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Biomedical subjects

R Koshy

Publications and source records attributed to R Koshy.

At least 19 recordsLinked to original sources
PubMed2004

Structure, surface excess and effective interactions in polymer nanocomposite melts and concentrated solutions.

The Polymer Reference Interaction Site Model (PRISM) theory is employed to investigate structure, effective forces, and thermodynamics in dense polymer-particle mixtures in the one and two particle limit. The influence of particle size, degree of polymerization, and polymer reduced density is established. In the athermal limit, the surface excess is negative implying an entropic dewetting interface. Polymer induced depletion interactions are quantified via the particle-particle pair correlation function and potential of mean force. A transition from (nearly) monotonic decaying, attractive depletion interactions to much stronger repulsive-attractive oscillatory depletion forces occurs at roughly the semidilute-concentrated solution boundary. Under melt conditions, the depletion force is extremely large and attractive at contact, but is proceeded by a high repulsive barrier. For particle diameters larger than roughly five monomer diameters, division of the force by the particle radius results in a nearly universal collapse of the depletion force for all interparticle separations. Molecular dynamics simulations have been employed to determine the depletion force for nanoparticles of a diameter five times the monomer size over a wide range of polymer densities spanning the semidilute, concentrated, and melt regimes. PRISM calculations based on the spatially nonlocal hypernetted chain closure for particle-particle direct correlations capture all the rich features found in the simulations, with quantitative errors for the amplitude of the depletion forces at the level of a factor of 2 or less. The consequences of monomer-particle attractions are briefly explored. Modification of the polymer-particle pair correlations is relatively small, but much larger effects are found for the surface excess including an energetic driven transition to a wetting polymer-particle interface. The particle-particle potential of mean force exhibits multiple qualitatively different behaviors (contact aggregation, steric stabilization, local bridging attraction) depending on the strength and spatial range of the polymer-particle attraction.

Computer Simulation↗
PubMed2001

Intracellular accumulation of middle hepatitis B surface protein activates gene transcription.

While the natural intact protein does not possess any transactivator function, C-terminal truncation of the middle hepatitis B surface (MHBs) protein yields a novel transactivator function. We have previously found that the truncated transactivator protein, MHBs(t167), is not secreted but retained within the secretory pathway. Here, we provide evidence that when full-length MHBs is coexpressed with the truncated MHBs(t167) protein, the secretion of the full-length protein is inhibited and both proteins accumulate within the cell. We further show that MHBs, forcibly retained in the cell by C-terminal fusion to the endoplasmic reticulum retention signal KDEL (MHBsKDEL), mimics the effects of MHBs(t167) in enhancing the nuclear-binding activity of transcription factors NFkappaB and AP-1, and activation of NFkappaB- and AP-1-dependent transcription of reporter genes. As is the case for MHBs(t167), MHBsKDEL-dependent activation of NFkappaB is inhibited by the antioxidant N-acetyl-L-cysteine indicating the involvement of reactive oxygen intermediates and suggesting a similar mechanism of activation. This study suggests that the intracellular retention and accumulation of the normally secreted MHBs leads to oxidative stress and activation of transcription. This may be an important but not exclusive mechanism in hepatocarcinogenesis.

Carcinoma, Hepatocellular↗
PubMed1999

Downregulation of DNA excision repair by the hepatitis B virus-x protein occurs in p53-proficient and p53-deficient cells.

Synergism between exposure to chemical carcinogens and infection with the hepatitis B virus (HBV) has been implicated in the high incidence of hepatocellular carcinoma. In this study we report that the HBV protein HBx, inhibits cellular DNA repair capacity in a p53-independent manner. Two alternative assays were used: the host cell reactivation assay, which measures the cell's capacity to repair DNA damage in a reporter plasmid, and unscheduled DNA synthesis, which measures the overall DNA repair capacity in damaged cells. Two p53-proficient cell lines, the hepatocellular carcinoma cell line HepG2 and liver epithelial cell line CCL13, were co-transfected with the pCMV-HBx reporter plasmid and the pCMV-CAT plasmid damaged with UVC radiation. Compared with cells transfected with control plasmid, the presence of HBx resulted in approximately 50% inhibition of the cell's capacity to reactivate CAT activity of UVC-damaged plasmid, and approximately 25% inhibition of unscheduled DNA synthesis in cells treated with either aflatoxin B1 epoxide or UVC radiation. Using the p53-deficient cell line Saos-2, we demonstrated that expression of HBx also resulted in diminished overall cellular DNA repair of damage induced by both aflatoxin B1 epoxide and UVC radiation, using both the host cell reactivation and unscheduled DNA synthesis assays. In summary, this study provides evidence for p53-independent regulation of DNA repair by HBx.

Aflatoxin B1↗
PubMed1998

Erk-independent partial activation of AP-1 sites by the hepatitis B virus HBx protein.

The hepatitis B virus X protein (HBx) is suggested to regulate transcription by stimulation of intracellular signalling pathways. We have analysed the effects of HBx on activation of the MAP kinase (Erk) and JNK/SAPK signalling pathways and confirm a stimulation of the Erk/MAP kinase in quiescent cells. However, a substantial Erk-independent activation of AP-1, and phosphorylation of c-Jun (serine-63), but not Erk-2, was induced by HBx in dividing, serum-maintained cells. These data suggest that HBx promiscuously activates Erk and JNK responsive pathways and that its overall effect on signalling may be influenced by external mitogenic stimuli.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

The hepatitis B virus MHBst167 protein is a pleiotropic transactivator mediating its effect via ubiquitous cellular transcription factors.

C-terminally truncated surface proteins of hepatitis B virus (HBV) are frequently translated from genomically integrated viral sequences. They may be relevant for hepatocarcinogenesis by stimulating gene expression. First, we examined the transactivating potential of middle hepatitis B surface protein truncated at amino acid (aa) position 167 (MHBst167) on the HBV regulatory element. In transient cotransfection assays using Chang liver or HepG2 cell lines and chloramphenicol acetyltransferase (CAT) reporter constructs only the HBV enhancer I, but no other HBV regulatory elements like the X promoter, the S1 or S2 promoter or the enhancer II/core promoter could be stimulated by MHBst167. Since there is no evidence for a direct interaction of MHBst167 with DNA, we subsequently analysed whether cellular transcription factors were involved in mediating transactivation. This was tested both with isolated transcription-factor-binding sites and in the natural context of viral and cellular promoter elements. Deletion analysis and electrophoretic mobility shift assays revealed that Sp1, AP1 and NF-kappa B can mediate transactivation by MHBst167. No involvement of CREB, NF1 or the liver-specific factor C/EBP was found. These data indicate that MHBst167 is a pleiotropic, non-liver-specific transactivator which exerts its effect via ubiquitous cellular transcription factors that are also involved in the regulation of expression of cellular genes relevant for proliferation and inflammation.

Enhancer Elements, Genetic↗
PubMed1996

Evaluation of hepatitis C virus protein epitopes for vaccine development.

Infection with hepatitis C virus (HCV) leads to viral persistence and chronic disease in a very high proportion of cases, despite a broad immunological response to viral proteins. These responses may thwarted by the high rate of mutation, which leads to the generation of 'escape' variants of HCV that persist as a quasi-species in infected individuals. The specificity of the immuno response of infected patients suggests that responses directed at certain viral epitopes may be associated with less aggressive disease and, possibly, good interferon response and virus clearance. The identification of such epitopes may hold the key for future development both of prophylactic and therapeutic vaccines.

Animals↗
PubMed1996

Hepatitis B virus transcriptional activators: mechanisms and possible role in oncogenesis.

The hepatitis B virus (HBV) genome encodes a 154 amino acid protein termed X (HBx, hepatitis B x protein), which is a promiscuous transcriptional activator of polymerase II and III promoters. HBx upregulates a wide range of cellular and viral genes and is thought to facilitate viral pregenome and mRNA transcription; however, its precise role in the viral replication cycle remains to be elucidated. The functional mechanisms of HBx appear very complex. It was shown to activate transcription factors AP-1 and NF-kappa B vis cytoplasmic pathways including ras-MAP kinase. In contrast, nuclear HBx is thought to activate the transcriptional machinery directly. A second transcriptional activator protein (Mst, middle s transactivator) is encoded by 3'-truncated preS2/S sequences of integrated HBV DNA, but not by the intact viral gene. HBx and Mst may contribute to the pathogenicity of chronic hepatitis B and are suggested to promote hepatocyte transformation via upregulation of cellular proto-oncogenes. Further, HBx may enhance HBV related carcinogenesis by inactivation of the tumour suppressor gene product p53.

Animals↗
PubMed1995

Mutant p53 but not hepatitis B virus X protein is present in hepatitis B virus-related human hepatocellular carcinoma.

Hepatitis B virus (HBV) X protein is thought to play an important role in the development of hepatocellular carcinoma. Recent studies on a transgenic mouse tumor model suggest that HBV X protein may contribute to transformation by binding to and inactivating the cellular growth suppressor protein p53. We have studied 31 hepatocellular carcinoma tissues from Chinese patients for the possible occurrence of such interactions. Although most of the samples contained markers of HBV infection, including free and/or integrated HBV DNA, there was no detectable expression of HBV X protein by Western blot, immunoprecipitation, or histochemical staining. There was also no evidence of HBV X protein associated with p53 immunoprecipitated from the tumors. These observations suggest that, in naturally occurring human hepatocellular carcinoma, such interactions are uncommon and, therefore, unlikely to be of relevance in the latter stages of tumor development. On the other hand, 29 of 31 (93%) samples contained mutated forms of p53, as determined by various antibodies that detect wild-type or mutant p53 or both, and by the association of heat shock protein 70 with immunoprecipitated p53. These results show that conformationally altered p53 protein is present in tumors at a much higher frequency than is suggested by the presence of known mutations in the gene. This mutant p53 is functionally inactive, as suggested by the lack of expression of the p53-induced M(r) 21,000 Cip1/Waf1 protein in the tumors. Because this inactivation of p53 was not correlated with the expression of HBV X protein, any interaction of HBV X protein with p53 may be relevant only during acute infection. Such an interaction could serve to relax cell growth control at a time when virus replication requires hepatocyte destruction to be balanced by regeneration.

Antibodies, Monoclonal↗
PubMed1995

Enzymatic properties of overexpressed HBV-mevalonate kinase fusion proteins and mevalonate kinase proteins in the human hepatoma cell line PLC/PRF/5.

We have previously reported a case of integration of HBV sequences in the 5'-noncoding region of the gene for mevalonate kinase (Mk) (EC.2.7.1.36) in the human hepatoma cell line PLC/PRF/5, resulting in overexpression of viral-cellular fusion transcripts and enhanced intracellular levels of the enzyme. Here, we present an evaluation of the functionalities of Mk and HBV/Mk fusion proteins derived from viral-cellular fusion- and Mk-transcripts, some of which lack 156 bp in the Mk coding region as a result of a differential splicing process. cDNA clones with a full-length Mk-ORF produce proteins which can metabolize mevalonate to its monophosphorylated form. Our results suggest that the enhanced and inappropriate expression of Mk may lead to increased metabolism of mevalonate and phosphorylation of hitherto unknown cellular proteins. This consequence of HBV-DNA insertion could thus be related to the activation of proteins that may be relevant in oncogenesis.

Alternative Splicing↗
PubMed1995

A mitochondrial elongation factor-like protein is over-expressed in tumours and differentially expressed in normal tissues.

The tissue-specific expression of an antigen (P43) ubiquitously expressed at high levels in a variety of tumours of human and animal origin was investigated using a monoclonal antibody to P43. Whereas low amounts of P43 are expressed in the spleen, skeletal muscle and pancreas, P43 is abundantly produced in the liver and in other tissues such as the kidney, heart and brain which have levels of oxidative metabolism. Interestingly, a related protein of higher molecular weight is abundantly expressed in the lung and in amounts which were higher than those observed with other tissues. The human cDNA for P43 was isolated from a human liver cDNA library and mapped to chromosome 16 between p11.2 and 12 and also to a position near the centromere on the long arm of chromosome 17. The deduced amino acid sequence of P43 is remarkably similar to that of E. coli EF-Tu and the mitochondrial EF-Tu of S. cerevisiae with the structurally and functionally important amino acids of EF-Tu being completely conserved in P43. A comparison of the distribution of P43 and a mitochondrial protein Hsp 60 among different cellular fractions indicated a likely mitochondrial localisation for P43. Taken together these results suggest that P43 is a human mitochondrial elongation factor.

Amino Acid Sequence↗
PubMed1994

Integrated hepatitis B virus X and 3' truncated preS/S sequences derived from human hepatomas encode functionally active transactivators.

The hepatitis B virus (HBV) frequently integrates into hepatocellular genomic DNA during viral infection. Transcriptional transactivators encoded by integrated HBV X and 3' truncated preS/S sequences are known to stimulate gene expression from homologous and heterologous promoters. Here we demonstrate that 21 of 26 (81%) hepatocellular carcinoma tissues/cell lines contain coding sequences for at least one of the two known transactivators. Four integrated X and three preS/S transactivator sequences contained in five isolates from three hepatoma primary tissues or cell lines were used as examples to prove functionality of the encoded transactivators. In one case, where both X and preS/S sequences were present, dissection of X and preS/S transactivator sequences showed independent functionality. The investigation of X- and preS/S-specific RNA and protein expression revealed the existence of carboxyterminally truncated viral-cellular fusion proteins that were able to stimulate gene expression from the c-fos proto-oncogene promoter five- to ten-fold. These results demonstrate that structurally intact HBV transactivator sequences are integrated in the majority of HBV-associated HCCs/hepatoma cell lines. In all tested examples integrated DNAs had retained functionality as transactivators. This data thereby support indirectly the hypothesis of a possible involvement of HBV transactivators in liver cell proliferation and hepatocarcinogenesis.

Base Sequence↗
PubMed1994

Effect of homeopathic drugs plumbum and opium on experimentally induced lead toxicity in rats.

Homeopathic drugs plumbum 1M and Opium 30 were partially effective in the recovery of delta ALAD activity of the lead (150 mg% lead acetate) intoxicated rats. Plumbum 1M did not exhibit protective effect when dietary lead at high concentrations (> 25 mg% lead acetate) were given concurrently as assessed by blood delta ALAD activity and hemoglobin concentration. However it was partially effective in the recovery of delta ALAD activity and relieving anemia caused by chronic exposure of low doses of lead (below 15 mg% lead acetate).

Animals↗
PubMed1994

Insertional activation of mevalonate kinase by hepatitis B virus DNA in a human hepatoma cell line.

Insertional mutagenesis of growth related genes by hepatitis B virus (HBV) DNA is presumed to play a role in hepatocarcinogenesis. Here, we report on insertional activation of the mevalonate kinase (MK) gene in the human hepatoma cell line PLC/PRF/5. Integration of HBV DNA dissociated the promoter and upstream regulatory elements of the gene from its coding sequences. This led to the over-expression of hybrid transcripts arising from an HBV promoter and the consequent over-production of functionally active mevalonate kinase. MK phosphorylates mevalonate, a major intermediate in the branched cholesterol/isoprenoid biosynthetic pathway. Isoprenylation is crucial to the functions of cellular proteins related to growth control, including the proto-oncogene ras. As the enzymes of these biosynthetic pathways are regulated at multiple points by negative feedback, both transcriptionally and at the protein level, the results discussed here support the idea that aberrant growth could result from deregulated overexpression of MK and, perhaps, other enzymes in the cholesterol pathway. These results invoke novel mechanisms by which cell transformation might occur.

Amino Acid Sequence↗
PubMed1993

Transactivating function of integrated hepatitis B virus.

HBV DNAs are often found in integrated forms in human hepatocellular carcinoma (HCC). Discovery of a transactivation function coded by a limited region of the HBV genome has promoted us to survey our collection of HBV integrants with flanking cellular sequences, asking whether they might exhibit a transactivation function. In transient cotransfection assays using the HepG2 cell line, six out of the twelve integrants showed transactivation effects on the expression of cellular genes such as c-fos. These results strongly demonstrate that the transactivating effects of integrated HBV DNA are widely distributed, and some of these effects might be correlated to hepatocarcinogenesis.

Carcinoma, Hepatocellular↗
PubMed1993

Transactivation of cellular promoters by an integrated hepatitis B virus DNA.

A new Hepatitis B virus(HBV) DNA integrant clone DA2-6, isolated from a human hepatocellular carcinoma(HCC) genomic library, was tested for its ability to transactivate expression of other genes. DA2-6 consists of 3.7 kb flanking cellular sequences and an integrated 2.8 kb HBV DNA which covers the region of preS, S, and the 3' truncated X. Using a chloramphenicol acetyltransferase (CAT) assay, a number of cellular and viral promoters were transactivated by DA2-6, and the spectrum of transactivational effect was the same as that by the wild type X gene of the virus. Deletion mutant analyses indicated that the transactivation function of DA2-6 is expressed by the region that encodes a truncated X-cell fusion product.

Amino Acid Sequence↗
PubMed1993

Effect of X protein on transactivation of hepatitis B virus promoters and on viral replication.

The X gene product of hepatitis B virus (HBV) transactivates a wide variety of promoters, including four promoters on the HBV genome (Rossner, 1992, J. Med. Virol. 36, 101-117). We compared their transactivation efficiencies and investigated whether the spatial organization of the promoters with respect to other cis-acting elements might influence their activities. Eight reporter plasmid constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene were designed such that four had the isolated HBV promoters linked to the CAT gene. In the other four, the CAT gene was inserted downstream to each of the four promoters retained in context in the HBV genome. Cells of the human hepatoblastoma line HepG2 were transfected with each one of these reporters together with an effector plasmid, pRSVX, which allowed expression of X protein. All of these promoters could be stimulated by X protein by approximately 2- to 3.5-fold irrespective of their spatial context in the HBV genome. Mutational analysis of in-frame ATG codons in the X gene provides evidence that transactivator product(s) are produced by internal initiation of translation. Transfection of HepG2 cells with HBV genomes bearing a stop mutation in the X gene at codon 118 resulted in poor production of all viral components. Their syntheses were restored upon transfection of the wild-type X gene.

Base Sequence↗
PubMed1992

A chromosome 17:7 translocation is associated with a hepatitis B virus DNA integration in human hepatocellular carcinoma DNA.

Chronic hepatitis B virus infection is often associated with major structural rearrangements of both the integrated viral DNA and the associated cellular sequences. We present here the structure of a single-copy hepatitis B virus insert cloned from human hepatocellular carcinoma DNA recently reported to encode a novel transcriptional trans-activator function. The hepatitis B virus portion of the clone consists of two colinear fragments covering the X gene with its promoter and enhancer (nucleotides 717 to 1796) and a 3' truncated pre-S/S gene (nucleotides 2703 to 423). The lack of the entire pre-C/C gene caused a fusion of the 3' end of the X gene with sequences upstream from the pre-S gene. The structure of the integrated viral DNA fragments suggests insertion of hepatitis B virus replication intermediates into cellular DNA and subsequent recombination between these primary integrations to generate the final structure of the clone. The 5' and 3' cellular flanking sequences mapped to the centromeric alpha-satellite DNA of chromosome 17 and to the short arm of chromosome 7 (p14-pter), respectively, indicating that chromosomal translocation was associated with the hepatitis B virus DNA integration. Because this is the fourth case reported in which hepatitis B virus-associated rearrangements have affected chromosome 17, it is conceivable that a loss of important cellular genes (such as the p53 antioncogene on chromosome 17) may be a crucial step in hepatitis B virus-related hepatocarcinogenesis.

Amino Acid Sequence↗