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Biomedical subjects

Y Ichikawa

Publications and source records attributed to Y Ichikawa.

At least 19 recordsLinked to original sources
PubMed1995

Electron paramagnetic resonance investigation of cytochrome P-450c21 from bovine adrenocortical microsomes: a new enzymatic activity.

Cytochrome P-450c21 (CYP21A1) purified from bovine adrenocortical microsomes was investigated by electron paramagnetic resonance (EPR) spectroscopy to clarify the interactions among heme active center, protein surroundings, water molecules and bound substrates or analogues. The low-spin EPR signals of the oxidized enzyme (as purified) consisted of two species; one at gz = 2.39, gy = 2.23, and gx = 1.925 (component A) and the other at gz = 2.42, gy = 2.23, and gx = 1.916 (component B). The component A is probably representing a product-bound form, whereas the component B indicates either occupation of the substrate-binding site with a substrate analogue or absence of steroid at the site. Upon addition of progesterone, the component A signal completely disappeared and the intensity of high-spin signal (g = 8.06, 3.54) increased. Addition of 17 alpha-hydroxyprogesterone caused a development of a new low-spin signal at gz = 2.42, gy = 2.21, and gx = 1.966 (component C) and a further increase in intensity of the high-spin signal (g = 8.06, 3.54). Addition of 20 beta-hydroxyprogesterone caused an increase in intensity of the component C signal (and the g = 8 high-spin signal) even stronger than did 17 alpha-hydroxyprogesterone. These observations suggest that 20 beta-hydroxyprogesterone binds to the cytochrome P-450c21 active center in a very similar manner as 17 alpha-hydroxyprogesterone does and, therefore, may be a metabolizable substrate. A new enzymatic pathway catalyzed by cytochrome P-450c21 was confirmed with a reconstituted enzymatic system consisting of cytochrome P-450c21, NADPH-cytochrome P-450 reductase and an NADPH-generating system. 20 beta-Hydroxyprogesterone was converted to progesterone via a putative 20 beta-oxidase reaction in a comparable turnover number to that of the 21-hydroxylation of 17 alpha-hydroxyprogesterone.

Adrenal Cortex↗
PubMed1995

Interaction of NADPH-adrenodoxin reductase with NADP+ as studied by pulse radiolysis.

The reduction of flavin in NADH--adrenodoxin reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced directly the flavin of the reductase to form a blue semiquinone of the enzyme. Subsequently, the semiquinone decayed by dismutation to form the oxidized and fully reduced forms of the enzyme with a second-order rate constant of 4.4 x 10(4) M-1 s-1. In the presence of equimolar NADP+, the decay of eaq- accompanied an absorption increase at 400 nm, the spectrum of which, formed transiently, is identical to that of NADP radical (NADP.). Subsequently, the transient species decayed concomitantly with the formation of the semiquinone. The rate constant in the formation of the semiquinone was independent of the concentration of the enzyme (6.1 x 10(4) s-1 at pH 7.5). From these results, it is concluded that eaq- reacts with NADP+ bound to the enzyme to form NADP. initially, and subsequently, an electron flows from the NADP. to the flavin by an intracomplex electron transfer. A similar result was obtained in the reaction of CO2- or N-methylnicotinamide radical with the NADP(+)-adrenodoxin reductase complex. These results suggest that the nicotinamide moiety of NADP+ bound to the enzyme is accessible to the solvent and masks the flavin completely.

Animals↗
PubMed1995

Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers.

p16Ink4 and p15Ink4B are cyclin-dependent kinase 4 inhibitors and link to the regulation of cell cycle in mammalian cells. The genes encoding these inhibitors are located at 9p21, which is a frequent site of allelic loss in various types of tumors. Twenty-five primary biliary tract cancers were examined for somatic mutations in p16Ink4/CDKN2, p15Ink4B/MTS2, p53, and K-ras genes and allelic loss of 9p21 by microsatellite analysis. Four biliary tract cancer cell lines were analyzed for homozygous deletions and point mutations. We found frequent homozygous deletions in p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in the biliary tract cancer cell lines. Each cancer cell line had alteration of either p16Ink4/CDKN2, p15Ink4B/MTS2, or p53 genes. In primary tumors, 16 of 25 (64%) biliary tract cancers had point mutations in the p16Ink4/CDKN2 gene. These include 14 missense and 2 silent mutations. The frequency of mutations in gall bladder cancer and hilar bile duct cancer were 80% (8 of 10) and 63% (5 of 8), respectively. Each of codons 1, 80, and 111 was changed in two cases of these cancers. One of three intrahepatic bile duct cancers, one of two common bile duct cancers, and one of two ampullary cancers had mutations in the p16Ink4/CDKN2 gene. In contrast, no mutation in the p15Ink4B/MTS2 gene, one base change in the K-ras gene, and one loss of heterozygosity at the IFN alpha locus in 25 cancers and one base change in the p53 gene in 19 cancers were observed. These results suggest that p16Ink4/CDKN2, rather than p15Ink4B/MTS2 or p53 genes, and its inactivation may be important in biliary tract carcinogenesis.

Base Sequence↗
PubMed1995

An essential lysyl residue (Lys208) in the substrate-binding site of porcine FAD-containing monooxygenase.

The substrate (amine)-binding site of porcine FAD-containing monooxygenase (FMO) (EC 1.14.13.8) was examined using pyridoxal 5'-phosphate (pyridoxal-P) to modify lysyl residues. The enzymic activity of the FMO was inhibited competitively by pyridoxal-P. Upon reduction of pyridoxal-P-treated FMO with NaBH4, a new characteristic absorption peak of substituted pyridoxal-P appeared at 325 nm. The amino acid residue compositions of the native and pyridoxal-P-treated FMOs indicated that the lysyl residues were modified by pyridoxal-P. The about 74% inactivation of the enzymic activity on covalent pyridoxal-P treatment of the FMO was nearly completely prevented in the presence of the substrate, N,N-dimethylaniline. The FMO covalently modified with pyridoxal-P in the presence or absence of N,N-dimethylaniline was digested with trypsin treated with tosylphenylalanylchloromethane and the resultant peptide fragments were separated with a reverse-phase high-performance liquid chromatography system; only one peptide was specifically labeled with pyridoxal-P and was detected at 325 nm in the absence of N,N-dimethylaniline. The modified peptide was analyzed and identified as that comprising the amino acid residues 186-208. These results suggest that Lys208 plays an important role in the substrate (amine)-binding site of FMO.

Amino Acid Sequence↗
PubMed1995

Identification of essential lysyl and cysteinyl residues, and the amino acid sequence at the substrate-binding site of retinal oxidase.

Retinal oxidase, which synthesizes all-trans and 9-cis retinoic acid from all-trans and 9-cis-retinal, has been purified from rabbit liver cytosol. The substrate-binding site of the retinal oxidase was investigated with some chemical modification reagents. Lysyl-specific pyridoxal 5'-phosphate (PLP) and cysteinyl-specific p-chloromercuribenzoate (PCMB) competitively inhibited the activity of the retinal oxidase, and the inhibition could be prevented by the presence of all-trans-retinal or its derivatives. Treatment with sodium borohydride (NaBH4) resulted in covalent attachment of PLP to the lysyl residue of the retinal oxidase and the PLP modified-retinal oxidase was cut with cyanogen bromide, and the polypeptides modified with PLP were further digested with trypsin. Two of the peptides modified with PLP were purified from the digested polypeptide mixture and their amino acid sequences were determined.

Aldehyde Oxidoreductases↗
PubMed1995

Inhibition of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolic activity of porcine FAD-containing monooxygenase by selective monoamine oxidase-B inhibitors.

The MPTP metabolic activity of porcine FAD-containing monooxygenase (FMO) (EC 1.14.13.8) was inhibited considerably by deprenyl and pargyline, selective MAO-B inhibitors, and they showed typical competitive inhibition. Deprenyl and pargyline, amine derivatives were also examined as to whether they are substrates for the FMO. It was found that deprenyl and pargyline are excellent substrates for the FMO. The Ki and Km values of deprenyl and pargyline for the FMO are 14 microM and 9 microM, and 14.3 microM and 11.6 microM, at pH 8.0 and 25 degrees C, respectively.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1995

Changing autoantibody profiles with variable clinical manifestations in a patient with relapsing systemic lupus erythematosus and polymyositis.

The production of autoantibodies characteristic of different autoimmune disease subsets is thought to be controlled primarily by genetic factors, whereas non-genetic factors are generally believed to be of secondary importance. A patient with systemic lupus erythematosus (SLE) and polymyositis (PM) who experienced frequent relapses associated with changing clinical manifestations and autoantibody specificities is reported. Her initial presentation as SLE with anti-Sm antibodies shifted to the onset of PM with temporal production of a different set of autoantibodies. The latter antibodies disappeared when myositis resolved, followed by the reappearance of autoantibodies and clinical manifestations characteristic of SLE. The shifts of autoantibody profiles in association with variable clinical manifestations in this patient argue that environmental factors may play a more important role in autoimmunity than previously supposed, and that interplay between environmental triggers and genetic predisposing factors may lead to the constellation of autoimmune disease manifestations exhibited at a particular time.

Adult↗
PubMed1995

Massive pericardial effusion in scleroderma: a review of five cases.

Medical records of five patients with scleroderma (SSc), each of whom had pericardial effusion with an estimated volume of more than 200 ml, were reviewed to study the clinical and immunological significance of massive pericardial effusion in SSc. Diffuse SSc (4/5), with a wide area of pigmentation (4/5), flexion contracture (4/5), oesophageal hypomotility (5/5), pulmonary fibrosis (4/5) and autoantibodies to topoisomerase I (3/5) were the common features in this group. High protein, lactate dehydrogenase and low white blood cell count were the characteristics of pericardial fluid. None of the patients had signs of acute pericarditis. Four of the five cases died within 9 months of the diagnosis of pericarditis; two with renal failure, one with cardiac tamponade and another with sudden death. The pericarditis in diffuse SSc, especially in cases with anti-topoisomerase I, may be characterized by a chronic form of pericarditis with poor prognosis, often complicated by renal failure.

Adult↗
PubMed1995

Thermoluminescence dosimetry of the Hiroshima atomic-bomb gamma rays between 1.59 km and 1.63 km from the hypocenter.

Gamma-ray kermas from the Hiroshima atomic bomb were measured by a thermoluminescence (TL) method using tile specimens collected from five locations on the rooftop of a building (Postal Saving Bureau), 1,591-1,635 m from the hypocenter. Four tile specimens each from five locations were sampled and quartz grains were extracted from them. TL of these grains were analyzed by the high temperature TL method and gamma-ray kermas were obtained. The results in tissue kerma, on average, are 21% higher (standard errors are 4.3-7.3%) than the DS86 estimation. The present data and the reported TL results indicate that the measured gamma-ray kermas begin to exceed the DS86 values at about 1.3 km and the discrepancy between them increases with ground distance. This discrepancy is attributed to errors in the DS86 neutron source spectrum and is supported by previous neutron activation measurements.

Gamma Rays↗
PubMed1995

Characterization of a novel human tumor necrosis factor-alpha mutant with increased cytotoxic activity.

Various novel recombinant human tumor necrosis factor-alpha (TNF) mutants were prepared using protein engineering techniques, and their cytotoxic activity was compared with that of the intact form of TNF (intact TNF). Mutant 471 (a TNF mutant molecule with the deletion of 7 amino acids at the amino-terminal and the substitution of Pro8Ser9Asp10 by ArgLysArg) had a 6-fold higher cytotoxic activity against murine L929 cells. The mutant TNF had an increased ability to bind to TNF receptor on murine L929 fibroblasts cells. A cross-linking study revealed that mutant 471 had an increased ability to form an active trimer. Mutant 471 also showed higher cytotoxic activity against human KYM myosarcoma cells and human MIA PaCa-2 pancreatic carcinoma cells. The possible cachectin activity of the mutant was almost the same as that of intact TNF. These results suggest that mutant 471 might be a more promising candidate as an anticancer agent than intact TNF.

Amino Acid Sequence↗
PubMed1995

Spontaneous production of various cytokines except IL-4 from CD4+ T cells in the affected organs of sarcoidosis patients.

We investigated surface antigens and spontaneous cytokine production of T cells from bronchoalveolar lavage fluid (BALF) and aqueous humor (AH) from pulmonary sarcoidosis patients for a better understanding of the role of T cells in granuloma formation. The levels of CD3, CD11b, and CD28 antigen expression on freshly isolated T cells in the BALF of patients were significantly lower than those in peripheral blood lymphocytes (PBL) of either sarcoidosis patients or healthy donors (HD). In contrast, the levels of CD80 (B7/B7-1) and CD86 (B70/B7-2) antigen expression were significantly higher on these T cells and alveolar macrophages in the BALF of patients. Fifty-three T cell clones (TCC) established from the BALF and AH of the three sarcoidosis patients displayed primarily either CD4+ CD11b+ CD28+ or CD4+ CD11b- CD28- phenotypes. Most (61-90%) of these TCC spontaneously produced greater amounts of IL-1 alpha, IL-10, tumour necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) than did TCC from the PBL from sarcoidosis patients or HD (P < 0.05). Interferon-gamma (IFN-gamma), IL-6, and IL-2, but not IL-4, were also produced by 40-48% of these TCC. These results suggest that CD4+ T cells of the affected organs of sarcoidosis patients are activated and involved in the immunopathogenesis of sarcoidosis through production of various cytokines.

Adult↗
PubMed1995

Sealing of a fabric vascular prosthesis with autologous adipose tissue: a preliminary report of its clinical application.

A new method of sealing fabric vascular prostheses with autologous adipose tissue was clinically applied as an alternative to preclotting with fresh blood. Thirty-six patients with peripheral arterial occlusive disease were implanted with highly porous fabric prostheses. The prostheses were prepared by sealing the fabric pores with autologous adipose tissue that had been chopped up into small pieces and enmeshed in the fabric by forceful injection of the tissue suspension through a syringe. There was no complication related to the sealed graft such as graft bleeding after implantation. In-hospital mortality occurred in 4 patients: 1 case each of pneumonia, pulmonary infarction, sepsis, and acute myocardial infarction. During the period of 274 +/- 190 days, 3 prostheses were found to be occluded. All the other grafts were patent. The overall patency rate was 91.4%. Postoperative angiography revealed neither intimal thickening at the anastomotic sites nor irregularity of the prosthetic surface. The method proved safe and useful for implantation of smaller caliber artificial grafts.

Adipose Tissue↗
PubMed1995

CYP2E1 and ALDH2 genotypes and alcohol dependence in Japanese.

The genotypes of the CYP2E1 and ALDH2 loci of alcoholic (alcohol dependence) and nonalcoholic (healthy) Japanese were investigated to examine the relationship between the polymorphism of CYP2E1 (C1/C2) and ALDH2 (ALDH2*1/ALDH2*2), and the susceptibility to alcoholism. There was no significant difference in C2 gene frequency between alcoholics (0.19) and nonalcoholics (controls) (0.20), whereas there was a significant difference in ALDH2 allele frequency, suggesting that, in Japanese, the C2 genotype of CYP2E1 may have nothing to do with the risk of developing alcohol dependence. However, the ALDH2*1 allele may influence drinking behavior and the development of alcohol dependence. Furthermore, racial interethnic differences in the frequency of the mutated allele of the CYP2E1 gene (C2) were found, like the ALDH2 gene. Japanese healthy controls showed a significantly higher frequency of the C2 allele than did Swedish healthy controls (0.05; reported by Persson et al., FEBS Lett. 319:207-211, 1993).

Alcohol Drinking↗
PubMed1995

Periarticular osteopenia in adjuvant induced arthritis: role of interleukin-1 in decreased osteogenic and increased resorptive potential of bone marrow cells.

OBJECTIVE: To clarify the local osteogenic and bone resorptive potential of periarticular bone in adjuvant induced arthritis (AIA). METHODS: Formation of fibroblast colony forming units (FCFU; osteogenic precursor cells) and osteoclast-like cells in bone marrow culture was studied in AIA rats. Osteoclast-inducing activity in the AIA rat bone marrow was assayed by the addition of the marrow supernatant from rats with AIA to control cultures. Bone mineral density was determined by dual x ray absorptiometry. RESULTS: Marrow from AIA rats and that from animals receiving recombinant human interleukin-1 (IL-1) beta for seven days grew significantly fewer FCFU than control marrow. Formation of osteoclast-like cells was increased in bone marrow cultures from rats with AIA, especially when bone marrow cells were cultured in the presence of marrow supernatant. Formation of resorption lacunae on ivory slices was increased in the marrow cultures from rats with AIA, especially from the right (adjuvant inoculated) tibia. AIA rat marrow supernatant promoted osteoclast-like cell formation in control culture, and this was significantly suppressed by an anti-IL-1 antibody. Rats with AIA showed a significant decrease in the bone mineral density of the periarticular regions of the tibia and femur. CONCLUSION: An uncoupled state in bone resorption-formation linkage, possibly mediated through an increase of IL-1 in the bone marrow, may contribute to the development of periarticular osteopenia in inflammatory arthritis.

Animals↗
PubMed1995

Production of granulocyte colony-stimulating factor by malignant mesothelioma.

We present the case of a male patient, aged 45 yrs, with malignant mesothelioma producing granulocyte colony-stimulating factor (G-CSF). The diagnosis was established by histopathological examination of the autopsied pleural tissues. Production of G-CSF was confirmed by immunoperoxidase staining, using a specific monoclonal antibody against recombinant G-CSF (rhG-CSF).

Granulocyte Colony-Stimulating Factor↗