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Biomedical subjects

R Ray

Publications and source records attributed to R Ray.

At least 109 records · Page 6Linked to original sources
PubMed1996

Hepatitis C virus core protein inhibits human immunodeficiency virus type 1 replication.

We previously demonstrated that hepatitis C virus (HCV) core protein is a strong repressor of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) basal transcription. In this study, we have localized the HCV core protein-response domain to a region between nucleotides -65 and +3 within the HIV-LTR. Thus, neither the upstream negative regulatory elements, or binding sites for various transcription factors (e.g. NF-kappa B, USF-1, IL2/IL-2R) nor the downstream TAR regions were involved in HCV core-mediated repression. HCV core protein mediated repression of the basal transcriptional activity of HIV-1 LTR was abrogated by the Tat protein. Furthermore, HeLa-T4 cells expressing HCV core protein showed inhibition of HIV-1 replication after acute infection with cell-free HIV. A similar observation was also noted in CD4+ and CD4-lymphocytic cell lines cotransfected with an infectious molecular clone of HIV-1 and the HCV core protein expression vector. Thus, a repression of basal transcription prior to the accumulation of threshold levels of Tat protein appears to restrict HIV-1 transcription and modulate viral replication.

Anti-HIV Agents↗
PubMed1996

Temperature-sensitive phenotype of the human parainfluenza virus type 3 candidate vaccine strain (cp45) correlates with a defect in the L gene.

We have previously demonstrated that the temperature sensitivity of a human parainfluenza virus type 3 (HPIV-3) candidate vaccine strain (cp45), which is currently under evaluation in humans, is associated with poor transcriptional activity of the virus at the nonpermissive temperature (R. Ray, K. Meyer, F. Newman, and R. B. Belshe, J. Virol. 69:1959-1963, 1995). In this study, the temperature sensitivity of cp45 virus was further investigated by the complementation of a specific gene function. CV-1 cells were transfected with cloned genes from wild-type HPIV-3 encoding the large protein (L), phosphoprotein (P), and nucleocapsid protein (NP), alone or together, for the expression of biologically active proteins. Only cells expressing the L gene were able to rescue cp45 replication when incubated at the nonpermissive temperature (39.5 degrees C), whereas cells transiently expressing NP or P were incapable of rescuing the virus. The virus titers obtained following complementation of the L protein were 190 to 2,300 PFU/ml of culture medium, compared with the undetectable growth of the cp45 temperature-sensitive mutant at the nonpermissive temperature. Rescued progeny virus still maintained the temperature-sensitive phenotype. Results from this study suggest that the temperature sensitivity of the cp45 candidate vaccine strain is associated primarily with L-protein function and that the defect can be complemented by transient expression of the wild-type protein. This study underscores the importance of the L protein in RNA polymerase activity and its critical role in virus replication.

Animals↗
PubMed1996

Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype.

We have previously demonstrated that hepatitis C virus (HCV) core protein regulates cellular protooncogenes at the transcriptional level; this observation implicates core protein in the alteration of normal hepatocyte growth. In the present study, the transforming potential of the HCV core gene was investigated by using primary rat embryo fibroblast (REF) cells which were transfected with or without cooperative oncogenes. Integration of the HCV core gene resulted in expression of the viral protein in REF stable transformants. REF cells cotransfected with HCV core and H-ras genes became transformed and exhibited rapid proliferation, anchor-independent growth, and tumor formation in athymic nude mice. Results from these studies suggest that the core protein plays an important role in the regulation of HCV-infected cell growth and in the transformation to tumorigenic phenotype. These observations suggest a possible mechanism for this viral protein in the pathogenesis of hepatocellular carcinoma in HCV-infected humans.

Animals↗
PubMed1996

Direct in situ nucleic acid amplification: control of artefact and use of labelled primers.

Aims-To evaluate factors which ameliorate false positive artefacts with direct in situ PCR using labelled dNTPs; to investigate the use of labelled primers to overcome this artefact whilst maintaining sensitivity.Methods-Sections of measles (RNA virus) infected Vero cells with cytoplasmic signal or cytomegalovirus (DNA virus) infected fibroblasts with nuclear signal were collected. In situ PCR (or in situ RT-PCR) was carried out by methods permitting evaporation. Reagents or conditions which may control false positive artefacts using labelled dNTPs were investigated systematically. Labelled primers were tested to overcome artefacts, with adjuncts which improve sensitivity.Results-No reagent nor condition investigated was able to control the artefact with labelled dNTPs. Excessive digestion and incomplete DNAse treatments exacerbated the artefact, whereas novobiocin decreased both specific signal and artefact. However, the artefact was controlled by labelled primers, albeit with relatively low sensitivity. Sensitivity using labelled primers could be increased using alcohol fixation, albumin or Perfectmatch.Conclusions-A repair process is implicated for the artefact using labelled dNTPs. Excessive digestion or DNAse treatment may exacerbate DNA damage by disrupting histones or the DNA, respectively. Labelled primers control this artefact, albeit with reduced sensitivity, which may be improved by precipitation fixatives (alcohol) and reagents which enhance specific reaction.

Journal Article↗
PubMed1996

Molecular recognition in vitamin D-binding protein.

Vitamin D-binding protein (DBP) or group-specific component (Gc) is a relatively abundant serum protein with multiple functions, the majority of which are initiated by the highly specific recognition and binding of a ligand by this protein. During the past decade and a half, several structure-functional studies have been carried out to shed light on the physiological significance of the multiple functions of DBP. Results of these studies are discussed.

Actins↗
PubMed1996

Gestational diabetes in Singaporean women: use of the glucose challenge test as a screening test and identification of high risk factors.

In this study, 1141 antenatal women attending 9 Government polyclinics were screened between 24 and 28 weeks of gestation for gestational diabetes mellitus (GDM) using the glucose challenge test (GCT). An attempt was made to determine whether 7.2 mmol/l or 7.8 mmol/l was an effective cut-off point as an indicator for GDM. The women with GCT levels of > or = 7.2 mmol/l had an oral glucose tolerance test (OGTT) for confirmation of GDM. The results showed that 420 (36.8%) had GCT levels > or = 7.2 mmol/l, including 299 (26.2%) with GCT levels of > or = 7.8 mmol/l. Of the 190 OGTT carried out, 8 (4.2%) were confirmed to have GDM, all of whom had GCT levels of > or = 7.8 mmol/l, indicating that a GCT level of 7.8 mmol/l was the effective cut-off point. Univariate analysis showed that epidemiological features associated with a raised GCT > or = 7.8 mmol/l were, older age that is 30 years and above, Chinese ethnic group, maternal obesity (body mass index > or = 25) and history of > or = 4 pregnancies. Multivariate analysis using stepwise logistic regression, showed that factors significantly and independently associated with raised GCT levels were older age, Chinese ethnic group and maternal obesity. All antenatal women should be screened for GDM using the GCT, and those with levels of > or = 7.8 mmol/l should be subjected to the OGTT for confirmation.

Adult↗
PubMed1996

Basaloid-squamous carcinoma. A distinct histopathological entity.

Basaloid Squamous Carcinoma (BSC) is an uncommon aggressive neoplasm occurring in the upper aerodigestive tract. Lung as a primary site has only recently been reported. A combination of microscopic features is required to make a diagnosis and to differentiate it from other more common malignancies in these sites. Three cases of BSC including one in the lung are reported. There are no prior published reports of this neoplasms in Indian literature. The study highlights the typical microscopic features and the diagnostic difficulties which may be encountered.

Aged↗
PubMed1995

sigma2 Site-mediated inhibition of electrically evoked guinea pig ileum longitudinal muscle/myenteric plexus contractions.

Functional and binding studies were performed in order to characterize the relative efficacy and affinity of a number of compounds that bind to sigma sites. The ability of sigma site ligands to inhibit electrically evoked contraction of the guinea pig ileum longitudinal muscle/myenteric plexus preparation was compared to the affinities of these compounds for sigma1 sites (assessed by displacement of [3H](+)-pentazocine) and sigma2 sites (assessed by displacement of [3H]1,3-di-o-tolylguanidine (DTG) in the presence of 5 microM dextromethorphan). It was shown that the rank order of potencies for suppression of electrically evoked contractions of guinea pig ileum perfectly matched the rank order of affinities of these compounds for the sigma2 binding site, while correlating poorly with the sigma1 binding site. In addition, no significant correlations were found between the efficacy of the tested compounds to inhibit contraction of the guinea pig ileum preparation and previously reported affinities for muscarinic, dopamine D2 or MK-801 binding sites. Thus, the present study represents the first functional bioassay selectively sensitive to agents interacting with the sigma2 receptor subtype binding site, and provides a means with which to further elucidate the functional role of sigma2 sites.

Animals↗
PubMed1995

25-Hydroxy[26,27-methyl-3H]vitamin D3-3 beta-(1,2-epoxypropyl)ether: an affinity labeling reagent for human vitamin D-binding protein.

Vitamin D-binding protein (DBP) is primarily involved in the binding and transportation of vitamin D3 and its various metabolites to target organs and tissues. This is manifested by the ability of DBP to bind vitamin D3 and its metabolites with high affinity. In the present study we developed 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)ether (25-OH-D3-epoxide) as an affinity labeling reagent of human DBP (hDBP). Competitive radioligand binding assays of 25-OH-D3-epoxide with hDBP demonstrated that the binding affinity of this analog was similar to that of 25-hydroxyvitamin D3 (25-OH-D3). Incubation of 25-hydroxy[26(27)-3H]-vitamin D3-3 beta-(1,2-epoxypropyl)ether [[3H]25-OH-D3-epoxide] with hDBP covalently labeled the protein. When the incubation was carried out in the presence of a large excess of 25-OH-D3, labeling was removed completely. When human Cohn IV fraction, containing hDBP, was incubated with [3H]25-OH-D3-epoxide a single protein band, corresponding to hDBP, was labeled. Labeling was completely obliterated in the presence of a large amount of 25-OH-D3. However, an equivalent amount of 7-dehydrocholesterol had no effect on labeling. These results demonstrated that [3H]25-OH-D3-epoxide most probably labeled the vitamin D sterol-binding domain of hDBP.

Affinity Labels↗
PubMed1995

Hepatitis C virus core protein: synthesis, affinity purification and immunoreactivity with infected human sera.

The genomic region encoding the core (C) protein (amino acids 1-162) of hepatitis C virus (HCV) was expressed in Escherichia coli as a recombinant (re-) protein with the maltose-binding protein (MBP) using the prokaryotic expression vector pMAL-CR1. The fusion protein (C::MBP) was identified as a approx. 62-kDa polypeptide by immunoblot analysis using antiserum to MBP and HCV-infected human sera. The size of C::MBP corresponded to the calculated combined molecular mass of the approx. 20-kDa HCV C protein and the approx. 42-kDa MBP. The approx. 62-kDa C::MBP was purified using amylose resin as a matrix in affinity chromatography, and showed specific reactivity with HCV-infected human sera. These results suggest that C::MBP may serve as a source of the core antigen for immunological studies on HCV infection.

ATP-Binding Cassette Transporters↗
PubMed1995

Trp-145 is essential for the binding of 25-hydroxyvitamin D3 to human serum vitamin D-binding protein.

Chemical modification of specific amino acid residues in a protein has been a valuable tool in identifying amino acid residues that are responsible for ligand binding of a protein. In the present investigation, we targeted Trp and His residues in human serum vitamin D-binding protein (hDBP) by modifying them with specific chemical modifiers. We also evaluated the results of these modifications in the binding of 25-hydroxy[26(27)-3H]vitamin D3 ([3H]25-OH-D3) to hDBP. We observed a dose-dependent loss of binding activity by N-bromosuccinimide (specific for Trp). Similar results were observed with diethylpyrocarbonate (specific for His). Furthermore, loss of [3H]25-OH-D3-binding was protected by preincubation of hDBP samples with an excess of 25-hydroxyvitamin D3. These results strongly emphasized the importance of Trp (single residue at position 145) and 1 His residue (out of a total of 6) in the vitamin D sterol-binding by vitamin D-binding protein.

Bromosuccinimide↗
PubMed1995

Metabolism of 3H-1 alpha,25-dihydroxyvitamin D3 in cultured human keratinocytes.

In the present investigation we studied the metabolism of 1 alpha,25-dihydroxy-[1 beta-3H]vitamin D3 (3H-1,25(OH)2D3) in culture-grown human keratinocytes (CHK). Our results showed that the cellular uptake of 3H-1,25(OH)2D3, upon incubation with CHK, occurred very rapidly; and it paralleled a decrease in the concentration of 3H-1,25(OH)2D3 in the medium. The amount of 3H-calcitroic acid, on the other hand, increased slowly in the medium, while the concentration of 3H-calcitroic acid in the cell remained undetectable during the whole period of incubation. When the cells were preincubated with 1,25(OH)2D3 (10(-8)M), conversion of 3H-1,25(OH)2D3 to 3H-calcitroic acid increased almost twofold, indicating that 1,25(OH)2D3 catalyzed its own catabolism.

Calcitriol↗
PubMed1995

Affinity purification of human plasma vitamin D-binding protein.

During the course of our studies to probe the vitamin D ligand-binding domains of vitamin D-binding protein and vitamin D receptor, we developed a synthetic procedure to modify the 3 beta-hydroxyl group of vitamin D3 and its 25-hydroxy- and 1,25-dihydroxy metabolites with a 3'-aminopropylether group. In the present study we have coupled 25-hydroxyvitamin D3-3 beta-3'-aminopropylether to an activated Sepharose matrix. Using this stable and reusable affinity matrix we have purified human vitamin D-binding protein from human plasma to homogeneity.

Binding Sites↗
PubMed1995

Sulfur mustard-induced increase in intracellular free calcium level and arachidonic acid release from cell membrane.

The mechanism of action of the alkylating agent bis-(2-chloroethyl)sulfide (sulfur mustard, SM) was studied using the in vitro mouse neuroblastoma-rat glioma hybrid NG108-15 clonal cell line model. Following 0.3 mM SM exposure, cell viability remained high (> 80% of untreated control) up to 9 hr and then declined steadily to about 40% of control after 20-24 hr. During the early period of SM exposure, when there was no significant cell viability loss, the following effects were observed. The cellular glutathione level decreased 20% after 1 hr and 34% after 6 hr. Between 2 and 6 hr, there was a time-dependent increase (about 10 to 30%) in intracellular free calcium (Ca2+), which was localized to the limiting membrane of swollen endoplasmic reticula and mitochondria, to euchromatin areas of the nucleus, and to areas of the cytosol and plasma membrane. Moreover, there was also a time-dependent increase in the release of isotopically labeled arachidonic acid ([3H]AA) from cellular membranes. Increase in [3H]AA release was 28% at 3 hr and about 60-80% between 6 and 9 hr. This increase in [3H]AA release was inhibited by quinacrine (20 microM), which is a phospholipase (PLA2) inhibitor. At 16 hr after SM exposure, there was a large increase (about 200% of control) in [3H]AA release, which was coincident with a 50% loss of cell viability. These results suggest a Ca(2+)-mediated toxic mechanism of SM via PLA2 activation and arachidonate release.

Animals↗
PubMed1995

Aminopropylation of vitamin D hormone (1 alpha,25-dihydroxyvitamin D3), its biological precursors, and other steroidal alcohols: an anchoring moiety for affinity studies of sterols.

In this communication we describe a simple two-step procedure for the conversion of several steroidal alcohols to their aminopropyl ether derivatives. To demonstrate the usefulness of this procedure we synthesized a second-generation photoaffinity labeling analog of 1 alpha,25-dihydroxyvitamin D3, and a 25-hydroxyvitamin D3 derivative containing a long and chemically stable tether. Utilities of these aminopropyl ether derivatives of steroids in various affinity studies of receptor proteins are discussed.

Alcohols↗
PubMed1995

In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymerase chain reaction.

Using single primer pairs, intracellular gene sequences of cytomegalovirus (CMV-Towne's strain) and alpha-tubulin were amplified (in situ PCR) from cells in human body fluids and in suspensions. Visualization of CMV amplificants was carried out by in situ hybridization (ISH), using both a biotinylated double-stranded DNA probe and a radiolabelled oligonucleotide probe. Visualization of alpha-tubulin amplificants was achieved using both radiolabelled single-stranded cRNA and oligonucleotide probes. Liberated amplificants were also identified by bands of expected size by gel electrophoresis. The specificity of the PCR products was confirmed by Southern blot analysis. Intracellular amplification was identified both in unfixed cells and, optimally, after brief alcohol fixation, whilst maintaining relative isotonicity in all working solutions. For CMV, enhanced signal was observed in cells (cultured fibroblasts or urine sediment) undergoing in situ PCR using either biotinylated or radiolabelled probes compared with controls undergoing ISH alone. For alpha-tubulin, radiolabelled riboprobes and oligoprobes only produced signals within cells (human peripheral lymphocytes, ascitic fluid and bladder washings from routine cytological specimens) after in situ PCR, but not after ISH alone. Morphological evaluation was superior with biotinylated probes, and minimal back-diffusion effect was found compared with radiolabelled probes. Up to 80% of cells survived thermal cycling. In situ PCR detected short sequence (100 bp) foreign DNA and low copy number genomic DNA, and was superior to ISH alone. In contrast to radiolabelled probes, very small CMV amplificants could be detected without a significant 'back-diffusion' effect when using the large biotinylated probe in this model system.

Animals↗