Biomedical subjects

R A Bhat

Publications and source records attributed to R A Bhat.

At least 19 recordsLinked to original sources

PubMed2006

Selective and opposing actions of progesterone receptor isoforms in human endometrial stromal cells.

Transcriptional regulation by progesterone is mediated primarily through the two progesterone receptor (PR) isoforms, PR-A and PR-B. Primary human endometrial stromal cell cultures, in which endogenous PR expression was lost, were infected with adenovirus expressing PR-A, PR-B, or both. Global gene expression analysis was conducted on vehicle and 30 nM progesterone (P4) treated cells following 12 h treatment. Interestingly, many genes regulated by PR-B alone or upon PR-A and PR-B co-expression, did not overlap with each other or with the PR-A expression group. Although many genes known to be progestin regulated in the uterus in vivo were also regulated in this study, markedly little overlap with published P4 regulated genes from human breast cancer cells was observed. Progesterone dose response curves were generated for several genes demonstrating gene selective potency and efficacy for each PR isoform. Furthermore, the PR isoforms opposed each other in regulation of tissue factor, with PR-B increasing and PR-A decreasing both mRNA and protein levels. Our data provide a view of global gene expression by PR isoforms in human endometrial cells and a comparison with other cell types. The specific genes and regulation patterns found provide groundwork to revealing the mechanism of PR isoform selectivity, and perhaps ultimately to the tissue selective properties these receptors appear to exhibit.

Cells, Cultured↗

PubMed2006

A re-look at the duration of human pregnancy.

INTRODUCTION: The duration of human pregnancy is arbitrarily taken as 280 days (40 weeks). Foetuses are considered to be at high risk once pregnancy goes beyond the expected date of confinement. This study was carried out with the aim of determining the mean gestation age of low-risk pregnancies that went into spontaneous labour and the incidence of adverse outcomes in relation to gestation. METHODS: Low-risk singleton pregnancies admitted in spontaneous labour at a single community hospital in the Udupi district of Karnataka in South India, from December 2002 to December 2003, were analysed for mean gestational age at the onset of spontaneous labour and rates of perinatal complications by gestational age. RESULTS: Among the 1,094 women who went into spontaneous labour, the mean gestational age was 272.1 +/- 9 days. A significantly increased incidence of meconium-stained amniotic fluid beyond 39 weeks of gestation was observed. 783 of 1,094 women (80 percent) had delivered during the period of 261-280 days of pregnancy (period of one standard deviation around the mean gestational age at delivery). There was significant increase in perinatal morbidity indicators and mortality rates once the pregnancy carried beyond 280 days. CONCLUSION: Mean gestational age at the onset of labour for women native to the area of study was 272 days (standard deviation 9 days). Pregnancies beyond a duration of 280 days showed significantly increased perinatal morbidity. It is suggested that there is a need for determining the length of gestation and to compile gestation-wise incidence of meconium-stained amniotic fluid as an indicator of foetal maturity or the undisclosed risk factor, in addition to other neonatal morbidity indicators for different populations.

Adolescent↗

PubMed1999

Enhanced immunogenicity of hepatitis B surface antigen by insertion of a helper T cell epitope from tetanus toxoid.

The currently marketed hepatitis B vaccines in the U.S. are based on the recombinant major hepatitis B surface antigen (HBsAg) of hepatitis B virus. Although a large majority of individuals develop protective immunity to HBV-induced disease after three immunizations, routinely a small but a significant percentage of the human population does not respond well to these vaccines. In this report, we describe the generation of a novel HBsAg molecule containing a Th epitope derived from tetanus toxoid (TT). Using recombinant DNA technology. the TT Th epitope (TTe) was inserted into the HBsAg coding sequence. Using a recombinant adenovirus expression system, HBsAg TTe chimeric protein was produced in A549 cells and found to be secreted into culture medium as 22 nm particles. The chimeric HBsAg particles were readily purified by immunoaffinity chromatography and their immunogenicity was evaluated relative to native HBsAg produced in an adenovirus expression system. When evaluated in inbred and outbred strains of mice, HBsAg TTe was shown to enhance several-fold the anti-HBs response relative to native HBsAg. Further enhanced responses were observed in mice primed with TT. This highly immunogenic form of HBsAg has promise as an improved HBsAg subunit vaccine.

Animals↗

PubMed1999

The hepatitis C virus NS2 protein generated by NS2-3 autocleavage is required for NS5A phosphorylation.

Hepatitis C virus (HCV) is dependent on NS2-3 autocleavage and NS5A phosphorylation for its life cycle. We demonstrate that NS5A, when released from the NS2-5 polyprotein of the BK virus strain, is phosphorylated as two distinct forms, pp56 and pp58. Deletion analysis indicates that the appearance of pp58 requires NS2 in cis, while pp56 is NS2 independent. Disruption of NS2-3 autoproteolysis by directed mutagenesis results in loss of pp58. Expression of a construct producing NS2-3 is sufficient to restore pp58 in trans. These data indicate that generation of functional NS2 via autocleavage of the NS2-3 precursor and NS5A phosphorylation are consecutive processes, suggesting coordinate regulation during virus propagation in vivo.

Animals↗

PubMed1998

Estrogen regulation of the apolipoprotein AI gene promoter through transcription cofactor sharing.

Estrogen replacement therapy increases plasma concentrations of high density lipoprotein and its major protein constituent, apolipoprotein AI (apoAI). Studies with animal model systems, however, suggest opposite effects. In HepG2 cells stably expressing estrogen receptor alpha (ERalpha), 17beta-estradiol (E2) potently inhibited apoAI mRNA steady state levels. ApoAI promoter deletion mapping experiments indicated that ERalpha plus E2 inhibited apoAI activity through the liver-specific enhancer. Although the ERalpha DNA binding domain was essential but not sufficient for apoAI enhancer inhibition, ERalpha binding to the apoAI enhancer could not be detected by electrophoretic mobility shift assays. Western blotting and cotransfection assays showed that ERalpha plus E2 did not influence the abundance or the activity of the hepatocyte-enriched factors HNF-3beta and HNF-4, two transcription factors essential for apoAI enhancer function. Expression of the ERalpha coactivator RIP140 dramatically repressed apoAI enhancer function in cotransfection experiments, suggesting that RIP140 may also function as a coactivator on the apoAI enhancer. Moreover, estrogen regulation of apoAI enhancer activity was dependent upon the balance between ERalpha and RIP140 levels. At low ratios of RIP140 to ERalpha, E2 repressed apoAI enhancer activity, whereas at high ratios this repression was reversed. Regulation of the apoAI gene by estrogen may thus vary in direction and magnitude depending not only on the presence of ERalpha and E2 but also upon the intracellular balance of ERalpha and coactivators utilized by ERalpha and the apoAI enhancer.

Adaptor Proteins, Signal Transducing↗

PubMed1998

A novel human estrogen receptor beta: identification and functional analysis of additional N-terminal amino acids.

A novel human estrogen receptor beta (hERbeta) was cloned from human testis mRNA, ovary and thymus cDNA utilizing PCR and 5' RACE methods. The 5' end of hERbeta contained an additional open reading frame, in-frame and upstream of the published clones. hERbeta encodes a protein of 530 amino acids with an approximate molecular weight of 63 kDa and is larger than the previously reported rat, mouse and human protein. To determine the functional role of additional N-terminal amino acids, we compared the transcription functions of receptor lacking (hERbetaT) and receptor containing (hERbetaL) this N-terminal extension. hERbetaL is more active than hERbetaT in transactivating ERE-based reporter genes. hERbetaL, but not hERbetaT, attenuated cytokine mediated NFkappaB activation. Taken together, the additional N-terminal amino acids appear to play a role in modulating estrogen responsive gene expression in vitro.

Amino Acid Sequence↗

PubMed1997

Functional properties of a conditionally phenotypic, estrogen-responsive, human osteoblast cell line.

Osteoblasts are established targets of estrogen action in bone. We screened 66 conditionally immortalized clonal human osteoblast cell lines for estrogen receptors (ERs) using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ER alpha mRNA and transactivation of adenovirus-estrogen response element (ERE)-tk-luciferase by 17 beta-estradiol (17 beta-E2) for functional ER protein. One of these cell lines, termed HOB-03-CE6, was chosen for further characterization. The cells, which were conditionally immortalized with a temperature-sensitive SV40 large T antigen, proliferated at the permissive temperature (34 degrees C) but stopped dividing at the nonpermissive temperature (> or = 39 degrees C). Alkaline phosphatase activity and osteocalcin secretion were upregulated by 1 alpha, 25-dihydroxyvitamin D3 in a dose-dependent manner. The cells also expressed type I collagen and other bone matrix proteins, secreted a variety of growth factors and cytokines, formed mineralized nodules based on alizarin red-S and von Kossa histochemical staining, and responded to dexamethasone, all-trans retinoic acid, and transforming growth factor-beta 1. This cell line expressed 42-fold less ER message than MCF-7 human breast cancer cells, as determined by quantitative RT-PCR. However, adenovirus-ERE-tk-luciferase activity was upregulated three- to fivefold in these cells by 17 beta-E2 with an EC50 of 64 pM. Furthermore, this upregulation was suppressed by co-treatment with the anti-estrogen ICI-182, 780. Cytosolic extracts of these cells specifically bound [125I]-17 beta-E2 in a concentration-dependent manner with a Bmax of 2.7 fmoles/mg protein (approximately 1,200 ERs/cell) and a Kd of 0.2 nM. DNA gel-shift analysis using a [32P]-ERE demonstrated the presence of ERs in nuclear extracts of these cells. Moreover, binding of the extracts to this ERE was blocked by a monoclonal antibody to the human ER DNA-binding domain. We evaluated these cells for 14 of 20 reported endogenous responses to 17 beta-E2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17 beta-E2 suppressed parathyroid hormone-induced cAMP production, as well as basal interleukin-6 mRNA expression; conversely, the steroid upregulated the steady-state expression of alkaline phosphatase message in these cells. In summary, we have identified a clonal, conditionally phenotypic, human osteoblast cell line that expresses functional ERs and exhibits endogenous responses to 17 beta-E2. This cell line will be a valuable in vitro model for exploring some of the molecular mechanisms of estrogen action in bone.

Aged↗

PubMed1997

Replication and immunogenicity of Ad7-, Ad4-, and Ad5-hepatitis B virus surface antigen recombinants, with or without a portion of E3 region, in chimpanzees.

Human adenovirus vectors containing intact or largely deleted E3 region were used to construct adenovirus-hepatitis B recombinant viruses (Ad-HepB) and shown to produce substantial amount of recombinant protein, hepatitis B surface antigen (HBsAg), in tissue culture. Previously we showed that these viruses were able to elicit good anti-HBs antibodies in a dog model. In the present study, the Ad-HepB viruses were evaluated for replication and immunogenicity in chimpanzees which sustain permissive infection by human adenoviruses. Recombinants containing entire E3 region showed better replication pattern than their E3 deleted counterparts as evidenced by longer duration and high titers of virus shedding. The effect of E3 region was also seen in the antibody titers against HBsAg in that the E3 containing viruses showed better response than the E3 deleted viruses. The importance of E3 region for the development of adenovirus vectored vaccines is further discussed.

Adenovirus E3 Proteins↗

PubMed1997

Regulated processing of hepatitis C virus core protein is linked to subcellular localization.

Posttranslational processing and subcellular localization of the HCV core protein are critical steps involved in the assembly of hepatitis C virus (HCV). In this study, both of these events were investigated by in vitro translation and transient COS-1 cell transfection of core protein expression constructs. Mutations at amino acid residues 173 to 174 and 191 to 192 disrupted processing events at the two putative cleavage sites in the C-terminal hydrophobic region of the core protein, indicating that these residues are implicated in the pathway of core protein maturation. As a result, two forms of core protein, C173 and C191, were detected by immunoblotting. Indirect immunofluorescence experiments showed that core proteins C173 and C191, when produced from HCV full-length protein or various polyprotein precursors, displayed a cytoplasmic localization. The C173 species, however, was translocated to the nucleus when expressed in the absence of C191. These findings indicate that preferential cleavage may occur during core protein maturation and that the association of the C191 with the C173 species may contribute to the distinct subcellular distribution of core protein. This may provide a possible mechanism for the control of the diverse biological functions of core protein during HCV replication and assembly.

Amino Acid Sequence↗

PubMed1994

Substrate specificity of the NS3 serine proteinase of hepatitis C virus as determined by mutagenesis at the NS3/NS4A junction.

Hepatitis C virus (HCV) encodes a polyprotein that is processed to produce the structural and nonstructural proteins of the virus. Nonstructural protein 3 (NS3) is a serine proteinase that cleaves the polyprotein to release the NS4A, NS4B, NS5A, and NS5B proteins. To characterize the substrate specificity of NS3, we synthesized by in vitro translation the polyprotein NS2*-NS3-NS4*P that includes 70% of the NS2 protein, the complete NS3 protein, and 25% of the NS4 protein region attached to substance P, an epitope tag. We demonstrated that NS3 cleaves at the NS3/NS4A junction to release the NS4*P protein. Subsequently, we used this reaction to evaluate the importance of conserved amino acids that flank the NS3/NS4A junction. We replaced amino acids in the P6, P1, and P1' positions of the scissile bond of this junction using site-directed mutagenesis. When the P6 aspartic acid was changed to asparagine, lysine, or serine, NS3-mediated cleavage occurred. When threonine in the P1 position was replaced with other polar amino acids or with amino acids having aliphatic side chains, cleavage occurred, although it was not detected when arginine or tyrosine was present. Replacement of serine in the P1' position with other polar amino acids, with amino acids having aliphatic side chains, or with arginine resulted in NS3-mediated cleavage. Thus, since fewer amino acids in the P1 position supported cleavage than in the P6 or P1' positions, the P1 position of the scissile bond may play a more important role in defining the substrate specificity of the HCV NS3 proteinase.

Amino Acid Sequence↗

PubMed1994

Efficacy of adenovirus-vectored respiratory syncytial virus vaccines in a new ferret model.

In the absence of an adequate small animal model for testing the efficacy of adenovirus-vectored respiratory syncytial virus (RSV) vaccines, a ferret model was established for this purpose. Recombinant adenovirus types 4, 5 and 7 expressing the RSV fusion glycoprotein (F), the attachment glycoprotein (G) or both F and G were constructed previously. These recombinants contain a deletion of a large portion of the E3 region of the respective adenovirus vector. In addition, an Ad7(E3+)F recombinant virus which contains an intact E3 region was constructed to assess whether E3 region functions might enhance vaccine immunogenicity. Evaluation of these viruses in the ferret model demonstrated that Ad4 and Ad5 recombinants, administered intranasally to ferrets, induce stronger seroresponses to RSV than do Ad7 recombinant viruses. Ad7(E3+)F did not show enhanced immunogenicity relative to E3-deleted recombinant viruses. However, measurement of RSV infectivity in nasal washes, following intranasal RSV challenge, showed that five different vaccination regimens, Ad7F/Ad4F, Ad7G/Ad4G, Ad7FG/Ad4FG, Ad4F/Ad7(E3+)F and Ad5F/Ad4F, protected ferrets from RSV infection in a dose-dependent manner.

Animals↗

PubMed1992

Immunogenicity of recombinant adenovirus-respiratory syncytial virus vaccines with adenovirus types 4, 5, and 7 vectors in dogs and a chimpanzee.

Recombinant adenovirus type 4, 5, and 7 expressing the fusion glycoprotein (F) gene, the attachment glycoprotein (G) gene, or both F and G genes of respiratory syncytial virus (RSV) was constructed. Intratracheal immunization of dogs with Ad7F induced moderate titers of RSV-neutralizing antibodies. After booster immunization with Ad4F, the dogs developed high titers of RSV-specific antibody. Subsequently, three two-dose vaccination regimens, Ad4F/Ad5F, Ad7G/Ad4G, and Ad7FG/Ad4FG, were compared with Ad7F/Ad4F for immunogenicity and protective efficacy. The results indicated that Ad4F/Ad5F was equal or greater in immunogenicity to Ad7F/Ad4F, but Ad7G/Ad4G and Ad7FG/Ad4FG were less effective than Ad7F/Ad4F in inducing RSV-neutralizing antibody. All vaccination regimens completely protected the lungs of dogs from RSV infection. A chimpanzee was sequentially immunized orally with Ad7F, Ad4F, and Ad5F. A low-level antibody response to RSV was induced after the primary immunization, but no significant increases were observed after booster immunizations.

Adenoviruses, Human↗

PubMed1991

Adenovirus vectors for gene expression.

Adenoviruses possess a combination of features that make them highly suitable as vectors for expression of heterologous genes. Non-conditional and non-defective adeno-vectors have been constructed to obtain high level expression of a number of foreign genes and some of them have been shown in animal models to exhibit excellent promise as vaccine candidates.

Adenoviridae↗

PubMed1990

Molecular characterization of a new variant of hepatitis B virus in a persistently infected homosexual man.

Based on the diversity of nucleotide sequences of cloned hepatitis B virus DNA genomes, we have predicted possible replication of genetic variants of human hepatitis B virus. This prediction is exemplified by studies of a chronic carrier of HBsAg/adw2, who lacked anti-HBc but carried exceedingly high levels of hepatitis B virus DNA in serum. Molecular characterization of a number of clones revealed a restriction map that deviated significantly from the typical pattern of the adw2 subtype, especially around the EcoRI site commonly used as a reference point. Mutations appearing consistently in the precore and core regions included (a) mutation in the precore region resulting in a termination codon after the initiation codon, (b) mutation of the core initiation codon and (c) an inframe insert of 36 nucleotides in the precore region with a new initiation site for the core protein. The 36-nucleotide insertion resulted in a new core protein with 12 extra amino acids at its amino-terminal end. A few scattered point mutations were clustered in the amino-terminal half of the core gene. Although the core protein of this hepatitis B virus variant carried immunologically detectable HBcAg, the absence of a humoral immune response to HBcAg could have been caused by previous infection with human immunodeficiency virus. This naturally occurring human hepatitis B virus variant replicated efficiently without expressing the precore region, confirming previous observations made of the artificial mutants of duck hepatitis B virus.

Adult↗

PubMed1990

A precore-defective mutant of hepatitis B virus associated with e antigen-negative chronic liver disease.

The pathogenesis of chronic liver disease (CLD) due to persistent hepatitis B virus (HBV) infection has not been defined, but the disease activity is believed to correlate with the presence of hepatitis B e-antigen (HBeAg) antigenemia and high viremia. The molecular characterization of an HBV mutant isolated from an HBeAg-negative patient with severe CLD required amplification of the circulating HBV DNA (2 pg/ml) by the polymerase chain reaction (PCR). Direct sequencing of the nucleotides from five independent amplifications of the conserved precore region consistently revealed a G to A mutation in each of the two terminal codons of the precore region. Codon 28 was mutated from tryptophan-encoding TGG to a translational stop codon, TAG; codon 29 preceding the core initiation codon was changed from GGC to GAC. For biologic evaluation of these mutations on HBV replication and expression of HBeAg in vitro, HepG2 cells were transfected with cloned, recircularized mutant HBV DNA. The transfected cells contained subviral core particles in the cytoplasm and secreted mature HBV, without HBeAg, into the medium. The findings present the first evidence that complete HBV genomes can be amplified by PCR and are replication-competent in vitro. The data also indicate that HBeAg is not necessary for replication of HBV and furthermore suggest that HBeAg is not required for the progression of HBV-induced CLD.

Adult↗

PubMed1989

Efficient expression of small RNA polymerase III genes from a novel simian virus 40 vector and their effect on viral gene expression.

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.

Animals↗

PubMed1989

Enzymatic amplification of hepatitis B virus DNA in serum compared with infectivity testing in chimpanzees.

The in vivo infectivity titration of hepatitis B virus (HBV) has been biologically standardized in terms of chimpanzee infectious dose (CID50). The polymerase chain reaction (PCR) was used for in vitro enzymatic amplification of HBV DNA and comparison with CID50. Serial dilutions of human serum specimens containing HBV adw (CID50 10(-7] and ayw (CID50 10(-7.5] were tested to determine the reproducibility and sensitivity of PCR for the detection of HBV DNA. The detection limit of HBV DNA in serum using PCR was 10(-8) for either amplification of HBV DNA extracted from serum or direct amplification of HBV DNA in proteolyzed serum. The amplification efficiency of PCR in proteolysates was not significantly reduced by using a 0.25% concentration (vol/vol) of nonionic detergents and 2.5% concentration (vol/vol) of digested human serum. Thus, PCR is a specific and rapid method for in vitro detection of HBV DNA that is more sensitive than the in vivo infectivity titration of HBV by chimpanzee inoculation. Therefore, if HBV-related DNA cannot be detected by PCR in specimens or biologic products, they probably do not contain infectious HBV.

Animals↗

PubMed1989

Functional dissection of adenovirus VAI RNA.

During the course of adenovirus infection, the VAI RNA protects the translation apparatus of host cells by preventing the activation of host double-stranded RNA-activated protein kinase, which phosphorylates and thereby inactivates the protein synthesis initiation factor eIF-2. In the absence of VAI RNA, protein synthesis is drastically inhibited at late times in infected cells. The experimentally derived secondary structure of VAI RNA consists of two extended base-paired regions, stems I and III, which are joined by a short base-paired region, stem II, at the center. Stems I and II are joined by a small loop, A, and stem III contains a hairpin loop, B. At the center of the molecule and at the 3' side, stems II and III are connected by a short stem-loop (stem IV and hairpin loop C). A fourth, minor loop, D, exists between stems II and IV. To determine sequences and domains critical for function within this VAI RNA structure, we have constructed adenovirus mutants with linker-scan substitution mutations in defined regions of the molecule. Cells infected with these mutants were analyzed for polypeptide synthesis, virus yield, and eIF-2 alpha kinase activity. Our results showed that disruption of base-paired regions in the distal parts of the longest stems, I and III, did not affect function, whereas mutations causing structural perturbations in the central part of the molecule containing stem II, the proximal part of stem III, and the central short stem-loop led to loss of function. Surprisingly, one substitution mutant, sub742, although dramatically perturbing the integrity of the structure of this central portion, showed a wild-type phenotype, suggesting that an RNA with an alternate secondary structure is functional. On the basis of sensitivity to single-strand-specific RNases, we can derive a novel secondary structure for the mutant RNA in which a portion of the sequences may fold to form a structure that resembles the central part of the wild-type molecule, which suggests that only the short stem-loop located in the center of the molecule and the adjoining base-paired regions may define the functional domain. These results also imply that only a portion of the VAI RNA structure may be recognized by the host factor(s).

Adenoviruses, Human↗