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Biomedical subjects

G Padmanaban

Publications and source records attributed to G Padmanaban.

At least 37 records · Page 2Linked to original sources
PubMed1992

de novo biosynthesis of heme offers a new chemotherapeutic target in the human malarial parasite.

The human malarial parasite, Plasmodium falciparum, has been found to synthesize heme de novo, despite the accumulation of large quantities of polymeric heme derived from the hemoglobin of the red cell host. The parasite delta-aminolevulinate dehydrase level is significantly lower than that of the host and its inhibition by succinylacetone leads to inhibition of parasite protein synthesis and viability.

5-Aminolevulinate Synthetase↗
PubMed1992

Identification and functional characterization of a cis-acting positive DNA element regulating CYP 2B1/B2 gene transcription in rat liver.

A positive cis-acting DNA element in the near 5'-upstream region of the CYP2B1/B2 genes in rat liver was found to play an important role in the transcription of these genes. An oligonucleotide covering -69 to -98 nt mimicked the gel mobility shift pattern given by the fragment -179 to +29 nt, which was earlier found adequate to confer the regulatory features of this gene. Two major complexes were seen, of which the slower and faster moving complexes became intense under uninduced and Phenobarbitone-induced conditions respectively. Minigene cloned DNA plasmid covering -179 to +181 nt in pUC 19 and Bal 31 mutants derived from this parent were transcribed in whole nuclei and cell free transcription extracts and mutants containing only upto -75 nt of the upstream were poorly transcribed. Transcription extracts from phenobarbitone-injected rat liver nuclei were significantly more active than extracts from uninduced rats in transcribing the minigene constructs. Addition of the oligonucleotide (-69 to -98nt) specifically inhibited the transcription of the minigene construct (-179 to +181 nt) in the cell free transcription system. It is therefore, concluded that the region -69 to -98 nt acts as a positive cis-acting element in the transcription of the CYP2B1/B2 genes and in mediating the inductive effects of phenobarbitone.

Amanitins↗
PubMed1991

Chloroquine inhibits heme-dependent protein synthesis in Plasmodium falciparum.

A cell-free protein-synthesizing system has been reconstituted using the S-30 fraction or ribosomes and the S-100 fraction from Plasmodium falciparum. Addition of heme in vitro stimulates cell-free protein synthesis strikingly. Chloroquine inhibits the heme-dependent protein synthesis in the parasite lysate. The drug has also been found to inhibit parasite protein synthesis in situ at therapeutic concentrations soon after addition to parasite cultures. Ribosomes as well as the S-100 fraction isolated from such chloroquine-treated cultures are defective in protein synthesis. Addition of hemin plus glucose 6-phosphate or high concentrations of GTP, cAMP, and an active preparation of eIF-2 to the parasite cell-free system restores protein synthesis to a significant extent in chloroquine-treated cultures. Under conditions of inhibition of protein synthesis in situ by chloroquine in the culture, the parasite eukaryotic initiation factor 2 alpha- (eIF-2 alpha) is phosphorylated in the parasite lysate to a greater extent than that observed in the control culture. Addition of hemin in vitro suppresses this phosphorylation. eIF-2 alpha kinase activity is present in the parasite lysate and is not a contaminant derived from the human erythrocytes used to culture the parasite. The heme-chloroquine interactive effects can also be demonstrated with purified eIF-2 alpha kinase from rabbit reticulocyte lysate. It is proposed that chloroquine inhibits heme-dependent protein synthesis in the parasite and this is an early event mediating the growth-inhibitory effects of the drug.

Animals↗
PubMed1991

Estradiol-17 beta induces polyaromatic hydrocarbon-inducible cytochrome P-450 in chicken liver.

A cDNA clone has been isolated from a chicken liver library prepared against messenger RNA isolated after chronic estradiol - 17 beta treatment. The clone, pP-450 IA - 61, has an insert of 900nt and the sequence shows high homology to CYPIA2 subfamily from four other species. A single injection of estradiol - 17 beta to immature chicken results in a striking induction of mRNA hybridizing to labeled pP-450IA - 61. The probe also hybridizes to mRNA induced by 3 - methylcholanthrene in chicken. These results offer direct proof for the similarity in the mode of action at the transcriptional level of polyaromatic hydrocarbons and estrogenic compounds.

Amino Acid Sequence↗
PubMed1991

Involvement of heme in the transcriptional activation of CYPIIB1/B2 gene by phenobarbitone in rat liver--studies with succinylacetone.

Earlier studies in this laboratory had implicated heme to function as a positive modulator of phenobarbitone-mediated activation of CYPIIB1/B2 gene transcription in rat liver. However, recent reports have indicated that succinylacetone, a specific inhibitor of delta-aminolevulinate dehydrase, does not affect this process. The present studies indicate that succinylacetone does inhibit the phenobarbitone-mediated increase in CYPIIB1/B2 mRNAs and their transcription in rat liver at early time points (45 min to 3 h), but the inhibition is not pronounced at later time points (16 h). Succinylacetone is a weaker inhibitor of heme biosynthesis than CoCl2, 3-amino-1,2,4-triazole, or thioacetamide used earlier in this laboratory. Succinylacetone induces delta-aminolevulinate synthase, whereas the other compounds depress the levels of the enzyme. There is a good correlation between the amount of freshly synthesized nuclear heme pool and the activation of CYPIIB1/B2 transcription by phenobarbitone. A model implicating a nuclear heme pool regulating the transcription of delta-aminolevulinate synthase, CYPIIB1/B2, and heme oxygenase genes is proposed.

Animals↗
PubMed1990

Dexamethasone negatively regulates phenobarbitone-activated transcription but synergistically enhances cytoplasmic levels of cytochrome P-450b/e messenger RNA.

Dexamethasone has a potentiating effect on phenobarbitone mediated induction of cytochrome P-450b + e mRNAs in adult rat liver. However, the glucocorticoid inhibits phenobarbitone-activated transcription of cytochrome P-450b + e mRNAs by 60-70%. This inhibitory effect is evident in run-off transcription of the endogenous genes as well as in the transcription of an added cloned gene fragment. Dexamethasone inhibits the phenobarbitone-mediated increase in the binding of a transcription factor(s) to the upstream region of the gene as evidenced by gel retardation and Southwestern blot analysis. The glucocorticoid does not stabilize the phenobarbitone-induced polyribosomal cytochrome P-450b + e mRNAs but appears to stabilize the nuclear transcripts. It is proposed that a negative element may mediate the action of dexamethasone at the level of nuclear transcription and stabilization of the nuclear transcript may account for the potentiating effect of the glucocorticoid on phenobarbitone-mediated increase in cytochrome P-450b + e mRNAs in the cytoplasm of the adult rat liver. However, the cytochrome P-450b protein levels are slightly lower in phenobarbitone + dexamethasone treatment than in phenobarbitone-treated liver microsomes.

Animals↗
PubMed1989

Haem as a multifunctional regulator.

Haem has long been known as the prosthetic group of haemoproteins such as haemoglobin, catalase and the cytochromes. Its biosynthesis is regulated by feedback mechanisms that ensure its adequate production but prevent its overaccumulation, which is highly deleterious as diseases such as porphyrias attest. However, recent years have seen rapid strides in our understanding of how haem (or more accurately haemin, its oxidized form) itself acts as an intracellular regulator of a variety of other metabolic pathways for systems that utilize oxygen.

Animals↗
PubMed1989

Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor.

The cloned DNA fragment of the cytochrome P-450b/e gene containing the upstream region from position -179 through part of the first exon is faithfully transcribed in freeze-thawed rat liver nuclei. Phenobarbitone treatment of the animal strikingly increases this transcription, and the increase is blocked by cycloheximide (protein synthesis inhibitor) or CoCl2 (heme biosynthetic inhibitor) treatment of animals. This picture correlates very well with the reported cytochrome P-450b/e mRNA levels in vivo and run-on transcription rates in vitro under these conditions. The upstream region (from position -179) was assessed for protein binding with nuclear extracts by nitrocellulose filter binding, gel retardation, DNase I treatment ("footprinting"), and Western blot analysis. Phenobarbitone treatment dramatically increases protein binding to the upstream region, an increase once again blocked by cycloheximide or CoCl2 treatments. Addition of heme in vitro to heme-deficient nuclei and nuclear extracts restores the induced levels of transcription and protein binding to the upstream fragment, respectively. Thus, drug-mediated synthesis and heme-modulated binding of a transcription factor(s) appear involved in the transcriptional activation of the cytochrome P-450b/e genes, and an 85-kDa protein may be a major factor in this regard.

Animals↗
PubMed1988

Heme is a positive regulator of cytochrome P-450 gene transcription.

Inhibitors of heme biosynthesis such as CoCl2, 3-amino-1,2,4-triazole, and thioacetamide block the 3-methylcholanthrene-mediated induction of cytochrome P-450 (c + d) messenger RNAs and their transcription in rat liver. This effect is specific, since the messenger RNA levels for albumin and glutathione transferase (Ya + Yc) and their transcription are not significantly influenced under conditions of heme depletion. Exogenous administration of heme at very low doses (50 micrograms/100 g body wt) is able to completely counteract the effects of the heme biosynthetic inhibitors on cytochrome P-450 (c + d) messenger RNA levels and their transcription. This constitutes a direct proof for the role of heme as a positive regulator of cytochrome P-450 gene transcription.

Amitrole↗
PubMed1988

A processed pseudogene with an intact coding sequence for rat liver cytochrome c oxidase subunit VIc.

The biogenesis of eukaryotic cytochrome c oxidase involves the coordinate expression of nuclear and mitochondrial genes. Very little information is available on the gene structure of nuclear-coded cytochrome c oxidase subunits in mammalian systems. We report here the isolation and complete nucleotide sequence determination of a processed pseudogene for cytochrome c oxidase subunit VIc from rat liver. The pseudogene lacks introns and the coding region is intact with no deleterious lesions; however, there are 7 amino acid (aa) differences when compared to the sequence derived from cDNA clones. The pseudogene has the potential to code for a protein of 76 aa, containing a putative 3 aa N-terminal presequence when compared to the mature bovine heart VIc subunit. Potential regulatory regions, including a TATA box, are present in the 5'-flanking region.

Amino Acid Sequence↗
PubMed1988

Heme regulates cytochrome P-450 gene transcription elongation.

Administration of 3-methylcholanthrene (MC) to rats results in a striking increase in the transcription of cytochrome P-450 (c + d) messenger RNA with isolated nuclei, which is blocked by the simultaneous administration of cobalt chloride, an inhibitor of heme biosynthesis. Transcription of cytochrome P-450 (c + d) mRNAs with nuclei isolated from MC treated rats shows a linear increase with time of incubation, whereas it shows a progressive decrease with incubation time in the case of nuclei isolated from MC+CoCl2 treated rats. Addition of heme in vitro (10(-6)M) to the latter nuclei results in a significant counteraction of the decreased cytochrome P-450 (c + d) mRNA transcription. The inhibition in transcription rates observed in MC+CoCl2 treated rat liver nuclei is more pronounced with the seventh exon probe than with the second exon probe. Once again, in vitro heme addition can counteract the inhibition observed with both the probes. Since run off transcription with isolated nuclei represents essentially elongation of the initiated transcripts, the data obtained can be interpreted on the basis that heme regulates cytochrome P-450 gene transcription elongation.

Animals↗
PubMed1987

Regulation of cytochrome P-450 messenger RNA and apoprotein levels by heme.

2-Allylisopropylacetamide, a porphyrinogen which decreases the microsomal and cytosolic heme pools, is a phenobarbitone-like inducer of cytochrome P-450(b + e) messenger RNAs in rat liver. The porphyrinogen, however, does not affect the nuclear heme pool and enhances the transcription of cytochrome P-450(b + e) messenger RNAs strikingly. Inhibitors of heme biosynthesis, such as CoCl2 and 3-amino-1,2,4-triazole, which decrease the total heme levels including that of the nuclear heme pool, block the 2-allylisopropylacetamide- or phenobarbitone-mediated increase in the transcription of cytochrome P-450(b + e) messenger RNAs. Administration of exogenous heme at a very low concentration (25 micrograms/100 g) is able to counteract the inhibitory effects of the heme biosynthetic inhibitors. Addition of heme in vitro to heme-depleted nuclei leads to a significant increase in the transcription rates for cytochrome P-450(b + e) messenger RNAs. 2-Allylisopropylacetamide, unlike phenobarbitone, fails to increase the levels of cytochrome P-450b protein at 12 h after the drug administration, although there is a striking increase in the messenger RNA levels. Under conditions of 2-allylisopropylacetamide treatment, the cytochrome P-450 messenger RNA is translated, but the newly synthesized apoprotein undergoes rapid degradation. It is concluded that heme is a positive modulator of cytochrome P-450 gene transcription and is also required to stabilize the freshly synthesized apoprotein.

Allylisopropylacetamide↗
PubMed1987

Differential effects of cycloheximide on rat liver cytochrome P-450 gene transcription in the whole animal and hepatoma cell culture.

The induction of cytochrome P-450 (c+d) messenger RNAs in rat liver by 3-methyl cholanthrene follows a biphasic pattern. Administration of cycloheximide blocks the induction of cytochrome P-450 (c+d) messenger RNAs by 3-methylcholanthrene as well as cytochrome P-450 (b+e) messenger RNAs by Phenobarbitone. Transcription of these messenger RNAs in isolated nuclei is also blocked by cycloheximide administration. Thus cycloheximide not only fails to mimic the superinduction effects reported in hepatoma cell cultures, but actually blocks the specific transcription process. Exogenous hemin, while counteracting the effects of CoCl2 (heme biosynthetic inhibitor) on cytochrome (c+d) messenger RNA induction by the hydrocarbon, fails to counteract the effects of cycloheximide. It is suggested that a positive labile transcription factor is involved in the regulation of cytochrome P-450 gene expression in vivo.

Animals↗
PubMed1987

Gossypol mediated DNA degradation.

Gossypol, a polyphenolic compound isolated from cotton plant was found to degrade pBR322 DNA in vitro in a reaction which required the presence of a metal ion, a reducing agent (2-mercaptoethanol) and oxygen as revealed after agarose gel electrophoresis. Fe3+ and Co2+ showed maximum degradation whereas addition of Ca2+ and Mg2+ prevented the gossypol mediated DNA damage. Gossypol caused degradation of rat liver DNA incubated in vitro even in the absence of added metal ions and 2-mercaptoethanol. Incubation of intact rat liver nuclei with gossypol revealed DNA degradation and nuclei isolated from rats treated with gossypol in vivo showed higher susceptibility to DNA fragmentation when incubated with gossypol in vitro than control nuclei. EcoR1 and AIuI digestion of DNA isolated from gossypol treated rats gave clear cut evidence for DNA degradation. These observations indicate that gossypol is genotoxic and considerable care has to be exercised in its use.

Animals↗
PubMed1987

Isolation of a cytochrome P-450e gene variant and characterization of its 5' flanking sequences.

A cytochrome P-450e gene variant has been isolated from the rat liver genomic library. It is a typical e gene clone but unique in having b-like single base substitutions at specific sites in the 5' flanking region. It also appears to have certain additional restriction sites in the introns. When compared with the cytochrome P-450b gene, the e gene has some of the repetitive motifs interrupted in the 5' flanking region. In addition, this region is characterized by the presence of alternating pyrimidine-purine stretch, steroid hormone regulatory elements, consensus eukaryotic enhancer sequence and sequences involved in general amino acid regulation.

Animals↗
PubMed1986

Expression of cytochrome P-450 and albumin genes in rat liver: effect of xenobiotics.

Thioacetamide, a hepatocarcinogen and an inhibitor of heme synthesis, blocks the phenobarbitone-mediated increase in the transcription of cytochrome P-450b+e messenger RNA in rat liver. This property is also shared by CoCl2 and 3-amino-1,2,4-triazole, two other inhibitors of heme synthesis. Thus, it appears feasible that heme may serve as a positive regulator of cytochrome P-450b+e gene transcription. Thioacetamide enhances albumin messenger RNA concentration, whereas phenobarbitone decreases the same. However, these changes in albumin messenger RNA concentration are not accompanied by corresponding changes in the transcription rates. Therefore, drug-mediated changes in albumin messenger RNA concentration are due to posttranscriptional regulation. The property of thioacetamide to enhance the albumin messenger RNA concentration is not shared by CoCl2 and 3-amino-1,2,4-triazole. Therefore, heme does not appear to be a regulatory molecule mediating the reciprocal changes brought about in the concentrations of cytochrome P-450b+e and albumin messenger RNAs.

Acetamides↗
PubMed1986

A comparative study of the regulation of cytochrome P-450 and glutathione transferase gene expression in rat liver.

A cDNA clone for the Ya subunit of glutathione transferase from rat liver was constructed in E. coli. The clone hybridized to Ya and Yc subunit messenger RNAs. On the basis of experiments involving cell-free translation and hybridization to the cloned probe, it was shown that prototype inducers of cytochrome P-450 such as phenobarbitone and 3-methylcholanthrene as well as inhibitors such as CoCl2 and 3-amino-1,2,4-triazole enhanced the glutathione transferase (Ya+Yc) messenger RNA contents in rat liver. A comparative study with the induction of cytochrome P-450 (b+e) by phenobarbitone revealed that the drug manifested a striking increase in the nuclear pre-messenger RNAs for the cytochrome at 12 hr, but did not significantly affect the same in the case of glutathione transferase (Ya+Yc). 3-Amino-1,2,4-triazole and CoCl2 blocked the phenobarbitone mediated increase in cytochrome P-450 (b+e) nuclear pre-messenger RNAs. These compounds did not significantly affect the glutathione transferase (Ya+Yc) nuclear pre-messenger RNA levels. The polysomal, poly (A)- containing messenger RNAs for cytochrome P-450 (b+e) increased by 12-15 fold after phenobarbitone administration, reached a maximum around 16 hr and then decreased sharply. In comparison, the increase in the case a glutathione transferase (Ya+Yc) messenger RNAs was sluggish and steady and a value of 3-4 fold was reached around 24 hr. Run-off transcription rates for cytochrome P-450 (b+e) increased by nearly 15 fold in 4 hr after phenobarbitone administration, whereas the increase for glutathione transferase (Ya+Yc) was only 2.0 fold. At 12 hr after the drug administration, the glutathione transferase (Ya+Yc) transcription rates were near normal. Administration of 3-amino-1,2,4-triazole and CoCl2 blocked the phenobarbitone-mediated increase in the transcription of cytochrome P-450 (b+e) messenger RNAs. These compounds at best had only marginal effects on the transcription of glutathione transferase (Ya+Yc) messenger RNAs. The half-life of cytochrome P-450 (b+e) messenger RNA was estimated to be 3-4 hr, whereas that for glutathione transferase (Ya+Yc) was found to be 8-9 hr. Administration of phenobarbitone enhanced the half-life of glutathione transferase (Ya+Yc) messenger RNA by nearly two fold. It is suggested that while transcription activation may play a primary role in the induction of cytochrome P-450 (b+e), the induction of glutathione transferase (Ya+Yc) may essentially involve stabilization of the messenger RNAs.

Amitrole↗