Production and characterization of Monoclonal antibodies to bluetongue virus

In the present study, a total of 24 MAbs were produced against bluetongue virus (BTV) by polyethyleneglycol (PEG) mediated fusion method using sensitized lymphocytes and myeloma cells. All these clones were characterized for their reactivity to whole virus and recombinant BTV-VP7 protein, titres, isotypes and their reactivity with 24 BTV-serotype specific sera in cELISA. Out of 24 clones, a majority of them (n = 18) belong to various IgG subclasses and the remaining (n = 6) to the IgM class. A panel of eight clones reactive to both whole BTV and purified rVP7 protein were identified based on their reactivity in iELISA. For competitive ELISA, the clone designated as 4A10 showed better inhibition to hyperimmune serum of BTV serotype 23. However, this clone showed a variable percent of inhibition ranging from16.6% with BTV 12 serotype to 78.9% with BTV16 serotype using 24 serotype specific sera of BTV originating from guinea pig at their lowest dilutions. From the available panel of clones, only 4A10 was found to have a possible diagnostic application.     

Identification and characterization of midgut proteases in <Emphasis Type="Italic">Achaea janata</Emphasis> and their implications

Insect midgut proteases are excellent targets for insecticidal agents such as Bacillus thuringiensis Cry toxins and protease inhibitors. The midgut proteases of Achaea janata have been characterized and Casein zymograms indicated at least five distinct activities corresponding to approx 17, 20, 29 and 80, and 90 kDa. Using a combination of synthetic substrates and specific inhibitors in casein zymograms, photometric assays and activity blots, three trypsin-like and one elastase-like serine proteases were identified but no chymotrypsin-like activity. Various proteinase inhibitors displayed differential inhibitory effects towards the midgut proteases.     

Comparison of the dynamics of bovine and human angiogenin: a molecular dynamics study

Molecular dynamics simulations have been carried out for 1 ns on human and bovine angiogenin systems in an effort to compare and contrast their dynamics. An analysis of their dynamics is done by examining the rms deviations, following hydrogen-bonding interactions and looking at the role of water in and around the protein. The C-terminus of bovine angiogenin moves appreciably during dynamics suggesting a better structure for ligand binding. However, we do not find any evidence of a conformation where the glutamate residue that obstructs the active site takes on a different conformation. We observe a differential hydrogen-bonding pattern in the active site regions of bovine and human angiogenins, which could have a bearing on the different catalytic activities of the proteins. We also propose that the differential binding of the monoclonal antibody toward the two proteins might be due sequential and not conformational differences. Water molecules might play an important functional role in both proteins given their subtle functional differences. A simple computation on the molecular dynamics data has been carried out to identify locations in and around the protein that are invariably occupied by water. The locations of nearly half the waters we have identified from the simulation as being invariant in bovine angiogenin occupy similar locations in the bovine angiogenin crystal structure. The positions of the waters identified in human angiogenin differ considerably from that of bovine angiogenin.     

Tools for comparative protein structure modeling and analysis

The following resources for comparative protein structure modeling and analysis are described (http://salilab.org): MODELLER, a program for comparative modeling by satisfaction of spatial restraints; MODWEB, a web server for automated comparative modeling that relies on PSI-BLAST, IMPALA and MODELLER; MODLOOP, a web server for automated loop modeling that relies on MODELLER; MOULDER, a CPU intensive protocol of MODWEB for building comparative models based on distant known structures; MODBASE, a comprehensive database of annotated comparative models for all sequences detectably related to a known structure; MODVIEW, a Netscape plugin for Linux that integrates viewing of multiple sequences and structures; and SNPWEB, a web server for structure-based prediction of the functional impact of a single amino acid substitution.     

Stress responses in two genotypes of mulberry (Morus alba L.) under NaCl salinity

Changes in biomass yield rates, cell membrane stability (CMS), malondialdehyde (MDA) content and in the levels of physiological stress markers such as proline and glycine betaine in two high yielding genotypes (S1 and ATP, salt tolerant and salt sensitive, respectively) of mulberry under NaCl salinity were studied. Biomass yield rates and CMS were significantly decreased in both the genotypes under stress conditions. Per cent of decrease in biomass yield rate and CMS was relatively less in S1 than in ATP. Salt stress results a significant increase in the accumulation of proline, by 6-fold in S1 and 4-fold in ATP. Glycine betaine content was also increased significantly in stressed plants. However, the per cent increase was more in S1 than in ATP. The level of lipid peroxidation as indicated by MDA formation was greater in ATP than in S1. These results clearly support the better salt tolerant nature of S1 compared to ATP genotype.     

Discovery of bacterial NAD⁺-dependent DNA ligase inhibitors: improvements in clearance of adenosine series

Optimization of clearance of adenosine inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. To reduce Cytochrome P-450-mediated metabolic clearance, many strategies were explored; however, most modifications resulted in compounds with reduced antibacterial activity and/or unchanged total clearance. The alkyl side chains of the 2-cycloalkoxyadenosines were fluorinated, and compounds with moderate antibacterial activity and favorable pharmacokinetic properties in rat and dog were identified.     

Aqueous two phase extraction of invertase from baker's yeast: Effect of process parameters on partitioning

Invertase (β-d-fructofuronoside fructohydrolase) is an industrially important enzyme useful for the hydrolysis of sucrose. The potential of aqueous two phase extraction for the isolation and purification of invertase from crude baker's yeast is explored. Influence of the process parameters such as type of phase forming salts, PEG molecular weight, concentration of salt and polymer, tie line length and volume ratio on partitioning of invertase was studied. PEG 3350/magnesium sulphate system was found most suitable for the extraction which has resulted in favorable pH (5 ± 0.2) for the enzyme extraction. Polymer and salt concentration were found to significantly affect the degree of purification and enzyme recovery of invertase. The purity of ∼8.81 fold was obtained compared to crude extract with recovery of 77% at the standardized process conditions. Overall results demonstrated the feasibility of aqueous two phase extraction for the isolation and purification of invertase without the need of multiple steps.