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H Tsukamoto

Publications and source records attributed to H Tsukamoto.

At least 109 records · Page 6Linked to original sources
PubMed1996

Expression of intracellular adhesion molecule 1 by activated hepatic stellate cells.

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. HSC activation results in numerous changes in cellular morphology, cellular metabolism, and in the pattern of gene expression. Many of the changes that are observed in activated HSCs in animal models of hepatic fibrosis are also seen when these cells are activated by culturing on plastic. These changes include morphological changes to a myofibroblast-like cell with the appearance of smooth muscle alpha-actin, a loss of the retinol stores, an increase in the rough endoplasmic reticulum, and increases in extracellular matrix production, including a dramatic increase in type I collagen. To identify additional genes that are induced or suppressed during HSC activation, we used the differential polymerase chain reaction (PCR) display technique. Using this technique, we isolated a complementary DNA (cDNA) fragment for the intercellular adhesion molecule 1 (ICAM-1). Northern blotting confirmed that the ICAM-1 messenger RNA (mRNA) was expressed in HSCs activated by culture, but not in quiescent, freshly isolated HSCs. The presence of ICAM-1 protein was demonstrated in culture-activated HSCs, but not in quiescent cells by Western blot analysis and immunohistochemical staining. A functional assay was performed, demonstrating that lymphocytes will adhere to activated HSCs and that treatment of these cells with tumor necrosis factor alpha (TNF-alpha) increases lymphocyte adherence. Furthermore, ICAM-1 mRNA levels were increased in HSCs activated in rats in vivo after 1 week of bile duct ligation (BDL). Together, these data indicate that ICAM-1 expression is induced following HSC activation and that the HSC may have a direct role in the transmigration of leukocytes from the hepatic sinusoid to sites of tissue damage during the inflammatory response in the liver.

Animals↗
PubMed1996

A new type of major aminopeptidase in bovine brain.

A new type of major aminopeptidase was purified from bovine brain by ammonium sulfate fractionation and TMAE-fractogel (anion exchange), arginine-Sepharose 4B, Sephadex G-150, and Sephadex G-100 column chromatography. The purified enzyme showed a maximum activity at pH 7.2, and its molecular size was estimated to be 98,000 by gel filtration and 104,000 by SDS-PAGE with or without 2-mercaptoethanol. Further properties were activation by thiol reagents; inhibition by EDTA, puromycin, bestatin, amastatin, actinonin, leuhistin and probestin; and very low concentrations of Cu2+, Cd2+, Pb2+, Al3+, Fe3+, and Zn2+ inhibited activity. The enzyme hydrolyzed several amino acyl-7-amido-4-methylcoumalin derivatives (amino acid-MCA). The order of MCA-substrate specificity expressed as kcat/Km is Lys-MCA > Arg-MCA > Leu-MCA > Met-MCA > Phe-MCA > Tyr-MCA > Ala-MCA >> Gly-MCA, Pro-MCA, Ser-MCA, Asn-MCA. Immunoreactivity of the antibody against the purified aminopeptidase was observed in human brain and most rat tissues examined including brain, liver, kidney, lung, heart, and skeletal muscle at the same molecular size as in bovine brain aminopeptidase. Most of the Lys-, Leu-, Met-, and Phe-MCA degrading activity in crude bovine and human brain extracts was absorbed by the aminopeptidase IgG, suggesting that this aminopeptidase is a major enzyme, sharing at least Lys-, Leu-, Met-, and Phe-MCA degrading aminopeptidase activities in the brains.

Amino Acids↗
PubMed1996

Glutathione peroxidase activity in endometrium: effects of sex hormones and cancer.

The aim of this study was to determine whether glutathione peroxidase (GSH-Px) activity in endometrial tissue is regulated by sex hormones and to compare the GSH-Px activity of normal and cancerous endometrium. The localization of GSH-Px in human normal endometrium and endometrial cancer was determined immunohistochemically. GSH-Px activity was assayed in endometrial tissue obtained from women with endometrial cancer and age-matched controls, as well as in rat uterine tissue. GSH-Px immunoreactivity was localized in the glandular epithelium of normal human endometrium and reached a maximum in the late proliferative and early secretory phases of the menstrual cycle. In spayed rats, uterine GSH-Px activity was significantly increased by exogenous estrogen (P < 0.01) and significantly reduced by exogenous progesterone (P < 0.01). GSH-Px activity in endometrial cancer tissue was significantly higher (P < 0.01) than that in endometrial tissue from age-matched healthy controls. Among endometrial cancer tissues, a significant increase in GSH-Px activity was associated with well-differentiated rather than moderately or poorly differentiated adenocarcinoma (P < 0.01), with slight rather than marked myometrial invasion (P < 0.01), and with the presence of concurrent endometrial hyperplasia (P < 0.01). These results show that endometrial GSH-Px activity is regulated by sex hormones, being stimulated by estrogen and suppressed by progesterone, and that the level of GSH-Px activity in endometrial cancer tissue may be a significant prognostic factor.

Adenocarcinoma↗
PubMed1996

Allele frequencies of intragenic, and 5' and 3' markers of the dystrophin gene in Japanese families afflicted with Duchenne or Becker muscular dystrophy.

Using the polymerase chain reaction method (PCR), we examined the allele frequencies and heterozygosities of 7 polymorphic sites (pERT87, and CA polymorphisms in the 5' and 3' regions) of the dystrophin gene in 20 Japanese Duchenne muscular dystrophy and Becker muscular dystrophy (DMD or BMD) families consisting of 36 males, including 23 cases of DMD and BMD, and 28 females. The allele frequencies of three primer and enzyme sets in the pERT87 locus were well comparable to those in the previously reported Japanese female cases but different from in other countries. The frequencies of 5' markers of the dystrophin gene in Japanes were different from the reported Caucasian frequencies. As for 5'DYS-I and 5'DYS-II, the numbers of alleles in our cases were less than in Caucasians, and the heterozygosities of all three markers (5'DYS-I, II and III) were lower than in Caucasians. However, the 3'CA polymorphisms showed almost the same frequencies and heterozygosities as in Caucasians. All of our females showed a heterozygous pattern for at least one locus, with the combination of the seven markers. The usefulness of linkage analysis involving PCR methods with these intragenic, and 5' and 3' markers of the dystrophin gene in the carrier and prenatal diagnosis of DMD and BMD was confirmed by the successful prenatal diagnoses in 15 fetuses, the exception being one case considered to have a new mutation.

Alleles↗
PubMed1996

Production of the third and fourth component of complement (C3, C4) by smooth muscle cells.

Production of the third and fourth components of complement (C3, C4) by smooth muscle cells was investigated by using normal human aortic smooth muscle cells (AoSMC), human smooth muscle cell line (G402) and vascular smooth muscle cells obtained from human umbilical cord vein (UVSMC). AoSMC spontaneously produced both C3 and C4 at 15 ng/10(6) cells/72 hr and 22 ng/10(6) cells/72 hr, respectively, and both were enhanced by interferon-gamma (IFN-gamma). Although phorbol 12-myristate 13-acetate (PMA) and tumour necrosis factor-alpha (TNF-alpha) enhanced C3 production, C4 production was reduced by these agents. On the other hand, G402 produced C4 but not C3 in a dose-dependent manner when cultured with IFN-gamma. UVSMC produced only a small amount of C3 and C4 compared with AoSMC or G402. C3 and C4 produced by AoSMC were confirmed to be identical with their human serum counterparts as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and measurement of haemolytic activity. Northern blotting analysis showed that the expression of mRNA of C3 and C4 was enhanced by TNF-alpha and IFN-gamma, respectively, in AoSMC. Our findings suggest the importance of smooth muscle cells as a source of components of complement in vascular diseases including vasculitis.

Aorta↗
PubMed1996

Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe's disease.

The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5' end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG-->TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3'-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.

Amino Acid Sequence↗
PubMed1996

Evidence for translational control elements within the 5'-untranslated region of GLUT1 glucose transporter mRNA.

Recent studies have indicated that the blood-brain barrier GLUT1 glucose transporter is under post-transcriptional regulation. To begin functional mapping of the GLUT1 transcript, in the present investigation we studied the translational efficiency of capped full-length synthetic GLUT1 mRNA, and both 5'- and 3'-untranslated regions (UTRs) deleted GLUT1 mRNAs. Deletion of 5'- and 5'-/3'-UTRs markedly reduced the translation efficiency of the human (h) GLUT1 transcript in the rabbit reticulocyte lysate (RRL), and this effect was not modified by addition of microsomes to the translation system. The putative role of these hGLUT1 5'-UTR cis-acting elements was studied using the luciferase expression vector pGL2. DNA corresponding to the hGLUT1 5'-UTR generated by PCR was subcloned at the HindIII site of the pGL2 located upstream of the luciferase 5'-UTR. Transfection of brain endothelial cultured cells with pGL2 containing most of the hGLUT1 5'-UTR (nucleotides 1-171) markedly increased the expression of luciferase, and disruption of luciferase-leading sequence with an unrelated 171-nucleotide fragment decreased its expression. Insertion of nucleotides 1-96 of the hGLUT1 5'-UTR retained most of the stimulatory effect, and nucleotides 123-171 produced 64% of maximal induction. On the contrary, clones containing nucleotides 79-171 and 154-171 of bGLUT1 5'-UTR had marginal effects on luciferase expression. The present data provide evidence suggesting that the 5'-UTR of the GLUT1 mRNA contains cis-acting elements involved in the translational control of the GLUT1 gene in mammalian cells.

Animals↗
PubMed1996

Partial deletion of the long arm of chromosome 11: ten Japanese children.

The clinical features of partial deletion 11q were correlated with the size of the deleted region. Ten Japanese children with partial deletion of 11q were investigated. They were divided into three groups. Three patients in the first group had interstitial deletions and preserved subband q24.1. Six patients in the second group demonstrated terminal deletion of 11q including subband q24.1, with typical features of 11q- syndrome (Jacobsen syndrome). The third group included only one patient, who had terminal deletion of 11q without characteristics of typical 11q- syndrome. Prominent features of patients in the first group included severe mental and motor developmental delay, seizures, cleft lip and palate, and ophthalmological findings. Patients in the second group showed mild to moderate developmental delays without deterioration. Abnormalities in neuroimages, high intensity in the cerebral white matter in T2-weighted magnetic resonance (MR) images, and recurrent infections were not observed after the age of 7 years. The subject in the third group, with the smallest amount of deleted chromosome, did not show developmental delays, suggesting that some unknown genes related to developmental delays may be located adjacent to subband q24.1. Variation in the deleted parts of 11q resulted in different clinical features in each group.

Abnormalities, Multiple↗
PubMed1996

Effects of dietary polyunsaturated fat on ethanol-induced Ito cell activation.

Ito cells, vitamin A-storing perisinusoidal cells, are believed to undergo myofibroblastic transformation in liver fibrogenesis. Our previous studies have shown that a diet high in polyunsaturated fat was key for induction of experimental alcoholic liver fibrosis. To investigate the cellular basis for this fibrogenic effect of a high-fat diet, we analyzed the content of vitamin A and cellular retinol binding protein (CRBP), the steady-state mRNA levels of procollagen-alpha1(I), transforming growth factor-beta1 (TGF-beta1), interleukin-6 (IL-6), and smooth muscle alpha-actin (alpha-SM) in freshly isolated Ito cells from rats given isocaloric amounts of ethanol and a low- or high-fat diet. After 10 wk, the Ito cell content of retinyl palmitate was severely reduced in both the high- and low-fat diet-ethanol-fed animals to 13-17% of those measured in respective pair-fed controls. On the other hand, the content of CRBP was reduced in the high-fat-ethanol rats but not in the low-fat-ethanol group. The cells from the high-fat-ethanol but not low-fat ethanol rats showed an 18-fold increase in procollagen-alpha1(I) mRNA at 17 wk, which was accompanied by 2.8- and 2.3-fold enhancement of TGF-beta1 and alpha-SM transcripts. IL-6 mRNA was not detected in the cells from any groups. These results demonstrate 1) myofibroblastic activation of Ito cells is evident in rats given a high-fat diet and ethanol but not in the low-fat-ethanol animals; 2) vitamin A depletion of Ito cells is the early and general effect of chronic ethanol intake but does not necessarily predict subsequent myofibroblastic activation; 3) reduced CRBP level is more closely associated with the subsequent cellular activation seen under the high-fat-ethanol regimen; and 4) IL-6 is not expressed in vivo by Ito cells from either normal livers or livers with alcoholic liver fibrosis.

Animals↗
PubMed1996

Effect of MCI-186 on postischemic reperfusion injury in isolated rat heart.

MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) is a newly developed antioxidant which has been shown to reduce brain edema in cerebral ischemia through inhibition of the lipoxygenase pathway of arachidonic acid. However, its effect on myocardial reperfusion injury after prolonged ischemia has not yet been demonstrated. We compared the mode of the effect of MIC-186 and recombinant human CuZn superoxide dismutase (rh-SOD) in isolated perfused rat hearts subjected to 60-min ischemia followed by 60-min reperfusion. Left ventricular developed pressure (LVDP), necrotic area and the release of creatine phosphokinase (CPK) and endogenous CuZn superoxide dismutase (endoge-SOD) were measured to evaluate myocardial damage. The decrease in left coronary flow (CBF) was measured as an index of the damage of left coronary circulation. MCI-186 (14.5 mg/L) was perfused for 10 min in the MCI group and rh-SOD (70 mg/L) was perfused during the reperfusion period in the SOD group starting 5 min prior to reperfusion. The release patterns of CPK and endoge-SOD were analyzed to elucidate the difference in the mode of protection of MCI-186 and rh-SOD. The LVDP remained higher in both MCI and SOD groups than that of control (76 +/- 1, 77 +/- 2 and 69 +/- 1% of preischemic value, respectively). The necrotic area was significantly attenuated in both MCI and SOD groups compared with that in the control group (16 +/- 1, 14 +/- 1 and 32 +/- 1%, respectively, p < 0.05). Total CPK release was lower in both MCI and SOD groups than in the control (78 +/- 7, 100 +/- 2 and 116 +/- 4 x 10(3) units/g myocardium respectively). The decrease in CPK release was more marked in the MCI group than that in the SOD group (p < 0.05). The reduction in CBF was significantly attenuated by the treatment with rh-SOD or MCI-186, but the effect was much higher in the SOD group than in the MCI group (69 +/- 5, 58 +/- 2, and 48 +/- 2% in SOD, MCI and control groups, respectively). The release pattern of endoge-SOD was identical to that of CPK and thus this did not distinguish the mode of effect of MCI-186 from that of rh-SOD. These results indicate that MCI-186 reduces reperfusion injury in isolated perfused hearts with prolonged ischemia and the effect is more closely related to the reduction of myocyte damage than the preservation of the coronary circulation.

Animals↗
PubMed1996

Genetic characterization of protein C deficiency in Japanese subjects using a rapid and nonradioactive method for single-stand conformational polymorphism analysis and a model building.

We studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5'-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phe139Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Arg169Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.

Amino Acid Sequence↗
PubMed1995

Urinary C4 excretion in systemic lupus erythematosus.

The fourth component of complement (C4) in urine was measured in 19 patients with systemic lupus erythematosus (SLE). Urinary C4 was detectable in all SLE patients using an enzyme linked immunosorbent assay. In 11 of 13 patients whose urinary C4 was serially measured, a decrease of urinary C4 was found in parallel with a decrease of disease activity of SLE. In the remaining 2 patients, the amount of C4 increased preceding a flare-up of the disease. The measurement of urinary C4 may be useful in evaluating the disease activity of SLE in individual patients and sometimes in predicting the flare-up of the disease. The specific hemolytic activity of excreted C4 and Western blotting analysis showed that urinary C4 consisted mainly of degraded fragments of C4. In two cases, camostat, a serine protease inhibitor, rapidly decreased the urinary C4 excretion, suggesting that the complement activation occurred in the glomerulus in lupus nephritis.

Adolescent↗
PubMed1995

Tumor-associated glycoantigen, sialyl Lewis(a) as a target for bispecific antibody-directed adoptive tumor immunotherapy.

The KM231 mAb recognizing sialyl Lewis(a) (sLe(a)) epitope of glycoprotein or glycolipid expressed on various human cancers was used to prepare bispecific antibody (BSAb) containing anti-CD3 x anti-sLe(a) mAb. The effect of anti-CD3 x anti-sLe(a) BSAb on the induction of cytotoxicity by activated T cells was investigated. The activated CD3+ T cells expressing CD8 or CD4 were induced from human peripheral blood mononuclear cells by culture with recombinant IL-2 plus immobilized anti-CD3 mAb. The activated CD8+ and CD4+ T cells showed marginal cytotoxicity against tumor cells by themselves. However, addition of anti-CD3 x anti-sLe(a) BSAb resulted in a great augmentation of their cytotoxicity against gastrointestinal tumor cells. The BSAb also triggered IL-2 production of CD4+ helper/killer T cells during lysis of tumor cells. Moreover, the BSAb was demonstrated to have a potent in vivo antitumor activity against human colon cancer implanted in nude mice by combination with CD4+ helper/killer cells. These results demonstrated that sLe(a) antigen might be a good target molecule for BSAb-directed adoptive tumor immunotherapy.

Animals↗
PubMed1995

Non-iron mediated alteration in hepatic transferrin gene expression in the nephrotic rat.

Both transferrin and the iron it carries are lost in the urine in the nephrotic syndrome. Patients may develop hypochromic microcytic anemia and synthesis of transferrin, a protein regulated in large part by iron availability, is increased. Transferrin synthesis has also been reported to be increased in liver slices from rats with hereditary analbuminemia, and their plasma transferrin levels are increased, suggesting that transferrin synthesis may be stimulated by processes other than iron depletion in this hypoalbuminemic condition. Transferrin metabolism was studied in rats with Heymann nephritis (HN), in a strain of Sprague-Dawley (SD) rats with hereditary analbuminemia [Nagase analbuminemic rats (NAR)], and in normal SD rats. Plasma transferrin concentration and mass was decreased significantly in HN, but increased in NAR. Transferrin synthesis was increased both in NAR (measured either as the disappearance of [125I] labeled transferrin or as the incorporation of [3H] phenylalanine) and in HN (incorporation of [3H] phenylalanine). The fractional rate of transferrin catabolism was unchanged in NAR. Thus transferrin mass was increased in NAR entirely as a consequence of increased synthesis. Transferrin and albumin synthesis correlated with one another in both HN and SD (P < 0.001). Transferrin mRNA was increased in both HN and NAR and was unaffected by administration of iron to HN. Hepatic transferrin and albumin mRNA levels were also correlated positively in HN and SD, suggesting that increased hepatic synthesis of both proteins might be responding to the same stimuli. Transferrin gene transcription was increased in both HN and NAR and was unaffected by administration of iron to HN. Transferrin mRNA was not increased in the testis in either HN or NAR, suggesting that augmentation in transferrin gene expression is driven by a non-iron dependent process and is confined to the liver.

Albumins↗
PubMed1995

Molecular defects in Krabbe disease.

Krabbe disease (globoid cell leukodystrophy) is an autosomal recessive neurodegenerative disorder that affects both the central and peripheral nervous systems due to an enzymatic defect of the galactocerebrosidase. In this study, molecular defects in Krabbe disease were investigated in 11 patients (seven Japanese and four non-Japanese) using cultured skin fibroblasts. A Japanese late infantile patient had a missense mutation of Pro at codon 302 to Ala and a non-Japanese patient had a missense mutation of Val at codon 550 to Gly. The reduced enzymatic activities expressed from the cDNAs with these missense mutations and from the previously reported nonsense mutation (E369X, Glu at codon 369 to stop codon) were confirmed. Genomic DNA analyses revealed that the P302A and E369X mutations were heterozygous and the V550G mutation was homozygous in these patients. A 12 base deletion with a 3 base insertion was found in three unrelated Japanese infantile patients, but not in 30 controls. The mutation was homozygous in two patients and heterozygous in one patient. We could not find any confirmed mutation in the coding region in the other six patients. These findings suggest that mutations in infantile and late infantile patients are relatively heterogeneous.

Amino Acid Sequence↗
PubMed1995

Roles of oxidative stress in activation of Kupffer and Ito cells in liver fibrogenesis.

An increasing body of experimental evidence is emerging to incriminate oxidative stress as a pivotal signal for liver fibrogenesis. This paper reviews the results from our studies testing this hypothesis. In the rat model of alcoholic liver disease, the importance of oxidative stress was supported by marked accentuation of liver fibrosis by dietary supplementation of iron, a pro-oxidant, and the significant correlation of the liver malondialdehyde (MDA) and 4-hydroxynonenal (4HNE) levels with the hepatic collagen accumulation. Both MDA and 4HNE adduct epitopes were detected intensely and diffusely in close association with collagen deposition. The direct cause and effect relationship between MDA/4HNE and Ito cell stimulation was indicated by the demonstration of Ito cell collagen gene induction by these aldehydes in culture. In primary cultures of rat Kupffer cells (KC), addition of antioxidants such as alpha-tocopherol acetate and succinate suppressed mRNA expression and the release of interleukin (IL)-6 and tumour necrosis factor alpha (TNF alpha). In rats with biliary fibrosis, an increase in the liver MDA level was accompanied by enhanced mRNA expression of procollagen alpha 1(I) and transforming growth factor beta 1 in Ito cells; and that of TNF alpha and IL-6 in KC. Furthermore, the gel shift assay of KC nuclear extracts showed enhanced NF-kB DNA binding activity. These results support the proposal that enhanced oxidative stress constitutes an important signal for activation of Kupffer and Ito cells in experimental liver fibrogenesis.

Aldehydes↗