Biomedical subjects

M Negishi

Publications and source records attributed to M Negishi.

At least 253 records · Page 14Linked to original sources

PubMed1992

[Clinical varicella-zoster virus reinfection observed in two advanced-age persons].

We observed two mild varicella cases of an advanced-age man and woman, although varicella in the adult generally presents a more severe course than in the child. The cases had been exposed to his great-grandchild and her grandchild with varicella in their home. They did not experience neuralgia as is typical from herpes zoster. Serological examinations revealed that they had high IgG-antibodies (Ab) against varicella-zoster virus (VZV), but low anti-VZV IgM-Ab. They were also found to have a high level of Ab against VZV soluble antigens (Ag) which are generally low in primary VZV infection. Immunoblotting analysis of anti-VZV IgG-Abs using sera from case 1 or 2 showed that their reaction patterns resembled those of sera children who contracted mild varicella after immunization with varicella vaccine and a herpes zoster-patient rather than those of sera from an immunocompetent adult with varicella. These results of serological examinations and their clinical course indicated that the cases had contracted varicella in the past and were again exposed to VZV resulting in clinically overt but mild varicella.

Aged↗

PubMed1992

[A case of perinatal transmission of human immunodeficiency virus--diagnosis of HIV infection by culture and PCR].

A child born from a mother with HIV infection was reported. We have followed the patient from 3 month old of age. Her HIV antibody disappeared at 7 month by EIA, and at 10 month by WR. But we confirmed her HIV infection by PCR and HIV culture, repeatedly at those times, PCR and HIV culture are thought to be necessary for sero-negative infants born from mothers positive for HIV antibody.

Blotting, Western↗

PubMed1992

Inflammatory cytokines and enzymes in synovial fluid of patients with rheumatoid arthritis and other arthritides.

Cytokines and lysosomal enzymes, which are produced by inflammatory cells, play a role in inflammation. We have found that synovial fluid (SF) in rheumatoid and septic arthritis contained a large number of white blood cells (WBCs) and high levels of cytokines and enzymes, while in contrast the SF of osteoarthritis and traumatic arthritis did not contain significant amounts. Measurements of WBCs, cytokines and enzymes in SF are useful for evaluating clinical disease activity. Assays for WBCs and enzymes are simple and rapid when compared to those for cytokines.

Adult↗

PubMed1992

Interleukin 1 pretreatment reduces the insulin response to glucose in chronic sucrose-fed rats.

The present studies were undertaken to determine whether interleukin 1 beta ([IL-1] 1.0 micrograms/kg, ip) pretreatment for 3 days impairs the adaptive response to sucrose feeding in rats. One week after the last IL-1 injection, when no differences in plasma glucose and serum immunoreactive insulin (IRI) levels were observed, sucrose feeding was started. Sucrose feeding for 4 weeks did not affect basal glucose levels, whereas basal IRI levels were increased in sucrose-fed rats without IL-1 pretreatment. Eight weeks later, plasma glucose levels were increased before and at 15 min after intravenous bolus of 0.5 g/kg of glucose in sucrose-fed rats with IL-1 pretreatment. Only in IL-1-treated sucrose-fed rats were basal and glucose-stimulated IRI levels significantly reduced, compared with those levels in sucrose-fed vehicle-treated rats. IL-1 decreased pancreatic IRI contents at 1 and 9 weeks after the injection. These data suggest that pancreatic damage by IL-1 attenuated insulin response to glucose stimulation after long-term sucrose feeding.

Analysis of Variance↗

PubMed1992

[Lung injury associated with bucillamine therapy].

We have reported previously a first case of interstitial pneumonitis in rheumatoid arthritis treated with bucillamine in 1989. Thereafter, a dozen cases with lung injury associated with bucillamine therapy have been reported. To explore the similarities among these patients, we tried to carry out a survey concerning the lung injuries in these cases in the present study. We obtained the following results. (1) Patients with lung injury consisted of 7 females and 3 males, aged 65 years on average (59-72). (2) The total amount of bucillamine used was 20-40 g on average (5.1-144 g) during approximately 100 days. (3) Serum rheumatoid factor was positive in 9 of 10 cases. (4) HLA-DR4 was significantly (p less than 0.01) detected in all cases of lung injury associated with bucillamine. (5) Interestingly the effect of bucillamine on rheumatoid disease activity was excellent in most cases. (6) Drug lymphocyte stimulation test (DLST) using bucillamine was performed in 6 cases. Positive DLST was found in 3 of 6 cases. These results suggest that cell-mediated hypersensitivity reaction (Coombs & Gell type IV) may contribute to the development of lung injury associated with bucillamine therapy.

Aged↗

PubMed1991

Mouse pulmonary cytochrome P-450 naphthalene hydroxylase: cDNA cloning, sequence, and expression in Saccharomyces cerevisiae.

We have isolated a cDNA clone, Nah-2, encoding the cytochrome P-450Nah (naphthalene hydroxylase) from a mouse lung lambda ZAP cDNA library using anti-cytochrome P-450Nah IgG as a probe. This same antibody selectively blocked [Nagata, K., Martin, B.M., Gillette, J.R., & Sasame, H.A. (1990) Drug Metab. Dispos. 18, 557-564] the cytochrome P-450 in mouse lung microsomes that catalyzed the conversion of naphthalene to (1R,2S)-naphthalene 1,2-oxide, which has been postulated as a causative agent in the naphthalene-induced tissue-specific necrosis of Clara cells in mouse lung. The toxic effect is seen in mouse and not in rat. The cDNA encodes a polypeptide of 491 amino acids with a molecular mass of 50 kDa. Northern blot analysis with an Nah-2-specific probe revealed that the mRNA is expressed in a species- and tissue-specific manner, present only in mouse lung and liver and not in that of rat. The mRNA encoding Nah-2 is constitutively expressed and is not induced by either phenobarbital, pyrazole, pregnenolone 16 alpha-carbonitrile, or 3-methylcholanthrene. Comparative amino acid sequence analyses with other documented members of the P-450 gene superfamily revealed that this encoded protein is in the IIF subfamily. To analyze its substrate specificity, the cDNA was inserted into the vector, pAAH5, and expressed in the Saccharomyces cerevisiae strain, AH22. The presence of cytochrome P-450Nah in the microsomes isolated from transformed cells and analyzed by Western blot was confirmed by immunocomplexing product with anti-cytochrome P450Nah IgG. Furthermore, activity toward naphthalene in the microsomes from the transformed cells established that this clone encodes a naphthalene hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Structural function of residue-209 in coumarin 7-hydroxylase (P450coh). Enzyme-kinetic studies and site-directed mutagenesis.

Residue-209 plays a critical role in determining the substrate and product specificity of cytochrome P450coh. In order to investigate further the structural function of residue-209 in coumarin 7-hydroxylase reaction, we measured the enzyme-kinetic properties of wild-type P450coh and its mutants in which residue-209 was substituted with various amino acids. In general, the Km and Vmax values for coumarin increased as the size of residue-209 became smaller and Vmax values decreased. The size of residue-209, therefore, was a principle factor determining Km, Kd, and Vmax values of P450coh. Although the polarity and charge also increased the Km value consistently, they altered Vmax and Kd values in an irregular manner. The substitution of serine for residue-209 increased the Vmax, while the substitution of lysine decreased it. Coumarin 7-hydroxylase activity was inhibited weakly by indan, but competitively and strongly by 2-coumaranone. Moreover, Ki values for the inhibitor were similar to Km values of the corresponding, mutated P450s. The results indicate, therefore, that residue-209 is localized in a proposed substrate-binding sequence 1 which binds to the 2-keto group of coumarin and directs its 7-position toward the sixth ligand of heme. Consequently, the identity of residue-209 determines not only the binding of coumarin in P450coh, but also the other reaction step(s) of coumarin 7-hydroxylation.

Amino Acids↗

PubMed1991

Posttranscriptional regulation of coumarin 7-hydroxylase induction by xenobiotics in mouse liver: mRNA stabilization by pyrazole.

The induction mechanism by pyrazole or phenobarbital of coumarin 7-hydroxylase (cytochrome P450coh) was investigated in DBA/2J male mice. The P450coh mRNA in the pyrazole-induced mice was increased gradually to a 20-fold higher level within 48 h, yet transcription of the P450coh gene was not affected. The half-life of P450coh mRNA, on the other hand, was at least 4-fold longer in the pyrazole-induced DBA/2J (approximately 6.0 h) than in control DBA/2J (approximately 1.5 h) male mice. The stabilization of P450coh mRNA, therefore, is the primary mechanism for the induction by pyrazole of coumarin 7-hydroxylase. Phenobarbital, on the other hand, regulates the induction either translationally or posttranslationally. This drug affected neither the P450coh mRNA nor the P450coh gene's transcription levels in the DBA/2J male mice, although Western blots showed approximately a 3-fold increase of the P450coh protein in the liver microsomes of the drug-treated mice. The results indicate, therefore, that both phenobarbital and pyrazole regulate the P450coh induction posttranscriptionally; the former inducer enhances the translational efficiency of P450coh mRNA or alters the degradation rate of P450coh aproprotein, while the latter stabilizes P450coh mRNA.

Animals↗

PubMed1991

Induction of a 31,000-dalton stress protein by prostaglandins D2 and J2 in porcine aortic endothelial cells.

Prostaglandin (PG) D2 and PGJ2 stimulated porcine aortic endothelial cells to synthesize a 31,000-dalton protein (termed p31) in a time- and concentration-dependent manner. The induction of p31 synthesis was specific for PGD2, PGJ2 and PGA1 among the various PGs tested. p31 was also synthesized in response to the thiol-reactive agent diethylmaleate and heavy metal sodium arsenite but not to high temperature treatment, platelet-derived growth factor, and 12-O-tetradecanoylphorbol 13-acetate. Using two-dimensional polyacrylamide gel electrophoresis, p31 induced by PGJ2 had an isoelectric point of 5.4, which overlapped exactly with that induced by by arsenite. These results taken together indicate that p31 represents one of the stress proteins whose expression is regulated primarily by thio-active compounds but not by hyperthermia. Furthermore, it was induced by PGD2 and PGJ2 in rat capillary endothelial cells, rat skin fibroblasts, and rat hepatocytes. The data obtained from this study suggest that p31 induced by PGD2 and PGJ2 may play a role in the metabolic regulation of many mammalian cells.

Animals↗

PubMed1991

Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells.

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.

Adenosine Diphosphate↗

PubMed1991

Specific binding of glycyrrhetinic acid to the rat liver membrane.

Glycyrrhetinic acid bound specifically to a particulate fraction of rat liver. The binding was dependent on time, temperature and pH, equilibrium being reached after 10 min at 37 degrees C. The equilibrium dissociation constant and the maximal concentration of the binding site, as determined by Scatchard plot analysis, were 31 nM and 43 pmol/mg protein, respectively, indicating a single binding site entity. The binding site was highly specific for glycyrrhetinic acid, glycyrrhizin, various steroids, various fatty acids and retinoids showing no or only very low affinity. The binding was inhibited by boiling or treatment with trypsin or phospholipases. The specific activity of glycyrrhetinic acid binding was the highest in the liver, followed by in the kidney. The results suggest that glycyrrhetinic acid plays a significant role in the rat liver through its specific binding protein.

Animals↗

PubMed1991

Desensitization by glycyrrhetinic acid of other stimulatory substance-induced increases in intracellular calcium in a variety of cell types.

The effects of glycyrrhizin and its aglycone, glycyrrhetinic acid, on the cytoplasmic free calcium concentration ([Ca2+]i) in mastocytoma P-815 cells, DNP-Ascaris (antigen) sensitized mast cells, hepatocytes, fibroblasts and endothelial cells were investigated. In these cell types, glycyrrhetinic acid in the concentration range of 20 to 100 microM caused an increase in [Ca2+]i and inhibited calcium increases induced by an antigen (mast cells), ATP, phenylephrine (hepatocytes) and thrombin (fibroblasts and endothelial cells). Stimulation with phenylephrine, in place of glycyrrhetinic acid, did not inhibit subsequent calcium increases induced by phenylephrine or ATP. On the other hand, glycyrrhizin at concentrations up to 100 microM neither caused an increase in [Ca2+]i nor inhibited calcium increases induced by other stimulatory substances. These results suggest that the inhibition of the calcium-mediated signal pathway may participate in the cytostatic actions of glycyrrhetinic acid.

Adenosine Triphosphate↗

PubMed1991

Differential regulation of thrombin- or ATP-induced mobilization of intracellular Ca2+ by prostacyclin receptor in mouse mastocytoma cells.

Thrombin induced an increase in [Ca2+]i in mouse mastocytoma P-815 cells. This increase was markedly reduced by prior exposure to pertussis toxin (PT) but not by removal of extracellular Ca2+, suggesting that thrombin stimulates phospholipase C via a PT-sensitive GTP-binding protein. ATP also induced an increase in [Ca2+]i. This increase was insensitive to PT but completely suppressed on removal of extracellular Ca2+, suggesting that ATP stimulates Ca2+ influx in a PT-insensitive manner. Iloprost, a stable prostacyclin analogue, increased the cellular cAMP level and dose-dependently inhibited the thrombin-induced increase in [Ca2+]i, whereas the ATP-induced increase in [Ca2+]i was markedly enhanced by iloprost. Cyclic AMP analogues, dibutyryl cAMP and 8-bromo cAMP, also inhibited the increase in [Ca2+]i induced by thrombin and promoted that by ATP, indicating that the inhibitory and stimulatory effects of iloprost are mediated by cAMP. These results suggest that the prostacyclin receptor differentially regulates two distinct Ca2+ mobilizing systems via cAMP in mastocytoma cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Cytosol promotes the guanine nucleotide-induced release of the alpha subunit of Gi2 from the membrane of mouse mastocytoma P-815 cells.

Translocation of the alpha subunit of Gi2 from the membrane to the cytosol was studied in mouse mastocytoma P-815 cells. To monitor Gi2 alpha the membrane (300,000 x g pellet) was [32P]ADP-ribosylated with pertussis toxin. Incubation of the [32P]ADP-ribosylated membrane with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a small release (10%) of [32P]ADP-ribosylated Gi2 alpha from the membrane. Whereas cytosol (300,000 x g supernatant) alone had no ability to release the [32P]ADP-ribosylated Gi2 alpha from the membrane, it markedly augmented the release induced by GTP gamma S, about 50% of the total [32P]ADP-ribosylated Gi2 alpha being released by 30 min. The GTP gamma S-induced release and its enhancement by the cytosol were specific for GTP and GTP gamma S. When the cytosol was boiled this promoting activity was lost. The [32P]ADP-ribosylated Gi2 alpha released by the cytosol plus GTP gamma S from the membrane was eluted as a single peak corresponding to a molecular weight of about 100,000 from an Ultrogel AcA 44 column. In contrast, the [32P]ADP-ribosylated Gi2 alpha released by GTP gamma S alone was eluted at the position of Mr = 40,000, but it was eluted at the position of Mr = about 100,000 when it was incubated with the cytosol. Furthermore, Gi2 alpha purified from bovine lung also behaved in a similar way on gel filtration. The addition of thrombin, a stimulant of histamine secretion from mast cells, to mastocytoma cells drastically induced the translocation of Gi2 alpha from the membrane to the cytosol in a pertussis toxin-sensitive manner. These results taken together demonstrate that the cytosol contains some factor(s) that promotes the release of GTP-activated Gi2 alpha from the membrane and that the released Gi2 alpha exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.

Adenosine Diphosphate Ribose↗

PubMed1991

Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450.

The identities of the amino acid at position 209 are most critical in determining specific coumarin 7- and steroid 15 alpha-hydroxylase activity in P450coh and P450(15)alpha, respectively. This system, therefore, provides us with an excellent model to study the structural basis for P450 specificity as a monooxygenase. We expressed in Saccharomyces cerevisiae a series of the mutated P450s in which residue 209 was substituted with the various amino acids and characterized the spectral property and hydroxylase activity of these mutated P450s. The positioning of a hydrophobic residue including Phe, Leu, and Val at position 209 resulted in shifting the P450 to the high-spin state, while a charged amino acid such as Lys or Asp produced the low-spin form. Moreover, a P450 with Asn or Gly in this position exhibited spectra indicating a mixture of the high- and low-spin forms. This spin alteration, depending upon the hydrophobicity and size of residue at position 209, indicates that this position is likely to reside close to the sixth axial ligand on the distal surface of the heme in these P450s. This proximity of residue 209 to the ligand may explain the critical role of this residue in determining the hydroxylase specificity and activity of these P450s.

Amino Acids↗

PubMed1991

Comparison between cobalt and pyrazole in the increased expression of coumarin 7-hydroxylase in mouse liver.

The data in this report show that administration of both cobalt and pyrazole results in an elevation in the amount of hepatic mRNA encoding for microsomal P45015 alpha/P450Coh, an increase in the amount of P450Coh protein, and an activation of COH and to a lesser extent testosterone 15 alpha-hydroxylase in two inbred strains of mice. Considerable quantitative differences between the two compounds and the two mouse strains in the response suggest that the effects of cobalt and pyrazole are mediated, at least partly, through different mechanisms. It is of interest that human hepatic COH resembles very closely that in the mouse liver.

Animals↗

PubMed1991

Structures and characterization of sex-specific mouse cytochrome P-450 genes as members within a large family. Duplication boundary and evolution.

We characterized two female-specific testosterone 16 alpha-hydroxylase mouse cytochrome P-450 genes, 16 alpha oh-a and 16 alpha oh-b. Gene 16 alpha oh-a, consisting of nine exons, is approximately 38 kbp in size. The exon sequence of this P-450 gene is identical to cDNA pf26 nucleotide sequence [Noshiro, M., Lakso, M., Kawajiri, K. & Negishi, M. (1988) Biochemistry 27, 6434-6443], which encodes female-specific testosterone 16 alpha-hydroxylase regulated by the murine Rip locus. Gene 16 alpha oh-b, containing nine exons with the same junctions as the 16 alpha oh-a, spans at least 20 kbp, and encodes a cytochrome P-450 whose deduced amino acid sequence is 90% similar to the hydroxylase. Nucleotide sequences revealed that duplication of the two genes occurred 4-22 million years ago, and that the 5' duplication boundary is located 1336 bp upstream from the putative transcription-start site. In the flanking regions of both genes, there is a long stretch (100 bp) of CA repeats in addition to other motifs, including TATA box, glucocorticoid-response-element-core and Simian-virus-40-enhancer sequences and IgG light-chain gene promoter. We isolated many genomic DNA clones which contain exon 1 sequences, and compared their restriction maps, cross-hybridization and nucleotide sequences. The results indicate that these genomic clones represent closely related genes in the 16 alpha oh family with a minimum of 16 members, which is further divided into classes a, b and c. 16 alpha oh-a and 16 alpha oh-b belong to the first and second classes, respectively. Moreover, extensive segmental gene conversion and nonreciprocal recombination were noted among the genes, particularly among those in class b. All genes in that class contain the long ATTT repeat sequences in intron 1, which may have triggered a rapid gene conversion and/or stabilize the duplicated genes.

Animals↗

PubMed1991

Involvement of interleukin-1 in the inhibition of in vivo insulin release: possible role of adrenal glands.

The effect of recombinant human interleukin-1 beta (rhIL-1b) on in vivo insulin secretion was examined in intact and adrenalectomized (ADX) rats under conscious and pentobarbital anesthetized conditions. In conscious rats, rhIL-1b dose-dependently decreased blood glucose levels at 2 h in both groups and significantly inhibited insulin release only at doses of 1 and 10 micrograms/kg in ADX rats. Under pentobarbital anesthesia, rhIL-1b administration decreased blood glucose levels 2 h later in both intact and ADX rats and resulted in severe hypoglycemia in ADX rats. At 2 h later, rhIL-1b significantly suppressed insulin release by pentobarbital injection in both intact and ADX rats. It is concluded that rhIL-1b suppresses in vivo insulin secretion in conscious ADX and anesthetized rats.

Adrenal Glands↗