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Biomedical subjects

P Roy

Publications and source records attributed to P Roy.

At least 163 records · Page 9Linked to original sources
PubMed1996

Oxidation of ascorbic acid by lipoxygenase: effect of selected chemicals.

The ability of soybean lipoxygenase to mediate ascorbic acid oxidation was examined. The oxidation of ascorbic acid was dependent on the concentration of linoleic acid, ascorbic acid and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 800 microM linoleic acid, 500 microM ascorbic acid and 25 nM soybean lipoxygenase in 50 mM Tris buffer, pH 8.3. The reaction displayed a K(m) value of 100 microM for ascorbic acid and an average specific activity of about 460 nmol/min/nmol enzyme under the optimal conditions. The effect of ascorbic acid on the lipoxygenase-catalysed co-oxidation of xenobiotics was also evaluated. Ascorbic acid markedly decreased the rate of oxidation of test xenobiotics by the lipoxygenase. In contrast, the rate of ascorbic acid co-oxidation was enhanced significantly by the presence of xenobiotics, which are co-oxidized simultaneously by lipoxygenase through the formation of free radicals. Superoxide generation was not observed during lipoxygenase-mediated ascorbic acid co-oxidation.

Ascorbic Acid↗
PubMed1996

Purification, kinetical and molecular characterizations of a serine collagenolytic protease from greenshore crag (Carcinus maenas) digestive gland.

A serine collagenolytic protease was purified from a water soluble fraction of greenshore crab digestive gland by acidic precipitation, gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a Fractogel TSK DEAE column, immobilized metal ion affinity chromatography (IMAC) on IDA (Cu2+) Sepharose 6B and ion-exchange chromatography on Hyper D column. The molecular mass of the monomeric Carcinus serine collagenase (CSC) was estimated to be 23,000 by SDS PAGE and its isoelectric point was found to be 4.0. The CSC is optimally active at pH 7 and 30 degrees C and is stable over a month at room temperature. The CSC activity is strongly inhibited by PMSF, 3,4-DCI, soybean trypsin inhibitor, alpha 1 proteinase inhibitor and elastatinal. The CSC hydrolyzes native collagen (Type I and III). CSC N-terminal sequence is similar to shrimp chymotrypsin-like protease and crab collagenolytic protease sequences. Kinetic parameters of the CSC were determined using some peptidyl-p-nitroanilides. The catalytic efficiency (kcat/Km) is Leu > Phe > Ala.

Amino Acid Sequence↗
PubMed1996

Assembly of macromolecular complexes in bacterial and baculovirus expression systems.

Many proteins exist normally as oligomers or complexes with other proteins. Recent advances in vector design have allowed this aspect of protein function to be mimicked in recombinant expression systems. Examples of the ordered oligomerization of a single protein through to the assembly of eight different proteins have been documented in recombinant Escherichia coli and recombinant baculovirus systems.

Animals↗
PubMed1996

Measurement of surgically induced corneal deformations using three-dimensional confocal microscopy.

The goal of this study was to develop and apply a new set of experimental techniques for measuring the local deformations induced by partial-thickness corneal incisions in situ. Eight adult cat eyes were enucleated and cannulated, with corneal viability maintained as close to in vivo conditions as possible and intraocular pressure (IOP) carefully controlled. Experimental measurements were made pre/post radial keratotomy (RK) surgery in situ at IOPs of 15, 30, and 45 mm Hg. Incision depth and cross-sectional profiles were measured at the midpoint of selected incisions using three-dimensional (3-D) tandem scanning confocal microscopy (TSCM); central corneal curvature was estimated using a commercial corneal topographical analysis system, and corneal thickness was assessed by both 3-D TSCM and ultrasonic pachymetry. Corneas were then processed for light microscopy and incision depth was measured histologically. Finite element models were developed for comparison with the experimental measurements. There was no significant change in central corneal thickness (-5.3 +/- 3.9%, n = 8) over the course of the experiments, demonstrating that normal endothelial cell function and normal stromal hydration was maintained. The in situ TSCM incision depth measurements were significantly correlated with the histological measurements (slope = 0.95, R = 0.854, p < 0.01, n = 13 incisions). Measured incision gape at the top (anterior) of the stroma was 64.9 +/- 13.4, 87.3 +/- 12.6, and 108.7 +/- 14 microns at IOPs of 15, 30, and 45 mm Hg, respectively. The 3-D incision profiles were nonlinear in shape; comparison with the finite element models suggests that the shape of the wound profile may provide unique information regarding the shear stiffness of the cornea. Overall, the data suggest that TSCM measurements of the cross-sectional profile of the incisions immediately after RK under controlled in situ conditions provide important data regarding the mechanical behavior of the cornea after refractive surgery. These data should provide the foundation for future studies into the relationships between local tissue mechanics and corneal wound healing.

Animals↗
PubMed1996

The non-structural proteins of bluetongue virus are a dominant source of cytotoxic T cell peptide determinants.

Virus-specific, CD8+ cytotoxic T lymphocytes (CTLs) were generated in two strains of mice (BALB/c, CBA/Ca) against bluetongue virus serotype 10 (BTV-10). Recombinant vaccinia viruses (VV) expressing the individual structural and non-structural proteins of BTV were used to infect syngeneic target cells. We found that in both BALB/c (H-2d) and CBA/Ca (H-2k) mice, polyclonal CTL populations recognized target cells expressing the non-structural proteins better than those expressing the structural proteins. CTLs generated against other BTV serotypes also predominantly recognized the non-structural proteins. However, the extent of cross-reactivity was dependent on the H-2 background of the animals immunized. No CTLs cross-reactive to the BTV-10 heterotype were demonstrated with the panel of molecularly cloned recombinants in the H-2d haplotype. The outer capsid proteins VP2 and VP5 which vary considerably between serotypes were not recognized by heterotypic CTLs. Using this murine model we have determined which BTV proteins are the major targets of the CTL response. The implications for the design and development of subunit vaccines are discussed.

Animals↗
PubMed1996

Full protection against African horsesickness (AHS) in horses induced by baculovirus-derived AHS virus serotype 4 VP2, VP5 and VP7.

African horsesickness virus serotype 4 (AHSV-4) outer capsid protein VP2, or VP2 and VP5 plus inner capsid protein VP7, derived from single or dual recombinant baculovirus expression vectors were used in different combinations to immunize horses. When the proteins were purified by affinity chromatography, the combination of all three proteins induced low levels of neutralizing antibodies and conferred protection against virulent virus challenge. However, purified VP2 or VP2 and VP5 in the absence of VP7 failed to induce neutralizing antibodies and protection. Immunization with non-purified proteins enhanced the titres of neutralizing antibodies. Again, the combination of the three proteins was able to confer total protection to immunized horses, which showed absence of viraemia. The antigenicity of recombinant VP2 was analysed with a collection of 30 MAbs. Both purified and unpurified recombinant VP2 proteins showed different antigenic patterns in comparison to that of VP2 on virions. An immunization experiment with four more horses confirmed these results. The vaccine described here would not only prevent the disease, but would drastically reduce the propagation of the virus by vectors.

African Horse Sickness↗
PubMed1996

Recombinant baculovirus-synthesized African horsesickness virus (AHSV) outer-capsid protein VP2 provides protection against virulent AHSV challenge.

African horsesickness virus serotype 4 (AHSV-4) outer-capsid proteins VP2 or VP2 and VP5, prepared from single or dual recombinant baculovirus expression vectors grown in Sf9 insect cells, were administered in different amounts to horses and the neutralizing antibody responses were measured. Control and vaccinated horses were challenged with virulent AHSV-4 6 months later and monitored post challenge. The results indicated that two inoculations of extracts containing VP2 and VP5, or VP2 alone, in doses of 5 micrograms VP2 or more per horse, were sufficient to elicit protection against African horsesickness (AHS) disease. The recombinant VP2 protein is a potential candidate vaccine for AHS in horses.

African Horse Sickness↗
PubMed1996

Crystal structure of the top domain of African horse sickness virus VP7: comparisons with bluetongue virus VP7.

The baculovirus-expressed core protein VP7 of African horse sickness virus serotype 4 (AHSV-4) has been purified to homogeneity and crystallized in the presence of 2.8 M urea. The X-ray structure has been solved to a 2.3-Angstroms (1 Angstrom = 0.1 nm) resolution with an Rfactor of 19.8%. The structure of AHSV VP7 reveals that during crystallization, the two-domain protein is cleaved and only the top domain remains. A similar problem was encountered previously with bluetongue virus (BTV) VP7 (whose structure has been reported), showing that the connections between the top and the bottom domains are rather weak for these two distinct orbiviruses. The top domains of both BTV and AHSV VP7 are trimeric and structurally very similar. The electron density maps show that they both possess an extra electron density feature along their molecular threefold axes, which is most likely due to an unidentified ion. The characteristics of the molecular surface of BTV and AHSV VP7 suggest why AHSV VP7 is much less soluble than BTV VP7 and indicate the possibility of attachment to the cell via attachment of an Arg-Gly-Asp (RGD) motif in the top domain of VP7 to a cellular integrin for both of these orbiviruses.

African Horse Sickness Virus↗
PubMed1996

Sequences within the VP6 molecule of bluetongue virus that determine cytoplasmic and nuclear targeting of the protein.

Genome segment 9 of bluetongue virus serotype 10 encodes the minor protein VP6. The protein is abundant with basic residues particularly in two regions of the carboxy half of the molecule. A series of amino- and carboxy-terminal deletion mutants was expressed in mammalian cells by using a vaccinia virus T7 polymerase-driven transient expression system, and the intracellular fate of the products was monitored by both immunofluorescence staining and cell fractionation techniques. Data obtained indicated clearly that VP6 has nuclear transportation signals which may be correlated with positively charged domains of the molecule. In the intact molecule, though, these signals are masked and the protein is retained in the cytoplasm. The biochemical and immunofluorescence data obtained indicate that sequences in the region of residues 33 to 80 of the 328-amino acid protein are required for the retention of VP6 within the cell cytoplasm while amino acids 303 to 308 in the carboxy-terminal half of the molecule appear to possess nuclear localization capabilities.

Amino Acid Sequence↗
PubMed1996

Genetically engineered particulate virus-like structures and their use as vaccine delivery systems.

The Orbivirus genus within the family Reoviridae consists of nonenveloped architecturally complex viruses. The icosahedral viruses are 810 A diameter in size and are comprised of two protein shells containing seven proteins (VP1-VP7), surrounding a genome of ten double-stranded RNA segments. The prototype virus, bluetongue virus (BTV), is the etiological agent of a disease that can reach epidemic proportions among sheep and cattle. To develop highly protective virus-like particles, we have developed novel baculovirus multigene expression vector systems which have allowed us to coexpress three, four or five BTV genes from single recombinant vectors. The resultant particulate structures resemble BTV virus-like and subvirus-like particles which are structurally and immunologically indistinguishable from the BTV, and preliminary clinical trials have verified this vaccines safety and efficacy. Unlike live virus vaccines, VLPs are noninfectious and lack virus (or other) DNA/RNA required for replication. VLPs do not replicate in host cells. However, sheep trials have shown that VLPs are more immunogenic than subunit vaccines (viral proteins), or viruses killed by chemical inactivation. In addition, they are effective at eliciting humoral, cell-mediated and mucosal immunities. Virus-like particles (VLPs) are safe to produce and handle. The baculovirus vector and host cells used to make VLPs do not come from mammalian sources (hence they do not contain mammalian-derived pathogens). The multicomponent VLPs have also been utilized as vaccine delivery systems for multiple immunogens including B and T cell epitopes. The expression system described here is a tool which may have a range of applications in industries employing biotechnology to produce vaccines, insecticides, diagnostic and protein reagents.

Animals↗
PubMed1996

Thyroid hormone induces the synthesis of a putative protein in the rat granulosa cell which stimulates progesterone release.

125I-3,5,3'-tri-iodothyronine (T3) binds specifically to a pure nuclear preparation from rat granulosa cells. A Scatchard analysis of T3 binding showed a Kd of 0.65 x 10(-9) mol/l and a Bmax of 1.57 pmol/mg DNA. The biological relevance of T3 binding to granulosa cells was evaluated by adding T3 (20 ng/incubation) to granulosa cells (1 x 10(6) cells/incubation) which greatly stimulated progesterone release. T3-stimulated progesterone release was significantly inhibited by actinomycin-D (P < 0.01) and cycloheximide (P < 0.01). T3 caused about a twofold increase in granulosa cell protein synthesis as compared with the control which was inhibited by actinomycin-D and cycloheximide. The addition of T3 to granulosa cell incubations also resulted in a more than 2.5-fold increase in mRNA. The results indicated that T3 stimulation of progesterone release is mediated via T3-induced protein(s) or TIP. TIP was located in the soluble supernatant fraction (100,000 g supernatant; 100 k sup) from T3-incubated cells but could not be detected in the 100 k sup from the control cells or LH-incubated cells. TIP was purified based on its biological activity, i.e. its addition to granulosa cell incubations stimulated progesterone release into the medium. The 100 k sup from T3-incubated granulosa cells was subjected to Sephadex G-75 gel filtration, FPLC Mono Q and FPLC Superose 6 chromatography which resulted a 273-fold purification over the starting material and a clearly homogeneous protein was obtained. SDS-PAGE of purified TIP showed it to be a 53 kDa monomer protein. Experiments conducted with radiolabelled TIP suggested internalization of TIP into the granulosa cell. The results therefore showed that T3 induces the synthesis of mRNA and proteins in rat granulosa cells and that one of the proteins is TIP which, in turn, stimulates progesterone release from the cell, suggesting thereby that this putative protein is a novel mediator of T3 function in the granulosa cell.

Animals↗
PubMed1996

Structure-function relationships of human cholesteryl ester transfer protein: analysis using monoclonal antibodies.

Cholesteryl ester transfer protein (CETP), a 476 amino acid glycoprotein, mediates cholesteryl ester (CE), triglyceride, and phospholipid transfer among plasma lipoproteins. A monoclonal antibody (mAb), TP2, specific for an epitope within the last 26 amino acids of CETP has been shown to block all CETP-mediated lipid transfer, apparently by limiting access to lipid-binding sites in the carboxy terminal of CETP. A new panel of 16 anti-human CETP mAbs has now been used to further probe the structure-function relationships of CETP. Of the new mAbs, 9 partially inhibit CETP-mediated CE transfer (24-43%) from HDL to LDL. The corresponding epitopes were mapped within the CETP primary structure by the reactivity of the mAbs with CETP variants having deletions or amino acid substitutions. Of the 9 new, neutralizing mAbs, 6 are specific for epitopes situated between residues 410-450 and two others for epitopes between residues 184-260 and 332-366, respectively. The epitope of one neutralizing mAbs could not be mapped. Therefore, binding of mAbs to epitopes situated in four non-overlapping regions within CETP primary structure that are separated by as many as 280 residues can neutralize CETP-mediated CE transfer. Epitopes of mAbs that do not influence CE transfer activity map to the regions 184-260, 261-331, and 367-409, respectively. When pairs of mAbs were tested for their abilities to mutually compete for binding to immobilized CETP, competition was observed for mAbs specific for epitopes that are distant in CETP primary structure. The cross-competition patterns demonstrate that the carboxy terminal 60% of CETP adopts a compact structure. Together with previous mutagenesis studies, the data suggests that a carboxy terminal neutral lipid binding domain may be in close proximity to a lipoprotein binding region within native CETP.

Animals↗
PubMed1996

Antifungal susceptibility pattern of non-albicans Candida species & distribution of species isolated from Candidaemia cases over a 5 year period.

A marked increase in the incidence of patients of candidaemia is reported from a tertiary care hospital in north India over the last five years (15 patients in 1991 and 275 in 1995). The distribution of Candida species isolated from January 1991 through December 1995 was investigated. Antifungal susceptibility against amphotericin B, 5-fluorocytosine, ketoconazole and fluconazole of 100 randomly selected non-albicans Candida species isolated during 1995 was determined by an in-house standardized disc diffusion method and the standard broth dilution procedure recommended by the National Committee for Clinical Laboratory Standards (NCCLS, USA). The disc diffusion method correlated well with NCCLS method except for ketoconazole. Resistance against any antifungal was confirmed only by results of NCCLS method. A shift to higher isolation of non-albicans Candida species was observed during this period (52.6% in 1992 to 89.5% in 1995). Resistance was observed against 5-fluorocytosine in 3 per cent strains of C. krusei; and against fluconazole in 24.2 per cent of C. krusei, 15.4 per cent of C. guilliermondii and 5.7 per cent strains of C. tropicalis. No resistance was detected against amphotericin B and ketoconazole. Thus Candida species with fluconazole resistance have become more prominent in recent years.

Antifungal Agents↗
PubMed1996

Evaluation of recombinant bluetongue virus antigens, using dot immunobinding assay with serum from sheep and cattle.

OBJECTIVE: To evaluate 2 genetically engineered group-specific antigens: baculovirus- and yeast-expressed VP7 and the conventionally produced group-specific antigen of bluetongue virus (BTV), using dot immunobinding assay (DIA). SAMPLE POPULATION AND PROCEDURE: A total of 260 serum samples of sheep and cattle from various livestock farms in different Indian states (eg, Haryana, Himachal Pradesh, Jammu and Kashmir, Punjab, and Rajasthan) were tested for the presence of BTV antibodies by DIA. RESULTS: Of 260 sera tested, 92 (35%) were positive for BTV antibodies using the baculovirus-expressed antigen, 96 (37%) were positive using the yeast-expressed antigen, and 103 (40%) were positive by use of conventionally produced antigen. CONCLUSION AND CLINICAL RELEVANCE: The overall agreement for all the 3 antigens was 92%, indicating that the recombinant group-specific proteins can be used for serodiagnosis of BTV, using DIA.

Animals↗