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H Tsukamoto

Publications and source records attributed to H Tsukamoto.

At least 73 records · Page 4Linked to original sources
PubMed1999

An NcoI polymorphism in the human complement component 7 (C7) gene.

A novel polymorphic site has been found in the 3' untranslated region (UTR) of the human complement component 7 (C7) gene. The polymorphic site at 14-bp down-stream from the TAG stop codon was either C or A (Nco I-digested), with allele frequencies of 0.660 and 0.340. This NcoI polymorphism would be useful to perform a DNA marker haplotype study in patients with deficiencies of the complement genes, such as C6, C7, C9, which are located closely on chromosome 5p13.

Alleles↗
PubMed1999

Mutation analysis of a Japanese patient with fucosidosis.

Fucosidosis is a rare autosomal recessive disorder resulting from a deficiency of alpha-L-fucosidase. Recently, various mutations have been reported in this disease, but it is difficult to elucidate the phenotype from the genetic mutations. We report a patient with chronic infantile type fucosidosis, with a compound heterozygote of a nonsense mutation (W148X, Trp at codon 148 to stop codon) and a large deletion, including all exons. This is the first report of a large deletion demonstrated in fucosidosis. It is interesting that this patient has a relatively mild clinical course despite the absence of the mRNA. This case also indicates the difficulty in determining the phenotype from the genotype in fucosidosis.

Adolescent↗
PubMed1999

Ethanol diet increases the sensitivity of rats to pancreatitis induced by cholecystokinin octapeptide.

BACKGROUND & AIMS: Although alcoholism is a major cause of pancreatitis, the pathogenesis of this disorder remains obscure. Failure to produce experimental alcoholic pancreatitis suggests that ethanol may only increase predisposition to pancreatitis. This study sought to develop a model of ethanol pancreatitis by determining if an ethanol diet sensitizes rats to pancreatitis caused by cholecystokinin octapeptide (CCK-8). METHODS: Rats were fed intragastrically either control or ethanol diet for 2 or 6 weeks. The animals were then infused for 6 hours with either saline or CCK-8 at a dose of 3000 pmol. kg(-1). h(-1), which by itself did not induce pancreatitis. The following parameters were measured: serum amylase and lipase levels, pancreatic weight, inflammatory infiltration, number of apoptotic acinar cells, pancreatic messenger RNA (mRNA) expression of cytokines and chemokines, and nuclear factor (NF)-kappaB activity. RESULTS: All measures of pancreatitis, as well as NF-kappaB activity and mRNA expression for tumor necrosis factor alpha, interleukin 6, monocyte chemotactic protein 1, macrophage inflammatory protein 2, and inducible nitric oxide synthase, were significantly increased only in rats treated with ethanol plus CCK-8. CONCLUSIONS: An ethanol diet sensitizes rats to pancreatitis caused by CCK-8. The combined action of ethanol and CCK-8 results in NF-kappaB activation and up-regulation of proinflammatory cytokines and chemokines in the pancreas. These mechanisms may contribute to the development of alcoholic pancreatitis.

Amylases↗
PubMed1999

Molecular heterogeneity of Krabbe disease.

Krabbe disease (globoid cell leukodystrophy) is an autosomal recessive neurodegenerative disorder that affects both the central and peripheral nervous system due to an enzymatic defect of galactocerebrosidase (GALC). Following its cloning, many mutations in the galactocerebrosidase gene have been reported, but the correlation between phenotype and genotype was not clear in many cases. In this study we further investigated the molecular defects in another 10 patients (6 Japanese and 4 non-Japanese), using cultured skin fibroblasts, and found 10 mutations, of which 8 were novel, including a nonsense mutation (W647X) and 7 missense mutations (G43R, S52F, T262I, Y319C. W410G, R515H, T652R) in the coding region. Some phenotype-specific mutations were found but the other mutations were private. Mutations reported so far have been distributed over the whole GALC gene and it is difficult to speculate on functional domains of the GALC protein and phenotypically specific regions.

Alanine↗
PubMed1999

Enhanced expression of recombinant dystrophin following intramuscular injection of Epstein-Barr virus (EBV)-based mini-chromosome vectors in mdx mice.

Gene transfer by direct intramuscular injection of naked plasmid DNA has been shown to be a safe, simple but relatively inefficient method for gene delivery in vivo. Eukaryotic plasmid expression vectors incorporating the Epstein-Barr virus (EBV) origin of replication (oriP) and EBNA1 gene have been shown to act as autonomous episomally replicating gene transfer vectors which additionally provide nuclear matrix retention functions. Prolonged expression of a LacZ reporter gene and recombinant human dystrophin was shown using EBV-based plasmid vectors transfected into C2C12 mouse myoblast and myotube cultures. Intramuscular injection of EBV-based dystrophin expression plasmids into nude/mdx mice resulted in significant enhancement in the number of muscle fibres expressing recombinant dystrophin compared with a conventional vector. This effect was observed for over 10 weeks after a single administration. These results indicate the potential advantage of EBV-based expression vectors for focal plasmid-mediated gene augmentation therapy in Duchenne muscular dystrophy (DMD) and a range of other gene therapeutic applications.

Animals↗
PubMed1999

Association of Fas/APO-1 gene polymorphism with systemic lupus erythematosus in Japanese.

OBJECTIVES: This study was undertaken to investigate the possible association of Fas gene mutation(s) or polymorphism(s) with systemic lupus erythematosus (SLE) in Japanese. METHODS: Screening for structural defects of the Fas gene was performed by using reverse transcriptase-polymerase chain reaction (RT-PCR)/single-strand conformation polymorphism (SSCP) analysis in 57 patients with SLE, followed by direct sequencing for the aberrantly migrating bands. The frequency of Fas polymorphism was determined by sequence-specific oligonucleotide probe (SSOP) hybridization in 82 SLE patients and 132 ethnically matched healthy individuals. RESULTS: We found a novel polymorphism at nucleotide 297 (T297C), which was linked to Fas polymorphism at nucleotide 416 (A416G). The 297C/416G genotype was present in four of the 132 (3.0%) healthy controls, none of whom was homozygous for the genotype. The allele frequency for 297C/416G in the controls was 1.5%. In contrast, 10 of the 82 (12.2%) SLE patients carried the 297C/416G allele, including one patient homozygous for the genotype. The allele frequency in SLE patients was 6.7%. The 297C/416G allele was significantly frequent in SLE patients (P = 0.01, chi2) with a relative risk of 5.00. CONCLUSION: As the polymorphism 297C/416G is silent at the amino acid level, it may affect the expression of Fas itself or be linked to a neighbouring genetic abnormality that is responsible for the pathogenesis of SLE.

Amino Acid Sequence↗
PubMed1999

Relationship between serum levels of anti-low-density lipoprotein-acetaldehyde-adduct antibody and aldehyde dehydrogenase 2 heterozygotes in patients with alcoholic liver injury.

We prepared low-density lipoprotein (LDL)-acetaldehyde-adduct (hereafter abbreviated as LDL-adduct) and anti-LDL-adduct antibody by using Watanabe hyperlipidemic rabbits, and determined values of serum anti-LDL-adduct antibody levels by the ELISA method in healthy adults and patients with alcoholic liver injury. In the nondrinking group in healthy adults, values of anti-LDL-adduct antibody levels were 25 +/- 13 microg/ml, and there was no significant difference between moderate drinkers without diseases and the nondrinking group in healthy adults. Values of anti-LDL-adduct antibody in alcoholic disease groups, 17 +/- 9 microg/ml for the patients with the fatty liver group, 21 +/- 14 microg/ml for the hepatic fibrosis group, 70 +/- 21 microg/ml for the alcoholic hepatitis group, 41 +/- 50 microg/ml for the alcoholic cirrhosis group, and 19 +/- 18 microg/ml for the alcoholic pancreatitis group. Examinations of aldehyde dehydrogenase 2 (ALDH2) genetic variations by the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method in the healthy group and the liver injury group revealed a tendency for patients with ALDH2(1)/2(2) in the liver injury group to have relatively mild liver lesions. When comparing anti-LDL-adduct antibody levels between ALDH2 genetic variations, those for the patients with ALDH2(1)/2(2) (36 +/- 40 microg/ml) were significantly higher than those for patients with ALDH2(1)/2(2) (11 +/- 5 microg/ml). Results of the present study suggest that genetic variation may influence the progression of liver injury.

Acetaldehyde↗
PubMed1999

The localization of glutathione peroxidase in the photoreceptor cells and the retinal pigment epithelial cells of Wistar and Royal College of Surgeons dystrophic rats.

INTRODUCTION: If degenerating photoreceptor outer segments not phagocytized by RPE cells in the retina of Royal College Surgeons (RCS) rats were to undergo peroxidation, the distribution of glutathione peroxidase (GSH-PO) in the mitochondria or cytoplasm of the retina might be altered. We evaluated the immunocytochemical localization of GSH-PO to identify subcellular organelles in sections of the retinas of RCS rats. METHODS: Immunoblot analysis confirmed the presence of GSH-PO molecules in the retinas of RCS and Wistar rats aged 3 weeks. Sections were reacted with the F(ab) fragment of anti-rat alphaGSH-PO and then examined by laser scanning microscopy (LSM) and transmission electron microscopy (TEM). RESULTS: The size of the GSH-PO molecule in the retina was about 21 KD in the mitochondria and 23 KD in the cytosol in both strains of rats. LSM revealed fluorescent granules in the photoreceptor inner segments of the Wistar rats, and immunohistochemical TEM revealed GSH-PO in the mitochondria of their photoreceptor inner segments and retinal pigment epithelial (RPE) cells. In the RCS rats, the degenerating photoreceptor outer segments were clearly seen to be positive for anti-GSH-PO by conventional light microscopy (CLM). However, the photoreceptor inner segments of the RCS rats were negative for staining with anti-GSH-PO by LSM, and no GSH-PO could be detected in the mitochondria of the photoreceptor inner segments or RPE cells by immuno-TEM. CONCLUSION: Degeneration of the photoreceptor outer segments induced mitochondrial damage in the photoreceptor inner segments, and as a result GSH-PO shifted from the photoreceptor inner segments to the degenerating outer segments.

Animals↗
PubMed1999

Iron primes hepatic macrophages for NF-kappaB activation in alcoholic liver injury.

NF-kappaB activation induced by lipopolysaccharide (LPS) in cultured hepatic macrophages (HM) may be abrogated by pretreatment of cells with a lipophilic iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1, deferiprone), suggesting a role for iron in this molecular event [M. Lin, M., R. A. Rippe, O. Niemelä, G. Brittenham, and H. Tsukamoto, Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G1355-G1364, 1997]. To ascertain the relevance in vivo of this hypothesis, HM from an experimental model of alcoholic liver injury were examined for the relationship between nuclear factor (NF)-kappaB activation and iron storage. HM showed a significant increase in nonheme iron concentration (+70%), accompanied by enhanced generation of electron paramagnetic resonance-detected radicals (+200%), NF-kappaB activation (+100%), and tumor necrosis factor-alpha (+150%) and macrophage inflammatory protein-1 (+280%) mRNA induction. Treatment of the cells ex vivo with L1 normalized all these parameters. HM content of ferritin protein, ferritin L chain mRNA, and hemeoxygenase-1 mRNA and splenic content of nonheme iron were increased, suggesting enhanced heme turnover as a cause of the increased iron storage and NF-kappaB activation. To test this possibility, increased iron content in HM was reproduced in vitro by phagocytosis of heat-treated red blood cells. Treatment caused a 40% increase in nonheme iron concentration and accentuated LPS-induced NF-kappaB activation twofold. Both effects could be abolished by pretreatment of cells with zinc protoporphyrin, a hemeoxygenase inhibitor. To extend this observation, animals were splenectomized before 9-wk alcohol feeding. Splenectomy resulted in further increments in HM nonheme iron storage (+60%) and NF-kappaB activation (+90%) and mononuclear cell infiltration (+450%), particularly around the iron-loaded HM in alcohol-fed animals. These results support the pivotal role of heme-derived iron in priming HM for NF-kappaB activation and expression of proinflammatory genes in alcoholic liver injury.

Animals↗
PubMed1999

Cytokine regulation of hepatic stellate cells in liver fibrosis.

Cytokines constitute a major class of mediators responsible for "activation" of hepatic stellate cells (HSCs) in vitro and in vivo. They are largely divided into mitogenic (transforming growth factor-alpha, platelet-derived growth factor, interleukin-1, tumor necrosis factor-alpha, and insulin-like growth factor) and fibrogenic (transforming growth factor-beta and interleukin-6) cytokines. In addition to their mitogenic (stimulation of cell proliferation) and fibrogenic (induction of matrix proteins) properties, they are also shown to confer in vitro unique cellular changes known to be the key features of HSC "activation," including loss of vitamin A, stimulation of migration, enhanced cellular contractility, and matrix metalloproteinase and tissue inhibitor of metalloproteinase induction. Potential cellular sources of the cytokines consist of hepatic macrophages, endothelial cells, biliary epithelial cells, lymphocytes, platelets, hepatocytes, and activated HSCs. To better understand the mode of actions and the pathogenetic significance of cytokines/chemokines involved in "activation" of HSCs, the following four questions need to be addressed: (1) What other cytokines are expressed by HSCs to establish critical autocrine stimulation? (2) What are endogenous or exogenous priming factors for HSC stimulation? (3) What is the mechanism of activation for transforming growth factor-beta, the pivotal fibrogenic cytokine? (4) How important are HSC-derived proinflammatory mediators in liver fibrosis? This review will discuss these questions, along with the current understanding of the role of cytokines in HSC activation.

Cytokines↗
PubMed1998

GLUT1 glucose transporter: differential gene transcription and mRNA binding to cytosolic and polysome proteins in brain and peripheral tissues.

The ratio of GLUT1 mRNA to actin mRNA in brain was 6- to 13- fold greater than the corresponding ratio in spleen, lung, testis, heart, and skeletal muscle in the rat. However, the ratio of GLUT1 transcription rate to actin transcription rate is comparable in brain and the five other organs. Organ extracts were fractionated into cytosol and polysomes, and ultraviolet light cross-linking studies were performed with these proteins and 32P-labeled GLUT1 mRNA containing the 3'-untranslated region (UTR) generated from transcription plasmids. The cytosol of brain, lung, spleen, C6 glioma cells in tissue culture, and freshly isolated bovine brain capillaries express a pair of 95 kDa and 74 kDa proteins, designated collectively as p88, and the polysome fraction of brain, testis, or C6 glioma cells express a 44-kDa protein, designated p44. In a middle cerebral artery occlusion model, brain cytosol p88 was up-regulated and p44 was down-regulated. These findings are consistent with the hypothesis that GLUT1 gene expression is subject to regulation at the post-transcriptional level.

Animals↗
PubMed1998

The carboxyl-terminal region of protein C is essential for its secretion.

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.

Amino Acid Sequence↗
PubMed1998

Polymorphism in the Ialpha1 germ-line transcript regulatory region and IgA productivity in patients with IgA nephropathy.

Enhanced in vivo and in vitro production of IgA has been reported in patients with IgA nephropathy (IgAN) and their family members. It is generally considered that IgA1 is a prominent subclass of IgA in IgAN. Although genetic mechanisms of IgA class switch recombination in IgAN have been studied enthusiastically, the critical factors that induce IgA1-specific class switching in IgAN have yet to be elucidated. A large body of data indicates that the germ-line transcript of Ig constant region (C(H)) genes that precedes actual class switching has regulatory effects on class switch recombination. To analyze structural abnormalities in the Ialpha1 germ-line transcript regulatory gene, a region about 1000 bp long located upstream of Ialpha1 exons was surveyed by the PCR-single strand conformation polymorphism method, and the polymorphism detected was confirmed by subsequent DNA sequencing. Three hot spots for point mutation were detected upstream of the promoter region of the Ialpha1 germ-line transcript, and the mutations were observed more frequently in patients than in controls. Patients with the mutations showed higher levels of serum IgA and higher in vitro IgA synthesis. In the luciferase assay, the regulatory gene with the mutations showed a potent effect for induction of the Ialpha1 germ-line transcript. The polymorphism in the Ialpha1 regulatory region possibly causes enhanced IgA production in some patients with IgAN.

Base Sequence↗
PubMed1998

Molecular mechanisms of type II factor XIII deficiency: novel Gly562-Arg mutation and C-terminal truncation of the A subunit cause factor XIII deficiency as characterized in a mammalian expression system.

To explore the biological and clinical implications of the structure/function relationships in factor XIII, mutations in two patients with type II deficiency were identified and characterized in a mammalian expression system. Nucleotide sequence analysis of the A subunit gene showed that case no. 1 had a deletion of 4 bp (AATT) in exon XI and that, in case no. 2, Gly562 (GGG) had been replaced by Arg(AGG). The deletion in case no. 1 leads to a premature termination at codon 464. Restriction digestion of amplified DNAs confirmed that both cases were homozygous for their respective mutations. Reverse transcription-polymerase chain reaction analysis demonstrated that the level of mRNA was greatly reduced in case no. 1, whereas the level of mutant mRNA expressed in case no. 2 was normal. Molecular modeling calculated that Arg562 changed the conformation of the A subunit, suggesting misfolding and/or destabilization of the molecule. To determine how these mutations impaired synthesis of the A subunit, recombinant A subunits bearing the mutations were expressed in mammalian cells. Pulse-chase experiments showed that the mutants were synthesized normally but disappeared rapidly, whereas the wild-type remained. These results indicate that both mutant proteins with an altered conformation become prone to rapid degradation, resulting in factor XIII deficiency in these patients.

Arginine↗
PubMed1998

Design and synthesis of a water-soluble taxol analogue: taxol-sialyl conjugate.

Glycosidation, using the methylthio derivative of N-acetylneuraminic acid 3, of linker alcohol 4 in DME with "long-range participation" produced the alpha-glycosyl linkage with high stereoselectivity. The alpha-linked sialic acid 2 was introduced in taxol without protection of the alcohol functionality in sialic acid.

Drug Design↗
PubMed1998

Expression of interleukin-10 by in vitro and in vivo activated hepatic stellate cells.

Activated hepatic stellate cells (HSC) participate in matrix remodeling and deposition in liver fibrosis. The present study demonstrates that interleukin (IL)-10 is expressed by HSC upon activation in vitro or in vivo and that autocrine effects of this cytokine include inhibition of collagen production. Culture activation of HSC caused a distinct increase in IL-10 mRNA level compared with freshly isolated quiescent HSC. Treatment of cultured HSC with tumor necrosis factor-alpha, transforming growth factor-beta, or lipopolysaccharide further increased IL-10 mRNA by 2-fold and resulted in the release of IL-10 protein into the medium. HSC isolated from rats after bile duct ligation (BDL) showed prominent increases in IL-10 mRNA (x 100) and protein (x 30) levels at 7 days after BDL, but such induction disappeared in advanced liver fibrosis (19 days after BDL). IL-10 expression correlated positively with mRNA expression of interstitial collagenase and inversely with that of alpha1(I) collagen. Addition of anti-IL-10 IgG to cultured HSC caused enhanced collagen production under a basal or stimulated condition with TGF-beta, tumor necrosis factor-alpha, or lipopolysaccharide. These effects were associated with increased alpha1(I) collagen mRNA and reciprocally reduced collagenase mRNA levels. Co-transfection of HSC with an IL-10 expression vector and collagen reporter genes showed a 40% inhibition of alpha1(I) collagen promoter activity. These results demonstrate that activation of HSC causes enhanced autocrine expression of IL-10 which possesses a negative autoregulatory effect on HSC collagen production mediated at least in part by alpha1(I) collagen transcriptional inhibition and stimulation of collagenase expression. These findings, along with the demonstrated early induction of HSC IL-10 expression and its late disappearance during biliary liver fibrosis, suggest its in vivo role in matrix remodeling and a possibility that failure for HSC to sustain IL-10 expression underlies pathologic progression to liver cirrhosis.

Animals↗