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Y Ishimura

Publications and source records attributed to Y Ishimura.

At least 127 records · Page 7Linked to original sources
PubMed1990

Molecular cloning of a cDNA encoding aldosterone synthase cytochrome P-450 in rat adrenal cortex.

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.

Adrenal Cortex↗
PubMed1990

Localization and release of immunoreactive vasoactive intestinal polypeptide in bovine adrenal medulla.

The concentration of immunoreactive (IR) vasoactive intestinal polypeptide (VIP) in extracts from bovine adrenal medulla was 29.9 +/- 7.2 pmol/g wet wt., which was about 100 times that of IR neurotensin and 30 times that of IR somatostatin. Chromatographic analysis showed that most of the IR-VIP was the same molecular size as synthetic VIP(1-28). On retrograde perfusion of isolated bovine adrenal gland, release of VIP with catecholamine (CA) was marked on stimulation with high K+, but slight on stimulation with acetylcholine, which induced marked release of CA. These results suggest that most of the VIP is localized not in CA storing granules in chromaffin cells, but in other intraadrenal neuronal components. In immunohistochemical studies, IR VIP fibers with large varicosities were observed around the vessels in the adrenal medulla.

Acetylcholine↗
PubMed1990

Adenosine receptor-mediated accumulation of cyclic AMP-induced T-lymphocyte death through internucleosomal DNA cleavage.

Incubation of mouse thymocytes with adenosine and its receptor site agonist, 2-chloroadenosine, induced a pronounced increase in the intracellular cAMP level and resulted in internucleosomal DNA fragmentation followed by cell lysis. Similar DNA fragmentation was induced in peripheral T-lymphocytes prepared from spleen cells but to a lesser extent than in the thymocytes. The DNA fragmentation in both thymocytes and splenic T-lymphocytes was prevented by the addition of actinomycin D and cycloheximide, indicating that this process required mRNA and protein synthesis. The inhibition was accompanied by a reduction in cell lysis as judged by the release of lactate dehydrogenase into the medium. Involvement of cAMP accumulation in inducing DNA fragmentation was supported by the results of experiments with cAMP analogs such as dibutyryl cAMP and 8-bromo-cAMP, and cAMP level-raising drugs including forskolin, cholera toxin, and isobutylmethyxanthine. The latter agents induced pronounced or sustained elevation of cellular cAMP followed by internucleosomal DNA cleavage in T-lymphocytes. These results suggest that adenosine receptor-mediated accumulation of cyclic AMP regulates T-lymphocyte death through inducement of internucleosomal DNA cleavage.

1-Methyl-3-isobutylxanthine↗
PubMed1990

CD4+CD8+ thymocytes are susceptible to DNA fragmentation induced by phorbol ester, calcium ionophore and anti-CD3 antibody.

Stimulation of murine thymocytes with phorbol ester or calcium ionophore for 18-24 h resulted in 70%-80% fragmentation of DNA into 180-200-bp multiples, followed by cell death. Experiments with fractionated subpopulations by panning or flow cytometry revealed that DNA fragmentation was selectively observed in CD4+CD8+ cells and in a portion of CD4-CD8+ cells. To investigate whether DNA cleavage is also inducible via antigen-specific receptors, thymocytes were incubated in wells precoated with anti-CD3 antibody. An approximately 20% increase of DNA fragmentation was constantly observed when unseparated thymocytes were stimulated with anti-CD3 antibody. In this anti-CD3-induced DNA degradation, CD4+CD8+ cells are probably the target cells, since (a) fetal thymocytes at day 18 of gestation were found vulnerable to anti-CD3-induced DNA cleavage and (b) flow cytometry analysis of viable cells recovered after cultivation in the anti-CD3-coated wells revealed that CD4+CD8+ cells were preferentially decreased. Further experiments with purified CD4+CD8+ cells, however, could not define a clear-cut increase of DNA fragmentation when isolated CD4+CD8+ cells were stimulated with anti-CD3 antibody. Addition of interleukin (IL) 1, IL 2, IL 3, IL 4 or interferon-gamma to the CD4+CD8+ cell cultures failed to yield a DNA cleavage similar to that of unseparated thymocytes.

Animals↗
PubMed1990

Conversion of cholesterol to pregnenolone mobilizes cytochrome P-450 in the inner membrane of adrenocortical mitochondria: protein rotation study.

Rotation of cytochrome P-450 was examined in bovine adrenocortical mitochondria before and after an enzymatic transformation of cholesterol into pregnenolone by cytochrome P-450scc in the presence of malate. Rotational diffusion was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate substrate-dependent intermolecular interactions of cytochrome P-450 with other redox components. Rotational mobility of cytochrome P-450 was significantly dependent on the decrease in cholesterol content by side chain cleavage reaction catalyzed by cytochrome P-450scc. In a typical experiment, the observed value for the normalized time-independent anisotropy r(infinity)/r(0) was decreased from 0.78 in control mitochondria to 0.60 after conversion of 21% of cholesterol to pregnenolone, while no significant change was observed for the average rotational relaxation time phi of about 700 microseconds. Significantly high values of r(infinity)/r(0) = 0.78 and 0.60 imply co-existence of mobile and immobile populations of cytochrome P-450. Since we observed that the heme angle tilted 55 degrees from membrane plane, 22% (control mitochondria) and 40% (after conversion of cholesterol to pregnenolone) of cytochrome P-450 in mitochondria are calculated to be mobile in the preparation. The significant mobilization of cytochrome P-450scc molecules caused by the conversion of cholesterol to pregnenolone is likely due to changes in protein-protein interactions with its redox partners, since the lipid fluidity was kept unchanged by the cholesterol depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1989

Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters.

Calcium ionophore, A23187, is known to be a comitogen, but it activates a suicide process characterized by DNA fragmentation at linker regions in mouse immature thymocytes. It did not induce DNA fragmentation in T lymphocytes prepared from lymph node and spleen cells. Induction of DNA fragmentation by A23187 depends on protein phosphorylation and synthesis of mRNA and protein, because an inhibitor of protein kinase, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7), actinomycin D, and cycloheximide, respectively, inhibits the DNA fragmentation and cell death. Studies adding the inhibitors at various times show that protein phosphorylation and mRNA synthesis occur within a few hours after incubation with A23187 followed by the protein synthesis responsible for inducing DNA fragmentation. Phorbol esters, 12-O-tetradecanoyl 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBD), which are capable of activating protein kinase C, also induced similar DNA fragmentation in immature thymocytes, followed by cell death. PBD committed the suicide process after 6 h of incubation, because the DNA fragmentation above the control level was not induced when PDB was removed from the medium before 6 h of incubation. A23187 or a phorbol ester alone induced DNA fragmentation followed by cell death, whereas the addition of TPA at low concentration inhibited the DNA fragmentation induced by A23187 accompanied with an increase in DNA synthesis. The result suggests that TPA switched a suicide process induced by A23187 to an opposite process: stimulation of DNA synthesis. Physiologic factors and mechanisms which regulate cell proliferation and death in the thymus are not known at present, but the signals by protein kinases and calcium ions may regulate both cell proliferation and death, independently, synergistically or antagonistically.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex.

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-45011 beta 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 11 beta- 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 11 beta- 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.

Adrenal Cortex↗
PubMed1989

Superoxide anion is the initial product in the hydrogen peroxide formation catalyzed by NADPH oxidase in porcine thyroid plasma membrane.

The plasma membrane fraction from porcine thyroid is known to exhibit an NADPH-dependent production of hydrogen peroxide (H2O2), which is utilized for the oxidative biosynthesis of thyroid hormones catalyzed by thyroid peroxidase. The H2O2 formation is cyanide-insensitive, ATP-activatable, and Ca2+-dependent (Nakamura, Y., Ogihara, S., and Ohtaki, S. (1987) J. Biochem. (Tokyo) 102, 1121-1132). It remains unknown, however, whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of superoxide anion (O2-). We therefore attempted to analyze the mechanism of H2O2 formation by utilizing a new method for the simultaneous measurement of O2- and H2O2, in which diacetyldeuteroheme-substituted horseradish peroxidase was employed as the trapping agent for both oxygen metabolites. When NADPH was incubated with the membrane fraction in the presence of the heme-substituted peroxidase, a massive O2 consumption was observed together with the formation of compound III, and O2- adduct of the peroxidase. The amounts of compound III formed and O2 consumed were stoichiometric with each other, while formation of compound II, an indicative of H2O2, was not observed during the reaction. On the other hand, when an excess amount of superoxide dismutase was included in the reaction mixture, compound II was produced with complete suppression of the compound III formation. NADH minimally supported both O2 consumption and formation of compound III or II. These results indicate that the NADPH oxidase in the plasma membrane of thyroid produces O2- as the primary metabolite of O2 and hence that H2O2 required for the thyroid hormone synthesis provided through the dismutation of O2-.

Animals↗
PubMed1989

Distal His----Arg mutation in bovine myoglobin results in a ligand binding site similar to the abnormal beta site of hemoglobin Zurich (beta 63 His----Arg).

Carbon monoxide binding to a myoglobin mutant with distal arginine in place of histidine has been examined. The mutant is derived from a cDNA clone for Mb mRNA from fetal bovine skeletal muscle. The mutation only slightly perturbs visible/Soret spectra whereas the infrared spectrum of liganded CO is greatly modified to become nearly identical to Hb Zurich beta-subunit spectrum. The mutant IR spectra differ substantially from spectra of wild-type MbCO and normal HbCO beta-subunit. For both the Mb and the Hb the distal His----Arg mutation increases the affinity for CO and reduces the number of observed conformers. These results demonstrate that this mutation greatly reduces the differences between Mb and Hb in the structure and properties of its ligand binding sites.

Animals↗
PubMed1989

Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation.

Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.

Alanine↗
PubMed1989

Expression of bovine myoglobin cDNA as a functionally active holoprotein in Saccharomyces cerevisiae.

We isolated a cDNA clone for myoglobin mRNA from fetal bovine skeletal muscle using a DNA fragment of human myoglobin exon 2 as a probe. The complete coding sequence of myoglobin as well as the 3'- and part of the 5'-nontranslatable sequences (546 and 66 basepairs, respectively) were determined. The amino acid sequence predicted from the nucleotide sequence was in agreement with that determined in the purified protein from adult bovine cardiac muscle (Han, K. K., Dautrevaux, M., Chaila, X., & Biserte, G. [1970] Eur. J. Biochem. 16, 465-471), except for eight amino acid residues: Val-99----Ile,Ile-101----Val, Asn-122----Asp, Ala-124----Gly, Gly-129----Ala, Ala-142----Met, Glu-144----Ala, and Lys-145----Gln. When the myoglobin cDNA was expressed in Saccharomyces cerevisiae under the control of the GAL7 promoter, myoglobin was synthesized as a functionally active holoprotein which bound molecular oxygen reversibly. The amount of myoglobin reached nearly 1% of the total extractable protein in the yeast. N-terminal sequence analysis of the produced myoglobin revealed a glycine residue at the terminus, indicating that as in native muscle the N-terminal Met was removed in yeast by processing.

Amino Acid Sequence↗
PubMed1989

Isolation of two distinct cytochromes P-45011 beta with aldosterone synthase activity from bovine adrenocortical mitochondria.

Two distinct forms of cytochrome P-45011 beta, 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-450(11) beta (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-450(11) beta were capable of catalyzing aldosterone synthesis as well as the 11 beta- and 18-hydroxylation of 11-deoxycorticosterone. Thus, at least two distinct cytochrome P-450(11) beta species exist in the adrenal cortex and participate in steroidogenesis.

Adrenal Cortex↗
PubMed1989

12-O-tetradecanoylphorbol 13-acetate induces DNA cleavage at linker regions in mouse thymocytes.

A phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), induced the cleavage of nuclear DNA at linker regions in cultured mouse thymocytes. Similar DNA fragmentation was induced by 1-oleoyl-2-acetyl-glycerol, a synthetic diacylglycerol, but not by 4 alpha-phorbol-12,13 didecanoate. The DNA fragmentation was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine dihydrochloride, an inhibitor of protein kinase C, as well as actinomycin D and cycloheximide. It appears that TPA induces DNA cleavage through activation of protein kinase C and synthesis of yet unidentified protein(s). That the inhibition of DNA fragmentation was accompanied by a reduction in cell lysis suggests a causal relationship between DNA fragmentation and cell death.

Animals↗
PubMed1988

Activation by saturated and monounsaturated fatty acids of the O2- -generating system in a cell-free preparation from neutrophils.

Saturated and monounsaturated fatty acids with appropriate chain length such as laurate and oleate activated an O2- -generating enzyme system in a cell-free preparation from porcine neutrophils. The activated preparation catalyzed a stoichiometric conversion of O2 to O2- by utilizing NADPH as the electron donor. The preparation contained both membrane and soluble fractions and, upon separation into subfractions, the O2- -generating activity resided exclusively in the membrane fraction. Polyunsaturated fatty acids including arachidonate also activated the system, but they concurrently stimulated NADPH-independent O2 consuming reactions which yield neither O2- nor H2O2. The amount of such a non-O2- -producing O2 consumption often reached twice as much as that of O2- production. For the activation of the O2- -generating system in the membrane, the presence of the soluble fraction was essential. However, the soluble fraction was no longer effective when once used for the activation, suggesting that the effective component(s) in the fraction was consumed or translocated to the membrane during the activation. When the activated membrane was incubated with delipidated albumin, the activity was lost with concomitant decreases in the amount of membrane-associated fatty acids. The lost activity was restored by the replenishment of the fatty acid in the presence of a fresh soluble fraction. We also found that Ca2+ augmented a non-O2- -producing O2 consumption in the cell-free preparation by unsaturated fatty acids and interfered with the activation of the O2- -generating system, especially that by saturated fatty acids.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1988

Inhibition by the antilipogenic antibiotic cerulenin of thrombin-induced activation of human platelets.

Upon incubation with an antibiotic cerulenin, human platelets lost their abilities to aggregate and to release serotonin or ATP in response to various stimuli such as thrombin, ADP, collagen and platelet activating factor. The loss of activities was dependent on both incubation time and cerulenin concentrations. As judged by 14C-serotonin release, concentrations of cerulenin required for the half-maximal inhibition of thrombin-induced activation were 5-10 and 70 micrograms/ml in the incubation for 120 min at 37 degrees C for washed platelets and those in platelet rich plasma, respectively. The cerulenin treatment also resulted in a significant inhibition of 14C-acetate incorporation into the lipid fraction of platelets, suggesting that de novo synthesis of fatty acids was inhibited by the treatment. No release of lactate dehydrogenase activity nor morphological changes in platelet structure was detected upon cerulenin treatment. When effects of cerulenin on intracellular Ca2+ concentration were examined, mobilization of intracellular Ca2+ by thrombin was significantly depressed in the cerulenin-treated platelets as judged by Fura2, Quin2 or chlortetracycline fluorescence. Since the influx of external Ca2+ is not essential to the thrombin-induced platelet activation (Rink, T. J. et al. FEBS Lett. 148 21-26, 1982), the results suggest that cerulenin-treatments affect the platelet function through the inhibition of intracellular Ca2+ mobilization.

Adenosine Triphosphate↗
PubMed1988

Rotation and protein-protein interactions of cytochrome P-450 in the inner membrane of adrenocortical mitochondria.

Rotational diffusion of the total cytochrome P-450 (P-450scc plus P-45011 beta) in bovine adrenocortical mitochondria was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the hemo.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions of cytochrome P-450 with other membrane proteins. The absorption anisotropy decayed within 1 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the presence and absence of deoxycorticosterone (DOC), a substrate for cytochrome P-45011 beta. The observed value for the normalized time-independent anisotropy r(infinity)/r(0) and the average rotational relaxation time phi are r(infinity)/r(0) = 0.88 and phi = 233 microseconds when DOC is absent, and r(infinity)/r(0) = 0.65 and phi = 350 microseconds when DOC is present. Judging from the phi value, rotating P-450 is not a monomeric molecule, but would be a small microaggregate with an average diameter of about 120 A. A significantly high value of r(infinity)/r(0) implies co-existence immobile populations of cytochrome P-450. Based on the assumption that the heme angle tilts 55 degrees from the membrane plane (Gut et al. (1983) J. Biol. Chem. 258, 8588-8594), 65% (when DOC is present) or 88% (when DOC is absent) of cytochrome P-450 in mitochondria is immobilized within the experimental time range of 2 ms due to the presence of immobile protein microaggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗