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Biomedical subjects

M Watanabe

Publications and source records attributed to M Watanabe.

At least 217 records · Page 12Linked to original sources
PubMed2001

Molecular cloning and expression of the novel splice variants of K(+) channel-interacting protein 2.

Two cDNAs encoding the splice variants of K(+) channel-interacting protein 2 (KChIP2) recently reported as human KChIP2 have been identified from rat, mouse, and human heart by RT-PCR. A longer variant, KChIP2L encodes a protein of 270 amino acids, which has a 50-amino-acid insertion in N-terminus in comparison with a shorter one, KChIP2S. Interestingly, both KChIP2S and KChIP2L (KChIP2S/L) but not the original KChIP2 were expressed in human heart and umbilical vein endothelial cells (HUVECs). KChIP2S transcripts but not KChIP2L were predominantly expressed in rat, mouse, and human heart and HUVECs, whereas both transcripts were expressed at low levels in other tissues such as brain, aorta, and kidney. Using chimeric proteins of green fluorescence protein (GFP) fused to the N-terminus of KChIP2S/L, the interactions between Kv4.3 and KChIP2S/L were analyzed in native and Kv4.3-expressed HEK293 cells. Specific localization of GFP-fused KChIP2S/L proteins on or near cell membrane was observed only in Kv4.3-expressed HEK293 cells.

Amino Acid Sequence↗
PubMed2001

A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor alpha coactivator through the N-terminal activation domain (AF-1) with an RNA coactivator, SRA.

One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identified a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor alpha (hER alpha) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hER alpha A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogen-bound hER alpha in MCF7 cells and in partially purified complexes associated with hER alpha from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hER alpha and TIF2 in the nucleus. The presence of p72/p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hER alpha. These findings indicate that p72/p68 acts as an ER subtype-selective coactivator through ER alpha AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.

Amino Acid Motifs↗
PubMed2001

Number and position of mutations in the interferon (IFN) sensitivity-determining region of the gene for nonstructural protein 5A correlate with IFN efficacy in hepatitis C virus genotype 1b infection.

To explore the relationship between responses to interferon (IFN) and the mutation patterns in the IFN sensitivity-determining region (ISDR; amino acid positions 2209-2248) in the NS5A gene of hepatitis C virus genotype 1b, a cohort of 334 patients was analyzed. The number of mutations in the ISDR was higher in patients with sustained response (SR) than in patients with transient or no response (P<.001). Patients with viruses mutated at positions 2209 (P=.02), 2216 (P=.01), or 2227 (P=.02) more frequently experienced SR than did those without these mutations. Mutation occurred most frequently at position 2218, where the presence of cysteine was significantly associated with SR. Thus, the mutation pattern in the ISDR affects the virologic response to IFN and reflects different influences on the function of the NS5A protein. ISDR sequence analysis would allow the prediction of clinical IFN efficacy in individual patients.

Amino Acid Sequence↗
PubMed2001

Improvement in atrophic gastritis and intestinal metaplasia in patients in whom Helicobacter pylori was eradicated.

BACKGROUND: Glandular atrophy and intestinal metaplasia are precancerous lesions; whether Helicobacter pylori eradication affects these lesions is controversial. OBJECTIVE: To determine whether H. pylori eradication is associated with improvement in glandular atrophy and intestinal metaplasia after at least 1 year. DESIGN: Single-blind, uncontrolled prospective trial. SETTING: Academic gastroenterology clinic in Japan. PATIENTS: 163 consecutive patients with dyspepsia and H. pylori infection. INTERVENTION: One-week course of a proton-pump inhibitor and antibiotic therapy. MEASUREMENTS: Endoscopic examination with antral and corporal biopsy was done before treatment and at 1 to 3 and 12 to 15 months after treatment. Gastritis, atrophy, and metaplasia were graded according to the updated Sydney System. RESULTS: In the 115 patients in whom H. pylori was eradicated, inflammation and mean neutrophil activity had decreased by 1 to 3 months, and both glandular atrophy in the corpus and intestinal metaplasia in the antrum had decreased by 12 to 15 months. Glandular atrophy in the corpus improved in 34 (89%) of 38 patients with atrophy before treatment, and intestinal metaplasia in the antrum improved in 28 (61%) of 46 patients who had metaplasia at baseline. In the 48 patients in whom eradication was unsuccessful, no significant histologic changes were observed. CONCLUSION: In the year after successful H. pylori eradication, precancerous lesions improved in most patients.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed2001

Regional expression of the splice variants of Kv4.3 in rat brain and effects of C-terminus deletion on expressed K+ currents.

In situ hybridization and RT-PCR analyses have revealed that, among three Kv4.3 splice variants (a, b, and c) with distinct C-terminal cytoplasmic domains, the mRNA for Kv4.3a is abundant in cerebral cortex, cerebellum, olfactory bulb, and medulla oblongata, whereas the mRNA for Kv4.3c is localized mainly to hippocampus. Three new distinct splice variants of Kv4.3 (Kv4.3d, e and f), which consist of 601, 635, and 628 amino acids, respectively, and have distinct C-terminal cytoplasmic domains, were isolated from rat brain by RT-PCR. Kv4.3b, d, e and f were expressed at much lower levels in brain. Mutagenesis which removed 149 amino acids in C-terminal domain of Kv4.3a significantly slowed its rate of recovery from inactivation as measured in heterologous expression in HEK293 cells. Surprisingly, however, neither the rate of inactivation nor voltage dependence of the activation and inactivation were changed.

Amino Acid Sequence↗
PubMed2001

Distinct roles for the N- and C-terminal regions in the cytotoxicity of pierisin-1, a putative ADP-ribosylating toxin from cabbage butterfly, against mammalian cells.

Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1-233 or 234-850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N- and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing "nicked" pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N- and C-terminal fragments with intracellular protease, and the N-terminal fragment then exhibits cytotoxicity.

ADP Ribose Transferases↗
PubMed2001

A repressor element in the 5'-untranslated region of human Pax5 exon 1A.

B-cell-specific activator protein (BSAP) encoded by the Pax5 gene plays a critical role during B-cell development. We have analyzed the 5'-flanking region plus the 5'-untranslated region (5'-UTR) of human Pax5 exon1A to clarify its regulatory mechanisms. Functional dissection of these regions by luciferase reporter assays indicated that a cluster of regulatory elements acts as a strong repressor between +320 and +453. Insertion of this segment between the heterologous simian virus 40 (SV40) promoter and the luciferase gene in both the sense and reverse orientation sharply reduced the luciferase activity, but insertion into the upstream of the SV40 promoter did not. This suggests that this segment must be located in the 5'-UTR to function effectively. A search through databases with the sequence of this segment did not reveal any known DNA binding factor site. Electrophoretic gel mobility shift assay (EMSA) experiments demonstrated that unknown factors bound to the fragment +408 to +429. Insertion of this fragment between the SV40 promoter and the reporter gene strongly suppressed the luciferase activity. Competitive EMSA indicated that the region between nucleotides +413 and +427 encompassed the binding site of the unknown factors and was hence regarded as a repressor element. Mutagenesis in this element significantly recovered reporter gene activity. These results suggest that the segment +320 to +453, especially the repressor element +413 to +427, in the 5'-UTR is involved in the regulation of Pax5 gene expression.

Base Sequence↗
PubMed2001

Co-infection by serologically-silent hepatitis B virus may contribute to poor interferon response in patients with chronic hepatitis C by down-regulation of type-I interferon receptor gene expression in the liver.

Intrahepatic mRNA levels of type-I interferon (IFN) receptor genes have been shown to correlate with the clinical efficacy of IFN therapy in patients with chronic hepatitis C. Recently, co-infection by serologically-silent hepatitis B virus (HBV) has been assumed to be associated with the poor IFN response in patients with chronic hepatitis C. The aim of this study was to investigate the relationship between the co-infection of serologically-silent HBV and type-I IFN receptor gene expression in the liver of patients with chronic hepatitis C. The intrahepatic mRNA levels of IFNAR2, one of the two subunits of the type-I IFN receptor, were quantified and compared with both the prevalence of HBV DNA and the hepatitis C virus (HCV) genotype in 45 patients with chronic hepatitis C, who were negative for hepatitis B surface antigen. Co-infection, as evaluated by a nested polymerase chain reaction, was present in 22 patients (48.9%), with dominance of the HCV genotype 1b (65.2%) over genotype 2a (31.8%). Co-infection was associated with lower IFNAR2 mRNA levels, higher levels of serum HCV RNA, and a poor IFN response, regardless of the HCV genotype. The findings suggest the possibility that co-infection by serologically-silent HBV is one of the factors that can lead to an unfavorable IFN response in chronic hepatitis C by down-regulation of IFN receptor gene expression in the liver.

Adult↗
PubMed2001

Purkinje cell-specific and inducible gene recombination system generated from C57BL/6 mouse ES cells.

Spatiotemporally restricted gene targeting is needed for analyzing the functions of various molecules in a variety of biological phenomena. We have generated an inducible cerebellar Purkinje cell-specific gene targeting system. This was achieved by establishing a mutant mouse line (D2CPR) from a C57BL/6 mouse ES cell line, which expressed a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR). The Purkinje cell-specific expression of CrePR was attained by inserting CrePR into the glutamate receptor delta2 subunit (GluRdelta2) gene, which was expressed specifically in the Purkinje cells. Using the transgenic mice carrying the Cre-mediated reporter gene, we showed that the antiprogesterone RU486 could induce recombinase activity of the CrePR protein specifically in the mature cerebellar Purkinje cells of the D2CPR line. Thus this mutant line will be a useful tool for studying the molecular function of mature Purkinje cells by manipulating gene expression in a temporally restricted manner.

Animals↗
PubMed2001

Neoadjuvant chemotherapy using low-dose consecutive intraarterial infusions of cisplatin combined with 5-fluorouracil for locally advanced cervical adenocarcinoma.

OBJECTIVE: The goal of this work was to evaluate response rate, toxicity, and survival in treatment with intraarterial 5-fluorouracil (5-FU) and cisplatin in a neoadjuvant setting; this combination was administered to patients with locally advanced cervical adenocarcinoma. METHODS: Eleven patients were treated with preoperative neoadjuvant chemotherapy. Those eligible included patients with previously untreated stage IB, II, or III adenocarcinoma with good performance status. Treatment consisted of bilateral internal iliac artery infusion of cisplatin (a total of 10 mg/day) for 30 min, followed by 5-FU (a total of 250 mg/day) given by 24-hour continuous infusion for 10 days. Treatment was repeated every 3 weeks for a total of two or three cycles. All except one patient with progressive disease underwent radical hysterectomy following neoadjuvant chemotherapy. Postoperative radiotherapy was given to the whole pelvis to 6 patients; 3 of the 6 patients with involved common iliac nodes received radiotherapy to a paraaortic field in addition to the whole pelvis. RESULTS: Among 11 eligible patients, 7 had a partial response (64%). Stable disease was observed in 3 cases (27%) and progressive disease in 1 (9%). Histopathological changes related to chemotherapy, however, revealed only mild effects. Of the 24 treatment cycles administered, no Grade 3 or 4 toxicity was observed and there were no therapy-related deaths. The median follow-up period was 30 months (range, 1-65 months). The mean survival period was 34.7 months and the 5-year survival rate was 21.2%. CONCLUSIONS: Intraarterial neoadjuvant chemotherapy effectively reduced tumor size in patients with locally advanced cervical adenocarcinoma; however, a survival advantage was not clear.

Adenocarcinoma↗
PubMed2001

Carcinosarcoma of the uterus: immunohistochemical and genetic analysis of clonality of one case.

BACKGROUND: Carcinosarcomas of the uterus are characterized by admixtures of malignant epithelial and stromal cells, and their histogenesis remains controversial. CASE: An operated case of carcinosarcoma of the uterus in a 49-year-old woman is reported with clonal analysis. The tumor was composed of carcinomatous, sarcomatous, and transitional elements in the frontal wall of the uterine body and therefore was diagnosed as a carcinosarcoma. On immunohistochemical analysis, the sarcomatous component proved negative for epithelial membrane antigen and keratin while both components were positive for vimentin. Analysis of X-chromosome inactivation showed the same pattern throughout and additionally, the same K-ras and p53 mutations were homogeneously detected. Microsatellite instability analysis showed loss of heterozygosity at D5S346 in the sarcomatous but not the carcinomatous component. CONCLUSIONS: This tumor appears monoclonal in line with the combination tumor theory, with late divergence in genetic alteration in the sarcomatous elements.

Carcinosarcoma↗
PubMed2001

Stage III endometrial cancer: analysis of prognostic factors and failure patterns after adjuvant chemotherapy.

OBJECTIVE: This study was performed to assess the prognostic factors and patterns of recurrence in stage III endometrial carcinoma treated with surgery and adjuvant chemotherapy. METHODS: A retrospective review of 61 stage III endometrial carcinoma patients treated between 1988 and 1998 at Niigata University Hospital was performed. All patients underwent surgery, followed by adjuvant chemotherapy consisting of intravenous cisplatin, doxorubicin, and cyclophosphamide. Multivariate analysis was performed for the prognostic factors and actuarial techniques were used for the survival and recurrence rates. RESULTS: The 5-year disease-free survival was 78.6%. Multivariate analysis revealed deep myometrial invasion and lymph-vascular space involvement correlated significantly with disease-free survival. Based on these two factors, the patients could be divided into low-risk and high-risk groups. The 5-year disease-free survival for the low-risk group was 100%, which was significantly better than the 59.1% for the high-risk group. Disease recurrence occurred in 13 of 30 high-risk patients, and there was no recurrence in the 31 low-risk patients. Looking at the patterns of recurrence for the high-risk group by lymph node metastasis, 5 recurrences were locoregional, 1 was locoregional/distant, and 1 was distant in 16 node-positive high-risk patients. In 14 node-negative patients, 5 had distant and 1 had locoregional/distant recurrences. CONCLUSIONS: The locoregional failure in the node-positive high-risk group deserves further attention. For improvement of locoregional control, it may be worthwhile to consider new strategies. The role of new adjuvant chemotherapy should be investigated to control distant failure in node-negative high-risk patients.

Adult↗
PubMed2001

Neoadjuvant chemotherapy for patients younger than 50 years with high-risk squamous cell carcinoma of the cervix.

OBJECTIVE: To evaluate the response rate and toxicity and to improve survival, neoadjuvant chemotherapy (NAC) was utilized in patients younger than 50 years with locally advanced cervical squamous cell carcinoma. METHODS: Twenty-one patients were treated with preoperative NAC. Eligibility included patients with previously untreated stage IB or IIA with deep stromal invasion assessed by magnetic resonance imaging or bulky tumor or IIB squamous cell carcinoma who were younger than 50 years. The NAC consisted of cisplatin (60 mg/m(2)) on day 1, vinblastine (4 mg/m(2)/day) on days 1 and 2, and peplomycin (10 mg/day) on days 1, 8, and 15 (PVP). Treatment was repeated every 3 weeks for a total of two cycles. All 21 patients underwent radical hysterectomy following NAC. Postoperative radiotherapy was given to 18 patients. We used 21 patients who underwent radical hysterectomy and postoperative radiation therapy as a nonrandomized control group. RESULTS: The response rate for NAC was 86% (18/21). Two patients required discontinuation of PVP treatment after one administration because of grade 4 neutropenia and thrombocytopenia, and decreased carbon monoxide diffusion capacity, respectively. In the NAC group, stromal invasion was significantly reduced (P = 0.0103), and the incidence of lymph node metastasis was decreased. No patients had positive parametrial and vaginal margins. The overall 5-year survival rate was 84.0% in the NAC group, which was significantly better than that in the control group (58.9%) (P = 0.0434). CONCLUSIONS: NAC for younger patients with locally advanced cervical carcinoma is thought to be safe, well tolerated, effective, and useful for increasing operability, decreasing pathological risk factors, and improving survival.

Adult↗
PubMed2001

Overexpression of cdk Inhibitor p16INK4a by adenovirus vector inhibits cardiac hypertrophy in vitro and in vivo: a novel strategy for the gene therapy of cardiac hypertrophy.

Cardiac hypertrophy is one of the serious complications which increase mortality due to cardiovascular diseases. However, only a partial reduction of cardiac hypertrophy has been successful using current drug therapy. We demonstrate here reduction of cardiac hypertrophy in vitro and in vivo using an adenovirus vector encoding cyclin-dependent kinase (cdk) inhibitor p16INK4a. Adenovirus-mediated overexpression of cdk inhibitor p16INK4a completely inhibited cardiac myocyte hypertrophy induced by endothelin (ET)-1, as evaluated by [3H]leucine incorporation into the cells and mRNA levels of skeletal alpha -actin (SK-A) or atrial natriuretic peptide (ANP) as well as by morphometric analyses. We then evaluated whether p16INK4a can suppress left-ventricular (LV) hypertrophy induced by aortic banding (AOB) in rats. Catheter-mediated gene transfer of AxCAp16 was performed according to the method reported by Hajjar et al. LV overload was produced by coarctation of the ascending aorta immediately after inoculation of the heart with adenovirus. Two weeks after the procedure, the left ventricular weight/body weight ratio (LVW/BW) increased in the AOB+LacZ group in comparison to that in controls. However, LVW/BW was identical in the AOB+p16 group and controls. Histologic analysis revealed that p16INK4a inhibited hypertrophy of cardiac myocytes. These results suggest that G1 cell cycle regulators may restrict cardiac hypertrophy, and offer a novel strategy for the gene therapy of cardiac hypertrophy.

Actins↗
PubMed2001

FR167653 improves survival and pulmonary injury after partial hepatectomy under ischemia/reperfusion in rats.

BACKGROUND: FR167653 is a potent suppressant of production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta, which play an important role in hepatic and pulmonary injury due to ischemia/reperfusion of the liver and in liver regeneration after hepatectomy. We examined the effects of FR167653 on hepatectomy under ischemia/reperfusion in rats. METHODS: After initial 15-min ischemia and 5-min reperfusion, 70% hepatectomy was performed during the second 15-min ischemia period in FR167653-treated (FR group) and saline-treated (saline group) rats. The survival rate, relative liver weight, TNF-alpha, IL-1 beta, DNA synthesis rate of the remnant liver, and histological change and adhesion molecule (ICAM-1) of the lung were examined. Serum glutamic pyruvic transaminase and hepatic malondialdehyde were also measured. RESULTS: Expressions of TNF-alpha and IL-1 beta in the remnant liver were significantly inhibited in the FR group compared to the saline group. The survival was significantly better and pulmonary damage was less in the FR group after hepatectomy under ischemia/reperfusion. ICAM-1 expression of the lung was not altered after hepatectomy and was not significantly different between the two groups. Liver regeneration and injury were not significantly different between the two groups. CONCLUSION: FR167653 does not affect liver injury and regeneration after hepatectomy under ischemia/reperfusion, while it ameliorates pulmonary injury and improves the survival.

Alanine Transaminase↗
PubMed2001

Postsynaptic modulation of AMPA receptor-mediated synaptic responses and LTP by the type 3 ryanodine receptor.

The precise function of ryanodine receptors (RyRs) in synaptic transmission is unknown, but three of their subtypes are expressed in the brain. We examined the roleof RyRs in excitatory synaptic transmission in hippocampal slices, using type 3 RyR (RyR3)-deficient mice. The alpha-amino-3-hydroxy-5-methyl-4-isoxozolepropionic acid (AMPA) receptor-mediated basal synaptic responses in the CA1 region of mutant mice were smaller than those of wild-type mice, while there was no difference in N-methyl-d-aspartate receptor-mediated responses, suggesting selective postsynaptic modification of AMPA receptors by RyR3. The expression of synaptic AMPA receptor subunits examined by Western blotting or immunohistochemistry was indistinguishable, suggesting that the smaller AMPA synaptic responses in mutant mice were not due to the reduced number of synaptic AMPA receptors. Although the initial potentiation following tetanic stimulation of afferent fibers was similar, long-term potentiation (LTP) was smaller in mutant mice. There were no differences in presynaptic electrophysiological properties. We thus conclude that RyR3 postsynaptically regulates the properties of AMPA receptors and LTP.

2-Amino-5-phosphonovalerate↗
PubMed2001

Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues.

The mechanisms leading to neurodegeneration in ALS (amyotrophic lateral sclerosis) are not well understood, but cytosolic protein aggregates appear to be common in sporadic and familial ALS as well as transgenic mouse models expressing mutant Cu/Zn superoxide dismutase (SOD1). In this study, we systematically evaluated the presence of these aggregates in three different mouse models (G93A, G85R, and G37R SOD1) and compared these aggregates to those seen in cases of sporadic and familial ALS. Inclusions and loss of motor neurons were observed in spinal cords of all of these three mutant transgenic lines. Since a copper-mediated toxicity hypothesis has been proposed to explain the cytotoxic gain-of-function of mutant SOD1, we sought to determine the involvement of the copper chaperone for SOD1 (CCS) in the formation of protein aggregates. Although all aggregates contained CCS, SOD1 was not uniformly found in the inclusions. Similarly, CCS-positive skein-like inclusions were rarely seen in ALS neurons. These studies do not provide strong evidence for a causal role of CCS in aggregate formation, but they do suggest that protein aggregation is a common event in all animal models of the disease. Selected proteins, such as the glutamate transporter GLT-1, were not typically observed within the inclusions. Most inclusions were positively stained with antibodies recognizing ubiquitin, proteasome, Hsc70 in transgenic lines, and some Hsc70-positive inclusions were detected in sporadic ALS cases. Overall, these observations suggest that inclusions might be sequestered into ubiquitin-proteasome pathway and some chaperone proteins such as Hsc70 may be involved in formation and/or degradation of these inclusions.

Amyotrophic Lateral Sclerosis↗