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Biomedical subjects

H Kawashima

Publications and source records attributed to H Kawashima.

At least 163 records · Page 9Linked to original sources
PubMed1997

Production of bioactive salmon calcitonin from the nonendocrine cell lines COS-7 and CHO.

To produce bioactive salmon calcitonin from the conventional nonendocrine cell lines, COS-7 and CHO, we devised a salmon calcitonin expression vector by combining the amino-terminus of human calcitonin precursor with a salmon calcitonin sequence, inserting the efficient furin-cleavable processing sequence Arg-X-Arg-X-Lys-Arg before salmon calcitonin, and deleting the carboxyl-terminal extension peptide. This chimeric calcitonin precursor terminates at glycine to easily receive an amidation reaction. COS-7 and CHO produced a high level of bioactive calcitonin by the resorption pit formation assay. Although amidating activity is highly expressed in CHO, but only a little in COS-7 cells, both cells produced a similar level of bioactive calcitonin. Thus, the engineered salmon calcitonin expression vector enables nonendocrine cells even with low amidation activity to produce bioactive calcitonin.

Animals↗
PubMed1997

Characterization of a 180 kDa molecule apparently reactive with recombinant L-selectin.

In the present study we identified a 180 kDa molecule (p180) in rat lymph nodes (LN) apparently reactive with silkworm derived recombinant L-selectin (LEC-IgG) in a Ca(2+)-dependent manner. Analysis of amino acid sequence revealed that p180 has a strong homology to the macrophage mannose receptor (MMR), which was corroborated by the observation that p180 reacted with polyclonal anti-alveolar MMR antibody and mannosyl-BSA-agarose. In agreement with this notion, the binding of p180 to the silkworm LEC-IgG was inhibited by alpha-methyl-D-mannoside. However, in sharp contrast to its reactivity against the silkworm LEC-IgG, p180 failed to bind LEC-IgG produced by COS-7 cells, suggesting that p180 reacted with the silkworm LEC-IgG through the recognition of oligomannose-type oligosaccharides expressed on the silkworm products and that the lectin activity of L-selectin was not involved in the interaction. These results, together with the immunohistochemical studies showing that p180 was absent from the majority of high endothelial venules (HEV) but present in medullary macrophages, led us to conclude that p180 obtained from LN lysates by the use of the silkworm LEC-IgG is not a physiological ligand for L-selectin, warning against the use of recombinant proteins expressed in the baculovirus/ silkworm expression system for the detection of carbohydrate ligands.

Amino Acid Sequence↗
PubMed1997

Induction of cell death by chimeric L-selectin-Fas receptors.

In the present study, we have established a system where engagement of an adhesion molecule triggers a death signal into cells. L-selectin, which is a well characterized adhesion receptor involved in the initial adhesion between lymphocyte and endothelium, was fused to the intracellular domain of an apoptosis-inducing molecule, Fas. Ligation of the chimeric receptors with a carbohydrate ligand for L-selectin, fucoidin or a mAb that recognizes the lectin domain of L-selectin, induced apoptosis in receptor-expressing cells. However, ligation with an anti-L-selectin mAb reactive with a non-ligand binding site did not induce apoptosis, indicating that stimulation through the lectin domain of L-selectin in the chimeric receptor leads to signal delivery. Upon activation L-selectin shows a unique proteolytic cleavage at the membrane proximal site on the extracellular (EC) domain, of which the significance is also unclear. We found that truncations in the EC domain which abrogate the proteolytic cleavage of L-selectin did not influence induction of apoptosis, suggesting that the cleavage on the EC domain itself is not important for the signaling function of the chimeric receptor. This is the first demonstration that an adhesion signal can be converted to a signal that leads to apoptotic cell death.

Animals↗
PubMed1997

Virus elimination and histologic improvement in patients with chronic hepatitis C treated with interferon alpha.

We evaluated the elimination of hepatitis C virus (HCV) by means of interferon alpha (IFN-alpha) by investigating both positive- and negative-stranded RNA forms in the peripheral blood mononuclear cells and liver tissue. We also assessed the long-term histologic improvement accompanying viral clearance. We studied 20 persons with HCV whose serum aminotransferase levels remained normal for more than 1.5 years after IFN-alpha treatment withdrawal. The presence of HCV RNA in their peripheral blood and of both positive and negative strands in the peripheral blood mononuclear cells and liver tissue was investigated using the reverse transcription polymerase chain reaction method. We examined the histologic findings using the European classification and the histology activity index scoring system. In 16 of 20 patients, both strands disappeared from the possible reservoirs. The histologic findings indicated reduced activity, and histology activity index scores (1, 2, 3, and total) also showed significant improvement. We confirmed that IFN-alpha therapy can induce the elimination of HCV RNA from the conceivable HCV reservoirs and effect histologic improvement. Therapy with IFN-alpha is effective for treating chronic hepatitis C.

Adult↗
PubMed1997

Production of 5,8,11-Eicosatrienoic Acid (Mead Acid) by a (Delta)6 Desaturation Activity-Enhanced Mutant Derived from a (Delta)12 Desaturase-Defective Mutant of an Arachidonic Acid-Producing Fungus, Mortierella alpina 1S-4.

Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3(omega)9) was attained by a mutant fungus, Mortierella alpina M209-7, derived from (Delta)12 desaturase-defective M. alpina Mut48. The 20:3(omega)9 production by M209-7 was 1.3 times greater than that by its parent strain, Mut48. This is thought to be due to its enhanced (Delta)6 desaturation activity, which was 1.4 times higher than that of Mut48. In both strains, 87 to 88% of the total lipids comprised triacylglycerol (TG) and 85% of 20:3(omega)9 was contained in TG. On optimization of the culture conditions for M209-7, earlier glucose feeding and shifting of the growth temperature from 28 to 19(deg)C on the second day were shown to be effective. Under the optimal conditions with a 10-liter jar fermentor, 20:3(omega)9 production reached 1.65 g/liter of culture medium (corresponding to 118 mg/g of dry mycelia and 28.9% of total fatty acids), which is about twice that reported previously (0.8 g/liter).

Journal Article↗
PubMed1997

[A new model of delayed healing of acetic acid ulcers in rats by indomethacin via osmotic pump].

Gastric ulcers were produced in male Donryu rats (8-weeks-old) by subserosal injection of 20 microliters of 20% acetic acid. Five days later, an indomethacin (IND)-filled osmotic pump (OP) was implanted into the dorsal subcutaneous space in the rats and maintained for 2, 3 or 4 weeks. The natural healing of the acetic acid-induced ulcers was significantly (P < 0.05) delayed by administration of IND via OP implanted for 4 weeks. Two, 3 and 4 weeks after OP implantation, the plasma IND levels was 1.87 +/- 0.13, 2.43 +/- 0.16 and 0.74 +/- 0.26 (micrograms/ml), respectively, showing that a steady state IND plasma level was maintained for 3 weeks. The gastric mucosal PGE2 levels in rats with ulcers for 3 weeks after implantation of OP were significantly (P < 0.05) reduced by IND (IND(-):576.6 +/- 83.9 pg/mg, IND(+):355.6 +/- 34.7 pg/mg). Repeated administration of enprostil, famotidine and rabeprazole at 25 micrograms/kg, 100 mg/kg and 10 mg/kg (p.o., b.i.d.), respectively, showed anti-ulcer activity. Teprenone and rebamipide had only slight anti-ulcer activity. These findings indicated that this model of delayed healing of acetic acid ulcers by IND using OP is useful for investigating the anti-ulcer activity of drugs.

Acetates↗
PubMed1997

Effects of vitamin K2 (menatetrenone) on atherosclerosis and blood coagulation in hypercholesterolemic rabbits.

Gamma-Carboxyglutamic acid (Gla)-containing protein, synthesized in the presence of vitamin K, has been found in atherogenic plaques, but the pharmacological effect of vitamin K on atherosclerosis is unclear. We examined whether vitamin K2 (menatetrenone) could affect the progression of both atherosclerosis and hypercoagulability in hypercholesterolemic rabbits. Vitamin K2 in daily doses of 1, 10 and 100 mg/kg was given with a 0.5% cholesterol diet for 10 weeks to 8 rabbits each. The plasma levels of total-cholesterol in the vitamin K2-treated groups were clearly lower than that of the hypercholesterolemic control group. The excessive dose of vitamin K2, even at the high dose of 100 mg/kg/day for 10 weeks, did not accelerate the progression of atherosclerosis and did not promote the coagulative tendency in the rabbits. In contrast, the vitamin K2 treatment (1 to 10 mg/kg/day) suppressed the progression of atherosclerotic plaques, intima-thickening and pulmonary atherosclerosis, the increase of ester-cholesterol deposition in the aorta, and both the elevation in plasma factor X level and increase in Hepaplastin test value in the rabbits. These results indicate that the pharmacological dose of vitamin K2 prevents both the progression of atherosclerosis and the coagulative tendency by reducing the total-cholesterol, lipid peroxidation and factor X activity in plasma, and the ester-cholesterol deposition in the aorta in hypercholesterolemic rabbits.

Animals↗
PubMed1997

[A case of primary malignant melanoma of the esophagus].

A case of primary and malignant melanoma of the esophagus was reported. A 64-year-old male complaining of discomfort of anterior chest pain was admitted to our hospital for operation. Findings of upper G-1 X-ray and endoscopic examination revealed suspiciously malignant melanoma. Subtotal thoracic esophagectomy with R III dissection was performed. Operative findings included A0 N2 Pl0 M0 Stage III. Macroscopically it showed black-grayish colored polypoid tumors, 7 cm in size. The typical finding of junctional activity adjacent to the tumor mass and melanocytes were microscopically found. The patient received postoperative systemic chemotherapy, but was died of multiple liver and bone metastases 125 days after surgery. Malignant melanoma of the esophagus has extremely poor prognosis and none of effective therapies has been reported.

Esophageal Neoplasms↗
PubMed1997

Detection of mumps virus genome directly from clinical samples and a simple method for genetic differentiation of the Hoshino vaccine strain from wild strains of mumps virus.

A simple and sensitive method was developed for the differentiation of the Hoshino vaccine strain from wild strains with a restriction fragment length polymorphism (RFLP) analysis in the part of hemagglutinin-neuraminidase (HN) gene. The virus genome was amplified by using a reverse transcriptase-polymerase chain reaction (RT-PCR) directly from clinical samples. The PCR product of the Hoshino vaccine strain was cleaved into 2 fragments after digestion with Sca I and Afl II. All wild strains showed 2 RFLP profiles, A and B, different from that of vaccine strain. Wild A strains were cut into 2 fragments after digestion with Sca I only, while wild B strains were cleaved neither with Sca I nor Afl II. This molecular approach provides an effective method for differentiation of the Hoshino vaccine strain from wild strains of mumps virus in patients after vaccination.

Animals↗
PubMed1996

Genomic cloning and protein expression of a novel rat brain cytochrome P-450 CYP2D18* catalyzing imipramine N-demethylation.

We have previously reported the isolation of two cDNA clones, designated 2d-29 and 2d-35, which have identical open reading frames and code for a novel brain cytochrome P-450 (P-450) belonging to the CYP2D subfamily, and noted that the mRNA of clone 2d-35 seems to be expressed in the brain but not in the liver (1). Although the deduced amino acid sequence of these clones differs from that of the liver CYP2D4 by only 5 amino acids distributed in the C-terminal region, this new P-450 cDNA clone contained a unique 5'-extension, and we posit in this report by analysis of a genomic clone that this 5'-untranslated sequence is derived from a gene distinct from that of CYP2D4. Thus, this novel P-450 was named P-450 2D18 according to the recommended nomenclature (2). The expressibility of this cDNA was confirmed by in vitro translation using a reticulocyte system, and protein expression was performed using COS-M6 cells. Immunoblot analysis showed a cross-reacting band of the predicted size range with anti-P-450 2D6 antiserum, which was not seen in control cells. Furthermore, the CYP2D18-expressed COS cell lysate showed N-demethylation activity toward imipramine, whereas another brain P-450 CYP4F6-expressed COS cell lysate showed 10-hydroxylation activity. This is the first report that associates an individual P-450 isozyme in brain with a particular metabolic alteration of the antidepressant imipramine.

Animals↗
PubMed1996

Pervanadate stimulation of wortmannin-sensitive and -resistant 2-deoxyglucose transport in adipocytes.

Pervanadate mimics several distinct insulin effects, including stimulation of hexose uptake in the in vitro system, and reduces the blood glucose level in streptozotocin-treated diabetic rats. It has been proposed that pervanadate induces insulin-like effects mediated through autophosphorylation and activation of insulin receptor (IR) even in the absence of insulin by inhibiting protein tyrosine phosphatases. This study focused on the mechanism of pervanadate action on hexose uptake. Both insulin (100 nM) and pervanadate (100 microM), a protein tyrosine phosphatase inhibitor, induced a marked increase in the phosphorylation at tyrosine residues of IR and insulin receptor substrate 1 (IRS-1) and in 2-deoxyglucose uptake in 3T3-L1 adipocytes. Wortmannin (1 microM), a specific phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, inhibited the increased 2-deoxyglucose uptake by insulin completely but that by pervanadate only partially. On the other hand, both insulin- and pervanadate-stimulated PI 3-kinase activities were inhibited completely by wortmannin (100 nM), suggesting that the pervanadate-induced wortmannin-resistant effect on hexose uptake may be mediated through a PI 3-kinase-independent pathway. This pervanadate-induced wortmannin-resistant effect was abolished by ST-638, a specific tyrosine kinase inhibitor. These data suggest that at least two distinct tyrosine phosphorylation pathways may be involved in the insulin-like effect of pervanadate.

3T3 Cells↗
PubMed1996

cDNA cloning of a novel CYP3A from rat brain.

One full length cDNA clone, designated 3aH15, was isolated from a control male rat brain cDNA library. 3aH15 encoded a protein composed of 503 amino acid residues. The deduced amino acid sequence of 3aH15 was 92% identical to Cyp3a-13 and had a 68.4% to 76.5% homology with the other reported CYP3A sequences. Clone 3aH15 was thus named CYP3A9. No significant induction of the CYP3A9 expression in rat brain by dexamethasone was observed by Northern blot analysis. CYP3A9 cDNA was expressed in E. coli and the expressed P450 3A9 is active in the demethylation of erythromycin as well as benzphetamine.

Amino Acid Sequence↗
PubMed1996

Inhibitory effects of alkyl gallate and its derivatives on fatty acid desaturation.

Alkyl gallate, which is known as an antioxidant, intensively inhibited delta 5 and delta 6 desaturation in both rat liver microsomes and an arachidonic acid-producing fungus Mortierella alpina 1S-4. The rat liver microsomal delta 5 and delta 6 desaturases were inhibited by gallic acid esterified with alcohols with various numbers of carbons, suggesting that the necessary structure in an esterified alcohol for the inhibition is not so strict. Among the three hydroxy groups in gallic acid, the m-hydroxy group was shown to be the necessary structure. Kinetic analyses revealed that propyl gallate is a noncompetitive inhibitor of delta 5 desaturase (Ki = 2.6.10(-5)M) and delta 6 desaturase (Ki = 1.7.10(-4) M). These data indicate that alkyl gallate is a new type of desaturase inhibitor and different from known natural inhibitors, i.e., sesamin and curcumin.

Animals↗
PubMed1996

A synthetic oestrogen antagonist, tamoxifen, inhibits oestrogen-induced transcriptional, but not post-transcriptional, regulation of gene expression.

Oestrogen (E2) regulates the expression of its target genes at transcriptional and post-transcriptional levels. To clarify the mechanism of E2-induced post-transcriptional regulation, with attention to the involvement of the oestrogen receptor (ER), we studied the effect of tamoxifen (TAM), a synthetic E2 antagonist that inhibits ER-mediated transcription, on E2-induced transcriptional and post-transcriptional regulation of the chicken ovalbumin (OVA) gene in chick oviducts. Run-on analysis with oviduct nuclei isolated from E2-treated chicks showed that TAM treatment completely blocked E2-induced transcription of the OVA gene within 24 h without affecting ER gene expression. Likewise, the rate of transcription fell to below the limit of detection after E2 withdrawal from the chicks. Reflecting the transcription rate, OVA mRNA accumulated linearly in E2-treated chicks, and E2 withdrawal caused a rapid loss of OVA mRNA. However, in the chicks treated with TAM and E2, OVA mRNA was degraded slowly over 48 h with a half-life of 24 h, suggesting that TAM does not inhibit E2-induced mRNA stabilization. Moreover, E2-induced mRNA stabilization was observed even when transcription of the OVA gene was blocked by a transcription inhibitor. Western-blot analysis showed that the remaining OVA mRNA was translatable. Thus the present study indicates that E2 regulates expression of the OVA gene via distinct pathways at transcriptional and post-transcriptional levels.

Animals↗
PubMed1996

Activation of Cl- channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts.

Ionic channels regulated by extracellular Ca2+ concentration ([Ca2+]o) were examined in freshly isolated rabbit osteoclasts. K+ current was suppressed by intracellular and extracellular Cs+ ions. In this condition, high [Ca2+]o evoked an outwardly rectifying current with a reversal potential of about -25 mV. When the concentration of extracellular Cl ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4',4-diisothiocyanostilbene-2' 2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl- channels. Cd2+ or Ni2+ caused a transient activation of the Cl- current in contrast to the sustained activation elicited by Ca2+. Intracellular 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl- current. Either when the osmolarity of extracellular medium was increased, or when 100 microM cAMP was dissolved in the patch pipette solution, high [Ca2+]o still elicited the Cl- current, indicating that the divalent cation-induced Cl- current was carried through Ca(2+)-activated Cl- channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl- current, indicating that the activation of Cl- current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl- channels under whole cell clamp. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activated the Cl current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl current was mediated through a G protein, and that an increase in [Ca2+]i was critical for the activation of Cl-channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca(2+)-activated Cl- channels.

Animals↗
PubMed1996

Polymerase chain reaction detection of the hemagglutinin gene from an attenuated measles vaccine strain in the peripheral mononuclear cells of children with autoimmune hepatitis.

We examined the measles H gene using reverse transcriptase-polymerase chain reaction in peripheral mononuclear cells obtained from 4 pediatric and 2 adult patients with autoimmune hepatitis and 12 healthy children who had been infected with measles or vaccinated with an attenuated measles vaccine in the past. All patients were positive for the presence of the gene. Only one healthy control, who had been vaccinated two weeks before the study, was positive, while the other 11 controls were negative for the presence of the gene. The restriction enzyme patterns of the products in the pediatric patients were different from those observed in adults. The sequences of amplified products from pediatric patients coincided with the vaccine strain, whereas those from adults were different from the vaccine strain. The sequence of those from one of two adult patients was similar to those of the isolates in 1990 and later. Our results demonstrated that children with autoimmune hepatitis can have persistence of the vaccine strain in vivo for many years after vaccination.

Autoimmune Diseases↗
PubMed1996

Uptake of radioactive octanoate in astrocytoma cells: basic studies for application of [11C]octanoate as a PET tracer.

Fatty acids are taken up and metabolized in the brain. In vitro uptake experiments on astrocytoma cells were carried out to assess the potential use of [1-11C]octanoate as a positron emission tomography (PET) tracer for astroglial functions. Uptake of [1-14C]octanoate increased in a time-dependent fashion until 60 min after application. The uptake of [1-11C]octanoate showed similar results to that of [1-14C]octanoate until 10 min. As for medium pH, [1-14C]octanoate uptake increased gradually with the decrease in pH. We also examined the effects of glutamate, glucose deprivation and hypoxia on the uptake of octanoate and found that these conditions did not bring about any change in the extent of [1-14C]octanoate uptake. These results show that the octanoate uptake was not influenced by any of several pathological conditions. When the number of astrocytes increases in the area of hypoglycemia or hypoxia near a brain lesion, the amount of octanoate uptake also increases, so this indicates the possibility that 11C-octanoate will detect a brain lesion.

Astrocytoma↗