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Biomedical subjects

R Padmanabhan

Publications and source records attributed to R Padmanabhan.

At least 55 records · Page 3Linked to original sources
PubMed1999

Pattern of central nervous system anomalies in a population with a high rate of consanguineous marriages.

Nine thousand six hundred and ten births were prospectively studied in the three major hospitals in Al-Ain, United Arab Emirates (UAE) between October 1995 and January 1997. Babies suspected of, or diagnosed, as having central nervous system (CNS) abnormalities were evaluated by a neonatologist, a clinical geneticist and a pediatric neurologist. Brain computerized tomography/magnetic resonance imaging (CT/MRI) was performed on all babies suspected of having CNS abnormalities. In addition, metabolic screening and chromosome analysis were also performed when indicated. Of the 225 babies with congenital anomalies identified, 31 had CNS abnormalities (3.2/1000). Syndromic abnormalities of the CNS were present in 13 cases (42%), chromosomal abnormalities in one case (3.2%) and the rest included: neural tube defect (NTD) in 11 cases (36%), holoprosencephaly in two cases (6.4%) and hydrocephalus in four cases (12.9%). Detailed analysis of the syndromic types revealed that out of the 13 cases, 12 were inherited as autosomal recessive (AR) and in one case the inheritance was undetermined. Consanguinity with high level of inbreeding was present in 12 cases and the majority of the syndromes identified were extremely rare. The study indicates that CNS anomalies are fairly common in the UAE, particularly, the recessive syndromic types. Careful and detailed analysis of such anomalies is required so that accurate genetic advice can be given.

Central Nervous System↗
PubMed1999

Crystallization, characterization and measurement of MAD data on crystals of dengue virus NS3 serine protease complexed with mung-bean Bowman-Birk inhibitor.

Crystallization and preliminary characterization of the essential dengue virus NS3 serine protease complexed with a Bowman-Birk-type inhibitor from mung beans are reported. As the structure proved resistant to solution by molecular replacement and multiple isomorphous replacement methods, multi-wavelength anomalous diffraction data at the LIII edge of a holmium derivative have been measured. Promising Bijvoet and dispersive signals which are largely consistent with expected values have been extracted from the data. The structure, when determined, will provide a structural basis for the design, synthesis and evaluation of inhibitors of the protease for chemotherapy of dengue infections.

Crystallization↗
PubMed1999

The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids.

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity.

Acid Anhydride Hydrolases↗
PubMed1999

Familial Lecithin:cholesterol acyltransferase deficiency with renal failure in two siblings. First case report from India.

We encountered 2 siblings with corneal opacities, anemia, decreased concentration of plasma esterified cholesterol, chronic renal failure and markedly reduced levels of plasma lecithin:cholesterol acyltransferase. Renal biopsy in 1 patient showed clear lacunae containing characteristic dense bodies in the glomerular and tubular basement membranes and the interstitium. One of the 2 siblings received a kidney transplant and is doing well after 9 years. This is the first report of this rare metabolic disease from India.

Adult↗
PubMed1998

Transactivation of the human cdc2 promoter by adenovirus E1A in cycling cells is mediated by induction of a 110-kDa CCAAT-box-binding factor.

Cyclin-dependent protein kinases (Cdks) are key regulatory proteins of the eukaryotic cell cycle. Cdc2 is expressed in late G1/S phase and functions in the G2 to M phase transition. Adenovirus E1A proteins are known to induce the expression of p34cdc2 and DNA synthesis in normal quiescent cells. In this study, mutational analysis of the human cdc2 promoter revealed that transactivation of the promoter by the E1A proteins in cycling cells is mediated through the two CCAAT box binding motifs. A 110-kDa protein (CBF/cdc2) was identified in nuclear extracts from monkey kidney (CV-1) cells stably expressing E1A as well as from adenovirus-transformed human 293 cells. Further, we show that this EIA-inducible CBF/cdc2 is related to the CBF which was shown to activate the heat shock protein 70 promoter. Analyses of the functional domain(s) of E1A required for the induction of the CBF and transactivation of the cdc2 promoter in these conditions revealed that E1A mutants which were defective in binding the pRB family of proteins or the cellular p300 protein were still active in assays measuring the induction of the CBF and transactivation of the cdc2 promoter, albeit with reduced efficiencies. But the E1A mutant which lost both functional domains was inactive in these assays. These results suggest that E1A has redundant functional domains for the induction of the 110-kDa CBF and activation of human cdc2 gene expression.

Adenovirus E1A Proteins↗
PubMed1998

Deregulation of cdc2 gene expression correlates with overexpression of a 110 kDa CCAAT box binding factor in transformed cells.

Eukaryotic cell cycle progression is regulated by an orderly and sequential activation of several cyclin-dependent kinases, which phosphorylate key substrates during this process. p34cdc2, the catalytic subunit of cdc2 kinase, is expressed at the late G1/S boundary and is required for the G2-->M phase transition. Transactivation of the human cdc2 promoter by the DNA tumor virus-encoded oncogenic protein SV40 large T antigen is mediated by induction of a novel 110 kDa CCAAT box binding factor (CBF/cdc2). To investigate whether induction of CBF/cdc2 is an intrinsic property of the viral oncoprotein or is a common event during transformation of normal cells, expression of CBF/cdc2 was analyzed in many human tumor cell lines and in rodent cells spontaneously transformed or stably expressing various oncogenes. Our results showed that CBF/cdc2 was overexpressed in all transformed cells examined, including human 293, MCF-7, HeLa and HepG2 cells. Moreover, expression of CBF/cdc2 was elevated in spontaneously transformed rat liver epithelial cells (C4T), but not detectable in the non-tumorigenic parental (RLE) cells. The elevated levels of CBF/cdc2 expression in C4T cells correlated well with increased cdc2 mRNA and p34cdc2 levels. CBF/cdc2 was also overexpressed in a rat liver epithelial cell line (WB) stably transfected with various oncogenes, v-myc, v-Ha-ras and mutated rat neu and v-src. Using an electrophoretic mobility shift assay, specific binding of CBF/cdc2 to the CCAAT box motifs of the human cdc2, cycA and cdc25C promoters was detected, suggesting that transcription of these cell cycle regulatory genes are coordinately activated by CBF/cdc2.

Animals↗
PubMed1997

Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3(Pro) of dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B.

Polyprotein processing of dengue virus type 2, a positive strand RNA virus, is carried out by the host signal peptidase and a novel two-component viral proteinase of the serine proteinase family, NS2B/NS3(Pro), in the endoplasmic reticulum. Using an in vitro processing system, we examined the cis and trans cleavages of the 2B/3 and 4B/5 sites by NS2B/NS3(Pro), respectively. Lysates of BHK-21 cells coexpressing NS2B and NS3(Pro) mediated trans cleavage of the 4B/5 site in vitro, and the protease activity was associated with the membrane fraction. To study the role of membranes in the protease activity of NS2B/NS3(Pro), labeled precursors, NS2B-NS3(Pro), and the mutant ndNS2B-NS3(Pro) in which the functional hydrophilic domain of NS2B was deleted, were analyzed using a coupled in vitro transcription/translation system (TnT). The results showed that cotranslational addition of microsomal membranes to the TnT reaction markedly enhanced the cis cleavage of the 2B/3 site in a dose-dependent manner. NS2B synthesized in the presence of membranes also facilitated trans cleavage of the 2B/3 site in the mutant precursor. The cleavage products, NS2B and NS3(Pro), were membrane-associated. Furthermore, this membrane requirement was dictated by the hydrophobic regions of NS2B. Deletion of hydrophobic regions of NS2B, leaving only the conserved hydrophilic domain of 40 amino acids, resulted in highly efficient processing of the 2B-3 site in vitro in the absence of microsomal membranes.

Amino Acid Sequence↗
PubMed1997

Hepatitis C virus NS5A protein is phosphorylated in vitro by a stably bound protein kinase from HeLa cells and by cAMP-dependent protein kinase A-alpha catalytic subunit.

Hepatitis C virus (HCV) has a positive-strand RNA genome that codes for a polyprotein precursor, which is processed co- and post-translationally by cellular and viral proteinases into three structural and at least six non-structural (NS) proteins. The NS5A protein, expressed in mammalian cells, exists in two phosphorylated forms of 56-kDa and 58-kDa. In this study, we provide evidence for a stable association between NS5A and a protein kinase from HeLa cells and hepatocellular carcinoma (HepG2) cells by co-immunoprecipitation and by affinity to immobilized glutathione-S-transferase (GST)-NS5A fusion protein produced in E. coli. This protein kinase could phosphorylate in vitro the native NS5A on serine residues, (GST)-NS5A, histone H1, and casein as substrates. In addition, the GST-NS5A was also phosphorylated in vitro by the cAMP-dependent protein kinase A-alpha catalytic subunit.

Binding Sites↗
PubMed1997

Early region 4 modulates adenovirus DNA replication by two genetically separable mechanisms.

Three viral proteins, all products of early region 2 (E2), participate directly in adenovirus DNA replication. Three products of early region 4 (E4) also affect viral DNA synthesis: the product of E4 ORF4 inhibits viral DNA accumulation, while the products of E4 ORFs 3 and 6 antagonize that effect of ORF4 expression. Because no E4 products are required for DNA synthesis, these proteins probably act indirectly. The E4 ORF3, 4, and 6 proteins all participate in aspects of the regulation of viral gene expression. To determine whether they modulate DNA replication by effects on expression of viral replication proteins, we examined E2 expression in E4 mutant-infected cells. In cells infected by ORF3-, 6- mutants, expression of ORF4 substantially depressed the steady-state levels of replication proteins and E2 mRNAs, reduced E2 transcription rates, and profoundly inhibited viral DNA replication. Thus, in the absence of E4 ORFs 3 and 6, ORF4 acts as a transcriptional regulator of E2 expression, and reduced replication protein levels largely account for the inhibition of DNA replication by ORF4. Cells infected by viruses that express ORFs 3 and 6 in addition to ORF4 accumulated much larger quantities of viral DNA than did cells infected by the ORF3-, 6-, 4+ mutant. Increased DNA accumulation was not accompanied by a comparable increase in E2 expression. Therefore, the ORF3 and 6 products counteract the ORF4-induced reduction of DNA replication by a mechanism other than reversing the inhibitory effect of ORF4 on E2 expression. The effect of ORF4 on E2 expression is consistent with its ability to regulate levels of the transcription factor AP-1 (Müller et al., 1992, J. Virol. 66, 5867-5878); the mechanism by which ORFs 3 and 6 enhance replication is unknown.

Adenovirus E2 Proteins↗
PubMed1997

The amino terminal deletion mutants of hepatitis C virus nonstructural protein NS5A function as transcriptional activators in yeast.

To investigate the biological function of hepatitis C virus (HCV)-NS5A, the NS5A was fused at its N-terminus with the DNA binding domain (DBD) of yeast transcriptional activator GAL4 (GAL4-DBD). The GAL4-DBD alone had no transcriptional activation function. However, a mutant of the GAL4-DBD/NS5A fusion protein, in which 129 amino acid residues were deleted from the N-terminus of NS5A, exhibited strong transcriptional activation in yeast cells, bearing the Escherichia coli lacZ reporter gene encoding the beta-galactosidase under the transcriptional control of GAL4 promoter and TATA box. Further mutational analysis of NS5A revealed that the region between the amino acid residues 130 to 352 were critical for optimal level of transactivation. This region includes two acidic domains and one proline-rich region which have been shown to be involved in the function of several transcriptional activators.

Gene Expression Regulation, Viral↗
PubMed1997

Teratogenic effects of vigabatrin in TO mouse fetuses.

Vigabatrin (VGB) is a relatively recently introduced antiepileptic drug that enhances the brain levels of gamma aminobutyric acid (GABA). Few data on its teratogenic effects appear to have been reported. Our objective was to determine if VGB was teratogenic in the TO mouse. Single doses of 300-600 mg/kg of VGB dissolved in saline were administered intraperitoneally (IP) to groups of TO mice on one of gestation days (GD) 7-12. The controls were saline treated or untreated. No maternal toxic effects were observed in the 300 or 450 mg/kg groups, and the 600 mg/kg dose was totally lethal to the mothers. Fetuses were collected on GD 18. Both 300 and 450 mg/kg doses induced a consistently significant intrauterine growth retardation irrespective of the developmental stage at administration. VGB did not augment the spontaneous incidence of neural tube defects characteristic of this strain, but accelerated destruction of the brain in spontaneous exencephalic embryos. Mandibular and maxillary hypoplasia, arched palate, cleft palate (two cases), limb defects (one case), and exomphalos were observed in the malformed fetuses. The high incidence of exomphalos appears to be a unique result of VGB treatment. Alizarin red-S/alcian blue-stained, skeletons revealed hypoplasia of mid facial bones, stage-dependent increase in the frequency of cervical and lumbar ribs, rib fusion, and sternal and vertebral malformations in the drug-treated fetuses. Middle and distal phalanges of the forepaw and mid phalanges and tarsals of the hindpaw failed to ossify in a significant number of experimental fetuses. Homeotic shift in terms of presacral vertebral number and a high incidence of lumbar and cervical ribs in the treated group are suggestive of treatment-related alterations in gene expression. In view of the paucity of human and animal data on the reproductive toxicologic effects of VGB, the results of the present study assume particular importance and suggest that VGB should be used in pregnancy with extreme caution.

Abnormalities, Drug-Induced↗
PubMed1997

Retinoic acid-induced abnormal development of hindlimb joints in the mouse.

Following a single oral dose of 200 mg/kg of retinoic acid (RA) to pregnant TO mice on day 12 of gestation, fetuses were collected on day 18. RA-treatment resulted in a modest increase in embryo resorption, a significant reduction in fetal body weight and a 96% incidence of limb malformations. Both fore- and hindlimbs were malformed in equal frequency. In this study, only the anomalies of the hindlimbs have been considered. Alizarin stained skeletal preparations revealed a reduction in both length and thickness as well as bowing of the long bones. Agenesis of most of the metatarsals and some toes and/or phalanges was also noted. Paraffin sections cut serially confirmed the gross observations. Additionally, the joint cavities were found to be absent and the cartilaginous ends of the articulating bones were continuous with each other. In some this union was partial and occasionally the joint was only represented by a fine slit. The synovial membrane and other intra-articular structures were either rudimentary or absent. There was a marked reduction in endochondral ossification. Bony union in syndactyly was occasionally observed. These data indicate that a high dose of RA, both inhibits skeletal growth and interferes with the differentiation of the inter-zone mesenchyme, thus resulting in abnormalities of the joints in the TO mouse fetuses.

Animals↗
PubMed1996

Characterization of the nuclear localization signal and subcellular distribution of hepatitis C virus nonstructural protein NS5A.

Hepatitis C virus (HCV) has a positive strand RNA genome that codes for a polyprotein that is processed co-translationally and post-translationally into three structural and at least seven nonstructural (NS) proteins. To investigate the function of NS5A, a recombinant vaccinia virus was constructed in which the NS5A gene was cloned under the control of T7 promoter and encephalomyocarditis virus 5'-untranslated region (EMCV-UTR) for cap-independent translation in mammalian cells. In addition, the NS5A gene was also cloned under the control of cytomegalovirus (CMV) early promoter. The NS5A expressed in monkey kidney (CV-1) cells was located predominantly in the cytoplasm. Using immunohistochemical analysis, the subcellular distribution of NS5A in liver biopsy samples from chronic HCV-infected patients was also found to be in the cytoplasm. However, the NS5A protein has a stretch of positively charged domain in the vicinity of proline and valine residues, (PPRKKRTVV), characteristic of a nuclear localization signal (NLS), in the COOH-terminal half of the protein. To investigate whether the putative NLS of NS5A is functional, chimeric expression plasmids were constructed in which regions containing the NLS were fused to the N-terminus of the E. coli beta-galactosidase (E. coli beta-Gal). The expression of the fusion proteins in CV-1 cells resulted in their nuclear localization, indicating that the putative NLS is functional in targeting the heterologous protein, E. coli beta-Gal, to the nucleus, although the native NS5A is retained in the cytoplasm.

Animals↗
PubMed1996

SV40 large T antigen transactivates the human cdc2 promoter by inducing a CCAAT box binding factor.

Cyclin-dependent protein kinases (Cdks) play a key role in the cell division cycle of eukaryotic cells. Cdc2, the first mammalian Cdk that was discovered, is expressed in S phase and functions in the G2 to M phase transition. By transfecting segments of the human cdc2 promoter linked to a reporter gene into monkey kidney (CV-1) cells, we identified the region containing the Sp1, E2F, and two CCAAT box binding sites as essential and sufficient for basal transcription. SV40 large T antigen (SV40-LT) is a viral oncoprotein that transactivates viral and cellular promoters and induces DNA synthesis in quiescent cells. SV40-LT transactivated wild-type cdc2 promoter/reporter constructs in a dose-dependent manner, coinciding with an increase in endogenous cdc2 mRNA. A mutant promoter from which the two CCAAT box motifs were deleted was 8-fold less sensitive to SV40-LT. Activation by SV40-LT did not require its ability to bind the retinoblastoma or p53 tumor suppressor proteins. SV40-LT induced a specific CCAAT box-binding factor (CBF) in CV-1 and COS-7 cells, as judged by gel shift and Southwestern analyses. Similar results were obtained in human fibroblasts expressing a conditional SV40-LT. The SV40-LT-inducible CBF appears to be novel and differs from the CBF that activates heat shock protein 70 gene expression.

Animals↗