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Biomedical subjects

T Kabashima

Publications and source records attributed to T Kabashima.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cloning, sequencing, and expression of the dipeptidyl peptidase IV gene from Flavobacterium meningosepticum in Escherichia coli.

The dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) gene from Flavobacterium meningosepticum was cloned by Southern and colony hybridizations using probes amplified by PCR, and expressed in Escherichia coli DH1. E. coli DH1 harboring pFDP-H1, which was a subclone derived from the positive clone pFDP-1, showed 3.5-fold higher activity than F. meningosepticum. Nucleotide sequencing analysis revealed an open reading frame of 2133 bp, coding for a protein of 711 amino acids with a predicted molecular weight of 80,626. The expressed enzyme in E. coli DH1/pFDP-H1 was purified about 345-fold with an activity recovery of 12.3%. The molecular weight of the purified enzyme was estimated to be 75,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160,000 by gel filtration, respectively, suggesting a dimeric form of the native enzyme. The deduced amino acid sequence of DP IV was homologous to those of the serine proteases of the "prolyl endopeptidase family." A sequence near the C-terminal region and the putative catalytic triad residues were well conserved among these enzymes.

Amino Acid Sequence↗
PubMed1995

Protease II from Moraxella lacunata: cloning, sequencing, and expression of the enzyme gene, and crystallization of the expressed enzyme.

A gene coding for protease II (basic amino acid specific oligoendopeptidase) from Moraxella lacunata was cloned and expressed in Escherichia coli DH1. The transformant harboring a hybrid plasmid, pMPROII-12, with a 3.0-kbp insert at the PvuII-SacI site in pUC19, showed 23-fold higher enzyme activity than M. lacunata. The expressed enzyme from E. coli DH1/pMPROII-12 was purified by 40-80% ammonium sulfate fractionation, chromatography on DEAE-Toyopearl, and Sephadex G-150 gel filtration. The enzyme was most active at pH 6.5 and stable at pH 6.5-9.5. It had an optimum temperature of 35 degrees C for 5 min of reaction and was stable to up to 35 degrees C for 30 min at pH 7.0. Its molecular weight was estimated to be 80,000 by SDS-PAGE and gel-filtration analyses. It enzyme was inhibited by diisopropyl fluorophosphate (DFP) and classified as a serine endoprotease. Its amino acid sequence was 38% homologous to that of the E. coli protease II. By alignment with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-534, Asp-619, and His-654. The enzyme was crystallized by the hanging drop vapor diffusion method using PEG 4000 as precipitant.

Amino Acid Sequence↗
PubMed1995

Prolidase from Xanthomonas maltophilia: purification and characterization of the enzyme.

Prolidase (iminodipeptidase, EC 3.4.13.9) was purified from an extract of Xanthomonas maltophilia, by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Toyopearl HW65C, FPLC-Hiload Superdex 200 pg, and FPLC-Hitrap Q columns, which an activity recovery of 2.3%. The enzyme was the most active at pH 7.5 with Leu-Pro as substrate. It was stable between pH 6.0 and 8.5 for 60 min at 37 degrees C and retained half of activity after 60 min at 37 degrees C. The isoelectric point of the enzyme was 3.7. Its molecular weight was estimated to be 100,000 by gel filtration on FPLC-Hiload Superdex 200 and 51,000 by SDS-PAGE, suggesting that it is a dimer. It hydrolyzed dipeptides only if proline is located at the carboxyl terminal position. The enzyme was inhibited by PCMB and o-phenanthroline, and was activated by Mn2+.

Chromatography, Ion Exchange↗
PubMed1993

Pyroglutamyl peptidase gene from Bacillus amyloliquefaciens: cloning, sequencing, expression, and crystallization of the expressed enzyme.

The pyroglutamyl peptidase [EC 3.4.11.8] gene from Bacillus amyloliquefaciens was cloned and expressed in Escherichia coli DH1. The transformant of E. coli DH1 harboring plasmid pBPG 1 with a 2.1 kb chromosomal DNA fragment showed 80-fold higher activity than B. amyloliquefaciens. The nucleotide sequence of a 0.9 kb fragment that contains the promoter and the mature protein coding region was determined by the dideoxy chain-termination method. An open reading frame of 648 bp starting with an ATG methionine codon was found, which encodes a protein of 215 amino acid residues with a deduced molecular weight of 23,286. The enzyme has two cysteine residues (Cys68 and Cys144) per subunit molecule. Substitution of Cys144 with Ser by site-directed mutagenesis resulted in a complete loss of the activity, while that of Cys68 with Ser did not affect the activity at all. This result and titration with DTNB suggest that Cys144 is concerned in the catalytic action and Cys68 is located inside the enzyme. The expressed enzyme was purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with an activity recovery of 42.7%. The enzyme was most active at pH 6.5 and stable at pH 7.0-9.0. Its molecular weight was estimated to be 51,000 by gel filtration, suggesting it to be a dimer. Big crystals of the wild and PCMB-modified enzymes were obtained by the hanging drop method.

Amino Acid Sequence↗
PubMed1987

Intravenous gammaglobulin therapy for thromboneutropenic neonates of mothers with systemic lupus erythematosus.

i.v. IgG therapy was used to treat thromboneutropenia in 2 male neonates of mothers with SLE. The mothers of both neonates were thrombocytopenic but not neutropenic during the last 3 months of their pregnancies. Anti-platelet and anti-neutrophil IgG directed against platelets and neutrophils from the neonates were detected in the sera of both mothers. The platelet and neutrophil counts increased significantly with i.v. IgG therapy in both neonates. i.v. IgG therapy is a safe and effective regimen for passive immune thromboneutropenia.

Agranulocytosis↗
PubMed1987

Local bone resorption induced by serially transplantable human renal cell carcinoma in nude mice.

The high incidence of metastatic bone disease in urological cancer makes it necessary for clinicians to look for a valid experimental model to investigate the basic interactions between cancer cells and bone in order to improve the treatment. A new model of bone resorption was defined, namely the subcutaneous injection of tumor cell suspensions of serially transplanted renal cell carcinoma in nude mice after disruption of the periosteum of the calvaria. The tumor induced osteolysis associated with osteoclast proliferation with reactive bone formation. The X-p film from nude mice calvaria showed the same multiple osteolytic punched-out lesions as those of the patient's skull.

Aged↗
PubMed1986

Monocyte-mediated suppression of T lymphocyte blastogenesis and its reversal by deoxyguanosine. Defects in patients with systemic lupus erythematosus.

We investigated monocyte-mediated suppression of T lymphocyte blastogenesis in normals and patients with systemic lupus erythematosus (SLE). When monocytes from normals were cocultured with autologous T lymphocytes with a ratio of 1:1 and stimulated with phytohemagglutinin (PHA), 3H-thymidine incorporation by T lymphocytes was suppressed. This monocyte-mediated suppression was reversed by purine nucleoside phosphorylase substrate, deoxyguanosine. In SLE patients, both monocyte-mediated suppression and its reversal by deoxyguanosine were defective. The defective function was observed both in patients with active and inactive diseases. The defective function was studied sequentially before and after change in the clinical status of patients. The defects remained unaffected regardless of the disease activity. The defects in monocyte-mediated suppression and its reversal by deoxyguanosine in SLE patients as demonstrated in our study suggest the presence of intrinsic monocyte dysfunction in SLE.

Adult↗
PubMed1986

Enhanced Fc receptor function of monocytes from patients with clinically active systemic lupus erythematosus: binding and degradation of soluble immune complexes in vitro.

The in vitro Fc receptor function of monocytes as measured by using soluble immune complexes (IC) consisting of ovalbumin and 125I-labeled antibody to ovalbumin was examined in serum-free medium. Adherent monocytes were obtained from peripheral blood of 18 patients with systemic lupus erythematosus (SLE; 6 active, 12 inactive) and 13 normal controls. 5 X 10(5) monocytes were incubated with soluble IC containing 20 ng IgG for 16 hr at 4 degrees C, and for 4 hr and 16 hr at 37 degrees C, and then cell-associated and acid-soluble radioactivities were counted. After 16-hr incubation at 4 degrees C, there was no significant difference in the amount of cell-associated IC between monocytes from SLE patients and those from normal controls. After 4 hr incubation at 37 degrees C, monocytes from patients with active SLE bound and phagocytized more IC than those from patients with inactive SLE or from normal controls (P less than 0.01). After 16 hr incubation at 37 degrees C, degradation of soluble IC by monocytes from patients with active SLE greatly increased (P less than 0.003). These findings were discussed in relation to the possible pathophysiologic role of the mononuclear phagocyte system in SLE.

Adult↗
PubMed1986

Monoclonal antibody against bacterial lipopolysaccharide cross-reacts with DNA-histone.

Monoclonal antibodies to bacterial lipopolysaccharide (LPS) were prepared by fusing spleen cells from BALB/c mice immunized with Salmonella Minnesota Re 595 LPS to the mouse myeloma cell line P3U1. One of them, designated RS01, revealed a strong positive antinuclear activity and reacted with DNA-histone. RS01 also bound specifically to Salmonella Minnesota Re 595 LPS and eliminated the biological activity of LPS. The Salmonella completely inhibited the ANA activity of RS01 and DNA-histone blocked the reactivity of RS01 with LPS. Thus, it is clear that an anti-LPS monoclonal antibody, RS01 cross-reacts with DNA-histone.

Animals↗
PubMed1985

Beneficial effect of topical fibronectin in patients with keratoconjunctivitis sicca of Sjögren's syndrome.

Fibronectin, a high molecular weight plasma protein, was applied locally to 12 patients with keratoconjunctivitis sicca of Sjögren's syndrome refractory to conventional treatment. All patients had ophthalmologically proved keratoconjunctivitis sicca of unknown etiology and were suffering from severe eye symptoms. Fibronectin was purified from autologous plasma and administered to the patients as tear drops 3 times a day. All patients responded to the therapy with fewer subjective symptoms and decreased fluorescein staining. All but 3 responded to the therapy as judged by rose bengal test. No adverse effects were encountered. We conclude that local application of fibronectin may benefit patients with keratoconjunctivitis sicca of Sjögren's syndrome.

Fibronectins↗
PubMed1984

Isolation of a novel antibody, which precipitates ribonucleoprotein complex containing threonine tRNA from a patient with polymyositis.

We have isolated and characterized a novel antibody from a patient with polymyositis, which precipitates threonine tRNA and an unknown small RNA molecule from 32P-labeled HeLa extract. Fingerprint analysis showed that the human threonine tRNA precipitated was nearly identical to the murine tRNA described by Harada [(1978) Seikagaku 50, 397-411]. Nucleotide analysis suggested that the other small RNA molecule might be transfer RNA. Since phenol extraction resulted in the loss of immunoprecipitability, the protein portion was presumed to be involved in the recognition of the antigen. Immunofluorescence staining of HeLa cells with the antibody clearly demonstrated that the antigen was located predominantly in the cytoplasm.

Autoantibodies↗
PubMed1984

Amegakaryocytic thrombocytopenia associated with systemic lupus erythematosus successfully treated by a high-dose prednisolone therapy.

A patient with systemic lupus erythematosus (SLE) developed amegakaryocytic thrombocytopenic purpura. The absence of a drug relationship or other identifiable underlying cause made us consider this to be a rare manifestation of SLE. Her thrombocytopenia, which initially appeared to be steroid-resistant, ultimately improved following a prolonged high-dose prednisolone therapy. To our knowledge, this is the second case of amegakaryocytic thrombocytopenia associated with SLE in the literature. The possibility that amegakaryocytic thrombocytopenia in SLE can be caused by cellular rather than humoral immunologic mechanism was discussed.

Bone Marrow↗