The amino acid sequence deduced from a cDNA clone of the bovine adrenal steroid 21-hydroxylase cytochrome P-450 has been utilized to align peptide sequences derived from the corresponding porcine enzyme. Homology analysis revealed that only fifty percent of the amino acid sequence predicted by the cDNA clone overlapped with peptide sequences from the porcine enzyme. The homology in the remaining portions of the bovine sequence was restored by considering amino acid sequences predicted by the two additional DNA reading frames of the cDNA sequence. Forty eight percent of the bovine sequences predicted by the two alternate reading frames showed strong homology with the porcine peptide sequences. A minimum of 4 nucleotide sequencing errors account for the observed reading frame alterations and the approximate position of each error in the bovine cDNA sequence has been established.

Adrenal Glands↗

PubMed1984

C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 alpha-hydroxylase/C17,20-lyase cytochrome P-450.

The properties and the purity of a cytochrome P-450 (17 alpha-hydroxylase) from porcine adrenal microsomes have been examined following a report that the corresponding enzyme from bovine adrenocortical microsomes is inactive as a 17 alpha-hydroxylase and fails to show a high spin spectrum on addition of substrate, once the enzyme has been purified (Bumpus, J. A., and Dus, K. M. (1982) J. Biol. Chem. 257, 12696-12704). The purity of the porcine enzyme was demonstrated by electrophoresis on polyacrylamide with sodium dodecyl sulfate, immunoelectrophoresis, and NH2-terminal amino acid sequence (16 residues). The pure enzyme shows Mr = 54,000, heme content of greater than 0.8 nmol/nmol of protein, and absorption spectra typical of cytochrome P-450. The enzyme is active with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates as a 17 alpha-hydroxylase and with the corresponding 17 alpha-hydroxysteroids as a C17,20-lyase. All four substrates produce typical type I spectra with the enzyme (so-called high spin form). We conclude that: 1) porcine adrenal microsomes contain a 17 alpha-hydroxylase/C17,20-lyase which is a single protein molecule readily purified to an enzymatically active form; 2) the C17,20-lyase activity is largely suppressed in the microsomes; and 3) the enzyme closely resembles that found in testicular microsomes. We propose that this enzyme be referred to as the adrenal C21 steroid side chain cleavage enzyme.

Adrenal Glands↗

PubMed1984

[Purification of nicotineamide adenine dinucleotide phosphate-cytochrome P-450 reductase from porcine adrenal microsomes and reconstitution of 17 alpha-hydroxylase, C17,20 lyase and 21-hydroxylase activities].

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Adrenal Glands↗

PubMed1984

[Studies on chemical protectors against radiation. XXIII. Radioprotective activities of ferulic acid and its related compounds].

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Animals↗

PubMed1984

[Effect of diuretic, azosemide (SK-110), in combination with antibiotic, cephaloridine, on kidney].

This study was performed to compare the acute-renal toxicity of azosemide (SK-110) or furosemide (FM) treatment in combination with cephaloridine (CER). 1) Initially, the acute-renal toxicity of CER was studied after a subcutaneous administration at dose levels of 500, 1000, 1500 and 2000 mg/kg. The values of BUN, creatinine and relative kidney weights were increased in rats given CER at dose levels above 1500 mg/kg. consequently, it was considered that the threshold of CER with regard to acute-renal toxicity was 1000 mg/kg. 2) Secondarily, the acute-renal toxicity of SK-110 or FM were studied after oral administration of 40, 80, 160 or 320 mg/kg doses alone or in combination with CER. Acute-renal toxicity was evident in the 320 mg/kg-treated rats after both SK-110 and FM alone, as revealed by analysis of a number of parameters, i.e., BUN, serum creatinine, urine sediment and composition and pathological data composing both kidney weights and studies at the microscope level. However, the increase of BUN and relative kidney weights values, elevated numbers of epithelial cells in the urine and necrosis observed in the uriniferous-tubular epithelium on histopathological examination of kidneys were noted in rats given at dose levels of only 80 mg/kg with SK-110 + CER, whereas they were seen in animals treated with FM even at the lowest applied dose of 40 mg/kg in combination with CER. In conclusion, this study showed that the minimum dose of SK-110 or FM in combination with CER which causes acute-renal toxicity were, respectively, 80 mg/kg or less than 40 mg/kg.

Administration, Oral↗

PubMed1984

[Effect of azosemide (SK-110) and its metabolites on mouse liver].

The effect of azosemide (SK-110), and its metabolites, 5-(2'-amino-4'-chloro-5'-sulfamoylphenyl)-tetrazole (M-1), 2-thiophenecarboxylic acid (TC), on mouse liver was investigated using biochemical and pathological parameters as indices of hepatotoxicity. The effects were compared with that of furosemide (FM) administration. A single dose of each compound was administered orally, or intraperitoneally, while multiple oral dosing was carried out once daily for a week. The results are summarized as follows: 1) SK-110 did not produce hepatotoxicity even after a single p.o. dose as high as 5000 mg/kg, a single i.p. dose of 400 mg/kg, or multiple p.o. doses of 700 mg/kg/day. 2) M-1 also did not produce hepatotoxicity even after a single p.o. dose of 4000 mg/kg or multiple p.o. doses of 550 mg/kg/day. TC did not exert hepatotoxicity after a single i.p. dose of 150 mg/kg or multiple p.o. doses of 250 mg/kg/day, but did produce hepatotoxicity after a single p.o. dose of more than 1000 mg/kg. However, it was presumed that, in vivo, TC formed as a metabolite of administered SK-110 would hardly produce hepatotoxicity. 3) FM produced hepatotoxicity after a single p.o. dose of more than 800 mg/kg, or a single i.p. dose of more than 200 mg/kg, but not after multiple p.o. doses of 700 mg/kg/day. Based on these findings, it was concluded that, in contrast to FM, SK-110 had no hepatotoxic effect on the mouse liver.

Administration, Oral↗

PubMed1984

[A case of advanced gastric cancer which disappeared histopathologically after short-time chemotherapy].

Poorly differentiated adenocarcinoma was confirmed by endoscopic biopsy. Anticancer therapy was performed preoperatively, but was discontinued after the second intravenous administration of MFC because she developed nausea, vomiting and pancytopenia. On Jan. 18, 1980, gastrectomy with extended lymph node dissection was performed. Histologically, the excised stomach showed non-specific active ulcer (ul-IV) at the side of the tumor without evidence of residual cancer cells. The cause for the disappearance of the advanced carcinoma remains unknown. Although the dosage of the anticancer chemotherapy was quite small, this treatment may have promoted the regression of the tumor in conjunction with activated antitumor immunity of the host.

Adenocarcinoma↗

PubMed1983

Purification and properties of 17 alpha-hydroxylase from microsomes of pig adrenal: a second C21 side-chain cleavage system.

We have purified a cytochrome P-450 from microsomes of pig adrenal glands to homogeneity (11.1 n moles heme/mg protein) as demonstrated by electrophoresis on polyacrylamide gels with sodium dodecyl SO4 and by line of identity with an antibody using double diffusion. The enzyme shows both 17 alpha-hydroxylase and C17,20-lyase activities and therefore constitutes a C21 side-chain cleavage system like that previously purified in this laboratory from neonatal pig testis. Antibody to the testicular enzyme cross-reacts (line of identity) with both enzymes. It is concluded that the adrenal enzyme is the same or very similar to the testicular enzyme, that each enzyme possesses two enzymatic activities, and that microsomes provide some regulatory device to limit the lyase activity of the enzyme in vivo. No evidence was found for the usually accepted existence of an adrenal steroid 17 alpha-hydroxylase without lyase activity.

Adrenal Glands↗

PubMed1983

Steroid 21-hydroxylase (cytochrome P-450) from porcine adrenocortical microsomes: microsequence analysis of cysteine-containing peptides.

The steroid 21-hydroxylase cytochrome P-450 from porcine adrenocortical microsomes was purified to homogeneity. The protein exhibited two NH2-terminal sequences, one of which was identical with the first but lacking the NH2-terminal methionine. The sequence was extremely hydrophobic but had little homology to the 17 alpha-hydroxylase/C17,20-lyase isolated from neonatal porcine testes or to rat or rabbit liver microsomal cytochromes P-450. The cysteine-containing fragments of the S-carboxymethylated protein were purified by high-performance liquid chromatography and sequenced. Three of the cysteine-containing peptides exhibited significant sequence homology with peptides from the major phenobarbital-induced rat liver cytochrome P-450 (P-450b) and two with peptides from cytochrome P-450cam (camphor methylene hydroxylase from Pseudomonas putida). The presence of conserved regions in the primary sequences of these proteins appears likely to provide clues to the nature of their heme-binding domains.

Adrenal Cortex↗

PubMed1983

Effect of various immunomodulators on the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) in mouse spleen cell cultures.

Among various immunomodulating agents examined, OK-432 (Streptococcus pyogenes cell bodies), BCG (bacillus Calmette-Guerin) and LPS (lipopolysaccharide) were effective in enhancing the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the cultures of mouse spleen cells. Control non-stimulated spleen cell cultures accumulated GM-CSF activity maximally on day 3 and lost it subsequently within a few days. On the other hand, a large amount of GM-CSF activity could be detected in the medium until day 9 when OK-432 was added in the culture at the start of incubation. The GM-CSF obtained from OK-432 or LPS stimulated cultures and the GM-CSF obtained from the control non-stimulated culture eluted from an Ultro-gel AcA 34 column at a similar position and inactivated similarly at 80 degrees C. However, these three GM-CSFs behaved in slightly different manners upon isoelectrofocusing. These results suggest that the GM-CSFs produced in increasing amounts by the stimulated spleen cells are similar to that produced by the control non-stimulated cells, but slightly different from one another in the extent of sialylation according to the difference of the stimulant.

Adjuvants, Immunologic↗

PubMed1983

Participation of cytochrome b5 electron transport system coupled with delta 5-3 beta-hydroxysteroid dehydrogenase on cytochrome P-450 monooxygenase reactions of guinea pig adrenal microsomes.

Not only cytochrome P-450 and cytochrome P-450 reductase, but considerable amount of cytochrome b5 and cytochrome b5 reductase were also contained in the adrenal microsomes of guinea pig. Addition of nicotinamide adenine dinucleotide (NADH) stimulates the activities of cytochrome P-450 monooxygenases such as 21-hydroxylase, 17 alpha-hydroxylase and C17,20 lyase supported with nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. In addition, substrate for delta 5-3 beta-hydroxysteroid dehydrogenase plus NAD+ also stimulate the same cytochrome P-450 monooxygenase activities. These observations were suggesting the participation of cytochrome b5 electron transport system on cytochrome P-450 monooxygenase (21-hydroxylase, 17 alpha-hydroxylase and C17,20 lyase) reactions associated with the biosynthesis of corticoids and adrenal androgens. Furthermore, it is also suggesting that cytochrome b5 electron transport system involves the reaction of delta 5-3 beta-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗

PubMed1983

Specificity of the suppressive action of glucocorticoids on the proliferation of monocyte/macrophages in the CSF-stimulated cultures of mouse bone marrow.

Addition of cortisol and its analogs to soft-agar culture of bone marrow cells markedly decreased the number of monocyte colonies in the presence of colony-stimulating factor. Steroids of other categories and cortisol metabolites were much less inhibitory than cortisol, and the shape of dose-response curve apparently differed between cortisol and other steroids. The action of cortisol was not influenced by excess progesterone or testosterone. Addition of cortisol succinate in liquid cultures of CSF-stimulated cells caused a gradual decrease of cellular uptake of [3H]thymidine. The steroid also suppressed CSF-independent [3H]thymidine uptake, but the time course of suppression obviously differed from that of CSF-dependent uptake. On day 7 of incubation, the steroid-treated cultures contained a smaller number of cells but higher activities of lysosomal enzymes than the control cultures. These results show that the glucocorticoids inhibit proliferation of the cells of monocyte/macrophage lineage.

Animals↗