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Biomedical subjects

V R Prasad

Publications and source records attributed to V R Prasad.

14 recordsLinked to original sources
PubMed1992

Mutagenesis of the Glu-89 residue in human immunodeficiency virus type 1 (HIV-1) and HIV-2 reverse transcriptases: effects on nucleoside analog resistance.

A Glu-89-->Gly alteration in the human immunodeficiency virus type 1 reverse transcriptase (RT) was previously shown to result in resistance to several dideoxynucleoside analogs and to phosphonoformic acid (PFA; foscarnet). This residue was altered to Ala, Val, Ser, Thr, Gln, Asp, Asn, or Lys, and the ddGTP and PFA sensitivities of the mutant RTs were measured. Replacements with Ala, Gly, Val, and Thr led to resistance to inhibition by ddGTP, while mutants with amino acid Ser, Gln, Asn, Asp, or Lys displayed only moderate or no resistance. A similar result was obtained with inhibition by PFA, except that the Asp-89 mutant also displayed resistance. Furthermore, the introduction of Glu-89-->Gly alteration into the RT of human immunodeficiency virus type 2 likewise rendered it resistant to both ddGTP and PFA.

Amino Acid Sequence↗
PubMed1992

Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities.

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a dideoxyguanosine triphosphate-resistant mutant of human immunodeficiency virus reverse transcriptase.

The appearance of drug-resistant strains of viral pathogens is a major difficulty confounding current efforts to block viral infections. The identification and analysis of mutations responsible for drug resistance can provide important clues helpful in understanding the mechanisms of resistance and in the eventual development of better therapies. We have used a direct screening method to scan libraries of mutagenized genes encoding the reverse transcriptase of human immunodeficiency virus type 1, and have recovered a variant enzyme that is resistant to the chain-terminator inhibitor 2',3'-dideoxyguanosine triphosphate. The single substitution mutation in this variant conferred broad crossresistance to a variety of other antiviral compounds currently in clinical trials. Virus carrying the mutation was fully infectious in cultured human lymphocytes. The replication of the mutant virus was highly resistant to phosphonoformic acid but did not show increased resistance to the prodrug dideoxyguanosine.

Antiviral Agents↗
PubMed1990

Expression of enzymatically active reverse transcriptase of simian immunodeficiency virus in bacteria: sensitivity to nucleotide analogue inhibitors.

A fragment of the SIVmac251 pol gene was expressed in Escherichia coli as a trpE fusion protein. Analysis of extracts from bacteria containing this expression plasmid revealed the presence of a reverse transcriptase activity dependent on Mg2+ as divalent cation and active on both poly(rA).oligo(dT) and poly(rC.oligo(dG) templates. In comparative studies, the SIV and HIV-1 reverse transcriptases expressed in bacteria displayed very similar high sensitivities to the chain terminator inhibitors AZTTP and ddTTP. The reverse transcriptase of Moloney murine leukemia virus and the DNA polymerase of E. coli were both more resistant to ddTTP, and the E. coli enzyme was significantly more resistant to AZTTP.

Dideoxynucleotides↗
PubMed1990

Structure-function studies of HIV reverse transcriptase.

The retroviral RT is properly under intensive study as the major target of antiviral therapy. The enzyme exhibits a number of features that make it an attractive target: it is crucial for viral replication; its RNA-dependent DNA polymerase activity is probably unique to viral replication, or if not unique, is generally unimportant in host cell function; its activities are readily monitored; and powerful lead compounds in the form of nucleotide analogues are already in hand. Our laboratory has been involved in studies to elucidate the structure and function of the HIV-1 RT and to develop a formal genetics of the enzyme. Working with constructs expressing RT in bacteria, we been able to use in vitro mutagenesis to localize functions on the molecule; by coupling mutagenesis with high-throughput screening of colonies, we have been able to isolate mutants with specific, rare, phenotypes. We believe that extensions of these efforts will help us to understand the functions of the protein and, coupled to a detailed three-dimensional structure, should facilitate the development of new and better inhibitors.

Antiviral Agents↗
PubMed1990

Endotoxin protection of rats from pulmonary oxygen toxicity: possible cytokine involvement.

Treatment with endotoxin protects rats against lung injury during hyperoxia (greater than 98% oxygen at 1 atmosphere absolute for 60 h). This study demonstrates that serum from endotoxin-treated donor rats also protects recipients from oxygen toxicity. Rats treated with serum from saline-treated donors were not protected, and protection was not explained by residual endotoxin in protective sera. Unlike endotoxin-protected rats (where lung antioxidant enzyme activity is elevated after hyperoxia), postexposure superoxide dismutase (SOD) and catalase (CAT) activities in the lungs of serum-protected rats were not affected. Levels of tumor necrosis factor (TNF) and interleukin 1 (IL-1) in protective sera were increased. This study demonstrates that increases in lung SOD and CAT activity are not required for endotoxin protection from hyperoxia and suggests that TNF and IL-1 may participate in the mechanism of endotoxin protection.

Animals↗
PubMed1990

Effect of ischemia reperfusion or hypoxia reoxygenation on lung vascular permeability and resistance.

The effect of ischemia reperfusion or hypoxia reoxygenation on pulmonary vascular permeability and resistance was studied in 25 isolated blood-perfused dog lungs. Vascular permeability, assessed by determining filtration coefficient (Kf), and vascular resistances were measured at the beginning and end of the experiment. Ischemia reperfusion was produced by occluding blood flow to the lung for 3 h and reperfusing for 1 h, whereas hypoxia reoxygenation was obtained by ventilating the lung with 95% N2-5% CO2 for 3 h and then ventilating with 95% O2-5% CO2 for 1 h with no interruption of perfusion. There was a significant increase in Kf in both ischemia reperfusion and hypoxia reoxygenation groups (51 and 85%, respectively), and total vascular resistance increased greatly in both groups (386 and 532%, respectively). Two additional groups were also studied in which the ischemia reperfusion or hypoxia reoxygenation lungs were pretreated with allopurinol (20 micrograms/ml). The Kf did not significantly increase in either the allopurinol ischemia reperfusion or the allopurinol hypoxia reoxygenation groups (22 and 6%, respectively). However, total vascular resistance significantly increased in both groups (239 and 224%, respectively). Although vascular permeability is modestly increased by both ischemia reperfusion and hypoxia reoxygenation, the predominant change in these conditions is the increased vascular resistance, which predominantly affects the postcapillary resistance and would result in a greater tendency for edema to develop in these slightly damaged lungs. Allopurinol, which inhibits xanthine oxidase, attenuated the permeability changes in both groups and may be useful in preventing ischemia reperfusion injury in certain conditions.

Animals↗
PubMed1989

A novel in situ colony screening method to detect human immunodeficiency virus reverse transcriptase activity expressed in bacteria. Isolation of pseudorevertants of reverse transcriptase mutants.

An in situ screening procedure is described that permits the detection of RNA-dependent DNA polymerase activity in bacterial colonies expressing the reverse transcriptase of the human immunodeficiency virus. The procedure has been applied to the isolation of rare pseudorevertants of an inactive parental mutant.

Amino Acid Sequence↗
PubMed1989

Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain.

A plasmid construct expressing the p66 version of the human immunodeficiency virus reverse transcriptase as a bacterial fusion protein was subjected to in vitro mutagenesis, and the resulting variant proteins were assayed to define the locations of the two major enzymatic activities. The DNA polymerase activity was localized to the N-terminal portion of the protein; mutations altering or eliminating the C-terminal portion had little or no effect on that activity. The results suggest that, in contrast with previous reports, the p51 subunit found in virions should exhibit DNA polymerase activity. Mutations in many parts of the protein eliminated RNase H activity, suggesting that several areas are needed for proper folding and generation of that activity.

DNA-Directed DNA Polymerase↗
PubMed1989

Pulmonary vascular permeability and resistance measurements in control and ANTU-injured dog lungs.

Because questions have arisen regarding pulmonary vascular permeability and resistance measurements in isolated, perfused lungs, we sought to determine the 1) stability of repeated measurements of permeability and resistance in control lungs; and 2) magnitude of change in these measurements when permeability was greatly increased. Using blood-perfused dog lungs, we measured filtration coefficient (Kf) and isogravimetric capillary pressure (Pci) as indexes of vascular permeability, and we also determined total vascular resistance (Rt) as well as the segmental resistances using the double-occlusion pressure (Pdo). In a control group (n = 8), the base-line measurement of Kf (0.21 +/- 0.02 ml.min-1.cmH2O-1.100 g-1) and Pci (10.2 +/- 0.9 cmH2O) did not change over 4 h, indicating no changes in endothelial barrier function. Base-line Rt (13.9 +/- 2.6 cmH2O.l-1.min.100 g) also did not significantly increase. In a second group (n = 5), alpha-naphthylthiourea (ANTU) increased the initial Kf more than eight times (from 0.17 +/- 0.03 to 1.40 +/- 0.32 ml.min-1.cmH2O-1.100 g-1) and decreased Pci by 56% (from 9.4 +/- 0.6 to 4.1 +/- 0.4 cmH2O) at 1 h, indicating severely damaged endothelium. In addition, the Pdo determined during isogravimetric conditions correlated very well with Pci not only in control lungs (observed previously) but also in very permeable lungs (not previously reported). We conclude that this experimental model provides an excellent means of assessing changes in pulmonary microvascular permeability, with a spectrum ranging from no changes in hourly measurements for 4 h to obvious changes in permeability by 1 h.

Animals↗
PubMed1988

Protective effects of O2 radical scavengers and adenosine in PMA-induced lung injury.

We have previously shown that phorbol myristate acetate (PMA) produces acute lung injury in blood-perfused lungs but not in plasma-dextran-perfused lungs. This is compatible with the concept that its major mechanism of injury is the stimulation of O2 radicals by neutrophils, which in turn increase permeability by damaging the endothelial cells. In this study we measured vascular permeability and resistance before and 1 h after PMA in five groups of blood-perfused dog lungs: PMA alone in one group and pretreatment with catalase, superoxide dismutase, deferoxamine, and adenosine each in four other groups. By the use of two indexes of permeability, the filtration coefficient and the isogravimetric capillary pressure, we found that, compared with PMA alone, catalase, deferoxamine, and adenosine provided significant protection, whereas the results with superoxide dismutase were variable. These four drugs also significantly attenuated the marked increased resistance seen with PMA alone. Although the effects seen with the first three can be explained by their scavenging of O2 radicals, adenosine appears to provide protection through a separate mechanism.

Adenosine↗
PubMed1979

Isolation and characterization of binding proteins for retinol from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens.

Protein fractions that bind retinol were isolated from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens. The proteins isolated from the three sources showed similar elution profiles on chromatography through Sephadex G-75 and G-50 columns, and comparable mobility during electrophoresis on sodium dodecyl sulphate/polyacrylamide gels. Their molecular weights were calculated to be around 14500. When oviducts from vitamin A-depleted and vitamin A-repleted immature chicks given oestrogen injections for 6 consecutive days were incubated with [3H]retinyl acetate, uptake of the radioactivity in the nuclei of the vitamin A-depleted tissue was severalfold higher than that in the nuclei from the vitamin A-repleted tissue.

Animals↗
PubMedDate not supplied

Structural requirements for bacterial expression of stable, enzymatically active fusion proteins containing the human immunodeficiency virus reverse transcriptase.

A collection of variant plasmids that express the human immunodeficiency virus (HIV) reverse transcriptase as trpE fusion proteins were generated and scored for their ability to produce stable, active proteins. Trimming portions of the viral pol gene resulted in dramatic increases in yield over earlier constructs; the accumulation of high levels of enzymatically active protein in this system was increased by the retention of the trpE sequences at the amino terminus. A new in situ gel activity assay was used to demonstrate that the major induced protein, containing approximately 68 kD of viral sequences, was the active species.

Cloning, Molecular↗