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Biomedical subjects

A K Roy

Publications and source records attributed to A K Roy.

At least 91 records · Page 5Linked to original sources
PubMed1991

Structural characterization of the major adducts formed by reaction of 4,5-epoxy-4,5-dihydro-1-nitropyrene with DNA.

The only available marker of DNA adducts formed from 1-nitropyrene (1-NP) and DNA, N-(deoxyguanosin-8-yl)-1-aminopyrene, is derived from the nitroreduction pathway. Our studies, as well as those of others, have indicated that multiple DNA adducts are formed from 1-NP in vivo and in vitro. Thus the need for additional DNA adduct markers was apparent. Therefore, it was our goal to characterize the DNA adducts formed from 4,5-epoxy-4,5-dihydro-1-nitropyrene, a metabolite of 1-NP. The epoxide was incubated with calf thymus DNA (pH 5.4). The DNA was enzymatically hydrolyzed to deoxyribonucleosides which were analyzed by reverse phase HPLC. Three major peaks were obtained in yields less than 5%. The structural assignment of these adducts was made by comparison of their proton nuclear magnetic resonance spectra with those of cis- and trans-4,5-dihydro-4.5-dihydroxy-1-nitropyrene, and by long range coupling constants, decoupling experiments, D2O exchange, partitions and acid hydrolysis. Two adducts result from trans and one from cis addition of the N2-exocyclic amino group of deoxyguanosine to the C5-benzylic carbon of the epoxide ring. This is the first report that describes the structure of the DNA adducts formed with a ring-oxidized metabolite of 1-NP. On the basis of this finding we suggest that K-region oxides of 1-NP may be responsible for the formation of the putative 1-NP-DNA adducts in vivo.

Chromatography, High Pressure Liquid↗
PubMed1991

Characterization of two neuroblastoma cell lines expressing recombinant nerve growth factor receptors.

In earlier studies, a 75,000-dalton glycoprotein (gp75) has been identified as a component of both low- and high-affinity nerve growth factor (NGF) receptors (NGFRs). Using an amphoteric expression vector, we have introduced the cDNA encoding the human gp75 into two neuroblastoma cell lines. SHEP is a human neuroblastoma cell line that lacks most neuronal characteristics and does not express NGFRs. The transformant line SHEP/NGFR expressed a single affinity class of NGF binding sites, did not display NGF-induced up-regulation of fos oncogene expression, and did not efficiently internalize NGF. LAN5 is a neuroblastoma cell line with neuronal characteristics, including expression of neurofilament and display of short neurites. This cell line expresses a small number of high-affinity NGFRs but no detectable low-affinity sites. The transformant line LAN5/NGFR expressed both high- and low-affinity NGFRs, displayed NGF-induced up-regulation of fos oncogene, and efficiently internalized NGF. The number of high-affinity NGF binding sites was nearly the same for LAN5 and LAN5/NGFR, a finding suggesting that there is a limiting number of some separately coded factor or subunit that is required for high-affinity NGFRs. Because NGF induction of fos oncogene expression correlated with expression of high-affinity NGFRs, the putative second factor may also limit NGF responsiveness.

Binding Sites↗
PubMed1991

Age-dependent reversal of the lobular distribution of androgen-inducible alpha 2u globulin and androgen-repressible SMP-2 in rat liver.

Hepatocytes situated at pericentral and periportal zones of the liver lobule show differences in the expression of several liver-specific genes, such as androgen-inducible alpha 2u globulin and androgen-repressible senescence marker protein-2 (SMP-2). A marked temporal difference in the expression of these two androgen-regulated genes has also been observed. The liver of the pre-pubertal male rat is insensitive to androgen, and during this period hepatocytes synthesize only SMP-2. During young adult life (greater than 40 days), the liver becomes androgen sensitive and concomitant synthesis of alpha 2u globulin and repression of SMP-2 occur. In the senescent male rat (greater than 750 days), the liver again becomes androgen insensitive when the decline in alpha 2u globulin is accompanied by an increase in SMP-2 synthesis. In this article we present results to show a correlation between the temporal and spatial (intralobular) changes in the expression of the androgen-inducible alpha 2u globulin and the androgen-repressible SMP-2 in rat hepatocytes. Results indicate that the temporal changes in hepatic androgen sensitivity are dictated by the intralobular location of the hepatocytes. Hepatocytes located around the central vein (pericentral/perivenous) may benefit from a paracrine advantage for the expression of a subset of genes, including the gene for the androgen receptor.

Aging↗
PubMed1991

Androgen receptor messenger ribonucleic acid (mRNA) in the rat liver: changes in mRNA levels during maturation, aging, and calorie restriction.

By means of RNAase protection assay with an antisense cRNA probe, we have shown that the liver of the young adult male rat contains androgen receptor (AR) mRNA to a level of 4% compared to the prostate. Steady state levels of AR mRNA in the liver show both sex and age specificity. Compared to that of the male, the female liver contains a markedly reduced amount of AR mRNA. AR mRNA is almost undetectable in livers of prepubertal male (less than 35 days old) and senescent male (greater than 750 days old) rats. Both prepubertal and senescent animals are relatively insensitive to the androgenic induction of alpha 2u-globulin, a hepatic secretory protein. The age-dependent decline in hepatic androgen sensitivity and AR mRNA level can be delayed considerably by a 40% reduction in the dietary calorie intake. Analysis of poly(A)-containing RNA from two liver cell populations, hepatocytes and nonhepatocytes, revealed that only the hepatocytes that express alpha 2u-globulin gene contain AR mRNA. From these results and our earlier observation of in vitro induction of alpha 2u-globulin in isolated rat liver, we conclude 1) that androgen can act directly on hepatocytes to promote alpha 2u-globulin synthesis; 2) that changes in the hepatic androgen sensitivity during maturation and aging are reflections of the age-dependent expression of the receptor gene; and 3) that retardation of the age-dependent loss of androgen sensitivity by calorie restriction is due to a concomitant delay in the decline of the hepatic AR mRNA level.

Aging↗
PubMed1991

Effects of aluminium salts on bone marrow chromosomes in rats in vivo.

Oral administration of aluminium sulphate to laboratory bred Rattus norvegicus for prolonged period induced dose dependent inhibition of dividing cells and an increase in chromosomal aberrations. The effect was not influenced by the duration of exposure. The toxicity of the two salts, aluminium sulphate and potassium aluminium sulphate, did not differ significantly at doses in which the metal contents were kept constant.

Alum Compounds↗
PubMed1991

Clinical spectrum of myasthenia gravis and problems of management.

Seventeen cases of myasthenia gravis, aged 20-79 years, including one case of ocular myasthenia gravis, seen over a 3 year period, were analysed. Dysphagia was the presenting symptom in two cases. One patient had ectopic thymus gland in relation to the cardiac border, which posed difficulty in diagnosis. A majority of the patients were on medical treatment, including neostigmine, prednisolone and azathioprim. Plasmapheresis was done in 4 cases during crisis, with significant benefit. One patient was put on intermittent ventilatory support for 14-16 hours a day for over 2 years; she died during a power failure. Seven patients were subjected to thymectomy.

Adult↗
PubMed1991

Epilepsy as a manifestation of neurocysticercosis.

Epilepsy is the commonest manifestation of neurocysticercosis. Epilepsy was observed in 127 of 150 cases (84.7%) of neurocysticercosis seen over a period of 17 years. The basis of diagnosis was clinical presentation and concomitant evidence of extraneural cysticercosis in the pre-computed tomography (CT) scan era, and typical CT findings in later years. Eighty one cases (54%) who primarily presented as epilepsy without any neurological deficit have been analysed in detail. In the pre-CT period the occurrence of epilepsy as a presenting feature in neurocysticercosis was 43.5% whereas in later years it was 61.4%. Primary generalised seizure (49 cases) was more common than partial seizure (29) and partial complex seizure (3). Status epilepticus was seen in 6 cases. Magnetic resonance imaging, done in 8 cases, proved to be more sensitive in demonstrating various stages in the development of noncalcified cysticercosis. The new larvicidal drugs offer a potential cure and cysticercosis should be entertained as a cause of epilepsy especially in endemic areas.

Brain Diseases↗
PubMed1990

Changes in hepatic androgen sensitivity and gene expression during aging.

The hepatic tissue of the male rat exhibits a gradual decline and ultimate loss in androgen responsiveness during in vivo aging. Appearance of the age-associated androgen insensitivity can be delayed by dietary calorie restriction, an effective means for life-span extension. The androgen receptor mRNA is detectable in the liver only in its androgen-responsive state. Pubertal appearance of hepatic androgen sensitivity is remarkably correlated with the concomitant appearance of a cytoplasmic androgen binding (CAB) protein. Androgen resistance during senescence is associated with the loss of hepatic CAB activity as well. We are investigating the molecular basis for the temporal modulation of this hormone sensitivity through studies on the differential expression of two androgen-responsive marker genes. These are the androgen-repressible SMP-2, and the androgen-inducible alpha 2u-globulin. Androgen resistance of hepatocytes during aging results in repression of the alpha 2u-globulin gene, and derepression of the SMP-2 gene. The structural organizations for both of these genes have been characterized. The role of nuclear transcription factors (androgen receptor and any other transacting factor(s) which may be involved) in the coordinate regulation of alpha 2u-globulin and SMP-2 during aging and nutritional manipulation is being explored to establish the molecular mechanism of andropause in the liver.

Aging↗
PubMed1990

The senescence marker protein (SMP-2) of the rat liver: purification, immunochemical characterization and age-dependent regulation.

In vitro translation of total rat hepatic mRNAs has identified a 31 kilodalton senescence marker protein (SMP-2) which is present in higher amounts in prepubertal and senescent males than in the post-pubertal adult male (more than 10-fold). SMP-2 is an androgen-repressible protein. The negative regulation of the SMP-2 gene activity by androgen accounts for its increased expression during the androgen insensitive states of the prepubertal and senescent livers, and its constitutive expression in the female liver. A combination of separation procedures including salt fractionation, chromatofocusing, ion-exchange chromatography and preparative gel electrophoresis have led to the purification of SMP-2 to apparent homogeneity. The purified protein showed the same electrophoretic mobility as the sex- and age-specific in vitro translation product of hepatic mRNAs. The polyclonal antibody to SMP-2 was produced in the rabbit. The antibody selectively reacted with the 31 kDa sex- and age-specific translation product of hepatic mRNAs. Western blot analysis of the liver cytosol confirms monospecificity of the antiserum, as well as age- and sex-dependent changes in the tissue level of SMP-2. Histochemical staining of liver sections with the antiserum reveals a preferential periportal localization of SMP-2 in the hepatocytes. This finding is in marked contrast to the androgen-inducible alpha 2u globulin which is preferentially synthesized and localized in the pericentral hepatocytes. Thus, the zonal distribution of SMP-2 correlates with polarized androgen sensitivity of the hepatocytes within the liver lobule.

Aging↗
PubMed1990

Structure and regulation of the senescence marker protein 2 gene promoter.

The liver-specific expression of the senescence marker protein 2 (SMP-2) in the male rat is markedly reduced during the androgen-sensitive state of young adulthood, whereas it is up-regulated during the androgen-insensitive phases of prepuberty and senescence. Nuclear runoff studies show that the age-dependent changes in SMP-2 expression are due to transcriptional regulation of the gene. In order to explore the mechanism of the regulatory process, we have cloned the upstream flanking regions of two distinct SMP-2 genes (SMP-2A and SMP-2B) and established their nucleotide sequence. These clones contain approximately 2.2 kb of the 5'-flanking sequence, exons 1 and 2, the first intron, and a portion of the second intron. The SMP-2 genes, as well as the upstream sequences, contain the sequence motifs for a number of cis-acting regulatory elements, such as the hepatocyte-specific element (HP1) and the androgen response element (ARE). S1 nuclease and primer extension analyses have established the transcription initiation sites for these genes. For functional analysis of the upstream sequences, we have constructed a hybrid plasmid containing the SMP-2A gene sequence (-1970 to +38 bases) fused to the structural gene for chloramphenicol acetyl-transferase (CAT). Upon transfection into rat hepatoma cells (FT02B), this construct was able to drive expression of the CAT gene. The same construct, however, failed to function in fibroblast-derived L cells, indicating tissue-specific regulation of the construct promoter.

Animals↗
PubMed1990

Expression of the human inhibin alpha-subunit gene in preovulatory granulosa-theca cells.

Inhibin, a gonadal peptide that suppresses pituitary follicle-stimulating hormone, with lesser or no effect on luteinizing hormone, has recently been purified and the complementary deoxyribonucleic acid sequences cloned. Inhibin contains two subunits, labeled alpha-subunit and beta-subunit. Here we report for the first time the detection of human inhibin alpha-subunit gene expression in preovulatory granulosa-theca cells by Northern analysis. The transcript is the same size as previously reported for human placenta and corpus luteum, suggesting that the same gene is being expressed in all three tissues. These findings are consistent with previously reported Southern analysis of deoxyribonucleic acid, which showed only one copy of the alpha-inhibin gene in the human genome. Thus current data strongly suggest that there is only one copy of the inhibin alpha-subunit gene in the human genome, and this same gene is expressed in granulosa-theca cells, corpus luteum, and placenta.

Animals↗
PubMed1990

Modification of clastogenicity of lead and aluminium in mouse bone marrow cells by dietary ingestion of Phyllanthus emblica fruit extract.

Extract of Phyllanthus emblica fruit and ascorbic acid were evaluated separately for protection against clastogenicity induced by lead (Pb) and aluminium (Al) salts on mouse bone marrow chromosomes. Oral administration of Phyllanthus fruit extract (PFE) for 7 days before exposure to both metals by intraperitoneal injection increased the frequency of cell division and reduced the frequency of chromosome breaks significantly. Comparable doses of synthetic ascorbic acid (AA) were less effective and could protect against the effects of Al and only a low dose of Pb (10 mg/kg body weight). AA administered before treatment in mice given higher doses of Pb (40 mg/kg body weight) enhanced the frequency of chromosome breaks, giving a synergistic effect. The higher protection afforded by PFE may be due to the combined action of all ingredients, rather than to AA alone.

Administration, Oral↗
PubMed1990

Transient expression of full-length and truncated forms of the human nerve growth factor receptor.

To facilitate the characterization of nerve growth factor (NGF) receptor and mutated forms of the receptor, we have set up a rapid, efficient transient expression system utilizing COS cells. The human NGF receptor is a 427 amino acid protein with a hydrophobic signal sequence, a 222 amino acid extracellular domain, a single transmembrane domain and a 155 amino acid intracellular domain. The NGF receptor and a truncated form lacking the cytoplasmic domain were expressed in a COS cell expression system. Both recombinant proteins were detected on the cell surface and at a perinuclear site. Specific binding of 125I-NGF to the recombinant proteins was detected by chemical cross-linking. The extracellular domain of the NGF receptor was also expressed in the same system and detected in the COS cell endoplasmic reticulum and in the culture supernatant. This recombinant protein also specifically binds NGF.

Animals↗
PubMed1990

Introduction of nerve growth factor (NGF) receptors into a medulloblastoma cell line results in expression of high- and low-affinity NGF receptors but not NGF-mediated differentiation.

Expression of the cloned human nerve growth factor receptor (NGFR) cDNA in cell lines can generate both high- and low-affinity binding sites. Since the inability to respond appropriately to differentiation factors such as NGF may contribute to determining the malignant phenotype of neuroblastomas, we sought to determine whether the same is true of medulloblastomas. To generate a human central nervous system neuronal cell line that would respond to NGF, we infected the medulloblastoma cell line D283 MED with a defective retrovirus carrying the cDNA coding for the human NGFR. The resultant cells (MED-NGFR) expressed abundant low- and high-affinity NGFRs, and NGF treatment induced a rapid transient increase of c-fos mRNA in the NGFR-expressing cells but not in the parent line or in cells infected with virus lacking the cDNA insert. However, the MED-NGFR cells did not internalize the NGFR at high efficiency, nor did they differentiate in response to NGF. Three important conclusions emerge from this study: (i) internalization of NGFRs is not necessary for some early rapid transcriptional effects of NGF; (ii) an unknown factor(s) that cooperates with the cloned NGFR in allowing high-affinity NGF binding is found in a primitive central nervous system cell line; and (iii) NGFRs introduced into and expressed by D283 MED (i.e., MED-NGFR) cells are partially functional but are unable to induce differentiation in these primitive neuron-like tumor cells, implying that high-efficiency receptor-mediated endocytosis of NGF and its receptor may be a necessary step in the cascade of events leading to NGF-mediated differentiation.

Animals↗
PubMed1990

Androgen regulated expression of the alpha 2u-globulin gene in pancreatic hepatocytes of rat.

Under a copper-deficient regimen, pancreatic cells in the adult rat can be found to undergo differentiation into hepatocytes. Pancreatic hepatocytes induced in male and female rats were examined for the expression of the androgen-inducible hepatic protein, alpha 2u-globulin. Alpha 2u-Globulin protein was demonstrable by immunoperoxidase method in all the pancreatic hepatocytes of male rats. Northern blot analysis confirmed the presence of 1.3 kb alpha 2u-globulin mRNA transcript in the pancreas of male rats with hepatocytes. Orchiectomy resulted in marked decrease of alpha 2u-globulin protein and its mRNA. Administration of dihydrotestosterone to castrated rats resulted in increased levels of alpha 2u-globulin mRNA and the amount of alpha 2u-globulin protein in the pancreatic hepatocytes. Unlike normal males, in intact and ovariectomized females alpha 2u-globulin was not detectable in pancreatic hepatocytes. These results indicate that similar to hepatic parenchymal cells pancreatic hepatocytes synthesize alpha 2u-globulin under androgenic regulation. Furthermore, unlike in liver where it is expressed predominantly in perivenular and midlobular hepatocytes, there is no localized difference in the expression of this gene in the transdifferentiated pancreatic hepatocytes.

Alpha-Globulins↗