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Biomedical subjects

M Okuda

Publications and source records attributed to M Okuda.

At least 127 records · Page 7Linked to original sources
PubMed1997

Overexpression in Escherichia coli of chemically synthesized gene for active 0.19 alpha-amylase inhibitor from wheat kernel.

A synthetic gene encoding 0.19 alpha-amylase inhibitor (alpha-AI) from wheat kernel was obtained by enzymatic assembly of 18 oligodeoxynucleotides which were chemically synthesized. The synthetic gene was introduced into vector pET15b for expression in Escherichia coli BL21(DE3) under the control of T7 promoter. However, in SDS-PAGE and Western blotting analyses of the E. coli cell lysate, the expression product could not be detected. Expression analysis for various partially deleted gene fragments suggested that the putative hairpin-like structure of mRNA in the 5'-terminal coding region might interrupt efficient expression. When the hairpin structure was eliminated by using degenerate codons, the resulting gene could be overexpressed in E. coli. Although the gene product was accumulated in an insoluble fraction as inclusion bodies, its inhibitory activity could be recovered by solubilization with 8 M urea, followed by refolding through two successive steps of dialysis at alkaline pH. After purification, the recombinant 0.19 alpha-AI showed the same characteristics as the authentic inhibitor in terms of N-terminal amino acid sequence, peptide mapping on reverse-phase HPLC, far-UV circular dichroism (CD) spectrum and have inhibition of human salivary alpha-amylase. Thus, we have established an overexpression system in E. coli for active recombinant 0.19 alpha-AI.

Amino Acid Sequence↗
PubMed1997

Structure and function of the long terminal repeats of feline leukemia viruses derived from naturally occurring acute myeloid leukemias in cats.

Long terminal repeats of feline leukemia viruses cloned from feline acute myeloid leukemias frequently contained direct repeats of 40 to 74 bp in the upstream region of the enhancer (URE). The repetitive URE conferred an enhancer function upon gene expression in myeloid cells, suggesting its association with tumorigenic potential in myeloid cells.

Acute Disease↗
PubMed1997

Arginine vasopressin increases nitric oxide synthesis in cytokine-stimulated rat cardiac myocytes.

We investigated the effects of arginine vasopressin (AVP) on nitric oxide (NO) synthase activity in cardiac myocytes by measuring the production of nitrite, a stable metabolite of NO, and the expression of inducible NO synthase (iNOS) mRNA and protein. Incubation of cultured neonatal rat cardiac myocytes for 24 hours with interleukin-1beta (IL-1beta) caused a significant increase in NO production. Both AVP and V1a receptor agonist [Phe2,Ile3,Orn8]vasopressin augmented NO synthesis in IL-1beta-stimulated, but not in unstimulated myocytes, in a dose-dependent manner. The V1a receptor antagonist [d(CH2)[5]1,O-Me-Tyr2,Arg8]vasopressin completely inhibited the effect of AVP. The AVP-induced NO production by IL-1beta-stimulated cells was accompanied by increased iNOS mRNA and protein accumulation. AVP caused a significant increase in cytosolic free Ca2+ levels of cardiac myocytes, whereas it showed no effect on cytosolic cAMP levels. After protein kinase C activity was functionally depleted by treating cells with phorbol 12-myristate 13-acetate for 24 hours, AVP did not augment IL-1beta-induced NO production. The effect of AVP was also inhibited in the presence of the protein kinase C inhibitor calphostin C. The addition of AVP increased protein kinase C activity in cardiac myocytes, and its effect was significantly inhibited in the presence of calphostin C. These results support the hypothesis that the heart may be a target organ for AVP and that AVP modulates IL-1beta-induced iNOS expression in myocytes through the V1a receptor, which is mediated at least partially via activation of protein kinase C.

Animals↗
PubMed1997

Gastric mucin secretion from cultured rat epithelial cells.

In order to establish the measurement of gastric mucin secreted from cultured mucous cells, rat gastric mucin was purified from secreted mucus with Sepharose CL-4B column chromatography. Gastric mucin was measured by dot blot analysis using an enzyme-linked lectin (soybean agglutinin) assay in a good concentration-dependent manner. Surface epithelial cells were dispersed by limited digestion of a rat everted stomach and collected by density gradient centrifugation with Percoll. These cells were inoculated onto gelled collagen dishes, then cultured in a medium supplemented with 10% fetal calf serum under a 5% CO2 atmosphere in air. Changing the medium after a 2-d culture, the cells were cultured for another 3 d. During the culture, the numbers of cells each day were almost equal, but mucin contents in the cells increased, and then dropped at day 5 after inoculation. At that time, the edge of the cell layer peeled off and the cells adhered to each other. Using 2-d cultured cells, the effects of some secretagogues on mucin secretion were investigated. Carbachol, secretin, CCK-8 and prostaglandin E2 (PGE2) strongly stimulated mucin secretion, and gastrin I weakly did. However, histamine offered no stimulation.

Animals↗
PubMed1997

[Effect of eosinophil major basic protein on histamine release from nasal epithelial mast cells].

Eosinophils and nasal epithelial mast cells (NEMCs) are closely related in nasal allergy. The activity of the eosinophil cationic protein, major basic protein (MBP) on histamine release from human NEMCs has not been documented. We examined the effects of MBP on histamine release from NEMCs. Nasal scrapings (containing NEMCs) from 59 patients with house dust mite nasal allergy were incubated with MBP or alkylated MBP. Specimens were also pre-incubated with MBP followed by incubation with house dust mite allergen (HDM) or preincubated with HDM followed by incubation with MBP or HDM. Histamine released in incubated media and remained in incubated mast cells was quantified by radio-immunoassay. MBP at the concentration of 10(-5) mol/L induced mild but statistically significant histamine release from the NEMCs. Preincubation with MBP at the concentration of 1.57 x 10(-5) mol/L produced slight inhibition of subsequent HDM-induced histamine release but preincubation with HDM showed no effect on the subsequent MBP-induced or HDM-induced histamine release from NEMCs. These results suggest that accumulated and activated eosinophils may release MBP resulting in clinical symptoms by interaction with NEMCs and inhibit of histamine release from NEMCs by subsequent interaction with allergen.

Adolescent↗
PubMed1997

[Long-term effects of a selective alpha 1-adrenergic inhibitor on right and left ventricular masses in patients with chronic pulmonary disease].

We examined the effect of one year of treatment with the selective alpha 1-adrenergic inhibitor doxazosin on right ventricular mass (RV mass), left ventricular mass (LV mass), and arterial blood gases. The subjects were 24 outpatients (18 men and 6 women, mean age 68.3 +/- 9.4 years) with chronic pulmonary disease complicated by hypertension who were clinically stable. One year of drug therapy was associated with significant decreases both in systolic pressure (159 +/- 15.2 vs 125.8 +/- 14.1 mmHg, p < 0.05, n = 24), and in LV mass index (101.0 +/- 13.4 vs 97.6 +/- 11.8 gm-2, p < 0.05), n = 24). We obtained the RV mass index by multiplying the thallium score RV/LV count and the LV mass index obtained by echocardiography. One year of drug therapy was associated with a significant increase in RV mass index (42.9 +/- 31.2 vs 53.6 +/- 30.5 gm-2, p < 0.05, n = 8). Vital capacity decreased (2.18 +/- 1.95 vs 1.95 +/- 0.57 l, p < 0.05, n = 24), but PaO2 improved (77.3 +/- 17.2 vs 82.2 +/- 2.4 mmHg, p < 0.05, n = 24). These data indicate that doxazosin can decrease blood pressure and can depress the left ventricle with no adverse effect on oxygenation in patients with chronic pulmonary disease complicated by hypertension. The worsening of RV hypertrophy may have been caused by a mechanism different from the one that caused LV hypertrophy, and by an increase in the work load on the right ventricles secondary to lung deterioration.

Adrenergic alpha-Antagonists↗
PubMed1997

Cloning of feline p21WAF1 and p27Kip1 cDNAs and search for their aberration in leukemias and lymphomas in cats.

For investigation of the relation of cell cycle regulation with tumorigenesis in cats, we cloned feline p21WAF1 and p27Kip1 cDNAs and searched for their aberration in feline spontaneous leukemias and lymphomas. The feline p21WAF1 cDNA (pCFW.31) clone obtained from the PCR amplified product appeared to cover approximately 75% of the open reading frame, and showed 81.6% and 76.8% sequence similarities with those of human and mouse counterparts, respectively. The pHFK.5 clone isolated by plaque hybridization contained the whole open reading frame of cat p27Kip1 cDNA encoding 198 amino acids, showing 93.4% and 90.4% sequence similarities with those of human and mouse counterparts, respectively. Southern-blot analyses using these clones as probes did not show any deletion or rearrangement of both the p21WAF1 and p27Kip1 genes in 19 feline spontaneous cases of leukemias and lymphomas examined. RT-PCR/SSCP (single strand conformation polymorphism) analysis of p27Kip1 cDNA indicated that there was no mutation resulting in amino-acid substitution in 10 feline leukemia and lymphoma cases.

Amino Acid Sequence↗
PubMed1996

Increasing cAMP antagonizes hypertrophic response to angiotensin II without affecting Ras and MAP kinase activation in vascular smooth muscle cells.

Angiotensin II (Ang II), a potent hypertrophic factor for vascular smooth muscle cells (VSMC), induces activation of the ras proto-oncogene product (Ras) and mitogen-activated protein (MAP) kinases, and tyrosine phosphorylation of a focal adhesion-associated protein, paxillin. Forskolin, a direct activator of adenylate cyclase, and dibutyryl cAMP (Bt2 cAMP), a membrane permeable cAMP analogue, potently inhibited Ang II-stimulated protein synthesis. However, they did not inhibit Ang II-induced activation of Ras and MAP kinases. Although both forskolin and Bt2 cAMP potently reduced background tyrosine phosphorylation of paxillin, they allowed Ang II to induce the same reaction. These results indicate that increasing cAMP antagonizes the hypertrophic response to Ang II without affecting Ras and MAP kinase activation in VSMC and suggest that it does not interrupt signaling from the Ang II receptor to focal adhesions.

Angiotensin II↗
PubMed1996

Differences in hypervariable region 1 quasispecies of hepatitis C virus between human serum and peripheral blood mononuclear cells.

The complexity of hypervariable region 1 (HVR1) quasispecies of hepatitis C virus (HCV) in serum and peripheral blood mononuclear cells (PBMCs) was assessed at several time points in 11 patients with chronic hepatitis C. Polymerase chain reaction and single-strand conformation polymorphism analysis revealed that four patients (36%) showed serial changes in the complexity of HVR1 quasispecies, which were identical in both serum and PBMCs; two patients (18%) showed both serial changes in quasispecies and differences in quasispecies between serum and PBMCs; and five patients (45%) showed neither serial changes in nor quasispecies differences between serum and PBMCs. These results demonstrate both the existence of different quasispecies between serum and PBMCs and the infection of PBMCs by HCV.

Adult↗
PubMed1996

cDNA cloning and functional expression of a novel rat kidney organic cation transporter, OCT2.

A cDNA encoding a novel organic cation transporter, designated as OCT2 was isolated from the rat kidney. The rat OCT2 cDNA (2,205 bp) had an open reading frame encoding for a 593-amino acid protein (calculated molecular mass of 66 kDa) which shows 67% identity with the rat OCT1. Northern hybridization and reverse transcription-PCR analyses revealed that the rat OCT2 mRNA transcript was expressed predominantly in the kidney, at higher level in the medulla than in the cortex, but this transcript was not detected in the brain, heart, lung, liver, small intestine or spleen. When expressed in Xenopus oocytes, rat OCT2 stimulated the uptake of tetraethylammonium, and the uptake was markedly inhibited by the presence of cimetidine, procainamide and quinidine. These findings suggest that OCT2 is responsible for the transport of cationic drugs in the kidney.

Amino Acid Sequence↗
PubMed1996

Thymic lymphoblastic lymphoma of committed natural killer cell precursor origin: a case report.

BACKGROUND: Recently, it was demonstrated that the human fetal thymocyte contains a bipotential progenitor capable of both T lymphocyte and natural killer (NK) cell differentiation. However, prior to this report a malignant neoplasm arising from these cells had not been documented. METHODS: A Japanese female age 38 years was examined by morphology of light and electron microscopy, immunohistochemistry, 3-color flow cytometry, cytotoxic assay, and Southern blotting. RESULTS: The patient presented with a mediastinal mass and pleural effusion. Leukemic progression was identified following chemotherapy and complete clinical remission. Immunophenotyping of lymphoma revealed CD45++, c-kit dim+, terminal deoxynucleotidyl transferase (TdT)-<+, CD38++, CD34+<++, CD33+<-, CD13dim+approximately+, HLA-DR+, CD7+, cytoplasmic CD3 (cCD3)+, surface CD3 (sCD3)-, CD2dim+, CD56+, CD16-, CD11b+, CD57-, CD1a-, CD5-, TCR alpha beta-, TCR gamma delta-, CD4-, CD8-, CD28-, CD10-, CD19-, CD20-, CD22-, surface immunoglobulins-, and CD14-. Functional NK activity of the lymphoma cells was extremely low. DNA analysis revealed no gene rearrangement in TCR beta, gamma, and delta or immunoglobulin heavy and light chain genes. CONCLUSIONS: Lymphoma cells of this case were derived from a distinct subtype of lymphocyte that originate from a thymic precursor committed to NK cell differentiation. This category is different from those of thymic T or precursor B cell pheno-/genotype.

Adult↗
PubMed1996

Molecular cloning and tissue distribution of rat peptide transporter PEPT2.

A cDNA encoding rat H(+)- coupled peptide transporter PEPT2 was isolated. The cDNA encoded a protein of 729 amino acids with 48% amino acid identity to the rat PEPT1. The mRNA expression of rat PEPT2 was predominant in the kidney. When expressed in Xenopus oocytes, rat PEPT2 stimulated the uptake of bestatin, a dipeptide-like drug.

Amino Acid Sequence↗
PubMed1996

Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil.

An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD50 value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4-9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent.

Animals↗
PubMed1996

Ultrastructural studies on paramyosin core filaments from native thick filaments in catch muscles.

Catch muscles of molluscs usually have thick filaments of about 100 nm in diameter. The filament is constructed of a paramyosin core filament and an outer layer of myosin molecules. Myosin molecules are situated on the paramyosin core filament towards both ends, and they consequently have polarity towards both ends. According to our observations on the paramyosin core filaments from the native thick filaments, they bear regular cross-striations on their surfaces of about 14.5 nm periodicity when incubated in a KCl solution for a short time. The periodic pattern is supposed to be representative of peculiar arrangements of paramyosin molecules in the core, but the periodic pattern disappeared during incubation in a solution of high concentration KCl for a prolonged time, and a 'Bear-Selby net' pattern appeared substitutionally. These 'Bear-Selby net' patterns were conveniently divided into 3 types among 4 paramyosin core filaments from 4 'catch' muscles; the adductors of a pecten, an oyster and a clam, and the byssus retractor of a mussel. The 'Bear-Selby net' of an oyster resembled that of a pecten. Purified paramyosin crystals from the 4 muscles showed a common periodicity of about 72.5 nm. Electrophoresis with SDS of the 4 paramyosins on 6% polyacrylamide gels revealed molecular weights at 104 kD from a pecten, 105 kD from an oyster, 103 kD from a clam and 105 kD from a mussel.

Actin Cytoskeleton↗
PubMed1996

A water-soluble antitumor glycoprotein from Chlorella vulgaris.

An active substance with antitumor activity (ARS2) was purified from the culture media of Chlorella vulgaris and found to be a glycoprotein with a molecular weight of 63,100 amu, as determined by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry. ARS2 contains 66.9% carbohydrate, mainly D-galactose, and 35.2% protein. The carbohydrate moiety has a beta-1,6-D-galactopyranose backbone, as determined by methylation analysis and 13C-NMR. Apparently, the protein moiety, whose 15 amino acid sequence at the NH2-terminus, we determined as DVGEAFPTVVDALVA, is necessary for the antitumor activity, as assessed by hydrazinolysis, periodate oxidation, and proteolysis.

Amino Acid Sequence↗