Biomedical subjects

H Nishihara

Publications and source records attributed to H Nishihara.

At least 37 records · Page 2Linked to original sources

PubMed1999

Inflammatory cytokines induced down-regulation of m-calpain mRNA expression in fibroblastic synoviocytes from patients with osteoarthritis and rheumatoid arthritis.

Our previous reports revealed that calpain has proteoglycanase activity and exists in synovial fluid in osteoarthritis and rheumatoid arthritis. We examined the effects of cytokines on expression of the calpain-calpastatin system in fibroblastic synoviocytes (FLS). Primary cultures of human FLS from osteoarthritis (OA) and rheumatoid arthritis (RA) patients were stimulated with inflammatory cytokines and the amounts of m-calpain and calpastatin mRNAs expressed were determined by Northern blotting. Northern blots were subjected to computerized densitometer and band intensities were determined. Interleukin-1 (IL-1) down-regulated m-calpain and tissue-type calpastatin mRNA expression in OA and RA FLS. In RA FLS, although IL-6 did not alter m-calpain mRNA expression, IL-1 + tumor necrosis factor (TNF) and IL-1 + transforming growth factor (TGF) down-regulated m-calpain mRNA expression. These results provide new information about the effects of inflammatory cytokines on calpain and calpastatin system in OA and RA pathology.

Arthritis, Rheumatoid↗

PubMed1999

Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins.

Human DOCK180, which was originally identified as a major protein bound to the Crk oncogene product, is an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. After DOCK180, at least three putative human proteins that manifest high amino acid sequence similarity to DOCK180 have been registered in the GenBank/EMBL database. We have designated one of them, KIAA0209, as DOCK2 and characterize here. DOCK2 mRNA was expressed mostly in peripheral blood cells, followed by slight expression in the spleen and thymus, whereas DOCK180 was expressed in all tissues tested except in peripheral blood cells. Immunostaining of human cadaver tissues revealed that the expression of DOCK2 was limited to the lymphocytes and macrophages of various organs. DOCK2 bound to and activated Rac1, as did DOCK180; however, DOCK2 did not bind to CrkII, which transduces signals at focal adhesions. Thus, DOCK180 and DOCK2 are regulators of Rac and function in adherent and non-adherent cells, respectively.

Amino Acid Sequence↗

PubMed1999

Cloning and characterization of mdc genes encoding malonate decarboxylase from Pseudomonas putida.

The DNA fragment encoding malonate decarboxylase, involved in malonate assimilation, was cloned from Pseudomonas putida. The 11-kb DNA fragment contained nine open reading frames, which were designated mdcABCDEGHLM in the given order. N-terminal protein sequencing established that the mdcA, mdcC, mdcD, mdcE and mdcH genes encoded subunits alpha, delta, beta, gamma and epsilon of the malonate decarboxylase, respectively. Malonate decarboxylase was functionally expressed in Escherichia coli from plasmid harboring the entire gene cluster or the mdc genes lacking the mdcL and mdcM genes. The mdcL and mdcM genes encode membrane proteins and disruption of the genes of P. putida by the insertion of a kanamycin resistance cassette reduced the malonate uptake activity of the organism. Thus, we conclude that MdcLM is a malonate transporter.

Carboxy-Lyases↗

PubMed1999

Adult Leigh syndrome with mitochondrial DNA mutation at 8993.

Adult onset Leigh syndrome with a nucleotide (nt) 8993 mutation in mitochondrial (mt) DNA is reported. A 43-year-old woman with a 6-year-history of insulin-resistant diabetes mellitus developed muscular weakness, intractable nausea and vomiting, and anemia. These were followed vertigo, blindness, and deafness with nystagmus. Magnetic resonance imaging (MRI) revealed abnormal high intensities in the bilateral medial regions of the thalamus and periaqueductal gray matters. Autopsy disclosed well-demarcated necrotizing lesions with prominent capillaries in the areas detected by MRI, which were sufficiently diagnostic for Leigh syndrome. MtDNA analysis performed on DNAs extracted from formalin-fixed tissues including liver, heart, brain, muscle, kidney and pancreas showed a T-->G mutation at nt 8993. This is the first case of adult Leigh syndrome demonstrating on mtDNA mutations.

Adult↗

PubMed1999

Analysis of hematopoietic cell specific protein, M-DOCK.

Full length cDNA of M-DOCK was isolated at Kazusa DNA Institute and its sequence has been deposited as KIAA0209. The cDNA sequence of M-DOCK contains a 5490 bp open reading frame that encodes a 1830-aa protein with a calculated molecular mass of 212 kDa. The amino acid sequence of M-DOCK has 62.3% identity with DOCK180, excluding the carboxyl terminal variable regions. DOCK180, which was originally identified as a major protein bound to Crk oncogene product, is an archetype of a family of proteins including Ced-5 of C. elegans and Mbc of D. melanogaster. DOCK180 is localized at focal adhesions and regulates cell morphology through the activation of Rac. Expression of DOCK180 is ubiquitous except in hematopoietic cells. Here, the author reports on the characterization of M-DOCK protein, which is closely related to DOCK180 but expressed only in the hematopoietic cells. We immunized rabbits with bacterially-expressed GST-M-DOCK protein, and obtained an anti-serum specific to M-DOCK. Tissue distribution of M-DOCK was examined by Northern blotting and Western blotting. M-DOCK was expressed abundantly in peripheral blood leukocytes. M-DOCK expression was detected also in the cell lines derived from T-cells, B-cells, and monocytes, but never in the adherent cells. Most of the lymphocytes and macrophages in human organs expressed M-DOCK diffusely in the cytoplasm, which was analyzed by immunohistochemistry using an anti-M-DOCK antibody. We previously reported that membrane-targeted form of DOCK180 induced spreading of NIH 3T3 cells. By contrast, overexpression of M-DOCK rounded up flat NRK cells. This suggests that M-DOCK may regulate cell detachment of adherent cells. These results demonstrate that M-DOCK is a new member of DOCK180-family proteins and regulates cell shape in suspension cells.

Amino Acid Sequence↗

PubMed1998

Malonate decarboxylase of Pseudomonas putida is composed of five subunits.

Two different forms of malonate decarboxylase were purified from Pseudomonas putida. The active form was composed of the five different subunits alpha (60 kDa), beta (33 kDa), gamma (28 kDa), delta (13 kDa), and epsilon (30 kDa) and the inactive form was composed of the four subunits lacking the epsilon subunit. The former catalyzed the decarboxylation of malonate to acetate, but the latter could not, although it retained both activities of acetyl-CoA:malonate CoA transferase and malonyl-CoA decarboxylase. The delta subunit of the active form was acylated by the incubation with [2-14C]malonyl-CoA, but the delta subunit of the inactive form was not labeled. From the above results and the N-terminal amino acid sequence analysis, it was concluded that the epsilon subunit was an essential subunit to function as malonyl-CoA:ACP transacylase, which was an indispensable component of the enzyme for the cyclic decarboxylation of malonate.

Acyl-Carrier Protein S-Malonyltransferase↗

PubMed1998

Thymic B-cell non-Hodgkin's lymphoma in a child.

A 13-year-old male developed thymic non-Hodgkin's lymphoma. Microscopically, the tumor was composed of large cells, resembling centroblasts. Immunohistochemically, the tumor demonstrated leukocyte common antigen+, L26 (B-cell)+, UCHL1 (T-cell)-, suggesting the B-cell phenotype. In contrast to the terminally differentiated phenotype (CD10-, surface immunoglobulin-) observed in adult cases, flow cytometric analysis showed that they were relatively immature: CD10+, CD19+, HLA-DR-, IgM+/-, kappa+. He was successfully treated with intensive chemotherapy. Since childhood thymic lymphomas are exclusively small non-cleaved cell lymphoma with T-cell phenotype, this case represents a unique entity in children.

Adolescent↗

PubMed1998

Phylogenetic position of an obligately chemoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, on the basis of 16S rRNA gene sequences and two form I RuBisCO gene sequences.

Two form ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes from the obligately autotrophic, marine hydrogen oxidizer Hydrogenovibrio marinus were sequenced. The deduced amino acid sequences of both RuBisCOs revealed that they are similar to those of sulfur oxidizers (Thiobacillus) and a purple sulfur bacterium (Chromatium vinosum). According to the 16S rRNA gene sequences, H. marinus is also affiliated with these microorganisms, members of Thiomicrospira being the closest relatives. Sequence similarities of the 16S rRNA genes and of the RuBisCO genes among these gamma-Proteobacteria suggest a common autotrophic ancestry. An ancestor of purple sulfur bacteria might be a common root of H. marinus and related sulfur oxidizers.

Bacterial Proteins↗

PubMed1998

Constitutive association of EGF receptor with the CrkII-23 mutant that inhibits transformation of NRK cells by EGF and TGF-beta.

Crk belongs to the adapter proteins that participate in many signalling pathways from cell surface receptors. We have characterised the CrkII-23 mutant that inhibits the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. To study the biochemical difference, cDNAs of the wild-type CrkII and the CrkII-23 mutant were introduced stably into NIH 3T3 cells expressing EGF receptor (EGFR). Both CrkII and CrkII-23 were phosphorylated on tyrosine upon EGF simulation with similar time course and dose dependency. Whereas the wild-type CrkII bound to EGFR only after EGF stimulation, CrkII-23 bound to EGFR from before stimulation. Mutation in the Src homology (SH) 2 or amino-terminal SH3 domain did not abolish the binding of CrkII-23 to EGFR in the quiescent cells, suggesting that the binding is mediated by a novel mechanism. These CrkII-23-derived mutants, however, did not suppress transformation of NRK cells by EGF and TGF-beta. Hence, both the SH2 and amino-terminal SH3 domains are required to inhibit transformation of NRK cells. These results suggest that persistent signalling from CrkII-23 bound to EGFR suppresses transformation by EGF and TGF-beta in NRK23 cells.

3T3 Cells↗

PubMed1998

Epstein-Barr virus-associated Hodgkin's disease in HTLV-I seropositive patients: a report of two cases.

Diagnosis of Hodgkin's disease (HD) is quite difficult in the patient with seropositivity for human T cell lymphotropic virus I (HTLV-I). Herein, two cases of Epstein-Barr virus (EBV)-associated HD, which occurred in males with seropositivity for anti-HTLV-I, are reported. One patient is alive and was diagnosed as having interfollicular HD with CD20+CD15-CD30-CD3-CD4-CD8-CD45RO-Read-Sternberg (R-S) cells. Positivity for EBV-encoded RNA 1 (EBER-1) and latent membrane protein 1 (LMP-1) was shown on follicular germinal center cells and R-S cells. In that case, neither T cell receptor (TCR) beta chain rearrangement nor integration of the HTLV-I provirus was demonstrated in the lymph nodes, although atyical lymphocytes (2%) were found in the peripheral blood. The other case pursued an aggressive clinical course and the patient was diagnosed as having an adult T cell leukemia/lymphoma (ATLL) because of the presence of anti-HTLV-I antibody, lymph node swelling, and the appearance of flower-like cells in the peripheral blood. However, an autopsy revealed no obvious ATLL cell infiltration in any of the organs examined. Multiple granulomatous lesions were found in the bone marrow, liver, kidneys, spleen, and lymph nodes. Reassessment of lymph node lesions in biopsies and granulomatous lesions in autopsy samples demonstrated that both lesions contained CD15+CD30+CD3-CD4-CD8-CD20-CD45RO-EBER-1+L MP-1+R-S cells, and they were considered to be a composite lymphoma of HD and ATLL. These two cases therefore suggest that EBV-associated HD can develop in patients with seropositivity for HTLV-I.

Antigens, CD↗

PubMed1998

Expression of cytotoxic molecule TIA-1 in malignant lymphomas mimicking fulminant hepatitis.

Involvement of malignant lymphoma in the liver inducing fulminant hepatic failure has rarely been reported. Therefore, a close association between some lymphoma types with severe liver damage and the mechanism underlying the liver damage is intriguing. Three malignant lymphoma cases, which were clinically diagnosed as fulminant hepatitis, were collected from the autopsy records of Kawasaki Medical School (Kurashiki, Japan). All three cases were characterized by the presence of hepatosplenomegaly without superficial lymph node swelling, high elevation of transaminase and lactate dehydrogenase (LDH; especially LDH-2), and a quite aggressive clinical course. Immunohistochemically, the tumor cells in all three cases were positive for T cell intracellular antigen (TIA-1), which is a cytolytic protein in cytotoxic T and natural killer (NK) cells. The lymphomas were CD8+ peripheral T cell lymphoma (case 1), CD56+ T/NK cell lymphoma (case 2), and T cell lymphoma in a patient with mosquito hypersensitivity (case 3). Epstein-Barr virus infection was demonstrated on the tumor cells of cases 2 and 3 using an in situ hybridization method and those cases showed high titers of serum interferon-gamma and Fas. Frequent apoptosis of liver cells, where the lymphoma cells had infiltrated, was revealed by a terminal deoxyribosyl transferase-mediated deoxyuridine nick end-labeling (TUNEL) method. The findings in this study suggest that fulminant hepatic injury is closely associated with cytotoxic molecule TIA-1 expression of the lymphoma cells and that some specific mechanism may be involved in liver damage.

Adult↗

PubMed1998

Changes in size of intracellular pools of coenzyme A and its thioesters in Escherichia coli K-12 cells to various carbon sources and stresses.

Intracellular pools of three CoA molecular species of coenzyme A, CoASH, acetyl-CoA, and malonyl-CoA, in Escherichia coli K-12 cells were studied by acyl-CoA cycling method in replacement culture. The sizes and compositions of CoA pools starved for a carbon source changed within minutes after the addition of one of various carbon sources. A large acetyl-CoA pool formed after the addition of D-glucose, D-fructose, D-mannose, glycerol, or sorbitol, but there was little change when L-glucose, sucrose, maltose, succinate, or acetate was added. The beta-anomer of D-glucose was assimilated 10 times faster than the alpha-anomer. Intracellular CoA pools also changed with stress: in the pH, incubation temperature, or with osmotic stress. The sizes and compositions of CoA pools were not affected by pH changing between 4 and 8, but the breakdown of acetyl-CoA and CoASH was greater at pH 9 than at pH 4 to 8. Production of acetyl-CoA was greatest at 40 degrees C, and at 50 degrees C, an acetyl-CoA pool did not form at all and the size of the CoASH pool declined. When the organism was stressed by the addition of NaCl at concentrations of more than 0.6 M, little acetyl-CoA was produced. The total CoA pool (the sum of the concentrations of CoASH, acetyl-CoA, and malonyl-CoA) remained within the limits of 0.83-1.40 nmol/mg of dry cell weight (0.30-0.52 mM). Whenever acetyl-CoA increased, CoASH decreased. Therefore, the acetyl-CoA/CoASH ratio is an important index of facultative anaerobes that reflects the state of carbon and energy metabolism in vivo.

Acetyl Coenzyme A↗

PubMed1997

Biotechnological potential of P450 monooxygenases high-level production of bovine cytochrome P450c17 monooxygenase during medium cell density culture of a recombinant yeast, Saccharomyces cerevisiae GRF 18 (YEp-Toku1).

Bovine cytochrome P450c17 monooxygenase was produced in a 30-L fermenter by Saccharomyces cerevisiae GRF18 (YEp-Toku1), harboring the GAL10 promoter, and using conditions of medium cell density culture. Upon addition of D-galactose as an inducer and FeCl3 as a cofactor, cells began to produce the P450 hemoprotein. The yield of this enzyme reached a maximum after 31 h but its formation continued for more than 60 h after induction. The amount of P450c17 produced was 4.7-fold as compared to shake flask experiments.

Cell Count↗

PubMed1997

Characterization of an extremely thermophilic and oxygen-stable membrane-bound hydrogenase from a marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus.

The membrane-bound hydrogenase from a marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus was characterized as highly oxygen-tolerant, extremely thermophilic and thermostable in its membrane-bound form. The optimum temperatures for H2 oxidation and H2 evolution were 90 and 80 degrees C, respectively. The enzyme retained 90% of its activity after heating at 70 degrees C for 50 min under air and retained full activity at 90 degrees C for 80 min under hydrogen. The optimum pH values were 5.5 (H2 evolution) and 9.4 (H2 oxidation), and the activity ratio (H2 evolution/H2 oxidation) was high (50.3) at pH 5.5. The hydrogen evolution from reduced methyl viologen continued at high temperatures and acidic pH with the continuous addition of sodium dithionite.

Cell Membrane↗

PubMed1997

Successful hematopoietic reconstitution by transplantation of umbilical cord blood cells in a transfusion-dependent child with Diamond-Blackfan anemia.

A 4-year-old boy with Diamond-Blackfan anemia and a history of multiple transfusions underwent umbilical cord blood transplantation from his HLA-identical female sibling born by vaginal delivery at 38 weeks. The patient was prepared with busulfan, cyclophosphamide and antilymphocyte globulin. Methotrexate and cyclosporin A were given for the prophylaxis of GVHD. Regimen-related toxicity was not observed and successful engraftment occurred, including the erythroid series. No evidence of acute or chronic GVHD has been observed for 14 months after transplantation. This is the first case of successful umbilical cord blood transplantation to a patient with Diamond-Blackfan anemia.

Blood Transfusion↗

PubMed1997

Changes in the size and composition of intracellular pools of nonesterified coenzyme A and coenzyme A thioesters in aerobic and facultatively anaerobic bacteria.

Intracellular levels of three coenzyme A (CoA) molecular species, i.e., nonesterified CoA (CoASH), acetyl-CoA, and malonyl-CoA, in a variety of aerobic and facultatively anaerobic bacteria were analyzed by the acyl-CoA cycling method developed by us. It was demonstrated that there was an intrinsic difference between aerobes and facultative anaerobes in the changes in the size and composition of CoA pools. The CoA pools in the aerobic bacteria hardly changed and were significantly smaller than those of the facultatively anaerobic bacteria. On the other hand, in the facultatively anaerobic bacteria, the size and composition of the CoA pool drastically changed within minutes in response to the carbon and energy source provided. Acetyl-CoA was the major component of the CoA pool in the facultative anaerobes grown on sufficient glucose, although CoASH was dominant in the aerobes. Therefore, the acetyl-CoA/CoASH ratios in facultatively anaerobic bacteria were 10 times higher than those in aerobic bacteria. In Escherichia coli K-12 cells, the addition of reagents to inhibit the respiratory system led to a rapid decrease in the amount of acetyl-CoA with a concomitant increase in the amount of CoASH, whereas the addition of cerulenin, a specific inhibitor of fatty acid synthase, triggered the intracellular accumulation of malonyl-CoA. The acylation and deacylation of the three CoA molecular species coordinated with the energy-yielding systems and the restriction of the fatty acid-synthesizing system of cells. These data suggest that neither the accumulation of acetyl-CoA nor that of malonyl-CoA exerts negative feedback on pyruvate dehydrogenase and acetyl-CoA carboxylase, respectively.

Acetyl Coenzyme A↗

PubMed1997

Random amplified polymorphic DNA (RAPD) analyses for discriminating genotypes of Microcystis cyanobacteria.

Random amplified polymorphic DNA (RAPD) analysis was used to discriminate genotypes in five species of Microcystis cyanobacteria. Strains of each group with the identical allozyme genotype (T. Kato et al., Algol. Stud., 1991, 129-140; M. Watanabe, in "Toxic Microcystis," ed. by M.F. Watanabe et al., CRC Press, Tokyo, 1966, pp. 13-34) gave similar RAPD patterns characterizing the respective group. On the other hand, no similarities in RAPD patterns were observed among strains of which allozyme genotypes were different. A good accordance between the RAPD analysis and allozyme divergence indicated a high reliability of both methods for discrimination of the affiliated groups of Microcystis. Several amplified DNA fragments, which were expected to be markers for a particular taxon with identical allozyme genotype, were also observed on the RAPD patterns. Genetic homogeneities of M. novacekii, M. viridis, and M. wesenbergii were shown by RAPD analysis as well as the allozyme genotype. However, significant variations were observed in M. aeruginosa and M. ichthyoblabe in the levels of DNA and proteins (allozymes).

DNA Primers↗

PubMed1997

Elevation of plasma lipid peroxides in non-insulin dependent diabetics with multiple lacunar infarcts.

Our purpose was to determine whether lipid peroxides are elevated in the plasma of patients with non-insulin dependent diabetes with multiple lacunar infarcts as detected by magnetic resonance imaging (MRI), and to confirm whether peroxide levels correlate with glycemic controls and blood lipid levels. The level of lipid peroxide (measured as thiobarbituric acid reactive substances (TBARS)) was measured in 23 healthy controls and 28 diabetics showing normal MRI findings and 22 diabetics with multiple lacunar infarcts. These groups were age-matched. In patients with multiple lacunar infarcts, systolic blood pressure, diastolic blood pressure and TBARS levels were significantly higher than in diabetics without such infarcts (p < 0.05). When the diabetic patients were divided into two groups according to the presence or absence of hypertriglyceridemia or hyperglycemia, in both groups plasma TBARS levels in patients with multiple lacunar infarcts were significantly higher than in patients without such infarcts. Multivariate analysis showed systolic blood pressure and plasma TBARS levels to be independent predictors of multiple lacunar infarcts. Among diabetics, total plasma TBARS levels were positively correlated with fasting blood glucose, HbA1c and triglyceride levels, but not with total cholesterol levels and age. In conclusion plasma lipid peroxides were elevated in diabetics with multiple lacunar lesions, and are related to the metabolic imbalance of plasma glucose and lipids.

Aged↗