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Biomedical subjects

M Okada

Publications and source records attributed to M Okada.

At least 253 records · Page 14Linked to original sources
PubMed2000

Immunoaffinity purification and characterization of native placental leucine aminopeptidase/oxytocinase from human placenta.

cDNA cloning of placental leucine aminopeptidase (P-LAP)/cystinyl aminopeptidase (CAP)/oxytocinase demonstrated that this enzyme is a type II integral membrane protein, which means that native P-LAP, found in placenta, is membrane-bound and that the soluble form of this enzyme, found in maternal sera, is most likely derived from the native form. The presence of the different forms of the protein makes it difficult to purify homogeneously. In the current study we prepared antibody specific to native P-LAP and used it to purify native P-LAP from microsomal fractions of human placenta to homogeneity, 5039-fold within 4 h, by immunoaffinity chromatography. Zn(2+)and Cu(2+)strongly inhibited the enzyme but Ca(2+)did not. Amastatin was a more potent inhibitor than bestatin and leupeptin. Using antibodies against native P-LAP, protein having 83 per cent of l -methionine insensitive Leu-p-nitroanilide cleaving activity, was immunoprecipitated from the microsomal fraction of human placenta. The availability of a specific antibody against native P-LAP permits the rapid purification and the preliminary immunoassay of the enzyme. Establishment of simple purification and assay methods for the native, membrane bound form of P-LAP pave the way to elucidating the roles and processing systems of this enzyme.

Amino Acid Sequence↗
PubMed2000

Isoflavonoids from Pueraria mirifica and their estrogenic activity.

Nine isoflavonoids including a new pterocarpene, puemiricarpene, were isolated from the tuberous root of Pueraria mirifica (Leguminosae). The structure of puemiricarpene was determined by spectroscopic means. Estrogenic activity of the isolated isoflavonoids was tested using MCF-7 human breast cancer cells. Moderate activity was observed for kwakhurin, a prenylated isoflavonoid.

Estrogens, Non-Steroidal↗
PubMed2000

Needlescopic surgery for palmar hyperhidrosis.

OBJECTIVE: The aim of this study was to develop a less invasive technique for video-assisted thoracic sympathectomy. METHODS: A newly designed trocar was used. A skin incision of 2.0 to 2.5 mm in length was made in the third or fourth intercostal space at the midaxillary line. After insertion of the trocar, thoracic sympathectomy with electrocautery was carried out at the level of the second and third ribs. RESULTS: From October 1998 to March 1999, 180 patients with palmar hyperhidrosis underwent this technique. No complications related to the technique occurred, and within 1 week the operative wound had almost completely disappeared. There were recurrences necessitating reapplication of the technique. CONCLUSION: The technique allowed healing without a scar and improved the patient's cosmesis.

Adolescent↗
PubMed2000

Safety and efficacy of oestriol for symptoms of natural or surgically induced menopause.

To assess the safety and efficacy of oestriol in relieving post-menopausal symptoms 53 post-menopausal Japanese women with climacteric symptoms, 27 with natural menopause (group I) and 26 with surgically induced menopause (group II), received oral oestriol, 2 mg daily for 12 months. Clinical parameters including Kupperman index (KI) and the degree of satisfaction with symptomatic relief; serum concentrations of oestradiol, FSH and LH; serum lipids; blood pressure; bone mineral density, serum calcium (Ca), alkaline phosphatase (ALP), and urinary Ca were compared between the two groups. Oestriol improved KI in groups I and II by 49 and 80% respectively. Satisfaction with treatment was 85% in group I and 93% in group II. For both parameters, values were significantly different between groups I and II (P < 0.05 for both). Serum concentrations of oestradiol, FSH and LH changed in group I versus group II 6 months after initiation. A significant decrease in serum ALP and Ca/Cr was observed in group I at 6 months. Except for serum triglycerides, oestriol had no significant effect on lipids. Systolic and diastolic blood pressures were significantly decreased in group I at 3 months versus baseline. Slight vaginal bleeding occurred in 14.3% of group I. Histological evaluation of the endometrium in all women of group I and ultrasound assessment of the breasts following 12 months of oestriol treatment found normal results in all women. Therefore, oestriol appeared to be safe and effective in relieving symptoms of menopausal women. The beneficial biochemical effects of oestriol were marked in the natural menopause. Overall, oestriol may serve as a good choice for hormone replacement therapy to protect against other climacteric symptoms in post-menopausal women who do not need medication for osteoporosis or coronary artery disease.

Adult↗
PubMed2000

Maintenance-type DNA methyltransferase is highly expressed in post-mitotic neurons and localized in the cytoplasmic compartment.

Maintenance-type DNA methyltransferase (Dnmt1) is usually down-regulated in non-proliferating cells. In the present study, we detected significant expression of Dnmt1 protein in adult mouse brain where the majority of the cells are in a post-mitotic state. A significant amount of Dnmt1 protein was fractionated into the post-nuclear fraction for both cerebrum and cerebellum. The Dnmt1 in this fraction was enzymatically active. An immunofluorescence study revealed that Dnmt1 protein was mainly expressed in neurons and seemed to be localized in the cytoplasmic compartment. Primary culturing of neurons confirmed the expression and localization of Dnmt1 in the cytoplasmic compartment. The findings that the Dnmt1 transcript in the brain utilized the somatic-type exon and that the apparent size of the Dnmt1 protein in the cytoplasm was identical to that in proliferating culture cells indicate that the cytoplasmic Dnmt1 in neurons was of the somatic-type.

Animals↗
PubMed2000

Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system.

Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta-D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure-function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.

Amino Acid Sequence↗
PubMed2000

The spectrum of beta ig-h3 gene mutations in Japanese patients with corneal dystrophy.

PURPOSE: This study was undertaken to identify beta ig-h3 gene mutations in Japanese patients with granular corneal dystrophy (GCD), Avellino corneal dystrophy (ACD), lattice corneal dystrophy (LCD), and Reis-Bücklers' corneal dystrophy (RBCD). R124H, R124C, R555W, and R555Q mutations have been reported in Europe to cause ACD, LCD type I, GCD, and RBCD, respectively. METHODS: In total, 91 Japanese patients who had been clinically diagnosed with GCD, LCD, or RBCD were investigated to determine whether they had mutations in the beta ig-h3 gene. Genomic DNA was amplified using the polymerase chain reaction and analyzed using single-strand conformation polymorphism techniques. Mutations were identified using the direct sequencing method. RESULTS: In 68 unrelated patients who had been diagnosed with GCD, 62 patients (91%) were found to have the R124H mutation, which has been reported to cause ACD, whereas only six patients (9%) had the R555W mutation. In LCD patients, 10 patients with type I disease had the R124C mutation, and 10 patients with type IIIA disease had a P501T mutation. One patient with atypical LCD had an L527R mutation. In two patients with RBCD, one had an R555Q mutation and the other patient with geographic opacities was found to have an R124L mutation. CONCLUSIONS: Depending on the specific mutation in the beta ig-h3 gene, the phenotypes of corneal dystrophy may differ. Our results indicate that assay of mutations in the beta ig-h3 gene is required to establish a correct diagnosis.

Corneal Dystrophies, Hereditary↗
PubMed2000

Marked reduction of mortality in salt-loaded Dahl salt-sensitive rats by the new, selective endothelin ETA receptor antagonist, J-105859.

OBJECTIVE: To examine the chronic effects of a newly synthesized, potent and selective endothelin (ET) ETA receptor antagonist, J-1 05859, on mortality in salt-loaded Dahl salt-sensitive (DS) rats and to confirm the potential of this compound as an ETA antagonist METHODS: Vehicle and J-105859 were administered to salt-loaded DS rats for 12 weeks. Throughout the experimental period, blood pressure was measured continuously using a telemetry system and the survival rate was determined. The surviving animals were subsequently sacrificed and autopsy was performed. Binding and functional assays were also carried out to characterize J-1 05859. RESULTS: The Ki values of J-1 05859 for cloned human ETA and ETB were 0.025 and 48 nmol/l, respectively. J-105859 inhibited ET-1-induced contractions in rabbit iliac artery (pA2 = 10.08) and BQ-3020 (ETB agonist)-induced contractions in pulmonary artery (pA2 = 7.63). The pressor response to intravenous (i.v.) ET-1 (0.5 nmol/kg) was significantly inhibited by J-1 05859 at a dose of 0.03 mg/kg i.v. Chronic treatment with J-1 05859 [0.1 and 1 mg/kg per day orally (p.o.)] from the prehypertensive stage decreased the mortality of salt-loaded DS rats and markedly inhibited the development of brain lesions. The survival rates in the control and J-1 05859 (0.1 and 1 mg/kg per day) groups were 34, 80 and 100%, respectively. Development of hypertension was markedly inhibited at a dose of 1 mg/kg per day. CONCLUSION: J-105859 is a selective, potent, orally active ETA-selective antagonist ETA antagonists may reduce morbidity as well as mortality in salt-sensitive hypertension.

Alkanes↗
PubMed2000

Interaction between monocytes and vascular smooth muscle cells induces expression of hepatocyte growth factor.

OBJECTIVE: To investigate the expression of hepatocyte growth factor (HGF)--a multifunctional factor implicated in tissue regeneration, wound healing and angiogenesis--that is induced by cell-to-cell interactions between monocytes and vascular smooth muscle cells (VSMCs), using coculture of human VSMCs and cells of the human monocytoid cell line, THP-1. METHODS: We collected supernatant from the coculture medium and measured HGF concentrations with an enzyme-linked immunosorbent assay. Northern blot analysis of HGF mRNA was performed using a specific cDNA. To explore which types of cells produce HGF, we performed immunohistochemistry. RESULTS: Coculture of VSMCs with THP-1 cells for 24 h caused a fivefold increase in HGF concentrations over that in control VSMC culture. Northern blot analysis showed an induction of HGF mRNA in the coculture with a peak at 3 h. Separated cocultures demonstrated that both direct contact and soluble factors contribute to the production of HGF. Immunohistochemistry demonstrated that both types of cells in the coculture produce HGF. Neutralizing antibodies against tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 inhibited the HGF production in THP-1 cells and VSMCs that was induced by the coculture conditioned medium. The protein kinase C inhibitors H-7, calphostin C and K252b, and the tyrosine kinase inhibitor, genistein, significantly inhibited the production of HGF in the coculture. CONCLUSIONS: Cell-to-cell interactions between monocytes and VSMCs induced HGF synthesis in both types of cells, suggesting that local HGF production induced by this cell-to-cell interaction has an important role in the pathogenesis of hypertension, atherosclerosis or vascular remodelling.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Antihypertensive effects of a mixed endothelin-A- and -B-receptor antagonist, J-104132, were augmented in the presence of an AT1 -receptor antagonist, MK-954.

The single oral administration of a mixed endothelin-A-and -B- (ETA/ETB) receptor antagonist, J-104132 (L-753,037) decreased the blood pressure in conscious spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and Dahl salt-sensitive hypertensive rats fed high-salt diet (DS-H) at doses of 3 and 10 mg/kg, with a duration of approximately 24 h. The magnitude of the antihypertensive effects was greater in DS-H than in SHR and SHRSP Preproendothelin-1 mRNA expression in the kidney and aorta was greater (about twofold) in DS-H than that in nonnotensive Dahl salt-resistant rats fed high-salt diet (DR-H), while there was no difference in preproendothelin-1 mRNA expression between SHR and Wistar Kyoto rats (WKY). An AT1-receptor antagonist, MK-954 (Losartan), also attenuated hypertension in SHR and SHRSP at oral doses of 3 and 10 mg/kg, but bad no effect in DS-H. The concomitant treatment with MK-954 and J-104132 showed additive antihypertensive effects in SHR and SHRSP. In DS-H, MK-954 potentiated the antihypertensive effects of J-104132. From these results, we suggest that: (1) the contribution of endothelin (ET) to the maintenance of hypertension is greater in salt-sensitive hypertensive models than in SHR and SHRSP; (2) the antihypertensive efficacy of ETA/ETB blockade is augmented by AT1-receptor blockade; (3) ET blockers alone or in combination with AT1 antagonists could be useful in the treatment of hypertension for patients who do not respond adequately to renin-angiotensin system inhibitors alone.

Angiotensin Receptor Antagonists↗
PubMed2000

Characterization of Vibrio parahaemolyticus manganese-resistant mutants in reference to the function of the ferric uptake regulatory protein.

In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation. In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron-repressible outer membrane proteins (IROMPs). Ten out of 25 manganese-resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium. PCR-direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes. Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V. parahaemolyticus Fur. Western blotting with this antiserum revealed that the intracellular abundance of the wild-type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the wild-type Fur. These data afford an additional insight into the structure-function relationship of Fur and imply its involvement in the iron acquisition systems of V. parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.

Bacterial Proteins↗
PubMed2000

Molecular cloning, expression, and antigenicity of Seto virus belonging to genogroup I Norwalk-like viruses.

The viral capsid protein of the Seto virus (SeV), a Japanese strain of genogroup I Norwalk-like viruses (NLVs), was expressed as virus-like particles using a baculovirus expression system. An antigen detection enzyme-linked immunosorbent assay based on hyperimmune antisera to recombinant SeV was highly specific to homologous SeV-like strains but not heterologous strains in stools, allowing us type-specific detection of NLVs.

Animals↗
PubMed2000

Sequential requirements of the N-terminal palmitoylation site and SH2 domain of Src family kinases in the initiation and progression of FcepsilonRI signaling.

Initial biochemical signaling originating from high-affinity immunoglobulin E receptor (FcepsilonRI) has been ascribed to Src family kinases. To understand the mechanisms by which individual kinases drive the signaling, we conducted reconstitution experiments: FcepsilonRI signaling in RBL2H3 cells was first suppressed by a membrane-anchored, gain-of-function C-terminal Src kinase and then reconstructed with Src family kinases whose C-terminal negative regulatory sequence was replaced with a c-myc epitope. Those constructs derived from Lyn and Fyn, which are associated with detergent-resistant membranes (DRMs), physically interacted with resting FcepsilonRI and reconstructed clustering-induced signaling that leads to calcium mobilization and ERK1 and -2 activation. c-Src-derived construct, which was excluded from DRMs, failed to interact with FcepsilonRI and to restore the signaling, whereas creation of palmitoylatable Cys3 enabled it to interact with DRMs and with FcepsilonRI and to restore the signaling. Deletion of Src homology 3 (SH3) domain from the Lyn-derived construct did not alter its ability to transduce the series of signaling. Deletion of SH2 domain did not affect its association with DRMs and with FcepsilonRI nor clustering-induced tyrosine phosphorylation of FcepsilonRI beta and gamma subunits, but it almost abrogated the next step of tyrosine phosphorylation of Syk and its recruitment to FcepsilonRI. These findings suggest that Lyn and Fyn could, but c-Src could not, drive FcepsilonRI signaling and that N-terminal palmitoylation and SH2 domain are required in sequence for the initial interaction with FcepsilonRI and for the signal progression to the molecular assembly.

Animals↗
PubMed2000

Corneal guttata associated with the corneal dystrophy resulting from a betaig-h3 R124H mutation.

AIMS: To investigate the frequency of corneal guttata in patients with a corneal dystrophy resulting from an Arg124His (R124H) mutation of betaig-h3 gene. METHODS: Slit lamp examination was performed on 30 eyes with corneal dystrophy from a genetically confirmed betaig-h3 R124H mutation and on 50 age matched control eyes. The stage of the corneal dystrophy was classified as stage 0, I, or II and the degree of guttata was classified as none, mild, or severe. Specular microscopic examinations were performed to evaluate the morphology of the corneal endothelium. RESULTS: Slit lamp examination disclosed the presence of corneal guttata in 21 eyes (70%) of the 30 eyes with the corneal dystrophy, but in only one (2%) of the 50 eyes in the age matched control group (p<0.001, chi(2) with Yates's correction). Of the 12 eyes with stage I betaig-h3 R124H corneal dystrophy, seven had no corneal guttata and five had a mild degree of guttata. Of the 18 eyes with stage II, the degree of guttata was none in two, mild in nine, and severe in seven. The degree of corneal guttata was significantly related to the stage of the corneal dystrophy (p<0.0001, Kruskul-Wallis test ANOVA on ranks). There was no significant differences between eyes with betaig-h3 R124H corneal dystrophy and normal eyes in cell density, coefficient of variation, and cell hexagonality of corneal endothelium. CONCLUSION: Corneal guttata are one of the characteristics of the corneal dystrophy resulting from betaig-h3 R124H mutation.

Aged↗
PubMed2000

Long-term administration of oral low-dose topoisomerase II inhibitors, MST-16 and VP-16, for refractory or relapsed non-Hodgkin's lymphoma.

It is known that the topoisomerase II inhibitors, MST-16 (sobuzoxane) and VP-16 (etoposide), are effective for the treatment of lymphoma. Five patients with refractory or relapsed non-Hodgkin's lymphoma (NHL) were treated with a combination of oral MST-16 and VP-16 over a long period. Two patients had severely refractory NHL. The remaining 3 patients could not be treated with intensive chemotherapy because of severe organ dysfunction or a poor hematopoietic reserve. All 5 are alive and well after MST-16 and VP-16 treatment. MST-16 and VP-16 are effective for NHL when intensive chemotherapy is ineffective or contraindicated.

Administration, Oral↗
PubMed2000

Function and X chromosome inactivation analysis of B lymphocytes in myelodysplastic syndromes with immunological abnormalities.

To investigate the pathogenesis of immunological abnormalities (IA) in myelodysplastic syndrome (MDS), we examined B cells for their ability to produce cytokine and their X chromosome inactivation pattern (XCIP). An IA was defined as being positive for at least one autoimmune laboratory test (e.g. antinuclear antibody, rheumatoid factor). Seventy-three MDS patients [65 with refractory anemia (RA), 3 with RA with excess blasts (RAEB), and 5 with RAEB in transformation] were examined; 47 had IA and 26 had no IA. To examine the function of B cells in MDS, the production of interleukin (IL)-6 and IL-10 was measured in cultures of purified B cells with or without stimulators. Both IL-6 and IL-10 production rates in patients with IA were significantly higher than in patients without IA and normal controls. The skewing of XCIP of B cells was analyzed by using the polymerase chain reaction, and the skewing rate of B cell XCIP was quantitatively assayed by compared to control T lymphocytes. The skewing rate of B cells was higher in patients with IA than in those without IA and normal controls. Therefore, a small population of B cells in patients with IA might be derived from MDS clones, and be associated with the induction of IA.

Adolescent↗