Biomedical subjects

U R Pendurthi

Publications and source records attributed to U R Pendurthi.

28 records · Page 2Linked to original sources

PubMed1992

Phorbol esters inhibit the dioxin receptor-mediated transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Tetradecanoyl phorbol acetate (TPA) has been shown to inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced mouse P450IA1 benzo[a]pyrene hydroxylase activity (Raunio, H., and Pelkonen, O. (1983) Cancer Res. 43, 782-786). When we co-administered TPA and TCDD to C57BL/6 mice, the accumulation of TCDD-inducible liver P450IA1 and P450IA2 mRNA, as well as kidney P450IA1 mRNA, was greatly inhibited. When nuclear run-on assays were conducted, maximal levels of transcriptional activation were achieved for both liver Cyp1a-1 and Cyp1a-2 with 1 micrograms/kg (approximately equal to 3.0 nmol/kg) TCDD. TCDD elicited a dose-dependent increase in the rates of gene transcription, which paralleled the induction of P450IA1 and P450IA2 mRNA. Only Cyp1a-1 gene transcription was elevated in kidney. When these experiments were repeated following the co-administration of TPA with TCDD, the levels of TCDD-mediated transcriptional increases in liver Cyp1a-1 and Cyp1a-2 and P450IA1 and P450IA2 mRNAs were dramatically inhibited. The reduction in Cyp1a gene transcription by TPA could be accounted for by reduced DNA binding of the dioxin receptor to the xenobiotic-responsive element (XRE) sequences, as measured by gel-retardation analysis. Analysis of nuclear [3H]TCDD dioxin receptor by sucrose density gradients demonstrated that the inhibition of Cyp1a gene transcription and DNA binding by TPA resulted from a reduction in nuclear dioxin receptor concentration.

Animals↗

PubMed1992

Characterization of a rabbit factor IX cDNA.

A rabbit factor IX cDNA was isolated and characterized. The cDNA was 2,676 bp in length and contained the coding region for the leader peptide, the entire mature factor IX protein and a long 3' untranslated region. The deduced amino acid sequence shows a high degree of homology with the sequences of human factor IX and factor IX from other species. Northern blot analysis of liver RNA showed a single mRNA species of 2.8 kb for the rabbit factor IX.

Amino Acid Sequence↗

PubMed1991

Mechanism for diminished tissue factor expression by endothelial cells cultured with heparin binding growth factor-1 and heparin.

We have extended our earlier observation that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with heparin binding growth factor 1 (HBGF-1) 20 micrograms/mL and heparin 12 U/mL inhibits expression of tissue factor (TF) activity on HUVC monolayers perturbed with thrombin. TF activity was measured as the ability of monolayers or cell lysates to support FVIIa-catalyzed activation peptide release from 3H-FX. TF antigen in HUVEC extracts was measured in an enzyme-linked immunosorbent assay (ELISA) that uses a double-antibody sandwich technique with rabbit and goat antibodies to human TF. TF-mRNA was measured by Northern blot hybridization with a 32P-TF cDNA probe. Cells growth with HBGF-1/heparin had both decreased surface and total TF activity as compared with HUVEC from the same endothelial cell pool grown without HBGF-1/heparin. Means +/- SD for TF antigen for four primary cultures were 4.4 +/- 0.9 ng/10(6) cells without HBGF-1/heparin and 0.6 +/- 0.3 ng/10(6) cells with HBGF-1/heparin. TF mRNA 4 hours after incubation with thrombin of HUVEC grown without HBGF-1/heparin was about sevenfold higher than TF mRNA of HUVEC grown with HBGF-1/heparin. These data establish that growing primary cultures of HUVEC with HBGF-1/heparin impairs their ability to synthesize TF apoprotein after perturbation. This may be part of a generalized response of endothelial cells to HBGF-1/heparin facilitating migration during angiogenesis.

Antigens↗

PubMed1990

Characterization of the CYP2C5 gene in 21L III/J rabbits. Allelic variation affects the expression of P450IIC5.

Rabbits exhibit two heritable phenotypes, 21L and 21H, with the latter displaying roughly 10-fold higher liver microsomal progesterone 21-hydroxylase activity. The higher activity of the 21H animals reflects the elevated expression of cytochrome P-450 1 (P450IIC5). Breeding studies indicate that this phenotypic difference is linked to allelic CYP2C5 genes (Johnson, E. F., Finlayson, M., Husjsak, C. M., Pendurthi, U. R., and Tukey, R. H. (1989) Arch. Biochem. Biophys. 273, 273-280). In this study, we report on the expression and structure of the CYP2C5 gene in the 21L inbred strain III/J. A cDNA encoding P450IIC5 was generated from III/J rabbit liver mRNA using polymerase chain reaction to amplify this sequence. DNA sequence analysis revealed that the cDNA encoded the same protein as the cDNA cloned previously from a 21H rabbit, demonstrating that structural differences in P450IIC5 do not underlie the phenotypic difference in the expression of 21-hydroxylase activity. DNA sequence analysis of the exons and surrounding intron regions in two III/J genomic clones that encode portions of the CYP2C5 gene showed that the exon sequences were identical to that of the P450IIC5 mRNA and that the codon positions of the exon/intron junctions are conserved with other class II genes. Southern blot analysis of DNA digested with BamHI and probed with a portion of DNA isolated from intron 5 revealed that the gene characterized in this study is an allele of the gene conferring the 21H phenotype. Primer extension analysis with a P450IIC5-specific oligonucleotide demonstrated that the CYP2C5 gene uses several transcriptional start sites in both 21H and 21L rabbits. Analysis of mRNA from livers of 21H and 21L rabbits with the P450IIC5-specific oligonucleotide showed a greater than 20-fold difference in the relative abundance of mRNA, which accounts quantitatively for the phenotypic difference in progesterone 21-hydroxylase activity.

Alleles↗

PubMed1989

Regulation of the rabbit cytochrome P-450 3c gene. Age-dependent expression and transcriptional activation by rifampicin.

The macrolide antibiotic rifampicin is a potent inducer of cytochrome P-450-mediated drug metabolism in humans and rabbits. In this report, we demonstrate that in immature rabbits, rifampicin activates the transcription of the Cyp3A6 gene which encodes P450IIIA6 (cytochrome P-450 3c). The maximum increase in transcription was seen at 12 h following administration of rifampicin. Northern and slot blot analyses indicate that mRNAs corresponding to P450IIIA6 accumulates during the period of increased transcription and persist at 18 h when the rate of transcription has returned to basal levels. P450IIIA6 protein accumulates in liver microsomes over this period. At 24 h a greater than 10-fold increase in microsomal P450IIIA6 protein is detected by immunoblotting using a monoclonal antibody to P450IIIA6. The rate of microsomal progesterone 6 beta-hydroxylase activity is also elevated when compared to untreated rabbits, and this enzyme is activated in vitro by alpha-naphthoflavone. To determine whether this enzyme is stimulated by alpha-naphthoflavone in intact cells, COS-1 cells were transfected with an expression vector harboring the coding region of a P450IIIA6 cDNA. Our results demonstrate that the transfected COS-1 cells exhibit progesterone 6 beta-hydroxylase activity that is stimulated by alpha-naphthoflavone added to the culture medium.

Aging↗

PubMed1989

Genetic contributions to the variation among rabbits of liver microsomal deoxycorticosterone synthesis.

Outbred New Zealand white rabbits exhibit two phenotypes, 21H and 21L, corresponding to rates greater than or less than 1 nmol/min/mg, respectively, for liver microsomal progesterone 21-hydroxylase activity. In contrast, the inbred strain III/J exhibits only the 21L phenotype. Two 21H male New Zealand white rabbits were mated with several female III/J rabbits to produce a total 46 progeny. Both the 21H and 21L phenotypes were evident among male and female offspring in roughly equal numbers. Backcrosses between 21L progeny and III/J rabbits exhibit only the 21L phenotype, whereas 21H offspring yield both 21H and 21L progeny when backcrossed to the 21L inbred strain III/J. These results are consistent with autosomal dominant inheritance of the 21H phenotype. Analysis of Southern blots of genomic DNA digested with the restriction endonuclease KpnI reveals 20-, 13-, and 9-kb fragments that hybridize with a probe derived from the 3'-untranslated region of the 21-hydroxylase cDNA. The 13-kb band is not observed for strain III/J or 21L progeny of strain III/J crossed with 21H rabbits, but it is detected for both 21H fathers and 21H progeny indicating that the genetically determined difference of 21-hydroxylase expression is inherited cis to the gene for P450IIC5, the hepatic progesterone 21-hydroxylase. Electrophoretic analysis of P450IIC5 synthesized in vitro from mRNA isolated from 21L and 21H rabbits reveals that little or no P450IIC5 is synthesized from 21L mRNAs. A second immunoreactive, electrophoretically distinct protein is synthesized from both 21L and 21H mRNAs to a similar extent but in lesser amounts than P450IIC5. The second protein could represent either an allozymic form of the enzyme or the product of a distinct locus. Thus, it is likely that distinct structural genes for P450IIC5 contribute to the differences in P450-mediated metabolism in 21L as compared to 21H rabbits.

Animals↗

PubMed1988

Molecular defect in factor IXBm Lake Elsinore. Substitution of Ala390 by Val in the catalytic domain.

Earlier studies with factor IXBm Lake Elsinore (IXBmLE), a nonfunctional variant of factor IX, suggested that the defect in this protein may reside in the catalytic domain of the molecule (Usharani, P., Warn-Cramer, B. J., Kasper, C. K., and Bajaj, S. P. (1985) J. Clin. Invest. 75, 76-83). In this report, genomic DNA fragments from normal IX and IXBmLE alleles were cloned into phage lambda EMBL3 and the recombinant phage identified using normal IX cDNA and synthetic oligonucleotides. Exons VI, VII, and VIII of normal IX and IXBmLE gene were also amplified using a newly developed primer-directed polymerase chain reaction method. All eight exons and flanking regions of the normal IX and IXBmLE gene were sequenced by the dideoxy chain termination method. Comparison of the normal IX and IXBmLE sequences revealed a single base substitution (C----T) in the exon VIII of the BmLE variant, which results in the replacement of Ala390 by Val in the variant molecule. Although this mutation is in the catalytic domain of the molecule, purified factor IXaBmLE is indistinguishable from normal IXa in its activity toward a small synthetic substrate, L-tosylarginine methyl ester. These data, coupled with the previous data, identify a region (around residue 390) in the normal factor IXa which appears to play a major role in the extended macromolecular substrate binding site.

Alanine↗

PubMed1987

Characterization of multiple human cytochrome P-450 1 cDNAs. The chromosomal localization of the gene and evidence for alternate RNA splicing.

Employing the rabbit liver progesterone-21-hydroxylase P-450 1 cDNA as a probe (Tukey, R.H., Okino, S., Barnes, H., Griffin, K.J., and Johnson, E.F. (1985) J. Biol. Chem. 260, 13347-13354), we have identified a highly homologous (81% within the coding region) human liver cDNA, termed Hp1-1, that encodes a 490-amino acid protein. Comparison of the predicted translation products between the human and rabbit homologues demonstrates that the two proteins are 73% homologous, while increasing to 82% similarity when allowing for conserved amino changes. The human P-450 1 is 82% homologous to the s-mephenytoin 4-hydroxylase (Umbenhauer, D. R., Martin, M. V., Lloyd, R. S., and Guengerich, F. P. (1987) Biochemistry 26, 1094-1099). Southern blot analysis using various portions of the human P-450 1 cDNA as probes indicates that the human P-450 1 gene is part of a larger gene family but can be selectively identified by using a 3'-noncoding portion of the cDNA. Identification of the gene from a panel of human-rodent somatic cell hybrids using the conserved 3' portion of the human P-450 1 cDNA as a probe places the location of the gene on human chromosome 10. Results are also presented which demonstrate that the human P-450 1 gene transcript is processed by an alternate RNA-splicing mechanism that generates two mRNA products, one which represents the functional transcript, and the other a form of mRNA that is not capable of encoding a functional P-450.

Amino Acid Sequence↗

PubMed1986

Human cytochrome P-450 4 mRNA and gene: part of a multigene family that contains Alu sequences in its mRNA.

Several overlapping lambda gt11 cDNA clones have been sequenced and shown to encode for the full-length human cytochrome P-450 4. The structure and location of the exons and flanking intron regions were also identified from a lambda EMBL-3 human genomic clone that encodes the full-length human P-450 4 gene. The human P-450 4 mRNA is flanked by 62 base pairs of 5'- and 1508 base pairs of 3'-noncoding sequence, with 1548 bases that encode a protein of 516 amino acids (Mr, 58,376). The predicted amino acid sequence of human P-450 4 is 69% and 70% homologous to its equivalent in mouse and rat, respectively, 75% homologous to rabbit P-450 4, and 68% homologous to human P1-450. The 7.6-kilobase gene encodes 3118 nucleotides of exon sequence that is separated by six introns into seven exons. Exon 7, which is 1802 nucleotides, contains three inverse/complement Alu sequences that are organized in tandem. Comparison of the genomic DNA sequence of the human P-450 4 gene with the human P1-450 and related genes in rat and mouse and the identification of the amino acid residues and triplet codon at each exon-intron junction show that the location of each intron in the human P-450 4 gene is conserved within this gene family. Although the length and homology of the introns within a related gene family may not be conserved, the location of intronic sequences may be an important determinant in the identification of related P-450 genes.

Amino Acid Sequence↗

PubMed1985

Cloning and isolation of human cytochrome P-450 cDNAs homologous to dioxin-inducible rabbit mRNAs encoding P-450 4 and P-450 6.

Human cytochrome P-450s structurally related to 2,3,7,8-tetrachloro-p-dioxin (TCDD)-inducible rabbit P-450 4 and 6 have been cloned from a human liver cDNA library. The human P-450 4 cDNA clone, hpP-450 4, and the human P-450 6 cDNA clone, hpP-450 6, were identified by hybridization to rabbit P-450 4 and P-450 6 cDNAs, respectively. DNA sequence analysis demonstrates that hpP-450 4 is 83% and 75% homologous to rabbit P-450 4 and P-450 6 mRNAs, respectively, whereas hpP-450 6 is 79% and 72% homologous to rabbit P-450 6 and P-450 4, respectively. A comparison of DNA sequence of the two human cDNA clones shows they are 80% homologous. This is similar to the homology found between the cDNA sequences of rabbit P-450 4 and P-450 6. Northern blot analysis has shown that the human P-450 4 mRNA is approximately 3000 bases, while the human P-450 6 mRNA is 2600 bases in length. Clone hpP-450 4 preferentially hybridizes to TCDD-inducible rabbit P-450 4 and mouse P3-450 mRNAs, whereas hpP-450 6 preferentially hybridizes to TCDD-inducible rabbit P-450 6 and mouse P1-450 mRNAs. Both hpP-450 4 and hpP-450 6 recognize different genomic fragments, indicating that each is encoded by different genes. These results indicate the existence of at least two P-450 genes in humans that are highly homologous to the TCDD-inducible P-450s in rabbits and mice.

Amino Acid Sequence↗