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J P Hardwick

Publications and source records attributed to J P Hardwick.

At least 19 recordsLinked to original sources
PubMed1997

Relationships between NADPH diaphorase staining and neuronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues.

The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent enzymes-neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450 reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue. However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver, sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea, zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms.

Animals↗
PubMed1997

Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system.

Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 x 10(-2)) and a vOR of 10- to 20-fold less. This activity was approximately 7- to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450.

Animals↗
PubMed1997

Catalysis of the cysteine conjugation and protein binding of acetaminophen by microsomes from a human lymphoblast line transfected with the cDNAs of various forms of human cytochrome P450.

We have previously found that for acetaminophen kinetic differences exist between the hepatic microsomal catalyzed protein binding and cysteine conjugation. We have also observed that the protein binding of acetaminophen is only to intralumenal proteins. Together these data suggested that two pools of the reactive metabolite, N-acetyl-p-benzoquinone imine (NABQI), are formed during the oxidative metabolism of acetaminophen: one on the cytosolic surface and the other within the lumen of the microsomes. This would indicate that some of forms of cytochrome P450 (CYP) catalyzing NABQI formation have their active site on the cytosolic surface and others on the lumenal surface. We have examined this question by comparing the rates of cysteine conjugation and protein binding of acetaminophen by microsomes from lymphoblasts transfected with the cDNAs for human CYPs. We found that CYP2D6 catalyzed only cysteine conjugation; CYP1A2 and 3A4 catalyzed only protein binding; CYP2E1 catalyzed both; and CYP1A1, CYP2A6 and CYP2B6 catalyzed neither. These data suggest that CYP2D6 has its active site only on the cytosolic surface; CYP1A2 and CYP3A4 only on the lumenal surface; and CYP2E1 has catalytic sites on both the lumenal and cytosolic surfaces of the membrane. In mouse studies we have found that ethanol administration increased acetaminophen protein binding by 265% but cysteine conjugation by only 61%. CYP2E1 and CYP2B increased, whereas CYP3A decreased and the others did not change. These data suggest that in control mice CYP2E1 catalyzes the bulk of protein binding, whereas CYP2D catalyzes slightly more cysteine conjugation than does CYP2E1.

Acetaminophen↗
PubMed1996

Regional distribution of cytochrome P450 2D1 in the rat central nervous system.

Cytochrome P450s are enzymes involved in the oxidative metabolism of numerous endogenous and exogenous molecules. The enzyme cytochrome debrisoquine/sparteine-type monoxygenase is a specific form of cytochrome P450 and is found in the liver and the brain (in the rat the enzyme is known as CYP2D1). CYP2D1 has no established role in the brain; however, it has been shown to share substrate and inhibitor specificities with the dopamine transporter and the enzyme monoamine oxygenase B. Using CYP2D-specific deoxyoligonucleotide probes and a polyclonal antibody to CYP2D1, we have mapped the distribution of CYP2D mRNA and CYP2D1-like immunoreactivity in the rat central nervous system. CYP2D1 immunoreactivity and the CYP2D1 mRNA signal were heterogenously distributed between brain areas. There were moderate to high levels of immunoreactivity and mRNA signal in the olfactory bulb, olfactory tubercle, cerebral cortex, hippocampus, dentate gyrus, piriform cortex, caudate putamen, supraoptic nucleus, medial habenula, hypothalamus, thalamus, medial mammilliary nucleus and superior colliculus. In the brainstem, strong CYP2D1 immunoreactivity and CYP2D mRNA signal were observed in the substantia nigra compacta, red nucleus, interpeduncular nucleus, pontine grey, locus coeruleus, cerebellum, and the ventral horn of the spinal cord. This study indicates that CYP2D1 is widely and constitutively expressed in neuronal and some glial populations in the rat brain. The localization of CYP2D1 in several regions known to harbor catecholamines and serotonin may suggest a role for CYP2D1 in the metabolism of monoamines.

Animals↗
PubMed1996

Specificity of cDNA-expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12-dimethylbenz[a]anthracene.

7,12-Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6- to 33-fold more activity than other P450s, 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0-2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0- to 37-fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9-diol, but human 2B6 and rat 2B1 preferentially formed the 5,6-diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOM12MBA) than 12-hydroxymethyl-7-methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DMBA and in most circumstances generated the metabolic profile DMBA trans-8,9-dihydrodiol > 7HOM12MBA > or = DMBA trans-5,6-dihydrodiol > or = 7,12-dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans-3,4-dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Metabolism of phenytoin by the gingiva of normal humans: the possible role of reactive metabolites of phenytoin in the initiation of gingival hyperplasia.

Gingival hyperplasia is a well-known complication of therapy with cyclosporine, calcium channel blockers, and phenytoin. It is characterized by the presence of inflammation and a marked fibrotic response. The mechanism of this adverse reaction is unknown. We propose that it may be initiated by the metabolic activation of these drugs to form reactive metabolites. These then cause cellular injury and lead to the gingival hyperplasia. To evaluate this hypothesis we examined phenytoin metabolism and the cytochrome P450 contents of gingival tissues from 10 patients undergoing surgery for various periodontal conditions. We found that microsomes obtained from the gingiva show significant phenytoin hydroxylase activity as determined by the production of 5-(4'-hydroxyphenyl)-5-phenylhydantoin (HPPH) (range, 12.8 pmol HPPH/min.mg microsomal protein to 276.9 pmol HPPH/min.mg microsomal protein; rat control, 133.7 +/- 11.5 pmol HPPH/min.mg microsomal protein). We also found that CYP1A1, CYP1A2, CYP2C9, CYP2E1, and CYP3A4 were present in these microsomes. We detected no CYP2B6 or CYP2D6. We believe that these data support our hypothesis that the proliferative inflammation observed with drugs such as phenytoin, nifedipine, and cyclosporine may be initiated by the formation of reactive metabolites and that the formation of these metabolites may be catalyzed by one or more CYPs found in the gingiva. These metabolites may then cause cellular injury and induce a reactive inflammatory response, followed by fibroblastic proliferation. This proliferation leads to the excess collagen deposition observed with gingival hyperplasia.

Adolescent↗
PubMed1995

Fatty acid discrimination and omega-hydroxylation by cytochrome P450 4A1 and a cytochrome P4504A1/NADPH-P450 reductase fusion protein.

The omega-hydroxylation of fatty acids by certain cytochrome P450 enzymes shows a degree of chain-length and regionspecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strength enzyme-substrate interactions. To investigate the chemical basis for these features of omega-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of omega-hydroxylation catalyzed by cytochrome P4504A1 and a cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein omega-hydroxylated methyl laurate and lauryl alcohol about 1/10th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via omega-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the omega-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid omega-hydroxylation by 43% at equimolar concentrations. omega-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.

Cytochrome P-450 CYP4A↗
PubMed1995

cDNA-directed expression of human cytochrome P450 CYP1A1 using baculovirus. Purification, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purification.

A recombinant baculovirus containing the human cytochrome P450 (CYP) 1A1 cDNA was constructed and used to express CYP1A1 in Spodoptera frugiperda (SF9) insect cells (0.14 +/- 0.04 nmol/mg protein, 53 +/- 14 nmol/liter, N = 30). The enzyme represented approximately 1% of total cellular protein and was partially purified by a three-column procedure to a specific content of 5.0 nmol/mg protein. Catalytic activity was reconstituted with both the purified enzyme using lipid and NADPH-P450 oxidoreductase, and the SF9 insect cell membrane fraction without purification using NADPH-P450 oxidoreductase and small amounts of detergent. Catalytic activity of the enzyme after reconstitution was optimum using molar ratios of CYP1A1 to NADPH-P450 oxidoreductase of 1:8. Cytochrome b5 had no additional stimulating effect. The enzyme metabolized substrates characteristic for CYP1A1:benzo[a]pyrene (4.0 +/- 0.3 nmol/min/nmol CYP), 7-ethoxy-4-trifluoromethyl- coumarin (36 +/- 2), ethoxyresorufin (37 +/- 1), but not pentoxyresorufin (0.77 +/- 0.02). Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically active CYP1A1. Because human CYP1A1 has never been isolated in a catalytically active state from human tissue, nor has recombinant unmodified human CYP1A1, this system is an excellent alternative for the isolation and characterization of this CYP.

Animals↗
PubMed1994

Localization of NADPH cytochrome P450 oxidoreductase in rat brain by immunohistochemistry and in situ hybridization and a comparison with the distribution of neuronal NADPH-diaphorase staining.

An antibody to cytochrome P450 oxidoreductase, purified from rat liver, has been used for the immunohistochemical localization of cytochrome P450 oxidoreductase-like immunoreactivity in the rat central nervous system. The distribution of this immunoreactivity has been confirmed using in situ hybridization with specific cytochrome P450 oxidoreductase antisense DNA probes. Cytochrome P450 oxidoreductase immunoreactivity was detected in neurons and was found in some glial populations. Immunoreactivity and in situ messenger RNA signals were present in many forebrain areas including the olfactory bulb, in the cerebral cortex, caudate-putamen, globus pallidus, hypothalamus, thalamus and hippocampus. Cytochrome P450 oxidoreductase was also detected in the nucleus of the posterior commissure, superior colliculus, intermediate gray layer, periaqueductal gray and in the molecular, Purkinje and granular layers of the cerebellum. In the brain stem, cytochrome P450 oxidoreductase was detected in the substantia nigra, nucleus locus coeruleus and raphe nucleus. Western blotting studies revealed the brain immunoreactive protein has a mol. wt of approximately 72,000, as reported for cytochrome P450 oxidoreductase purified from rat brain microsomes. The distribution of cytochrome P450 oxidoreductase immunoreactivity was compared with the distribution of cells exhibiting NADPH diaphorase activity, which has been established as a histochemical marker for neuronal nitric oxide synthase, an enzyme which has a C-terminus with some structural similarity with cytochrome P450 oxidoreductase and catalyses a complex reaction resulting in the synthesis of nitric oxide from arginine. In general, cytochrome P450 oxidoreductase immunoreactivity and nitric oxide synthase diaphorase activity did not co-localize; however, some neuronal populations did express nitric oxide synthase and exhibit cytochrome P450 oxidoreductase immunoreactivity. Results of immunohistochemistry and in situ hybridization experiments suggest cytochrome P450 oxidoreductase is widespread in the rat central nervous system. The distribution pattern of cytochrome P450 oxidoreductase did not match with those of any one neurotransmitter; however, it did coincide with some brain regions known to harbour central catecholaminergic neurons. The general distribution of cytochrome P450 oxidoreductase was similar to the distribution reported for haeme oxygenase 2 and several cytochrome P450 enzymes. It is possible that malfunctions in cytochrome P450 enzyme systems and/or the haeme oxygenase 2 pathways, both of which involve cytochrome P450 oxidoreductase, may have implications in neurodegenerative diseases.

Animals↗
PubMed1993

Induction of drug-metabolizing enzymes in human pancreatic cancer and chronic pancreatitis.

Chronic pancreatitis and pancreatic cancer have both been linked with occupational exposure to organic chemicals. These chemicals are known to be metabolized within the liver by the cytochrome P-450 family of enzymes, and indeed are able to induce levels of these enzymes as evidence of their interaction. The purpose of this study was therefore to see if these enzyme systems were altered in chronic pancreatitis and pancreatic cancer. Immunocytochemistry of four phase I drug-metabolizing enzymes (cytochromes P-450 IIIA1, P-450 IIE, P-450 IA2, and NADPH cytochrome P-450 oxido-reductase) and one phase II enzyme [glutathione S-transferase (GST) 5-5] was therefore performed on pancreas and/or liver biopsy samples from organ donors and compared with patients with chronic pancreatitis or pancreatic cancer. In samples from donor subjects, the types and levels of drug-metabolizing enzymes in hepatocytes were similar to those seen in pancreatic acinar cells. In material from patients with chronic pancreatitis or pancreatic cancer, cytochrome P-450 enzyme levels were greater in both the liver and the pancreas than those seen in the donor group, while GST levels were unchanged. Islets of Langerhans showed high levels of P-450 IA2 in the donor group, with clear induction of P-450 IIIA1 and NADPH cytochrome P-450 oxidoreductase in patients with chronic pancreatitis but not in the pancreatic cancer group. Levels of GST 5-5 were also induced in the islets. The present findings raise the possibility of an aetiological relationship between elevated levels of drug-metabolizing enzymes and the subsequent development of disease.

Aryl Hydrocarbon Hydroxylases↗
PubMed1993

Identification of a new P450 subfamily, CYP4F1, expressed in rat hepatic tumors.

The expression of the rat cytochrome P450 CYP4 family was studied in hepatic tumors. In most of the primary and transplantable hepatic tumors studied, lauric acid omega-hydroxylase activity associated with the CYP4A subfamily enzymes decreased. The expression of CYP4A proteins and mRNAs in these tumors as assessed by Western and Northern blot was undetectable. However, while RNA analysis revealed the absence of 4A1, 4A2, and 4A3 mRNAs, the expression of CYP4 gene(s) was detected. A Uni-ZAP cDNA library was constructed from a 2-acetylaminofluorene-induced transplantable rat hepatic tumor and screened with a CYP4 family probe. A full-length sequenced cDNA of 1977 bp isolated contained a 23-bp 5' untranslated region, a 1572-bp open reading frame, and a 382-bp 3' untranslated region. This cDNA sequence deduced amino acid sequence encodes a P450 protein having a conserved region of the CYP4 family exhibiting 44-45% amino acid identity to rat CYP4A subfamily members, 43% to human CYP4B1, 35 and 32% to insect CYP4C1 and CYP4D1, respectively. This new P450 was thus named CYP4F1. RNA blot analysis with CYP4F1 cDNA and CYP4F1-specific oligonucleotide probes revealed the expression of CYP4F1 in all tumors. This is the first example of a P450 constitutively expressed in rat hepatomas at levels exceeding those in the parental liver tissue. These results suggest that there is differential regulation of CYP4 genes during hepatic carcinogenesis.

2-Acetylaminofluorene↗
PubMed1990

The dopamine transporter and cytochrome P45OIID1 (debrisoquine 4-hydroxylase) in brain: resolution and identification of two distinct [3H]GBR-12935 binding proteins.

Two [3H]GBR-12935 binding proteins, identified as the dopamine transporter and cytochrome P45OIID1, were solubilized in digitonin from canine striatal membranes, and were resolved following wheat germ agglutinin (WGA)-lectin column chromatography. Protein adsorbed to and specifically eluted from WGA-lectin with N-acetylglucosamine displayed saturable, high affinity (KD approximately 3 nM), and sodium-dependent binding of [3H]GBR-12935, which was inhibited in a concentration-dependent and stereoselective manner by dopamine uptake blockers and substrates with a pharmacological profile indicative of the dopamine uptake site. Protein not adsorbed to WGA-lectin also bound [3H]-GBR-12935 with high affinity (approximately 7 nM), in a sodium-independent manner, and was insensitive to classical dopamine uptake blockers and substrates such as mazindol or dopamine, corresponding to the so-called "piperazine acceptor" site seen in native membranes. [3H]GBR-12935 binding to this latter protein was, however, inhibited by various compounds with a pharmacological profile indicative of a form of cytochrome P450 designated P45OIID1 (debrisoquine/sparteine monooxygenase) with the following rank order of inhibitory potency: GBR-12909 greater than budipine greater than alpha-lobeline greater than quinidine greater than alpha flupenthixol greater than SKF-525A greater than sparteine greater than quinine. Ki values obtained for inhibition of [3H]-GBR-12935 binding to neuronal WGA passthrough fractions by these drugs correlate well with their respective Ki values for liver P45OIID1 activity. Western blotting and immunoprecipitation analysis with rabbit anti-rat P45OIID1 antibody also supported the identity of the mazindol-insensitive [3H]GBR-12935 binding site (or piperazine acceptor site) as P45OIID1. Furthermore, a [3H]GBR-12935 binding protein with pharmacological and immunological characteristics similar to those of P45OIID1 was solubilized from both bovine and human liver membranes, and GBR-12909 was found to be a potent competitive inhibitor (Ki approximately 100 nM) of sparteine monooxygenase activity in human liver microsomes. These data clearly indicate that [3H]GBR-12935 and its analogs display similar affinities for both the dopamine transporter and neuronal P45OIID1, and that this radioligand may be a useful probe of P45OIID1 activity in brain and liver. The exact molecular and functional association (if any) between these two distinct binding protein populations remains to be established; however, it is tempting to speculate that P45OIID1 is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells.

Animals↗
PubMed1990

Sparteine metabolism capacity in human liver: structural variants of human P450IID6 as assessed by immunochemistry.

An antibody raised against rat P450dbl was used to examine the heterogeneity of the human enzyme involved in the sparteine/debrisoquine polymorphism. The extent to which the antibody was able to inhibit sparteine metabolism varied in different human livers (10-80%, n = 9) and reflected the amount of sparteine metabolism carried out by the polymorphic P450IID6 in individual liver specimens. The individual sample variation in inhibition by the antibody correlated with the inhibition caused by quinidine, a prototype competitive inhibitor of the P450IID6 enzyme active site. Western immunoblots of the liver microsomes confirmed that the variation in the inhibition of sparteine metabolism by this antibody reflected the amount of P450IID6 protein. In addition, a detailed study of one of the livers (K19) which demonstrated a lack of inhibition by the antibody was performed which confirmed the lack of P450IID6 in this liver specimen and suggested that the nascent sparteine metabolism activity was due to other forms of P450.

Animals↗
PubMed1990

Clofibrate-inducible rat hepatic P450s IVA1 and IVA3 catalyze the omega- and (omega-1)-hydroxylation of fatty acids and the omega-hydroxylation of prostaglandins E1 and F2 alpha.

Cytochromes P450IVA1 and IVA3 display 72% amino acid sequence similarity and are expressed in livers of rats treated with the hypolipidemic drug clofibrate. The catalytic activities of IVA1 and IVA3 were examined by cDNA-directed expression using vaccinia virus. cDNA-expressed IVA1 and IVA3 had relative Mrs of 51,500 and 52,000, respectively, on SDS-polyacrylamide gels. Both enzymes displayed reduced, CO-bound absorption spectra with lambda max of 452.5 nm. IVA1 and IVA3 hydroxylated lauric acid at the omega and omega-1 positions with equivalent omega/omega-1 ratios of about 12.5. IVA1 had a substrate turnover of 21 min-1 which was about fourfold higher than that of IVA3. The omega and omega-1 hydroxylation of palmitic acid was also catalyzed by these P450s with combined turnover numbers for both metabolites of 45 min-1 or 18 min-1 for IVA1 and IVA3, respectively. The omega/omega-1 oxidation ratio of IVA1 for palmitate was 1.25 which was almost fourfold higher than that obtained for IVA3. These enzymes also catalyzed omega oxidation of the physiologically important eicosanoids prostaglandins E1 and F2 alpha with turnover numbers of about one-tenth those calculated for fatty acid oxidations. No omega-1 hydroxy metabolites were produced. These studies indicate that the P450 enzymes IVA1 and IVA3 are able to catalyze the oxidations of both fatty acids and prostaglandins.

Alprostadil↗
PubMed1989

The CYP2D gene subfamily: analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats.

The DA rat has been proposed as an animal model for the human debrisoquine 4-hydroxylase/bufuralol 1'-hydroxylase genetic deficiency. To determine the mechanism of this deficiency, we isolated and sequenced five cDNAs in the CYP2D gene subfamily including a new IID1 allele and two cDNAs of novel P450s, designated IID3 and IID5. IID3 and IID5 cDNA-deduced amino acid sequences contained 500 and 504 residues with calculated molecular weights of 56,683 and 57,081, respectively. IID5 displayed 20 amino acid differences with the IID1, yet bore only 72% and 76% similarity to IID2 and IID3. Despite an overall nucleotide similarity of 80-98% between the 4 cDNAs, a region of 134 nucleotides of sequence exists that contains only 1 base difference. This region is probably the result of gene conversion events between the P450 IID genes. Although all IID cDNAs were expressed into immunodetectable proteins using the COS cell SV40-based expression system, only IID1 could effectively catalyze the oxidation of the prototype substrate bufuralol. Expression of a cDNA isolated in an earlier study [Gonzalez, F. J., Matsunaga, T., Nagata, K., Meyer, U. A., Nebert, D. W., Pastewka, J., Kozak, C. A., Gillette, J., Gelboin, H. V., & Hardwick, J. P. (1987) DNA 6, 149-161], previously called db1 and now designated IID1v, produced a protein with a drastically reduced activity as compared to cDNA-expressed IID1 despite only four amino acid differences between the two cDNA-deduced protein sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1989

Alteration of the c-mos locus in "normal" tissues from humans exposed to radium.

The structure of a number of human protooncogenes of persons with internal systemic exposure to radium was analyzed by restriction enzyme digestion and Southern blotting of their DNA. Two extra c-mos EcoRI restriction fragment length bands of 5.0 and 5.5 kilobases were found in tissue DNA from six of seven such individuals. The extra c-mos bands were detected in DNA from many, but not all, of the tissues of the individuals exposed to radium. Kidney DNA, however, from three of four individuals exposed to radium contained these alterations; kidney DNA from six age-matched controls did not. The 5.0- and 5.5-kilobase bands, which were of a similar intensity, varied in their intensity with respect to that of the normal 2.5-kilobase band of the c-mos gene. The DNAs that have the polymorphic bands also appear to have a more complex c-mos methylation pattern. Our results suggest that the c-mos restriction fragment length alterations found in individuals exposed to radium were induced rather than inherited, are epigenetic in origin, and most likely result from changes in the methylation of bases surrounding the single exon of the c-mos protooncogene.

Aged↗