Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Tadashi Ogishima Last modified date:2021.04.18

Associate Professor / organic and biological chemistry / Department of Chemistry / Faculty of Sciences


Papers
1. Akihiro Higuchi, Hiroyoshi Inoue, Tetsuya Kawakita, Tadashi Ogishima, Kazuo Tsubota, Selenium Compound Protects Corneal Epithelium against Oxidative Stress, PloS one, 10.1371/journal.pone.0045612, 7, 9, 2012.09, The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium..
2. Cristia Volpe, Anders Höög, Tadashi Ogishima, Kuniaki Mukai, Ming Lu, Marja Thorén, Bertil Hamberger, Immunohistochemistry improves histopathologic diagnosis in primary aldosteronism, Journal of Clinical Pathology, 10.1136/jclinpath-2012-201287, 66, 4, 351-354, 2013.04, Background In primary aldosteronism (PA) the main source of aldosterone hypersecretion is an aldosteroneproducing adenoma (APA) or a bilateral hyperplasia. Histopathology of the adrenal gland from patients with PA has been difficult, as there are no morphological criteria to ascertain which are the cells that produce aldosterone. We therefore applied new specific antibodies to explore which cells in the adrenal contain the enzymes for aldosterone and cortisol production, respectively. Methods Adrenals from 24 patients with PA were studied. After routine preparation, consecutive sections were stained with antibodies for CYP11B1 (cortisol) and CYP11B2 (aldosterone) enzymes. Results APA had a strong immunoreactivity for CYB11B2. In adrenals from seven patients, we found no APA, but several nodules with strong CYB11B2 immunoreactivity, indicating aldosterone-producing nodular hyperplasia. Conclusions Immunohistochemistry of adrenal steroidogenic enzymes provides novel diagnostic information. This may become an important part of routine histopathology, and contribute to improved clinical management in PA..
3. Andrea Vecchiola, Carlos F. Lagos, Cristóbal A. Fuentes, Fidel Allende, Carmen Campino, Carolina Valdivia, Alejandra Tapia-Castillo, Tadashi Ogishima, Kuniaki Mukai, Gareth Owen, Sandra Solari, Cristian A. Carvajal, Carlos E. Fardella, Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro, Reproductive Biology and Endocrinology, 10.1186/1477-7827-11-76, 11, 1, 2013.08, Background: Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes.Methods: We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids.Results: In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients.Conclusions: Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy..
4. Koshiro Nishimoto, Ken Nakagawa, Dan Li, Takeo Kosaka, Mototsugu Oya, Shuji Mikami, Hirotaka Shibata, Hiroshi Itoh, Fumiko Mitani, Takeshi Yamazaki, Tadashi Ogishima, Makoto Suematsu, Kuniaki Mukai, Adrenocortical zonation in humans under normal and pathological conditions, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2009-2010, 95, 5, 2296-2305, 2010.05, Context: Aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1) catalyze the terminal steps for aldosterone and cortisol syntheses, respectively, thereby determining the functional differentiationofhumanadrenocortical cells. Little isknown, however, about how the cells expressing the enzymes are actually distributed in the adrenals under normal and pathological conditions. Objective: The objective of the study was to determine the localization of CYP11B2 and -B1 in human adrenal specimens by using developed antibodies capable of distinguishing the two enzymes from each other. Results: Under normal conditions, CYP11B2 was sporadically detected in the zona glomerulosa, whereas CYP11B1 was entirely detected in the zonae fasciculata-reticularis. Adrenocortical cells lacking both enzymes were observed in the outer cortical regions. In addition to conventional zonation, we found a variegated zonation consisting of a subcapsular cell cluster expressing CYP11B2, which we termed aldosterone-producing cell cluster,andaCYP11B1- expressing area. Aldosterone-producing adenomas differed in cell populations expressing CYP11B2 from one another, whereas CYP11B1-expressing and double-negative cells were also intermingled. Adenomas from patients with Cushing's syndrome expressed CYP11B1 entirely but not CYP11B2, resulting in atrophic nontumor glands. The nontumor portions of both types of adenomas bore frequently one or more aldosterone-producing cell clusters, which sustained CYP11B2 expression markedly under the conditions of the suppressed renin-angiotensin system. Conclusion: Immunohistochemistry of the human normal adrenal cortex for CYP11B2 and CYP11B1 revealed a variegated zonation with cell clusters constitutively expressing CYP11B2. This technique may provide a pathological confirmatory diagnosis of adrenocortical adenomas..
5. Tadashi Ogishima, Yoshiie Okada, Shiro Kominami, Shigeki Takemori, Tsuneo Omura, Partial amino acid sequences of two mitochondrial and two microsomal cytochrome p-450's from adrenal cortex, Journal of Biochemistry, 94, 5, 1711-1714, 1983.11, Partial amino acid sequences of two mitochondrial cytochrome P-450's, P-450 (SCC) and P-450 (11β), and two microsomal cytochrome P-450's, P-450 (C-21), and P-450 (17α, Lyase), from adrenal cortex were analyzed and compared. Mitochondrial P-450's and microsomal P-450's were different in the amino acid sequences at their NH2-terminals. The sequences of microsomal P-450's started from terminal meth-ionine and were highly hydrophobic, whereas those of mitochondrial P-450's lacked NH2-terminal methionine and were not hydrophobic. These findings strongly suggest that the NH2-terminal portions of newly synthesized P-450's determine their intracellular localization to different cell organelles..
6. Tadashi Ogishima, Yoshiie Okada, Tsuneo Omura, Presence of a prepiece at the NH2-terminai end of pre-cytochrome P-450(SCC) peptide, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a134762, 95, 5, 1525-1528, 1984.01, Mild acid treatment of in vitro translated cytochrome P-450(SCC) (pre-P-450(SCQ) peptide cleaved the peptide into two fragments. Comparison of the sizes and the NH2-terminal amino acids of the fragments with those of the corresponding fragments from mature P-450(SCC) suggested that the prepiece of pre-P-450(SCC) was present at the NH2-terminal end of the peptide. This conclusion was confirmed by radio-sequencing of the NH2-terminal portion of pre-P-450(SCC)..
7. S. Kominami, H. Hara, Tadashi Ogishima, S. Takemori, Interaction between cytochrome P-450 (P-450(C21)) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system, Journal of Biological Chemistry, 259, 5, 2991-2999, 1984.01, The interaction between P-450(C21) and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450(C21) via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450(C21) reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450(C21) and the reductase. The stable complex formation between P-450(C21) and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration..
8. Tadashi Ogishima, Yoshiie Okada, Tsuneo Omura, Fractionation of mammalian tissue mRNAs by high-performance gel filtration chromatography, Analytical Biochemistry, 10.1016/0003-2697(84)90813-3, 138, 2, 309-313, 1984.05, Total RNA was prepared from the free polysomes of bovine adrenal cortex and fractionated by high-performance gel filtration chromatography using a TSK-GEL G4000SW or G5000PW column. The former column gave better separation of 28 and 18 S rRNA than did the latter column when a suitable elution condition was selected. The separation of mRNAs by gel filtration chromatography was examined by in vitro translation of the fractionated RNA samples, and the fractionation of mRNAs roughly according to their sizes was confirmed. Significant enrichment of the mRNA of cytochrome P-450(SCC), whose in vitro-synthesized peptide had a molecular weight of 55,000, was achieved..
9. Tadashi Ogishima, Yoshiie Okada, Tsuneo Omura, Import and processing of the precursor of cytochrome P-450(SCC) by bovine adrenal cortex mitochondria, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a135335, 98, 3, 781-791, 1985.01, Isolated bovine adrenal cortex mitochondria imported in vitro synthesized pre-P-450(SCC) and processed it to the mature form. Partial radio-sequencing of the processed P-450(SCC) gave a result identical with that for authentic P-450(SCC). Rat liver mitochondria also imported pre-P-450(SCC) and processed it to the mature form, whereas bovine heart mitochondria were unable to import and process pre-P-450(SCC) although both mitochondrial preparations imported and processed pre-adrenodoxin. The pre-P-450(SCC) processing activity of bovine adrenal cortex mitochondria was associated with the matrix side surface of the inner membrane. The processing protease could be solubilized by sodium cholate and partially purified by ammonium sulfate fractionation. The partially purified processing protease cleaved pre-P-450(SCC) at the correct position. It was also active in processing pre-P-450(11β) but inactive toward pre-adrenodoxin. Bovine heart mitochondria lacked the processing activity to pre-P-450(SCC).The localization of pre-P-450(SCC) and mature P-450(SCC) in bovine adrenal cortex mitochondria was examined. Mature P-450(SCQ processed by the mitochondria was found associated with the matrix-side surface of the inner membrane, which is the correct location of P-450(SCC) in the cell. In the presence of o-phenanthroline, pre-P-450(SCC) was imported into the organelles without being processed and remained soluble in the matrix. The incorporation of newly processed mature P-450(SCC) into the inner membrane was also observed when pre-P-450(SCC) was incubated with inner membrane vesicles. Mature P-450(SCC) generated in vitro from pre-P-450(SCC) by the partially purified processing protease was incorporated not only into the inner membrane vesicles but also into bovine adrenal cortex microsomes. These findings suggested that the processing of pre-P-450(SCC) occurred prior to the incorporation of mature-P-450(SCC) into the inner membrane..
10. Akio Ito, Tadashi Ogishima, Weija Ou, Tsuneo Omura, Haruhiko Aoyagi, Sannumn Lee, Hisakazu Mihara, Nobuo Izumiya, Effects of synthetic model peptides resembling the extension peptides of mitochondrial enzyme precursors on import of the precursors into mitochondria, Journal of Biochemistry, 10.1093/oxfordjournals.jbchem.a135426, 98, 6, 1571-1582, 1985.01, One common and characteristic feature of the extension peptides of mitochondrial enzyme precursors is the presence of repeating short stretches of uncharged amino acids linked by basic amino acids. We synthesized several model peptides having this particular feature of the extension peptides. The peptides contained arginine or lysine as a basic amino acid residue linking sequences of two to four residues of leucine and alanine. We examined the effects of the peptides on the import of the precursors of two mitochondrial enzymes, cytochrome P-450(SCC) and adrenodoxin, and found that the peptides were generally inhibitory to the import of the precursors into mitochondria. The effective concentrations of some of the inhibitory peptides were as low as a few μM. The peptides containing lysine instead of arginine had an essentially similar inhibitory effect on the import. The peptides did not inhibit the binding of pre-P450(SCC) to the surface of mitochondria. The synthetic model peptides uncoupled oxidative phosphorylation of mitochondria prepared from either rat liver or bovine adrenal cortex, and induced leakage of enzymes from the inner compartments of mitochondria. However, the synthetic model peptides did not solubilize membrane-bound enzymes from mitochondria, suggesting that their effect on the membranes is different from that of detergents. The synthetic model peptides seem to bind to the membranes causing significant perturbation in the membrane structure, which is possibly related to the functions of the particular common sequence found in the extension peptides of mitochondrial enzyme precursors..
11. Tadashi Ogishima, Kyuichiro Okuda, An improved method for assay of cholesterol 7α-hydroxylase activity, Analytical Biochemistry, 10.1016/0003-2697(86)90613-5, 158, 1, 228-232, 1986.10, An improved assay method for cholesterol 7α-hydroxylase which is accurate, sensitive and yet still simple is described. The method consists of two parts: the first part is cholesterol 7α-hydroxylation in liver microsomes utilizing cholesterol in situ as the substrate, and the second part is conversion of the product, 7α-hydroxycholesterol, into 7α-hydroxy-4-cholesten-3-one having an intense absorption at 240 nm by the action of cholesterol oxidase. The converted sterol is then analyzed by high-performance liquid chromatography. During the second enzyme reaction, the first enzyme reaction is halted and further metabolism of the product is prevented. In consequence, the method had more than 10-fold increase in the sensitivity than the previous one..
12. T. Ogishima, S. Deguchi, K. Okuda, Purification and characterization of cholesterol 7 alpha-hydroxylase from rat liver microsomes., Journal of Biological Chemistry, 262, 16, 7646-7650, 1987.06, Cholesterol 7 alpha-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7 alpha-hydroxylating, EC 1.14.13.17) was purified from liver microsomes of cholestryramine-fed male rats by using high-performance ion-exchange chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000), and its dithionite-reduced CO complex exhibited an absorption maximum at 450 nm. The specific content of the enzyme was 9 nmol of cytochrome P-450/mg of protein. Upon reconstitution with NADPH-cytochrome P-450 reductase, the enzyme showed a high activity of cholesterol 7 alpha-hydroxylation with the turnover number of 50 min-1 at 37 degrees C. The reaction was inhibited neither by aminoglutethimide nor by metyrapone, but inhibited markedly by iodoacetamide and disulfiram. The reaction was also inhibited significantly by CO. The enzyme catalyzed hydroxylation of cholesterol with strict regio- and stereoselectivity and was inert toward other sterols which are intermediates in the conversion of cholesterol to bile acids, i.e. 7 alpha-hydroxy-4-cholesten-3-one (12 alpha-hydroxylation), 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (25-hydroxylation), and taurodeoxycholate (7 alpha-hydroxylation). Unlike other cytochromes P-450 isolated from rat liver microsomes, the enzyme showed no activity toward testosterone and xenobiotics such as 7-ethoxycoumarin and benzo[a] pyrene. The NH2-terminal amino acid sequence of the enzyme was Met-Phe-Glu-Val(Ile)-Ser-Leu-, which was distinct from those of any other cytochromes P-450 of rat liver microsomes hitherto reported. These results indicate that the enzyme is a novel species of cytochrome P-450 so far not isolated from liver microsomes..
13. Tadashi Ogishima, F. Mitani, Y. Ishimura, Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex, Journal of Biological Chemistry, 264, 19, 10935-10938, 1989, A cytochrome P-450 capable of producing aldosterone from 11-deoxycorticosterone was purified from the zona glomerulosa of rat adrenal cortex. The enzyme was present in the mitochondria of the zona glomerulosa obtained from sodium-depleted and potassium-repleted rats but scarcely detected in those from untreated rats. It was undetectable in the mitochondria of other zones of the adrenal cortex from both the treated and untreated rats. The cytochrome P-450 was distinguishable from cytochrome P-450(11β) purified from the zonae fasciculata-reticularis mitochondria of the same rats. Molecular weights of the former and the latter cytochromes P-450, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 49,500 and 51,500, respectively, and their amino acid sequences up to the 20th residue from the N terminus were different from each other at least in one position. The former catalyzed the multihydroxylation reactions of 11-deoxycorticosterone giving corticosterone, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and a significant amount of aldosterone as products. On the other hand, the latter catalyzed only 11β- and 18-hydroxylation reactions of the same substrate to yield either corticosterone or 18-hydroxydeoxycorticosterone. Thus, at least two forms of cytochrome P-450, which catalyze the 11β- and 18-hydroxylations of deoxycorticosterone, exist in rat adrenal cortex, but aldosterone synthesis is catalyzed only by the one present in the zona glomerulosa mitochondria..
14. Tadashi Ogishima, Fumiko Mitani, Yuzuru Ishimura, Isolation of two distinct cytochromes P-45011β with aldosterone synthase activity from bovine adrenocortical mitochondria, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a122694, 105, 4, 497-499, 1989.01, Two distinct forms of cytochrome P-45011β, with apparent molecular weights of 48,500 (48.5K) and 49, 500 (49.5K), have been isolated from bovine adrenocortical mitochondria. Their amino acid sequences up to the 19th position from the N-terminus were only different at the 6th position (Val and Ala for the 48.5K and 49.5K enzymes, respectively). Each sequence was assignable to a distinct cDNA clone for cytochrome P-45011β (Kirita, S., et al. [1988] J. Biochem. 104, 683-686), indicating that the two proteins originate from different genes in bovine adrenocortical cells. Both forms of cytochrome P-45011βwere capable of catalyzing aldosterone synthesis as well as the 11β- and 18-hydroxylation of 11-deoxycorticosterone. Thus, at least two distinct cytochrome P-45011β, species exist in the adrenal cortex and participate in steroidogenesis..
15. Michiyo Imai, Hideo Shimada, Yukiko Okada, Yuko Matsushima-Hibiya, Tadashi Ogishima, Yuzuru Ishimura, Molecular cloning of a cDNA encoding aldosterone synthase cytochrome P-450 in rat adrenal cortex, FEBS Letters, 10.1016/0014-5793(90)81398-8, 263, 2, 299-302, 1990.04, Using an oligonucleotide probe designed on the basis of the N-terminal amino acid sequence of purified rat aldosterone synthase cytochrome P-450 [(1989) J. Biol. Chem. 264 10935] we have isolated from rat adrenal cDNA library a 2687 base pair cDNA that encodes a protein of 500 amino acid residues. The deduced amino acid sequence contained the regions well conserved among all cytochrome P-450s sequenced to date, and also a portion (residues 25-44) which was identical to the N-terminal peptide sequence of rat aldosterone synthase cytochrome P-450. These results indicate that the cDNA encodes a precursor form of rat aldosterone synthase cytochrome P-450..
16. Hirotaka Shibata, Tadashi Ogishima, Fumiko Mitani, Hiromichi Suzuki, Marohito Murakami, Takao Saruta, Yuzuru Ishimura, Regulation of aldosterone synthase cytochrome P-450 in rat adrenals by angiotensin II and potassium, Endocrinology, 10.1210/endo-128-5-2534, 128, 5, 2534-2539, 1991.05, Changes in the levels of aldosterone synthase cytochrome P-450, a recently identified enzyme in rat adrenals, were studied in response to the renin-angiotensin system and K stimuli. As examined by an immunoblot technique, the zona glomerulosa mitochondria from rats fed on a low Na-normal K diet (8.6 mmol Na+and 207 mmol K+/kg of diet) or a low Na-high K (0.2 M KC1 in drinking water) diet for 4-10 days con-tained significantly higher amounts of aldosterone synthase cytochrome P-450 than those from rats fed on a normal diet (86 mmol Na+and 207 mmol K+/kg of diet). Activities of the enzyme were also found to increase by about 10-fold on day 10. In concert with these changes, both plasma renin activity and plasma aldosterone concentration increased, indicating that the renin-angiotensin system was activated in these rats. Feeding with a normal Na-high K diet also induced significantly higher levels of both amount and activity of aldosterone synthase cytochrome P-450 together with an elevated serum K concentra-tion on day 4, though they all decreased to near the control level on the following days. On the other hand, when enalapril malate, an angiotensin I-converting enzyme inhibitor, was administered to the low Na-normal K rats, the increases in the amount and activity of the enzyme as well as in plasma aldosterone concen-tration were suppressed altogether. However, the enalapril ad-ministration to the low Na-high K rats suppressed the increases only partially. These results indicate that the aldosterone syn-thase cytochrome P-450 is an ultimate target of the regulation of aldosterone biosynthesis by angiotensin II and K..
17. T. Ogishima, H. Shibata, H. Shimada, F. Mitani, H. Suzuki, T. Saruta, Y. Ishimura, Aldosterone synthase cytochrome P-450 expressed in the adrenals of patients with primary aldosteronism, Journal of Biological Chemistry, 266, 17, 10731-10734, 1991.09, A human cytochrome P-450 with aldosterone synthase activity was purified from the mitochondria of an aldosterone-producing adenoma. It was recognized by an anti-bovine cytochrome P-450(11β) IgG and by a specific antibody raised against a portion of the CYP11B2 gene product, one of the two putative proteins encoded by human cytochrome P-450(11β)-related genes (Mornet, E., Dupont, J., Vitek, A., and White, P. C. (1989) J. Biol. Chem. 264, 20961-20967). A similar and probably the same aldosterone synthase cytochrome P-450 was detected in the adrenal of a patient with idiopathic hyperaldosteronism. These aldosterone synthases were distinguishable from cytochrome P-450(11β), the product of another cytochrome P-450(11β)-related gene, i.e. CYP11B1, by their catalytic, molecular, and immunological properties and also by their localization. The latter enzyme was unable to produce aldosterone and did not react with the specific antibody against the CYP11B2 gene product. It was present both in tumor and non-tumor portions of the adrenals carrying the adenoma and in normal adrenal cortex. On the other hand, aldosterone synthase cytochrome P-450 localized in the tumor portion of the adrenals or in the adrenal of a patient with idiopathic hyperaldosteronism. Thus aldosterone synthase cytochrome P-450, a distinct species from cytochrome P-450(11β), is responsible for the biosynthesis of aldosterone in the human, at least in patients suffering from primary aldosteronism..
18. Kuniaki Mukai, Michiyo Imai, Hideo Shimada, Yukiko Okada, Tadashi Ogishima, Yuzuru Ishimura, Structural differences in 5′-flanking regions of rat cytochrome P-450aldo and P-45011β genes, Biochemical and Biophysical Research Communications, 10.1016/S0006-291X(05)81321-X, 180, 3, 1187-1193, 1991.11, Two rat genomic clones, one for cytochrome P-450aldo and the other for P45011β, were isolated and characterized. The two genes, encoding structurally homologous proteins, were closely similar in their intron-exon organizations. Their 5′-flanking regions, however, contained only a few homologous regions. A putative cyclic AMP responsive element, TGACGTGA, was found in the P-450aldo gene, but this sequence was altered at two positions in the P-45011β gene. S1 nuclease protection assay revealed a single transcription initiation site for the P450aldo gene, while multiple sites were found for the P-45011β gene. These results suggest that transcriptional regulation of the rat P-450aldo and P-45011β genes is due to differences in the sequences of their 5′-flanking regions..
19. Michiyo Imai, Tadashi Ogishima, Hideo Shimada, Yuzuru Ishimura, Effect of dietary sodium restriction on mrna for aldosterone synthase cytochrome P-450 in rat adrenals, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a123776, 111, 4, 440-443, 1992.04, Changes in the level of mRNA for aldosterone synthase cytochrome P-450 (cytochrome P-450aldo) in rats on dietary sodium restriction were studied by means of Northern and slot blot hybridization using an oligonucleotide probe that allowed differentiation of the message for this enzyme from that for cytochrome P-45011β. These two enzymes have been shown to be highly homologous with each other, exhibiting 88% homology in their nucleotide sequences in the coding region. Upon sodium restriction for 2 weeks, cytochrome P-450aldo mRNA in rat adrenals increased 7-fold, whereas the cytochrome P-45011β mRNA level in the same adrenals did not change significantly. The increase in cytochrome P-450aldo mRNA paralleled that in cytochrome P-450aldo protein, as analyzed by immunoblot technique. These results, together with our previous finding that angiotensin II induced cytochrome P-450aldO in rat adrenocortex [Shibata, H., Ogishima, T., Mitani, F., Suzuki, H., Murakami, M., Saruta, T., &Ishimura, Y. (1991) Endocrinology 128, 2534-2539], suggest that the production of cytochrome P-450aldo is regulated by angiotensin II at the pretranslational level, most likely at the transcriptional level..
20. Tadashi Ogishima, Hiroshi Suzuki, Jun Ichi Hata, Fumiko Mitani, Yuzuru Ishimura, Zone-specific expression of aldosterone synthase cytochrome p-450 and cytochrome p-45011β in rat adrenal cortex
Histochemical basis for the functional zonation, Endocrinology, 10.1210/endo.130.5.1572304, 130, 5, 2971-2977, 1992.05, Zonal distribution of aldosterone synthase cytochrome P-450 and cytochrome P-45011β in rat adrenocortex was investigated immunochemically using specific antibodies to these enzymes. Localization of aldosterone synthase cytochrome P-450 (cytochrome P-450aldo), a recently identified enzyme that converts deoxycorticosterone to aldosterone in rat adrenocortex was strictly confined to two or three outermost cell layers in the zona glomerulosa. In contrast, cytochrome P-45011β, which forms corticosterone, but not aldosterone, from deoxycorticosterone, was localized in the zona fasciculata-reticularis and not in the zona glomerulosa. Neither enzyme was detected in the medulla or the capsule. The functional zonation of adrenocortex with respect to aldosterone and corticosterone syntheses is, thus, ascribable to the localization of cytochromes P-450aldo and P- 45011β in the respective zones. When rats were maintained under Na-depleted conditions for 10 days, the zona glomerulosa cells containing cytochrome P-450aldo proliferated to 10-15layers, the thickness of which was 5-7-fold that in the nonstimulated rats. Proliferation of the cytochrome P-450aldo-positive cells into the zona fasciculata-reticularis was also observed along with arterial walls. Under these conditions, no significant change in the distribution of cytochrome P-45011β was noted. These results indicate that the angiotensin-II stimuli, which had been elicited by the low Na treatment, promoted proliferation of the glomerulosa cells, resulting in increased expression of cytochrome P-450aldo in rat adrenocortex..
21. H. Shibata, H. Suzuki, T. Ogishima, Y. Ishimura, T. Saruta, Significance of steroidogenic enzymes in the pathogenesis of adrenal tumour, Acta Endocrinologica, 10.1530/acta.0.1280235, 128, 3, 235-242, 1993.05, We examined both activities and amounts of steroidogenic cytochrome P-450s at the posttranslational protein level and steroid contents in the adrenocortical adenoma from patients with primary aldosteronism and Cushing's syndrome. Aldosterone synthase cytochrome P-450 (human P-450(aldo)) was detected in the tumour portion of aldosterone-producing adenoma, but not in the normal control adrenals, at the protein level. Neither the activities nor the amounts of other P-450s in the tumour portion of aldosterone-producing adenoma were significantly different from those in the non-tumour portion in the adenoma and the normal control adrenals. The aldosterone content was significantly elevated, while the androstenedione content was significantly decreased in the tumour portion of the adenoma compared with that in the normal control adrenals. In Cushing's syndrome, both the activities and amounts of P-450(17α) and P-450(C21) were significantly elevated in the tumour portion compared with the non-tumour portion of the adenoma and the normal control adrenals, while those of P-450(scc) and P-450(11β) in the tumour portion were not significantly different from the normal control adrenals. The cortisol content was significantly elevated, while the amounts of aldosterone and 18-hydroxydeoxycorticosterone in the tumour portion of the adenoma were significantly decreased compared with those in the normal control adrenals. These results demonstrate that overexpression of P-450(aldo) in aldosterone-producing adenoma, and those of P-450(17α) and P-450(C21) in cortisol-producing adenoma may play some role in the pathogenesis of primary aldosteronism and Cushing's syndrome, respectively..
22. Sakae Kitada, Takuro Niidome, Takashi Nagano, Tadashi Ogishima, Akio Ito, Molecular Cloning of the Smaller Subunit (P52) of Rat Liver Mitochondrial Processing Protease, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1993.1044, 190, 1, 289-293, 1993.01, A cDNA encoding the smaller subunit (P52) of mitochondrial processing protease was isolated from a rat liver cDNA library, using cDNA fragment for yeast MAS1 as the probe. The deduced amino acid sequence is highly homologous to those of PEP from Neurospora crassa and MAS1 from Saccharomyces cerevisiae. After in vitro transcription and translation, the precursor peptide was imported into isolated rat liver mitochondria and processed to its mature form..
23. T. Niidome, S. Kitada, K. Shimokata, T. Ogishima, A. Ito, Arginine residues in the extension peptide are required for cleavage of a precursor by mitochondrial processing peptidase. Demonstration using synthetic peptide as a substrate, Journal of Biological Chemistry, 269, 40, 24719-24722, 1994.01, Mitochondrial processing peptidase (MPP) specifically recognizes a large variety of mitochondrial precursor proteins and correctly cleaves off the extension peptides. To determine the structure common to all the extension peptides that is required for specific recognition by MPP, we synthesized various oligopeptides of different chain lengths and amino acid sequences, based on the amino acid sequence of the extension peptide of pre-malate dehydrogenase, and determined kinetic parameters of the cleavage reactions. The minimal length of peptides for effective cleavage was 16 amino acid residues consisting of 11 and 5 residues from the cleavage site to the amino- and carboxyl-terminal sides, respectively. Two sets of basic amino acids in the peptide, the distal arginine residue at position -10 and the proximal ones at positions -3 and -2 relative to the cleavage site, were necessary for effective hydrolysis. Of these two, the residue at position -2 was more important for effective cleavage than the one at position -3 and could not be replaced by a lysine residue. The replacement of the distal arginine by lysine had no effect on the cleavage. Our study demonstrates that use of peptides with the proper length is essential for performing kinetic analyses on the cleavage reaction by MPP and that an arginine residue at position -2 to the cleavage site is necessary for the recognition and cleavage of the extension peptide..
24. Fumiko Mitani, Hiroshi Suzuki, Jun Ichi Hata, Tadashi Ogishima, Hideo Shimada, Yuzuru Ishimura, A novel cell layer without corticosteroid-synthesizing enzymes in rat adrenal cortex
Histochemical detection and possible physiological role, Endocrinology, 10.1210/endo.135.1.8013381, 135, 1, 431-438, 1994.01, A stratum of cells that did not contain both aldosterone synthase cytochrome P450 (cytochrome P450aldo) and cytochrome P45011 beta was found immunohistochemically between the zona glomerulosa and the zona fasciculata of the rat adrenal cortex. As cytochromes P450aldo and P45011 beta are the enzymes responsible for the biosynthesis of aldosterone and corticosterone, respectively, the cells there are considered to be incapable of synthesizing both aldosterone and corticosterone. Furthermore, the cells are regarded as inert in producing adrenal androgens, because rat adrenal cortex is known to lack steroid 17 alpha-hydroxylase. Thus, the stratum is composed of cells that do not synthesize any of the major corticosteroids in significant quantities. It was 5-10 cells thick under normal feeding conditions, but diminished to 4-5 cells thick when animals were maintained under Na restriction, which is known to stimulate the secretion of angiotensin-II. When the distribution of 5-bromo-2′-deoxyuridine-labeled nuclei in the adrenocortex from BrdU-administered rats was examined, the stained nuclei were concentrated in and around the cell stratum. The pulse-chase experiments showed that the labeled cells migrated out of this layer and into the zonae fasciculata-reticularis. On the basis of these findings, we suggest that the newly discovered cell layer is the progenitor cell zone of the rat adrenal cortex..
25. Fumiko Mitani, Hiroshi Suzuki, Jun Ichi Hata, Tadashi Ogishima, Hideo Shimada, Yuzuru Ishimura, A novel cell layer without corticosteroid-synthesizing enzymes in rat adrenal cortex
Histochemical detection and possible physiological role, Endocrinology, 10.1210/endo.135.1.8013381, 135, 1, 431-438, 1994.07, A stratum of cells that did not contain both aldosterone synthase cytochrome P450 (cytochrome P450aldo) and cytochrome P45011β was found immunohistochemically between the zona glomerulosa and the zona fasciculata of the rat adrenal cortex. As cytochromes P450aldo and P45011β are the enzymes responsible for the biosynthesis of aldosterone and corticosterone, respectively, the cells there are considered to be incapable of synthesizing both aldosterone and corticosterone. Furthermore, the cells are regarded as inert in producing adrenal androgens, because rat adrenal cortex is known to lack steroid 17α-hydroxylase. Thus, the stratum is composed of cells that do not synthesize any of the major corticosteroids in significant quantities. It was 5-10 cells thick under normal feeding conditions, but diminished to 4-5 cells thick when animals were maintained under Na restriction, which is known to stimulate the secretion of angiotensin-II. When the distribution of 5- bromo-2'-deoxyuridine-labeled nuclei in the adrenocortex from BrdU administered rats was examined, the stained nuclei were concentrated in and around the cell stratum. The pulse-chase experiments showed that the labeled cells migrated out of this layer and into the zonae fasciculata-reticularis. On the basis of these findings, we suggest that the newly discovered cell layer is the progenitor cell zone of the rat adrenal cortex..
26. F. Mitani, Tadashi Ogishima, H. Miyamoto, Y. Ishimura, Localization of p450aldo and p45011β in normal and regenerating rat adrenal cortex, Endocrine Research, 10.3109/07435809509030457, 21, 1-2, 413-423, 1995.01, A novel layer of cells that do not contain both P450aldo and P45011β has been discovered between the zonae glomerulosa and fasciculata of the rat adrenal cortex. Since P450aldo and P45011β are the enzymes responsible for the formation of aldosterone and corticosterone, respectively, the cells in that zone are presumably inert in synthesizing both aldosterone and corticosterone, in other words, the layer is composed of cells that have no zone-specific endocrine function as an adrenocortical component. Cytologically, the layer consists of tightly packed cells, which contain a lesser amount of lipid droplet than the cells in the other zones, and appears as a white ring or a white zone in the double immunostaining with anti P450aldo and and P45011β Upon angiotensin II-stimulation evoked by Na-deficiency, the number of the zona glomerulosa cells expressing P450aldo increases for the initial 2 or 3 days and then the P450aldo-containing zona glomerulosa cells begin to proliferate. Thus angiotensin II serves as a proliferator of the zona glomerulosa cells of the rat adrenal cortex. During the period, the thickness of the white zone decreases for initial 3 days and becomes constant after 5 or 6 days, being about 5 % of the total cell number of the adrenal cortex. When localization of replicating cells was examined in the adrenal cortex, they were found to be concentrated in and around the white zone. Then the pulse-chase experiments with BrdU showed that the labeled cells migrated out of the white zone and into the zonae fasciculata and rencularis. The localization of the replicating cells in the regenerating adrenal cortex was also around the region between the zonae glomerulosa and fasciculata. On the basis of these findings, we suggest that the newly discovered cell layer (the white zone) is the stem cell zone of the rat adrenal cortex..
27. Sakae Kitada, Kunitoshi Shimokata, Takuro Niidome, Tadashi Ogishima, Akio Ito, A putative metal-binding site in the β subunit of rat mitochondrial processing peptidase is essential for its catalytic activity, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a124836, 117, 6, 1148-1150, 1995.01, Mitochondrial processing peptidase (MPP) consists of α- and β-subunits (α-MPP and β-MPP). β-MPP has a putative metal-binding sequence (HXXEH). To determine whether the sequence of β-MPP is essential for the enzymatic activity, we individually mutated the histidines and glutamic acid to arginines and glutamine, respectively. The wild-type and mutated β-MPPs were co-expressed with α-MPP in Escherichia coli. All three mutants had completely lost the activity, whereas the lost activity was recovered on the addition of wild-type β-MPP. The activity of the wild-type enzyme was reduced by the mutant β-MPPs. We conclude from these observations that the HXXEH region is involved in the formation of the active site and that β-MPP is the catalytic subunit of MPP..
28. Tadashi Ogishima, Takuro Niidome, Kunitoshi Shimokata, Sakae Kitada, Akio Ito, Analysis of elements in the substrate required for processing by mitochondrial processing peptidase, Journal of Biological Chemistry, 10.1074/jbc.270.51.30322, 270, 51, 30322-30326, 1995.12, We have recently demonstrated that synthetic peptides modeled on the extension peptide of malate dehydrogenase can be a good substrate of mitochondrial processing peptidase and that arginine residues present at positions -2 or -3 and distant from the cleavage point were important for recognition by the enzyme (Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24719-24722). We further investigated the elements required for substrates of the protease. To analyze the reaction by a more rapid yet quantitative method, we have developed intramolecularly quenched fluorescent substrates. Using the fluorogenic substrates we demonstrated that at least one of the proline and glycine between the distal and proximal arginine residues was also important while other connecting sequences were dispensable. In addition, the protease showed considerable preference for aromatic and, to a lesser extent, hydrophobic amino acids in the P1'-position. These results together with the previous daryl suggest that the proximal and distal arginine residues, proline and/or glycine between them, and P1' amino acid could be critical determinants for the specific cleavage of the substrates by the protease..
29. Myeong Cheol Song, Kunitoshi Shimokata, Sakae Kitada, Tadashi Ogishima, Akio Ito, Role of basic amino acids in the cleavage of synthetic peptide substrates by mitochondrial processing peptidase, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a021536, 120, 6, 1163-1166, 1996.01, Our recent experiments using model peptides of rat malate dehydrogenase (MDH) indicated that a proximal arginine and a distal basic amino acid are important for processing by mitochondrial processing peptidase (MPP). To elucidate if the recognition elements apply to other precursor proteins, we analyzed cleavage of model peptides of human ornithine aminotransferase (OAT). Purified peptidase cleaved peptides that corresponded to N-terminal 1-25 and 3-25 at the correct site (Gly17-Val18) at nearly equal rates. Replacement of Arg15 (-2 position) with lysine or alanine reduced the processing efficiency by 95- and 380-fold, respectively. Either deletion from Met1 to Arg10 or replacement of the basic amino acids between them decreased the processing efficiency considerably. A peptide containing Arg7 in addition to Lys4 and Arg10 was more effective than the control peptide. However, a peptide with one and two consecutive basic amino acids in the distal region had a processing efficiency close to the control peptide. These results indicated that processing of OAT was enhanced by an increase in the number of basic amino acids with a suitable distance between them. In other respects, the processing signal of OAT was essentially the same as that of MDH..
30. Kunitoshi Shimokata, Takenori Nishio, Myeong Cheol Song, Sakae Kitada, Tadashi Ogishima, Akio Ito, Substrate recognition by mitochondrial processing peptidase toward the malate dehydrogenase precursor, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a021841, 122, 5, 1019-1023, 1997.01, Mitochondrial processing peptidase (MPP) cleaves the extension peptides of precursor proteins newly imported into the mitochondria. Using synthetic oligopeptides modeled on the extension peptide of malate dehydrogenase, the critical elements of the substrate for the processing of MPP were determined. In the present study, we constructed mutant precursors and compared the processing reaction with that of the peptide substrates to confirm the validity of use of peptide substrates. In both cases, the arginine residue presents at a proximal (-2) position relative to the processing site proved to be important for the processing. The distal arginine residue at position 7 was replaceable with alanine with no significant loss in cleavage efficiency if the precursor protein contained two consecutive arginine residues at a proximal position, although the arginine residue at a position 7 was indispensable in the model peptide. The proline residue, lying between the distal and proximal arginine residues, which is assumed to break a continuous α-helix region in the extension peptide, was needed for the processing. This peptidase has a preference for aromatic amino acids at the P1' site. These results were essentially the same as those obtained with model peptides except for the role of the distal arginine. We also found that amino acids at P2' and P3' sites had some effects on the processing. Thus we concluded that an effective combination of model peptides with precursor proteins is needed for the studies on MPP responsible substrate-recognition mechanisms..
31. S. Wakino, M. Meguro, H. Suzuki, T. Saruta, T. Ogishima, H. Shimada, Y. Ishimura, T. Shinki, T. Suda, Evidence for 54-kD protein in chicken kidney as a cytochrome P450 with a high molecular activity of 25-hydroxyvitamin D3 1β-hydroxylase, Gerontology, 10.1159/000213826, 42, 1, 67-77, 1996.01, Conversion of 25-hydroxyvitamin D3 (25(OH)D3) to the active vitamin D3, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) is catalyzed by 25(OH)D3,1α-hydroxylase (1α-hydroxylase). It has been suggested that this enzyme is cytochrome P450 (P450). We purified 1α-hydroxylase 430-fold from cholate-solubilized kidney mitochondria of vitamin D-deficient chickens by utilizing hydrophobic and ion-exchange column chromatographies. Enzymatic activity was assessed by measuring on HPLC the formation of 1α,25(OH)2D3 from 25(OH)D3 in the assay mixture containing NADPH, adrenodoxin reductase, adrenodoxin as a reducing system. The purified enzyme showed a CO-difference spectrum characteristic of P450. The molecular activity of this preparation was calculated to be 8.7 pmol/min/pmol P450. This value was higher by more than 87-fold than those reported so far. The present preparation was found to contain several proteins on SDS-PAGE. Among them, only the 54-kD protein became undetectable when kidney mitochondria from normal and vitamin D-replete chickens, where 1α-hydroxylase activities were 15 and 0% of that found in vitamin D-deficient chicken, respectively, were used as the starting enzyme sources. Furthermore, the band intensity of the 54-kD protein accounted for the spectrophotometrically determined amount of P450 in the preparation. These results suggest that the 54-kD protein is 1α-hydroxylase..
32. A. Ito, Tadashi Ogishima, S. Kitada, Processing of mitochondrial protein precursors, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 42, 14 Suppl, 2362-2367, 1997.01.
33. Myeong Cheol Song, Tadashi Ogishima, Akio Ito, Importance of residues carboxyl terminal relative to the cleavage site in substrates of mitochondrial processing peptidase for their specific recognition and cleavage, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a022198, 124, 5, 1045-1049, 1998.01, We previously identified distal and proximal arginine residues in the N-terminal portion and an aromatic amino acid at position 1 (P1' site3) relative to the cleavage site as important recognition signals in substrates of mitochondrial processing peptidase. To further elucidate the elements required for the specific recognition and cleavage by the enzyme, we synthesized synthetic peptides that possessed only the distal and proximal arginine residues and phenylalanine at the P1' site in a poly alanine sequence, and analyzed the processing reaction toward them. They were not cleaved by the peptidase although they inhibited the peptidase activity. However, when serine was introduced into the C-terminal portions of the sequence, processing was observed. The efficiency of the resultant peptides improved as the number of serine residues was increased. A peptide with serine or histidine at P2' and threonine at P3' was processed most efficiently. These results indicate that the processing reaction catalyzed by the peptidase depends not only on the N-terminal portion but also on the C-terminal portion from the cleavage site in the substrates..
34. Kaori Moriwaki, Tadashi Ogishima, Akio Ito, Analysis of recognition elements for mitochondrial processing peptidase using artificial amino acids
Roles of the intervening portion and proximal arginine, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a022529, 126, 5, 874-878, 1999.01, We recently demonstrated, using synthetic peptides modeled on the extension peptide of malate dehydrogenase, that amino acid residues present at the proximal and distal positions relative to the cleavage site are critical determinants for the recognition of substrates by mitochondrial processing peptidase [Niidome et al. (1994) J. Biol. Chem. 269, 24719-24722). While the proximal arginine is unexceptionally located at the -2 position, the position of the distal residue varies among mitochondrial precursor proteins. Between the proximal and distal residues, proline and/or glycine are present in most mitochondrial precursor proteins, and they are considered to play a role in the specific recognition of a substrate by the peptidase. To elucidate the role of the intervening portion, we introduced a non-natural amino acid [2-(2-aminoethoxy)acetic acid] between the distal and proximal residues. We also analyzed the functional elements in the proximal arginine by replacing the residue with various arginine or lysine analogs. The results of kinetic studies indicated that the intervening portion should be flexible for efficient processing, and that the guanidino group of the proximal arginine is recognized by the peptidase through hydrogen and ionic bonds..
35. Yumiko Nagao, Sakae Kitada, Katsuhiko Kojima, Hidehiro Toh, Satoru Kuhara, Tadashi Ogishima, Akio Ito, Glycine-rich region of mitochondrial processing peptidase α-subunit is essential for binding and cleavage of the precursor proteins, Journal of Biological Chemistry, 10.1074/jbc.M003110200, 275, 44, 34552-34556, 2000.11, Mitochondrial processing peptidase, a metalloendopeptidase consisting of α- and β-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off amino-terminal extension peptides. The α-subunit has a characteristic glycine-rich segment in the middle portion. To elucidate the role of the region in processing functions of the enzyme, deletion or site-directed mutations were introduced, and effects on kinetic parameters and substrate binding of the enzyme were analyzed. Deletion of three residues of the region, Phe289 to Ala291, led to a dramatic reduction in processing activity to practically zero. Mutation of Phe289, Lys296, and Met298 to alanine resulted in a decrease in the activity, but these mutations had no apparent effect on interactions between the two subunits, indicating that reduction in processing activity is not due to structural disruption at the interface interacting with the β-subunit. Although the mutant enzymes, Phe289Ala, Lys296Ala, and Met298Ala, had an approximate 10-fold less affinity for substrate peptides than did that of the wild type, the deletion mutant, Δ289-291, showed an extremely low affinity. Thus, shortening of the glycine-rich stretch led to a dramatic reduction of interaction between the enzyme and substrate peptides and cleavage reaction, whereas mutation of each amino acid in this region seemed to affect primarily the cleavage reaction..
36. K. Kojima, E. Yamasaki, S. Kitada, T. Ogishima, A. Ito, Recognition of mitochondrial protein precursor lacking arginine at position -2 by Mitochondrial Processing Peptidase
Processing of bovine cytochrome p450(SCC) precursor, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a003012, 130, 4, 497-502, 2001.10, Mitochondrial processing peptidase (MPP) specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Previous studies demonstrated that Arg at position -2 from the cleavage site, which is found among many precursors, plays a critical role in recognition by MPP. We analyzed the structural elements of bovine cytochrome P450 side-chain cleavage enzyme precursor [pre-P450(SCC)], which has Ala at position -2, for recognition by MPP. Replacement of Ala position -2 of pre-P450(SCC) with Arg resulted in an increase in the cleavage rate. Replacement with Gly caused a reduction in the cleavage rate and the appearance of an additional cleavage site down-stream of the authentic site. A pre-P450(SCC) mutant with Met at position -2 retained cleavage efficiency equal to that of the wild type. These results indicate that -2 Ala of pre-P450(SCC) is recognized by MPP as a determinant for precise cleavage, and that the amino acid at -2 is required to have a straight methylene chain for interaction with the S2 site. The preference for distal basic residues, a hydrophobic residue at +1, and hydroxyl residues at +2 and +3, was almost the same as those of the precursors with Arg at -2, indicating that the recognition mechanism of pre-P450(SCC) by MPP is essentially the same as that of the precursors with Arg at position -2..
37. Fumiko Mitani, T. Ogishima, K. Mukai, R. Hoshino, K. Watanabe, M. Suematsu, Possible participation of outer mitochondrial membrane cytochrome B 5 in steroidogenesis in zona glomerulosa of rat adrenal cortex, Endocrine Research, 10.1081/ERC-200043911, 30, 4, 639-644, 2004.12, Outer mitochondrial membrane cytochrome b5 (OMb) originally found in rat liver is an isoform of cytochrome b5 (b5) of the endoplasmic reticulum. In contrast to accumulated data on the physiological roles of b5, functions of OMb have not been well characterized except for its involvement in regeneration of ascorbic acid [i.e., in a semidehydroascorbate reductase (SDAR) system]. By using highly specific antibodies against rat OMb, we found immunohistochemically that OMb in the rat adrenal gland was most abundant in the zona glomerulosa (zG) among the three cortical zones, and the expression level was enhanced on angiotensin II-stimulation. SDAR activity was found in zG and inhibited by anti-OMb antibody. Moreover, the increase in plasma aldosterone concentration under Na+-deficiency was suppressed by limited ascorbic acid (Asc) availability in rat mutants unable to synthesize Asc, while plasma corticosterone concentration was not affected. These data suggest that OMb, present abundantly in zG, participates in aldosterone formation in zG of rat under angiotensin II-stimulation through regeneration of Asc..
38. Tsutomu Oshima, Eiki Yamasaki, Tadashi Ogishima, Koh Ichi Kadowaki, Akio Ito, Sakae Kitada, Recognition and processing of a nuclear-encoded polyprotein precursor by mitochondrial processing peptidase, Biochemical Journal, 10.1042/BJ20041396, 385, 3, 755-761, 2005.02, The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14. After the preSDHB-RPS14 polyprotein is transported into the mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14. Here we report that the general MPP (mitochondrial processing peptidase) plays an essential role in processing of the polyprotein. Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14. Moreover, the connector region was processed more rapidly than the presequence. When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP processing motif that has also been shown to be present in the N-terminal presequence. Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence. In addition, MPP preferentially recognized the unfolded structure of preSDHB-RPS14. In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before processing of the presequence..
39. Sakae Kitada, Tsuneo Uchiyama, Tomoyuki Funatsu, Yumiko Kitada, Tadashi Ogishima, Akio Ito, A protein from a parasitic microorganism, Rickettsia prowazekii, can cleave the signal sequences of proteins targeting mitochondria, Journal of bacteriology, 10.1128/JB.01261-06, 189, 3, 844-850, 2007.02, The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found to resemble the α and β subunits of mitochondrial processing peptidase (MPP), which cleaves the transport signal sequences of mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with β-MPP but barely contained any of the glycine-rich loop region characteristic of α-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover, RPP appeared to activate yeast β-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria..
40. Tomonori G. Nishino, Ken Kitano, Katsuhiko Kojima, Tadashi Ogishima, Akio Ito, Sakae Kitada, Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer, Journal of biochemistry, 10.1093/jb/mvm095, 141, 6, 889-895, 2007.06, Mitochondrial processing peptidase (MPP), which is composed of heterodimeric α-MPP and β-MPP subunits. It specifically recognizes mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4′-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser84 and -Lys156 of β-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 Å, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of α-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide..
41. Junko Taketoh, Junpei Mutoh, Tomoki Takeda, Tadashi Ogishima, Shuso Takeda, Yuji Ishii, Takumi Ishida, Hideyuki Yamada, Suppression of fetal testicular cytochrome P450 17 by maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin
A mechanism involving an initial effect on gonadotropin synthesis in the pituitary, Life Sciences, 10.1016/j.lfs.2006.12.029, 80, 14, 1259-1267, 2007.03, The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the fetal expression of testicular cytochrome P450 17 (CYP17), one of the enzymes necessary for sex steroid synthesis, was studied in Wistar rats. Fetal testicular CYP17 exhibited reduced mRNA and protein levels following exposure of the dams at gestational day 15 to 1 μg/kg TCDD. In support of this, CYP17 activity catalyzed by fetal testis homogenate was also reduced by maternal exposure to TCDD. The reduction in CYP17 expression seemed to be specific for fetal stages, because 7 day-old pups born from TCDD-treated dams did not exhibit any reduction in CYP17. In sharp contrast to the in vivo observations, TCDD failed to reduce CYP17 expression in cultured fetal testis, although CYP17 could be induced by activating cAMP-dependent signaling. To assess the role of pituitary luteinizing hormone (LH) on TCDD-induced reduction in fetal testicular CYP17, a further investigation was performed to examine whether the direct injection of LH into fetuses restores the altered CYP17 expression. The results showed that in utero injection of equine chorionic gonadotropin, an LH-mimicking hormone, completely abolishes the TCDD-produced reduction in fetal CYP17. However, neither the α- nor β-subunits of LH in cultured fetal pituitary was reduced by TCDD. These results suggest that 1) maternal exposure to TCDD impairs the expression of testicular CYP17 in a fetal stage-specific manner; 2) this effect is due, at least partially, to a TCDD-produced reduction in circulating LH; and 3) TCDD exerts such an effect by affecting the upstream mechanism regulating the pituitary synthesis of LH..
42. Kazuhisa Yoshino, Hiroshi Munakata, Osamu Kuge, Akio Ito, Tadashi Ogishima, Haeme-regulated degradation of δ-aminolevulinate synthase 1 in rat liver mitochondria, Journal of biochemistry, 10.1093/jb/mvm159, 142, 4, 453-458, 2007.10, Protein turnover, which occurs at various rates, is critical for the homeostasis of cellular protein levels. However, the proteolysis systems that determine the turnover rate of mitochondrial proteins are largely unknown. Delta-aminolevulinic acid synthase (ALAS) 1, a rate-limiting enzyme in the haeme biosynthesis, is one of the mitochondrial proteins that have a very short lifetime. In this study, to reveal the regulatory mechanisms for ALAS1 degradation, we examined the turnover rates of ALAS1 in rat liver under several conditions. In primary rat hepatocytes, the degradation of ALAS1 was stimulated by haeme, and suppressed by inhibition of haeme biosynthesis. Furthermore, the haeme-stimulated degradation of ALAS1 was observed in the isolated mitochondria. These results suggested that, in mitochondria, there exists an ALAS1 degradation system that is regulated by cellular haeme level and plays a crucial role in the regulation of haeme biosynthesis..
43. Dai Chida, Shinichi Nakagawa, So Nagai, Hiroshi Sagara, Harumi Katsumata, Toshihiro Imaki, Harumi Suzuki, Fumiko Mitani, Tadashi Ogishima, Chikara Shimizu, Hayato Kotaki, Shigeru Kakuta, Katsuko Sudo, Takao Koike, Mitsumasa Kubo, Yoichiro Iwakura, Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0706953104, 104, 46, 18205-18210, 2007.11, ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus-pituitary-adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R-MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD..
44. Tadashi Ogishima, Fumiko Mitani, Makoto Suematsu, Cytochrome P-45017α in β-cells of rat pancreas and its local steroidogenesis, Journal of Steroid Biochemistry and Molecular Biology, 10.1016/j.jsbmb.2008.04.008, 111, 1-2, 80-86, 2008.07, We have found cytochrome P-45017α in the islets of Langerhans of rat pancreas. Its existence coincided with that of insulin and demarcated those of glucagon and somatostatin, demonstrating the localization in β-cells. The enzyme has not only 17α-hydroxylase activity but also lyase one, which is a prerequisite for androgen biosynthesis. The pancreatic microsomes converted progesterone mainly to androstenedione with a minor production of 17α-hydroxyprogesterone. Due to a low activity of the built-in lyase, cytochrome P-45017α requires a sufficient electron-transfer from P-450 reductase or presence of an activator to promote the C-C bond cleavage. In β-cells, P-450 reductase was abundant and could efficiently transfer electrons to P-45017α. Actually, inhibition with anti-P-450 reductase or limitation of NADPH preferentially reduced the lyase activity. Androstenedione was accumulated when its further metabolism was suppressed. We also found localization of cytochrome P-450scc and 3β-hydroxysteroid dehydrogenase in β-cells. These results indicate that the immediate substrate for androgen formation, progesterone, is intracellularly produced and is converted mainly to androstenedione with support by an efficient electron supply from P-450 reductase. The product was supposed to be further metabolized to the reduced derivatives such as testosterone, 5α-androstanedione, and dihydrotestosterone, which would act as local steroids in the islets of Langerhans..
45. Koshiro Nishimoto, Ken Nakagawa, Dan Li, Takeo Kosaka, Mototsugu Oya, Shuji Mikami, Hirotaka Shibata, Hiroshi Itoh, Fumiko Mitani, Takeshi Yamazaki, Tadashi Ogishima, Makoto Suematsu, Kuniaki Mukai, Adrenocortical zonation in humans under normal and pathological conditions, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2009-2010, 95, 5, 2296-2305, 2010.05, Context: Aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1) catalyze the terminal steps for aldosterone and cortisol syntheses, respectively, thereby determining the functional differentiationofhumanadrenocortical cells. Little isknown, however, about how the cells expressing the enzymes are actually distributed in the adrenals under normal and pathological conditions. Objective: The objective of the study was to determine the localization of CYP11B2 and -B1 in human adrenal specimens by using developed antibodies capable of distinguishing the two enzymes from each other. Results: Under normal conditions, CYP11B2 was sporadically detected in the zona glomerulosa, whereas CYP11B1 was entirely detected in the zonae fasciculata-reticularis. Adrenocortical cells lacking both enzymes were observed in the outer cortical regions. In addition to conventional zonation, we found a variegated zonation consisting of a subcapsular cell cluster expressing CYP11B2, which we termed aldosterone-producing cell cluster,andaCYP11B1- expressing area. Aldosterone-producing adenomas differed in cell populations expressing CYP11B2 from one another, whereas CYP11B1-expressing and double-negative cells were also intermingled. Adenomas from patients with Cushing's syndrome expressed CYP11B1 entirely but not CYP11B2, resulting in atrophic nontumor glands. The nontumor portions of both types of adenomas bore frequently one or more aldosterone-producing cell clusters, which sustained CYP11B2 expression markedly under the conditions of the suppressed renin-angiotensin system. Conclusion: Immunohistochemistry of the human normal adrenal cortex for CYP11B2 and CYP11B1 revealed a variegated zonation with cell clusters constitutively expressing CYP11B2. This technique may provide a pathological confirmatory diagnosis of adrenocortical adenomas..
46. Akihiro Higuchi, Hiroyoshi Inoue, Tetsuya Kawakita, Tadashi Ogishima, Kazuo Tsubota, Selenium Compound Protects Corneal Epithelium against Oxidative Stress, PloS one, 10.1371/journal.pone.0045612, 7, 9, 2012.09, The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium..
47. T. Dekkers, M. Ter Meer, J. W.M. Lenders, A. R.M. Hermus, L. Schultze Kool, J. F. Langenhuijsen, K. Nishimoto, T. Ogishima, K. Mukai, E. A.B. Azizan, B. Tops, J. Deinum, B. Küsters, Adrenal nodularity and somatic mutations in primary aldosteronism
One node is the culprit?, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2013-4255, 99, 7, E1341-1351, 2014.07, Context: Somatic mutations in genes that influence cell entry of calcium have been identified in aldosterone-producing adenomas (APAs) of adrenal cortex in primary aldosteronism (PA). Many adrenal glands removed for suspicion of APA do not contain a single adenoma but nodular hyperplasia. Objective: The objective of the study was to assess multinodularity and phenotypic and genotypic characteristics of adrenals removed because of the suspicion of APAs. Design and Methods: We assessed the adrenals of 53 PA patients for histopathological characteristics and immunohistochemistry for aldosterone (P450C18) and cortisol (P450C11) synthesis and for KCNJ5, ATP1A1, ATP2B3, and CACNA1D mutations in microdissected nodi. Results: Glands contained a solitary adenoma in 43% and nodular hyperplasia in 53% of cases. Most adrenal glands contained only one nodule positive for P450C18 expression, with all other nodules negative. KCNJ5 mutations were present in 22 of 53 adrenals (13 adenoma and nine multinodular adrenals). An ATP1A1 and a CACNA1D mutation were found in one multinodular gland each and an ATP2B3 mutation in five APA-containing glands. Mutations were always located in the P450C18-positive nodule. In one gland two nodules containing two different KCNJ5 mutations were present. Zona fasciculata-like cells were more typical for KCNJ5 mutation-containing nodules and zona glomerulosa-like cells for the other three genes. Conclusions: Somatic mutations in KCNJ5, ATP1A1, or CACNA1D genes are not limited to APAs but are also found in the more frequent multinodular adrenals. In multinodular glands, only one nodule harbors a mutation. This suggests that the occurrence of a mutation and nodule formation are independent processes. The implications for clinical management remain to be determined..
48. Cristia Volpe, Anders Höög, Tadashi Ogishima, Kuniaki Mukai, Ming Lu, Marja Thorén, Bertil Hamberger, Immunohistochemistry improves histopathologic diagnosis in primary aldosteronism, Journal of Clinical Pathology, 10.1136/jclinpath-2012-201287, 66, 4, 351-354, 2013.04, Background In primary aldosteronism (PA) the main source of aldosterone hypersecretion is an aldosteroneproducing adenoma (APA) or a bilateral hyperplasia. Histopathology of the adrenal gland from patients with PA has been difficult, as there are no morphological criteria to ascertain which are the cells that produce aldosterone. We therefore applied new specific antibodies to explore which cells in the adrenal contain the enzymes for aldosterone and cortisol production, respectively. Methods Adrenals from 24 patients with PA were studied. After routine preparation, consecutive sections were stained with antibodies for CYP11B1 (cortisol) and CYP11B2 (aldosterone) enzymes. Results APA had a strong immunoreactivity for CYB11B2. In adrenals from seven patients, we found no APA, but several nodules with strong CYB11B2 immunoreactivity, indicating aldosterone-producing nodular hyperplasia. Conclusions Immunohistochemistry of adrenal steroidogenic enzymes provides novel diagnostic information. This may become an important part of routine histopathology, and contribute to improved clinical management in PA..
49. Tadashi Ogishima, Andrea Vecchiola, Carlos F Lagos, Cristóbal A Fuentes, Fidel Allende, Carmen Campino, Carolina Valdivia, Alejandra Tapia-Castillo, Kuniaki Mukai, Gareth Owen, Sandra Solari, Cristian A Carvajal, Carlos E Fardella, Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro, Reproductive Biology and Endocrinology, 10.1186/1477-7827-11-76, 76, 1-11, 2013, 11:76, 2013.08, Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE..
50. Tadashi Ogishima, Anders Höög, A, Mukai, K, Lu, M., Thorén, M, Hamberger, B, Immunohistochemistry improves histopathologic diagnosis in primary aldosteronism, J Clin Pathol., 66, 351-354, 2013.01.
51. Tadashi Ogishima, Higuchi, A, Inoue, H, Kawakita, T, Tsubota, K, Selenium Compound Protects Corneal Epithelium against Oxidative Stress , PLoS ONE, 7, 9, 45612, 2012.09, The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium..
52. Koshiro Nishimoto, Minae Koga, Tsugio Seki, Kenji Oki, Elise P. Gomez-Sanchez, Celso E. Gomez-Sanchez, Mitsuhide Naruse, Tomokazu Sakaguchi, Shinya Morita, Takeo Kosaka, Mototsugu Oya, Tadashi Ogishima, Masanori Yasuda, Makoto Suematsu, Yasuaki Kabe, Masao Omura, Tetsuo Nishikawa, Kuniaki Mukai, Immunohistochemistry of aldosterone synthase leads the way to the pathogenesis of primary aldosteronism, Molecular and Cellular Endocrinology, 10.1016/j.mce.2016.10.014, 441, 124-133, 2017.04, Our group previously purified human and rat aldosterone synthase (CYP11B2 and Cyp11b2, respectively) from their adrenals and verified that it is distinct from steroid 11β-hydroxylase (CYP11B1 or Cyp11b1), the cortisol- or corticosterone-synthesizing enzyme. We now describe their distributions immunohistochemically with specific antibodies. In rats, there is layered functional zonation with the Cyp11b2-positive zona glomerulosa (ZG), Cyp11b1-positive zona fasciculata (ZF), and Cyp11b2/Cyp11b1-negative undifferentiated zone between the ZG and ZF. In human infants and children (<12 years old), the functional zonation is similar to that in rats. In adults, the adrenal cortex remodels and subcapsular aldosterone-producing cell clusters (APCCs) replace the continuous ZG layer. We recently reported possible APCC-to-APA transitional lesions (pAATLs) in 2 cases of unilateral multiple adrenocortical micro-nodules. In this review, we present 4 additional cases of primary aldosteronism, from which the extracted adrenals contain pAATLs, with results of next generation sequencing for these lesions. Immunohistochemistry for CYP11B2 and CYP11B1 has become an important tool for the diagnosis of and research on adrenocortical pathological conditions and suggests that APCCs may be the origin of aldosterone-producing adenoma..
53. Nishimoto,K., Nakagawa, K., Li, D., Kosaka, T., Oya, M., Mikami, S., Shibata, H., Itoh, H., Mitani, F., Yamazaki, T., Ogishima , T., Suematsu, M., & Mukai, M, Adrenocortical Zonation in Humans under Normal and Pathologic Conditions., J. Clin. Endocrinol. Metab, 95, 2296 - 2305, 2010.08.
54. Tadashi Ogishima, VARIEGATED ZONATION IN HUMAN ADRENAL CORTEX, 2010.05.
55. Ogishima, T., Mitani, F., & Suematsu, M. , Cytochrome P-45017alpha in beta-cells of Rat Pancreas and its Local Steroidogenesis.
. , J. Steroid Biochem. Mol. Biol. , 110, in press
, 2008.07.
56. Yoshino, K., Munakata, H., Kuge, O., Ito, A., & Ogishima, T. , Heme-regulated Degradation of delta-Aminolevulinate Synthase 1 in Rat Liver Mitochondria.
, J. Biochem, 142, 453-467, 2007.09.
57. Nishino, T., Kitano, K., Kojima, K., Ogishima, T., Ito, A., and Kitada, S. , Spatial orientation of mitochondrial processing peptidase and a preproteinrevealed by fluorescencer esonance energy transfer., J. Biochem., 2007.06.
58. Taketoh, J., Mutoh, J., Takeda, T., Ogishima, T., Takeda, S., Ishii, Y., Ishida, T., & Yamada, H. , Suppression of fetal testicular cytochrome P45017 by maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: A mechanis involving an initial effect on gonadotropin synthesis in the pituitary., Life Sci., 80, 1259-1267, 2007.02.
59. Kitada, S., Uchiyama, T., Funatsu, T., Kitada, Y., Ogishima, T., & Ito, A., A Protein from a Parasitic Microorganism Rickettsiae can Cleave the Signal Sequences of Proteins Targeting Mitochondria., J. Bacteriol., 189, 844-850, 2007.02.
60. Fumiko Mitani, Tadashi Ogishima, Kuniaki Mukai and Makoto Suematsu, Ascorbate Stimulates Monooxygenase-dependent Steroidogenesis in Adrenal Zona Glomerulosa., Biochem. Biophys. Res. Commun., 10.1016/j.bbrc.2005.08.156, 338, 1, 483-490, 338, Issue 1, 2005.12.
61. Ogishima, T., Kinoshita, J., Mitani, F., Suematsu, M., & Ito, A., Identification of Outer Mitochondrial Membrane Cytochrome b5 as a Modulator for Androgen Synthesis in Leydig Cells., Journal of Biological Chemistry, 10.1074/jbc.M301698200, 278, 23, 21204-21211, 278, 21202-21211, 2003, 2003.07.
62. Kojima, K., Yamazaki, E., Kitada, S., Ogishima, T., & Ito, A., Recognition of Mitochondrial Protein Precursor Lacking Arginine at Position -2 by Mitochondrial Processing Peptidase., Journal of Biochemistry, 130, 4, 497-502, 130, 497-502, 2001, 2001.03.
63. Nagao,Y., Kitada, S., Kojima, K., Toh, H., Kuhara, S., Ogishima, T., & Ito, A., Possible Function of Glycine-rich Region in Mitochondrial Processing Peptidase a-subunit., Journal of Biological Chemistry, 275, 34552-34556, 2000, 2000.10.
64. Kojima, K., Kitada, S., Ogishima, T., & Ito, A.:, A Proposed Common Structure of Substrates Bound to Mitochondrial Processing Peptidase., Journal of Biological Chemistry, 10.1074/jbc.M003111200, 276, 3, 2115-2121, 276, 2115-2121, 2001, 2001.07.
65. Ogishima, T., Niidome, T., Shimokata, K., Kitada, S., & Ito, A.:, Analysis of Elements in the Substrate Required for Processing by Mitochondrial Processing Peptidase., J. Biol. Chem., 270, 51, 30322-30326, 270, 30322-30326, 1995.09.
66. Kitada, S., Shimokata, K., Ogishima, T., & Ito, A., Glutamate Residues Required for Substrate Binding and Cleavage Activity in Mitochondrial Processing Peptidase., Journal of Biological Chemistry, 10.1074/jbc.273.49.32547, 273, 49, 32547-32553, 273, 32547-32553, 1998, 1998.10.