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Biomedical subjects

R Srivastava

Publications and source records attributed to R Srivastava.

At least 19 recordsLinked to original sources
PubMed1999

Cell surface expression of HLA-E: interaction with human beta2-microglobulin and allelic differences.

The formation of a trimeric complex composed of MHC class I heavy chain, beta2-microglobulin (beta2m) and peptide ligand is a prerequisite for its efficient transport to the cell surface. We have previously demonstrated impaired intracellular transport of the human class Ib molecule HLA-E in mouse myeloma X63 cells cotransfected with the genes for HLA-E and human beta2m (hbeta2m), which is most likely attributable to inefficient intracellular peptide loading of the HLA-E molecule. Here we demonstrate that cell surface expression of HLA-E in mouse cells strictly depends on the coexpression of hbeta2m and that soluble empty complexes of HLA-E and hbeta2m display a low degree of thermostability. Both observations imply that low affinity interaction of HLA-E with beta2m accounts to a considerable extent for the observed low degree of peptide uptake in the endoplasmic reticulum. Moreover, we show that the only allelic variation present in the caucasoid population located at amino acid position 107 (Gly or Arg) greatly affects intracellular transport and cell surface expression upon transfection of the respective alleles into mouse cells. No obvious difference was found with regard to the sequence of the peptide ligand.

Alleles↗
PubMed1999

Green fluorescent protein as a noninvasive stress probe in resting Escherichia coli cells.

We constructed and characterized three stress probe plasmids which utilize a green fluorescent protein as a noninvasive reporter in order to elucidate Escherichia coli cellular stress responses in quiescent or resting cells. Cellular stress levels were easily detected by fusing three heat shock stress protein promoter elements, those of the heat shock transcription factor sigma32, the protease subunit ClpB, and the chaperone DnaK, to the reporter gene gfpuv. When perturbed by a chemical or physical stress (such as a heat shock, nutrient [amino acid] limitation, or addition of IPTG [isopropyl-beta-D-thiogalactopyranoside], acetic acid, ethanol, phenol, antifoam, or salt [osmotic shock]), the E. coli cells produced GFPuv, which was easily detected within the cells as emitted green fluorescence. Temporal and amplitudinal mapping of the responses was performed, and the results revealed regions where quantitative delineation of cell stress was afforded.

Blotting, Western↗
PubMed1998

Green fluorescent protein as a reporter in rapid screening of antituberculosis compounds in vitro and in macrophages.

The development of new drugs against Mycobacterium tuberculosis is impeded by slow growth and highly infectious nature of the organism that warrants the need to work under highly stringent biosafety conditions. These problems can be overcome by use of reporter genes and surrogate strains. A strain of rapidly growing M. aurum has been recommended as test organism to screen inhibitors of mycobacteria to preselect compounds for progression into testing against M. tuberculosis. We have investigated the application of recombinant M. aurum expressing green fluorescent protein in rapid screening of antituberculosis compounds in vitro and in infected macrophages. Recombinant M. aurum[pGFM-11] expressing green fluorescent protein was constructed. The assay is based on measurement of fluorescent intensity at 509 nm. A good correlation was found between fluorescence and growth. Fluorescence of recombinant M. aurum was inhibited in vitro within 8 to 24 h by frontline antimycobacterial drugs at their reported MICs whereas inhibition in infected macrophages was observed in 72 h. Therefore green fluorescent reporter system provides a convenient screen to test antimycobacterial compounds that are active in vitro and within infected macrophages.

Animals↗
PubMed1998

Divergent evolutionary constraints on mitochondrial and nuclear genomes of malaria parasites.

Genetic variation among malaria parasites has important consequences with regard to drug resistance, pathogenicity, immunity, transmission, and speciation. In this regard, malaria parasites have been shown to display a high degree of inter- and intra-species genetic divergence. The nuclear genomes of Plasmodium falciparum, Plasmodium yoelii, and Plasmodium gallinaceum are vastly divergent yet share a similar codon usage and total A/T content of approximately 82%. This is in contrast to other primate-specific species including P. vivax which have an A/T content of approximately 67%. To assess the effects of this evolutionary divergence on the conservation of gene content, organization, and codon usage in the mitochondrial DNA (mtDNA) of malaria parasites, we have cloned and sequenced the mitochondrial genome of Plasmodium vivax, and compared it with the mtDNAs of P. falciparum, P. yoelii, and P. gallinaceum. The P. vivax mitochondrial genome was found to be 5990 base pairs in length, and displayed a gene organization identical to that of P. falciparum, P. yoelii, and P. gallinaceum. Furthermore, there was a remarkable 90% conservation of sequence identity between the mitochondrial genomes of all four species. As an example of intra-species conservation, comparison of mtDNAs from two independently cloned P. falciparum isolates, Malay Camp and C10, revealed only a single nucleotide substitution. A/T content of the P. vivax mitochondrial genome was found to be identical to other species of Plasmodium, hence, we have postulated that the mitochondrial genomes of malaria parasites were refractory to the evolutionary shifts in nucleotide content seen among the nuclear genomes of malaria parasites. Among different Plasmodium species, the second position of mitochondrial codons were found to be the least prone to substitutions and displayed a significant bias in pyrimidines. These aspects of mitochondrial codon usage were distinct from the nuclear genome and may reflect functional aspects of decoding by the mitochondrial translational system.

Amino Acid Sequence↗
PubMed1998

Interaction of HLA-E with peptides and the peptide transporter in vitro: implications for its function in antigen presentation.

The assembly of MHC Ia molecules in the endoplasmic reticulum requires the presence of peptide ligands and beta2m and is facilitated by chaperones in an ordered sequence of molecular interactions. A crucial step in this process is the interaction of the class I alpha-chain/beta2m dimer with TAP, which is believed to ensure effective peptide loading of the empty class I molecule. We have previously demonstrated impaired intracellular transport of the class Ib molecule HLA-E in mouse myeloma cells cotransfected with the genes for HLA-E and human beta2m, which is most likely attributable to inefficient intracellular peptide loading of the HLA-E molecule. We therefore analyzed the ability of HLA-E in the transfectant cell line to bind synthetic peptides by means of their ability to enhance cell surface expression of HLA-E. Peptide binding was confirmed by testing the effect on the thermostability of soluble empty HLA-E/human beta2m dimers. Two viral peptides binding to HLA-E were thus identified, for which the exact positioning of the N terminus appeared critical for binding, whereas the contribution of the length of the C terminus seemed to be minor, allowing peptides as short as seven amino acids and up to 16 amino acids to exhibit considerable binding activity. Furthermore, we demonstrate that HLA-E interacts with TAP and that this interaction can be prolonged by the proteasome inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucinal, which reduces the intracellular peptide pool. The presented data indicate that HLA-E is capable of presenting peptide ligands similar to the repertoire of HLA class Ia molecules.

Amino Acid Sequence↗
PubMed1998

Differential expression of proteins during healing of cutaneous wounds in experimental normal and chronic models.

Recent studies have demonstrated growth factors and other cellular proteins as being important in the healing process. In this study, we have investigated the differential expression of proteins in wound tissues of normal and chronic animal models. Proteins were identified by specific antibodies, partial N-terminal amino acid sequence, and molecular weight homology. In normal wound tissues de novo synthesis of a heat shock protein, platelet derived growth factor (PDGF), and fibroblast growth factor (FGF) was induced within 24 h of skin injury. Proteins resembling vascular endothelial growth factor, receptors for PGDF, FGF, and epidermal growth factor, were synthesized. The elevated synthesis declined to a basal level in 7 to 14 days after skin injury which coincided with healing of wounds. These changes occurred only in wound site tissues but not in distal tissues. In contrast, the chronic wounds presented a different picture. The expressions of these proteins were either delayed or inhibited. This suggested the role of these proteins during normal and chronic wound healing. The proteins which were down regulated in chronic wounds may be used in the management of wounds and exploited as targets for therapeutic development.

Animals↗
PubMed1998

Spectral sensitivity of cones in the goldfish, Carassius auratus.

The spectral sensitivities of retinal cones isolated from goldfish (Carassius auratus) retinas were measured in the range 277-737 nm by recording membrane photocurrents with suction pipette electrodes (SPE). Cones were identified with lambda max (+/- S.D.) at 623 +/- 6.9 nm, 537 +/- 4.7 nm, 447 +/- 7.7 nm, and about 356 nm (three cells). Two cells (lambda max 572 and 576 nm) possibly represent genetic polymorphism. A single A2 template fits the alpha-band of P447(2), P537(2), and P623(2). HPLC analysis showed 4% retinal:96% 3-dehydroretinal. Sensitivity at 280 nm is nearly half that at the lambda max in the visible. The lambda max of the beta-band (in nm) is a linear function of the lambda max of the alpha-band and follows the same relation as found for A1-based cone pigments of a cyprinid fish.

Animals↗
PubMed1998

Genetic rearrangements leading to disruption of heterologous gene expression in mycobacteria: an observation with Escherichia coli beta-galactosidase in Mycobacterium smegmatis and its implication in vaccine development.

Different mycobacteria carrying cloned genes for heterologous protective antigens have been proposed as vaccine vehicles. In this study, the stability of the expression of beta-galactosidase was studied in Mycobacterium smegmatis using integrative (pMV361::lacZ) and replicative (pMV261::lacZ) vectors. Recombinant M. smegmatis forms blue colonies on X-gal plates. Occasional white mutants encountered while plating on X-gal plates were genetically analysed. The loss of lacZ phenotype was due to insertion of an IS element in lacZ gene of integrative vector whereas in case of replicative vectors, loss of lacZ phenotype was due to deletions of different sizes in the lacZ gene and the Phsp60 promoter region. The frequency of such events was rare, 1.7 x 10(-5) in integrative vector and 2 x 10(-3) in the case of replicative vector. The integrative vector seemed more stable in terms of expression of foreign genes in mycobacteria. Hence, the rearrangements reported in the present study warrant serious consideration before implementing mycobacteria as recombinant vaccines.

Bacterial Vaccines↗
PubMed1998

Changing scenario of therapeutic apheresis in India in the last 14 years.

There has been a remarkable change in the scenario of therapeutic apheresis in the last 14 years in India. The crude method of manual removal of blood followed by separation of plasma by gravity, keeping it in the bottle for a long time, has now been totally replaced by plasmapheresis, centrifugation, membrane filtration, and immunoadsorption techniques. The indications for use have also changed from a list of limited indications in the beginning to include all immune complex disorders. The clinical beneficiaries have also increased from blood bankers to nephrologists and immunologists in addition to oncologists. Efforts are now underway with the help of the Indian Society for Apheresis (founded in 1985) to popularize the newer methods of cryofiltration, photopheresis and heparin extracorporeal low-density lipoprotein (HELP) and DALI apheresis systems besides the specialized techniques of immunoadsorption using filters, columns, or ligands. This is suggestive of a positive trend for the treatment of immune complex disorders.

Blood Component Removal↗
PubMed1998

A cluster of cases of severe cardiotoxicity among kala-azar patients treated with a high-osmolarity lot of sodium antimony gluconate.

In India, sodium antimony gluconate is the drug of choice for kala-azar. Due to increasing unresponsiveness to this drug in the current epidemic that began in the early 1970s, daily doses of 20 mg/kg/day for 30 days or more is recommended as opposed to the 10 mg/kg/day dose for 6-10 days used in the past. Of the 130-150 patients treated annually at our center with locally made sodium antimony gluconate, serious cardiotoxicity has occurred in less than 10%. During April 1995 at the University Hospital in Varanasi, we encountered life-threatening cardiotoxicity after 3-28 days of therapy in each of the eight patients being treated with a new lot of this drug made by a different manufacturer. Of the eight patients, six each developed congestive heart failure and/or prolongation of the corrected QT interval (QTc), and three died as a direct consequence of drug-induced toxicities. In three instances, the life-threatening complications occurred with a cumulative dose of less than 300 mg/kg. In patients with prolonged QTc, ventricular premature beats and ventricular tachycardia were recorded; in one patient, the ventricular tachycardia progressed to torsade de pointes, culminating in ventricular fibrillation and death. Since switching to different lots of this drug, we have not seen further clustering of dangerous cardiotoxicity. The antimony content of the implicated drug was comparable with that in lots from other manufacturers that did not show overt toxicity, but the osmolarity was approximately 300 mOsm/L higher. The simple technique of measuring of osmolarity may help identify inappropriately manufactured drug.

Adult↗
PubMed1998

Diagnosis of tuberculous meningitis by detection of antigen and antibodies in CSF and sera.

OBJECTIVE: To evaluate diagnostic potential of three immunological tests, namely, detection of H37Rv antigen of M. Tuberculosis in CSF, detection of antibodies (IgG) against H37Rv in CSF and detection of antibodies (IgG) against H37Rv in serum for diagnosis of tuberculous meningitis in children. SUBJECTS: 50 children diagnosed as patients of tuberculous meningitis were included as cases and 48 children with CNS diseases of nontubercular etiology [pyogenic meningitis (n = 31), encephalitis (n = 10), seizure disorder of unknown etiology (n = 5), brain tumor (n = 2)] served as controls. METHODS: H37Rv antigen of M. tuberculosis was detected in CSF by Dot ELISA, and antibodies (IgG) against H37Rv in CSF and serum were detected by Plate ELISA. RESULTS: Detection of H37Rv antigen in CSF was the most sensitive (90%) and specific (95.83%) with positive and negative predictive values of 95.74% and 90.19%, respectively, followed by detection of antibodies in CSF (sensitivity-74%, specificity-89.58%, positive predictive value-88.10%, negative predictive value-76.78%). Detection of antibodies in serum had low sensitivity (50%), specificity (91.67%), positive predictive value (86.21%) and negative predictive value (63.76%). CONCLUSIONS: Detection of antigen in CSF is a rapid, sensitive and specific test for diagnosis of tuberculous meningitis in children. Detection of antibody in CSF may be useful in some cases but needs further evaluation. Detection of antibody in serum does not appear to be useful for diagnosis of tuberculous meningitis.

Antigens, Bacterial↗
PubMed1997

Recombinant Mycobacterium aurum expressing Escherichia coli beta-galactosidase in high throughput screening of antituberculosis drugs.

Mycobacterium aurum is considered a surrogate of M. tuberculosis and recently has been proposed as test organism in high throughput screening of antituberculosis drugs (3). In this investigation, we suggest use of a recombinant M. aurum expressing E. coli lacZ gene, in which beta-galactosidase production is the reporter system as recently reported by us (6). The assay is based on production of beta-galactosidase in presence of drugs during growth. Enzyme production was inhibited within 4 h by frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their MICs. The assay could be performed conveniently in 96-well microtiter plate format.

Antitubercular Agents↗
PubMed1997

beta-Galactosidase reporter system in mycobacteria and its application in rapid antimycobacterial drug screening.

Pathogenic mycobacteria are generally slow growing organisms and it takes several weeks to evaluate inhibitors of growth. Therefore, for rapid screening of the inhibitors of mycobacterial growth, a beta-galactosidase reporter system has been described which utilises a recombinant Mycobacterium smegmatis mc(2)155 expressing E. coli lacZ gene as the test organism. The assay is based on production of beta-galactosidase in presence of drugs during growth. A correlation between beta-galactosidase production and colony forming ability of mycobacteria was obtained. beta-galactosidase production was inhibited within 6 h by front line standard antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, pyrazinamide and ofloxacin at their reported MICs. The assay was performed on mycobacterial cells permeabilized with chloroform and sodium dodecyl sulfate.

Anti-Bacterial Agents↗
PubMed1997

Characterization of simian malarial parasite (Plasmodium knowlesi)-induced putrescine transport in rhesus monkey erythrocytes. A novel putrescine conjugate arrests in vitro growth of simian malarial parasite (Plasmodium knowlesi) and cures multidrug resistant murine malaria (Plasmodium yoelii) infection in vivo.

A stage-dependent increase in the level of putrescine, spermidine, and spermine during intraerythrocytic growth of Plasmodium knowlesi in rhesus monkey erythrocytes was observed. Further, intraerythrocytic P. knowlesi-induced putrescine influx was found in trophozoite stage-infected erythrocytes and process was time- and temperature-dependent and showed saturable kinetics. Characteristics of induced putrescine influx appears in infected erythrocytes to be close to the normal erythrocytes in terms of affinity of putrescine to the putrescine transporter (Km 34.6 +/- 3.8 microM as normal erythrocytes and Km 37.2 +/- 5.2 microM in infected erythrocytes). However, the difference involves the significant increase in the putrescine influx rate after infection (Vmax = 4.21 nmol/min/10(10) normal erythrocytes, compared with 11.6 nmol/min/10(10) infected erythrocytes). Energy dependence, involvement of -SH group, and noninterference by amino acid, spermidine, and spermine in the putrescine influx process clearly demonstrate the presence of a distinct transporter for putrescine in infected erythrocytes. A putrescine conjugate N1,N4-bis(7-chloroquinoline-4-yl)butane-1, 4-diamine (BCBD) was synthesized, which inhibits the putrescine influx in the P. knowlesi infected erythrocytes (Ki of 43.2 microM) as well as in vitro growth of P. knowlesi (IC50 value, 7.64 +/- 0.97 ng/ml BCBD, 10.8 +/- 0.45 ng/ml chloroquine). Addition of exogenous polyamines failed to reverse the inhibitory effect of BCBD in vitro. Administration of BCBD (24 mg/kg body weight, intraperitoneal, twice a day for 4 days) cured the Swiss mice infected with multidrug-resistant infection of Plasmodium yoelii. Therefore, inhibition of putrescine transport in malaria-infected erythrocytes offers a lead in the search of a new class of chemotherapeutic molecules against malaria.

Aminoquinolines↗
PubMed1997

Screening of TnphoA mutants of Vibrio cholerae O139 for identification of antigens involved in colonisation.

A new serogroup of Vibrio cholerae non-O1, designated as O139, has emerged causing cholera-like disease among adults. Laboratory and field studies clearly show that there is no cross-protection between O1 and O139 pathogenic strains. Since colonisation of the intestine is a most important step in the pathogenesis of cholera caused by O1 strains and colonising antigens are known to be protective, investigation of the colonising antigens of O139 strain was initiated. By TnphoA mutagenesis, mutants were generated with insertions in the genome encoding membrane spanning or secretory proteins. Screening of the mutants for adherence to rabbit intestinal surface and colonisation in 5-day-old mice resulted in the identification of mutant clones, which were less adhesive than was the wild-type parent strain and which could not efficiently colonise the gut. Such non-colonising strains were attenuated in virulence. Analysis of the proteins by SDS-PAGE revealed that the non-colonising mutants did not express a 40-kDa outer-membrane protein.

Animals↗
PubMed1997

Comparison of immunomodulation by cyclosporin A and azathioprine on pancreatic xenograft survival.

Prolonged survival of pancreatic xenografts in the muscles of diabetic rabbits under the immunomodulation of cyclosporin A and azathioprine was achieved. The mean graft survival time (as assessed by euglycemic status and histopathological findings) in Group A (azathioprine treated) was 36 days, in Group B (cyclosporin A treated) it was 69 days, and the difference was significant (p < 0.001). Adequate immunosuppression helps the pancreatic xenograft to normalize the blood glucose level (BGL) by prolonging its survival time. Our study demonstrates that xenotransplanted islet cells in a natural environment (pancreatic tissue in the form of thin slices) survive and function adequately in immunosuppressed recipients, and addition of cyclosporin A significantly prolongs survival of the xenograft. This discordant xenotransplantation model may be useful for future xenotransplantation studies.

Adjuvants, Immunologic↗
PubMed1997

On the mechanism of isomerization of ocular retinoids by the crayfish Procambarus clarkii.

The eyes of some crustaceans store substantial amounts of retinyl esters, with most of the retinol in the 11-cis configuration. Earlier work in this laboratory suggested that in lobster and crayfish the mechanism of isomerization of retinol to the 11-cis form involves the hydrolysis of all-trans retinyl esters. Although this is the same process as that occurring in the vertebrate eye, it is different from the retinal photoisomerase reaction known in other arthropods, specifically diurnal insects (Hymenoptera and probably Diptera). Using homogenates of crayfish, we have tested this proposed mechanism by inhibiting retinyl ester synthetase activity in the presence of exogenous all-trans retinol. Inhibition of lecithin:retinol acyl transferase with 5 mumol l-1 retinyl bromoacetate or 2 mmol l-1 phenylmethylsulfonyl fluoride blocks the formation of both all-trans and 11-cis retinyl esters as well as 11-cis retinol, as shown by direct assay and by the decrease in counts derived from tritiated all-trans retinol. The similarity of this isomerization to the mechanism in vertebrate pigment epithelium is thus an interesting example of convergent evolution in the biochemistry of visual pigments, in which the pigments themselves (the opsins) are largely conserved across phyla.

Animals↗