reported. Prior hit-to-lead efforts resulted in the identification of 1 as a robust MCHr1 antagonist. Further delineation of the structural parameters essential for MCHr1-binding affinity of this class of nontraditional GPCR ligands resulted in the identification of compounds such as 33, 34 and 37, which demonstrate single digit nanomolar antagonism of MCHr1-mediated Ca(2+) release. The synthesis and biological evaluation of these compounds are reported.     

Changes in Fatty Acid Composition of Chromohalobacter israelensis with Varying Salt Concentrations

The adaptation of fatty acid composition of Chromohalobacter israelensis, a euryhalophilic bacterium, grown at different salt concentrations was studied. C. israelensis tolerated NaCl up to concentrations of 20% (w/v) and showed optimal growth at 7% (w/v). Major fatty acids of this bacterium were palmitic acid (16:0), stearic acid (18:0), palmetoleic acid (16:1cis9), and cis-vaccenic acid (18:111). The salt concentration strongly influenced the fatty acid composition. In the presence of sub-optimal salt concentrations, the degree of saturation decreased, suggesting the importance of salt in maintaining the osmotic balance of the cell with its environment.     

A database for Plasmodium falciparum protein models

There is an urgent need for developing alternate strategies to combat Malaria caused by Plasmodium falciparum (P. falciparum) because of growing drug resistance and increased incidents of infection in humans. 3D models of P. falciparum annotated proteins using molecular modeling techniques will enhance our understanding about the mechanism of host parasite interactions for the identification of drug targets and malarial vaccine design. Potential structural templates for P. falciparum annotated proteins were selected from PDB (protein databank) using BLASTP (basic local alignment search tool for proteins). This exercise identified 476 Plasmodium proteins with one or more known structural templates (>or= 40 % identity) for further modeling. The pair-wise sequence alignments generated for protein modeling were manually checked for error. The models were then constructed using MODELLER (a comparative protein modelling program for modelling protein structures) followed by energy minimization in AMBER force field and checked for error using PROCHECK. AVAILABILITY: http://bioinfo.icgeb.res.in/codes/model.html.     

Aminopiperidine indazoles as orally efficacious melanin concentrating hormone receptor-1 antagonists

The synthesis and biological evaluation of novel 3-amino indazole melanin concentrating hormone receptor-1 antagonists are reported, several of which demonstrated functional activity of less than 100nM. Compounds 19 and 28, two of the more potent compounds identified in this study, were characterized by high exposure in the brain and demonstrated robust efficacy when dosed in diet-induced obese mice.     

Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries

Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.     

Crystal structure of the von Willebrand factor modulator botrocetin

The binding of von Willebrand factor (vWF) to the platelet receptor, glycoprotein (GP) Ib-IX-V complex, has a key role in the initiation of thrombus formation and is regulated by interactions with extracellular matrix components under the influence of hemodynamic forces. To a certain extent, these effects can be mimicked in vitro by two nonphysiologic modulators, ristocetin and botrocetin. The latter, isolated from the venom of the snake Bothrops jararaca, is a 31-kDa heterodimeric protein that forms a soluble complex with vWF. As an initial step toward understanding the mechanisms that regulate vWF function, we have solved the crystal structure of botrocetin at 1.8 A resolution. Botrocetin exhibits homology with other snake proteins, but contains only one metal binding site as compared to two in Factor IX binding protein and Factor IX/X binding protein and none in flavocetin. A distinctive feature of botrocetin is the presence of a negatively charged surface that may play a role in the association with the vWF A1 domain.     

Scale up studies for the production of biosurfactant in packed column bioreactor

Biosurfactants capable of emulsifying pesticides have great potential to assist in microbial degradation of the pesticides. Solid State Fermentation (SSF) due to several advantages, is one of the efficient ways of producing these surfactants and seldom receives attention for commercial exploitation. In this study, a packed column bioreactor with wheat bran as the raw material and Bacillus subtilis has been used to produce a biosurfactant specific to disperse Fenthion, an organophosphrous pesticide. The emulsifier activity (EA) and surface tension from the packed column bioreactor were compared with flask fermentation experiments, which served as control. Airflow rate in the packed column bioreactor was varied from 10-20 l/min. Maximum EA and minimum surface tension occurred at airflow rate of 20 l/min. Peak EA in the control was 1.2 at 29 h while it was 1.9 in the bioreactor. The least surface tension of 24 dynes/cm was noticed at 54 h in the bioreactor, which was 33% better than the control at the same time period. The results indicate that the packed column bioreactor can become a more acceptable solid state fermentation system for commercial exploitation of Fenthion specific biosurfactant production.