Molecular modeling and conformational analysis of native and refolded viral genome-linked protein of Cardamom mosaic virus.

The viral genome-linked protein (VPg) of Potyviruses is covalently attached to the 5’ end of the genomic RNA. Towards biophysical characterization, the VPg coding region of Cardamom mosaic virus (CdMV) was amplified from the cDNA and expressed in E. coli. Most of the expressed VPg aggregated as inclusion bodies that were solubilized with urea and refolded with L-arginine hydrochloride. The various forms of CdMV VPg (native, denatured and refolded) were purified and the conformational variations between these forms were observed with fluorescence spectroscopy. Native and refolded CdMV VPg showed unordered secondary structure in the circular dichroism (CD) spectrum. The model of CdMV VPg was built based on the crystal structure of phosphotriesterase (from Pseudomonas diminuta), which had the maximum sequence homology with VPg to identify the arrangement of conserved amino acids in the protein to study the functional diversity of VPg. This is the first report on the VPg of CdMV, which is classified as a new member of the Macluravirus genus of the Potyviridae family.

Subclassification and targeted characterization of prophage-encoded two-component cell lysis cassette.

Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies.The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E.coli and the purified protein was functional, exhibiting lytic activity against E.coli and Salmonella typhi cell wall substrate. Such targeted sequence- structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.

Molecular modelling of epitope presentation using membrane protein OmpC.

Three-dimensional models of the chimeric S. typhi OmpC protein carrying an epitope from rotavirus VP4 capsid protein on either of two exposed loops (fourth and sixth) were constructed separately, using computer-aided homology modelling. The theoretical model of S. typhi OmpC was used as a template. The monomers were initially energy minimized. The trimers were generated for both the chimeric S. typhi OmpC proteins and the structures were optimized after several cycles of minimization. The surface accessibility calculations for the resulting models show that epitope recognition should be more effective in the fourth loop than in the sixth loop, in accordance with the experimental results on the immunogenic nature of the rotaviral epitope inserted into the two putative loops of S. typhi OmpC.

Prediction of B-cell epitopes for Salmonella typhi OmpC.

The possible B-cell epitopes of the outer membrane porin OmpC of Salmonella typhi have been identified, using the primary structure of the protein, by means of multiple sequence alignment and the known molecular structure of two other porins. From the analysis, 8 regions were identified as immunodominant and these were ranked based on antigenic index and the ratio of the number of nonconserved residues to the fragment length. Model building of the top two ranked regions show the tendency to form loop structures supporting the possibility of these being candidate epitopes.

Aortic valve replacement with concomitant open mitral valvotomy--early results and long term followup in 72 consecutive patients.

Experience with 72 consecutive subjects, who underwent aortic valve replacement with open mitral valvotomy is presented. Mean age was 30 years and history of rheumatic fever was forthcoming in 80%. 91.7% patients had aortic regurgitation--with or without aortic stenosis. 45% patients had pulmonary hypertension. Starr Edwards (51.4% and Bjork Shiley valves (45.8%) were commonly used. There were no early deaths. All patients were followed up for 1 to 19 years (mean = 9 years). There were 4 (5.5%) late deaths. 4.1% patients developed mild restenosis of the mitral valve over a mean period of 8 years and 1.04% developed mitral regurgitation mandating mitral valve replacement. A notable feature of this series is the low incidence of thromboembolism-free lives at upto 10 years followup despite receiving only aspirin in place of coumarin anticoagulation. We believe aortic valve replacement and open mitral valvotomy is a worthy alternative to double valve replacement in view of its zero hospital mortality, low late mortality, low incidence of restenosis, low thromboembolic episodes and the avoidance of coumarin anticoagulation.

Surgical management of coarctation of aorta.

Between 1961 and 1988, 68 patients underwent operation for coarctation of the aorta. The average age of presentation of these subjects was higher than in other series. 7.1 percent were asymptomatic, a finding which is not seen in reports from the west. Operative procedures included resection and end to end anastomosis, resection and graft interposition, bypass grafting, patch aortoplasty and subclavian flap aortoplasty. However, the technique of patch aortoplasty, routinely performed in the last 24 years of this series seemed by far the most satisfactory procedure. Subclavian flap aortoplasty was carried out in a selected group of younger children. Associated cardiac anomalies influenced the results adversely. The overall operative mortality was 5.8 percent. Hypertension did not regress in 11.7 per cent of patients inspite of a successful operation as judged by the return of peripheral pulses in the lower limbs. Re-coarctation was not seen in this series. The overall results of operation for coarctation of the aorta have been very satisfactory and comparable with those in other published series.