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Biomedical subjects

S N Sheikh

Publications and source records attributed to S N Sheikh.

8 recordsLinked to original sources
PubMed2001

The synthetic retinoid AGN 193109 but not retinoic acid elevates CYP1A1 levels in mouse embryos and Hepa-1c1c7 cells.

The synthetic retinoid AGN 193109 is a potent pan retinoic acid receptor (RAR) antagonist. Treatment of pregnant mice with a single oral 1 mg/kg dose of this antagonist on day 8 postcoitum results in severe craniofacial (median cleft face or frontonasal deficiency) and eye malformations in virtually all exposed fetuses. Using differential display analysis, we have determined that CYP1A1 mRNA levels are elevated in mouse embryos 6 h following treatment with AGN 193109. Similarly, an elevation in CYP1A1 mRNA levels, protein levels, and aryl hydrocarbon hydoxylase activity occurs in Hepa-1c1c7 cells, with the maximal elevation observed when the cells were treated with 10(-5) M AGN 193109 for 4 to 8 h. Elevation in CYP1A1 mRNA levels in mouse embryos and Hepa-1c1c7 cells does not occur upon treatment with the natural retinoid, all-trans-retinoic acid. Finally, elevation in CYP1A1 mRNA levels was not observed when mutant Hepa-1c1c7 cells, which are defective in either the aryl hydrocarbon receptor (AhR) or aryl hydrocarbon receptor nuclear translocator (ARNT), were treated with AGN 193109. This suggests that the AhR/ARNT pathway and not the RAR/RXR pathway is mediating the elevation of CYP1A1 mRNA levels by AGN 193109, at least in the Hepa-1c1c7 cells. This is the first example of a retinoid that displays the abililty to regulate both the RAR/RXR and AhR/ARNT transcriptional regulatory pathways.

Animals↗
PubMed1999

Regional distribution and relative amounts of glutamate decarboxylase isoforms in rat and mouse brain.

The levels of the two isoforms of glutamate decarboxylase (GAD) were measured in 12 regions of adult rat brain and three regions of mouse brain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting with an antiserum that recognizes the identical C-terminal sequence in both isoforms from both species. In rat brain the amount of smaller isoform, GAD65, was greater than that of the larger isoform, GAD67, in all twelve regions. GAD65 ranged from 77-89% of total GAD in frontal cortex, hippocampus, hypothalamus, midbrain, olfactory bulb, periaqueductal gray matter, substantia nigra, striatum, thalamus and the ventral tegmental area. The proportion of GAD65 was lower in amygdala and cerebellum but still greater than half of the total. There was a strong correlation between total GAD protein and GAD activity. In the three mouse brain regions analysed (cerebellum, cerebral cortex and hippocampus) the proportion of GAD65 (35,47, and 51% of total GAD) was significantly lower than in the corresponding rat-brain regions. The amount of GAD67 was greater than the amount of GAD65 in mouse cerebellum and was approximately equal to the amount of GAD65 in mouse cerebral cortex and hippocampus.

Amino Acid Sequence↗
PubMed1998

Elevation of brain GABA levels with vigabatrin (gamma-vinylGABA) differentially affects GAD65 and GAD67 expression in various regions of rat brain.

Previous studies have demonstrated that the expression of one of the isoforms of glutamate decarboxylase, GAD67, is selectively reduced in cultured cortical neurons and in rat cerebral cortex when the concentration of GABA is elevated. We asked whether the expression of GAD67 was similarly affected by elevated GABA throughout the brain. The concentration of GABA in rat brain was increased by inhibiting GABA transaminase (GABA-T) with vigabatrin (gamma-vinylGABA, GVG), an antiepileptic drug and selective inhibitor of GABA-T. Rats were injected with saline or vigabatrin (150 mg/kg) daily for 5 days, and the effects of accumulated GABA on total GAD activity and the expression of GAD65 and GAD67 proteins were determined in twelve brain regions. The GABA concentration was significantly elevated in all regions except amygdala and olfactory bulb after vigabatrin treatment. Total GAD activity was significantly lower than controls in six regions: cerebellum, frontal cortex, thalamus, substantia nigra, ventral tegmentum, and the remaining midbrain. The decrease in GAD activity was largest in cerebellum and thalamus (33% and 29%), while the changes in the other four areas were 15-18%. Vigabatrin treatment significantly reduced GAD67 protein in all regions except olfactory bulb, whereas GAD65 protein decreased significantly only in cerebellum. The failure to detect significant changes in GAD activity in regions having a significant change in GAD67 levels is attributable to the small contribution of GAD67 to total GAD in those regions. It is evident that there are marked regional differences in the effects of tissue GABA levels on the expression of GAD67.

Animals↗
PubMed1998

p53, but not p16 mutations in oral squamous cell carcinomas are associated with specific CYP1A1 and GSTM1 polymorphic genotypes and patient tobacco use.

Inactivation of tumor suppressor genes like p53 and p16 play a key role in tumor progression, with a high incidence of mutations existing for both genes in oral squamous cell carcinomas. Previous studies have demonstrated, (i) a correlation between the prevalence of p53 mutations and tobacco use [Brennan et al. (1995) New Engl. J. Med., 332, 712-717; Lazarus et al. (1996) Carcinogenesis, 17, 733-739], and (ii) a link between genotypes in specific xenobiotic metabolizing enzymes and oral cancer susceptibility [Park et al. (1997) Cancer Epid. Biomarkers Prev., 6, 791-797). In this paper, we present results of our examination of a series of 80 oral squamous cell carcinomas for p53 exons 5-9 and p16 exons 1-2 mutations, and the potential association of these mutations with specific genotyping patterns. p53 mutation prevalence in oral tumors was linked with increased patient tobacco use using several stratification criteria. There was a significantly higher prevalence of p53 mutations in OCSCCs from patients who smoked > 30 pack-years as compared to tumors from patients who smoked < or = 30 pack-years (OR = 2.8; CI = 1.1-7.2). No significant association was observed with patient alcohol consumption. There was a significant association between the prevalence of p53 mutations in oral tumors and CYP1A1 genotyping patterns in these oral cancer patients, with the highest p53 mutation prevalence observed in subjects with the CYP1A1 [val]/GSTM1 [+] genotype (OR = 6.0; CI = 1.2-29.7). A significant association was not observed between the prevalence of p16 mutations in oral tumors and tobacco use, or CYP1A1 [val] or GSTM1 (0/0) genotypes. These data suggest that the induction of mutations in specific tumor suppressor genes or oncogenes in oral tumors may be associated with specific carcinogen exposures, and that this association may be linked to specific polymorphic genotypes in xenobiotic-metabolizing enzyme genes.

Benzo(a)pyrene↗
PubMed1997

Re-usable DNA template for the polymerase chain reaction.

DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.

Carrier Proteins↗
PubMed1996

Heteromers of glutamate decarboxylase isoforms occur in rat cerebellum.

The subunit structure of brain glutamate decarboxylase in cerebellum was investigated by using gel electrophoresis and antisera that specifically recognize the individual isoforms of brain glutamate decarboxylase (termed GAD65 and GAD67). The antisera were prepared against peptides that corresponded to amino acid sequences specific to each isoform. Each antiserum reacted specifically with the appropriate peptide in an ELISA and with the appropriate form of GAD on immunoblots. Nondenaturing gradient gel electrophoresis indicated that GAD is principally multimeric with monomeric forms comprising < 3% of the total. Immunoprecipitation and immunoaffinity chromatography experiments were performed with antisera W624 and W883, which were prepared against peptides specific to GAD65 and GAD67, respectively. Immunoprecipitates prepared from cerebellar supernatants with W624 contained both GAD65 and GAD67, whereas some GAD67 was left in the supernatant. In a similar manner, immunoprecipitates prepared with W883 contained both GAD65 and GAD67, whereas some GAD65 remained in the supernatant. In addition, immunoaffinity columns prepared with either W624 or W883 retained both GAD65 and GAD67 even after extensive washing. These results are consistent with the presence of heteromultimers of GAD65 and GAD67 in cerebellum in addition to homomers of each form.

Amino Acid Sequence↗
PubMed1993

Effects of increased gamma-aminobutyric acid levels on GAD67 protein and mRNA levels in rat cerebral cortex.

Rats were injected with saline or the gamma-aminobutyric acid (GABA) transaminase inhibitor gamma-vinyl-GABA for 7 days and the effects on GABA content and glutamic acid decarboxylase (GAD) activity, and the protein and mRNA levels of the two forms of GAD (GAD67 and GAD65) in the cerebral cortex were studied. gamma-Vinyl-GABA induced a 2.3-fold increase in GABA content, whereas total GAD activity decreased by 30%. Quantitative immunoblotting showed that the decline in GAD activity was attributable to a 75-80% decrease in GAD67 levels, whereas the levels of GAD65 remained unchanged. RNA slot-blotting with a 32P-labeled GAD67 cDNA probe demonstrated that the change in GAD67 protein content was not associated with a change in GAD67 mRNA levels. Our results suggest that GABA specifically controls the level of GAD67 protein. This effect may be mediated by a decreased translation of the GAD67 mRNA and/or a change in the stability of the GAD67 protein.

Aminocaproates↗