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Biomedical subjects

H Watanabe

Publications and source records attributed to H Watanabe.

At least 595 records · Page 33Linked to original sources
PubMed1999

Perlecan is essential for cartilage and cephalic development.

Perlecan, a large, multi-domain, heparan sulfate proteoglycan originally identified in basement membrane, interacts with extracellular matrix proteins, growth factors and receptors, and influences cellular signalling. Perlecan is present in a variety of basement membranes and in other extracellular matrix structures. We have disrupted the gene encoding perlecan (Hspg2) in mice. Approximately 40% of Hspg2-/- mice died at embryonic day (E) 10.5 with defective cephalic development. The remaining Hspg2-/- mice died just after birth with skeletal dysplasia characterized by micromelia with broad and bowed long bones, narrow thorax and craniofacial abnormalities. Only 6% of Hspg2-/- mice developed both exencephaly and chondrodysplasia. Hspg2-/- cartilage showed severe disorganization of the columnar structures of chondrocytes and defective endochondral ossification. Hspg2-/- cartilage matrix contained reduced and disorganized collagen fibrils and glycosaminoglycans, suggesting that perlecan has an important role in matrix structure. In Hspg2-/- cartilage, proliferation of chondrocytes was reduced and the prehypertrophic zone was diminished. The abnormal phenotypes of the Hspg2-/- skeleton are similar to those of thanatophoric dysplasia (TD) type I, which is caused by activating mutations in FGFR3 (refs 7, 8, 9), and to those of Fgfr3 gain-of-function mice. Our findings suggest that these molecules affect similar signalling pathways.

Abnormalities, Multiple↗
PubMed1999

Mice lacking link protein develop dwarfism and craniofacial abnormalities.

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of aggrecan and hyaluronan, giving cartilage its tensile strength and elasticity. Cartilage provides the template for endochondral ossification and is crucial for determining the length and width of the skeleton. During endochondral bone formation, hypertrophic chondrocytes die and the cartilage is replaced with bone matrix. Here, we have generated targeted mutations in mice in the gene encoding LP (Crtl1). Homozygotes showed defects in cartilage development and delayed bone formation with short limbs and craniofacial anomalies. Most Crtl1(tm1Nid/tm1Nid) mice died shortly after birth due to respiratory failure, but some survived and developed progressive dwarfism and lordosis of the cervical spine. They showed small epiphysis, slightly flared metaphysis of long bones and flattened vertebrae, characteristic of spondyloepiphyseal dysplasias. The cartilage contained significantly reduced aggrecan depositions in the hypertrophic zone, and decreased numbers of prehypertrophic and hypertrophic chondrocytes. Reduced Indian hedgehog (Ihh) expression was observed in prehypertrophic chondrocytes, and apoptosis was inhibited in hypertrophic chondrocytes. These results indicate that LP is important for the formation of proteoglycan aggregates and normal organization of hypertrophic chondrocytes, and suggest that cartilage matrix has a role in chondrocyte differentiation and maturation.

Animals↗
PubMed1999

Bcl-2 expression related to altered p53 protein and its impact on the progression of human pancreatic carcinoma.

p53 and Bcl-2 are two important factors related to apoptosis and tumorigenesis. In this study, a series of 52 cases of pancreatic carcinoma (PC) were investigated using an immunohistochemical assay to determine whether altered expression of Bcl-2 and p53 has an impact on the progression of this malignancy. Cytoplasmic immunoreactivity for Bcl-2 and nuclear staining of p53 was found in 12 (23.1%) and 32 (63.5%) cases of PC respectively. Furthermore, an inverse correlation between the expression of p53 and Bcl-2 existed in this series (P < 0.01). In a subgroup, the proportion of tumours showing that p53-positive and Bcl-2-negative staining was increased with increasing histological grade and clinical stage (P < 0.05), and moreover, the survival period of those patients whose tumour had this staining was shorter than those with other staining patterns of combined p53 and Bcl-2 (P < 0.05). Therefore, it is concluded that simultaneously aberrant expression of Bcl-2 and p53 may confer PC with more malignant clinicopathological characteristics.

Adenocarcinoma↗
PubMed1999

Endothelium-derived relaxing, contracting and hyperpolarizing factors of mesenteric arteries of hypertensive and normotensive rats.

Differences in the acetylcholine (ACh)-induced endothelium-dependent relaxation and hyperpolarization of the mesenteric arteries of Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) were studied. Relaxation was impaired in preparations from SHRSP and tendency to reverse the relaxation was observed at high concentrations of ACh in these preparations. Relaxation was partly blocked by NG-nitro-L-arginine (L-NOARG, 100 microM) and, in the presence of L-NOARG, tendency to reverse the relaxation was observed in response to higher concentrations of ACh, even in preparations from WKY. The relaxation remaining in the presence of L-NOARG was also smaller in preparations from SHRSP. The tendency to reverse the relaxation observed at higher concentrations of ACh in preparations from SHRSP or WKY in the presence of L-NOARG were abolished by indomethacin (10 microM). Elevating the K+ concentration of the incubation medium decreased relaxation in the presence of both indomethacin and L-NOARG. Relaxation in the presence of L-NOARG and indomethacin was reduced by the application of both apamin (5 microM) and charybdotoxin (0.1 microM). This suggests that the relaxation induced by ACh is brought about by both endothelium-derived relaxing factor (EDRF, nitric oxide (NO)) and hyperpolarizing factor (EDHF), which activates Ca2+-sensitive K+ channels. Electrophysiological measurement revealed that ACh induced endothelium-dependent hyperpolarization of the smooth muscle of both preparations in the presence of L-NOARG and indomethacin; the hyperpolarization being smaller in the preparation from SHRSP than that from WKY. These results suggest that the release of both NO and EDHF is reduced in preparations from SHRSP. In addition, indomethacin-sensitive endothelium-derived contracting factor (EDCF) is released from both preparations; the release being increased in preparations from SHRSP.

Acetylcholine↗
PubMed1999

Relationship between NaF- and thapsigargin-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. In isolated rat mesenteric artery with endothelium, NaF caused slowly developing hyperpolarization. The hyperpolarizing effect was unchanged in the presence of N(G)-nitro-L-arginine (L-NOARG) and indomethacin, but was markedly reduced by high K+. In Ca2+ -free medium or in the presence of Ni2+, NaF failed to produce hyperpolarization. 2. NaF-induced hyperpolarization was substantially unaffected by deferoxamine, an Al3+ chelator, okadaic acid and calyculin A, phosphatase inhibitors, and preincubation with pertussis toxin, suggesting that neither the action of fluoroaluminates as a G protein activator nor inhibition of phosphatase activity contributes to the hyperpolarizing effect. 3. The selective inhibitors of the Ca2+ -pump ATPase of endoplasmic reticulum, thapsigargin and cyclopiazonic acid, elicited hyperpolarization, whose properties were very similar to those of NaF. When intracellular Ca2+ stores had been depleted with these inhibitors, NaF no longer generated hyperpolarization. 4. In Ca2+ -free medium, NaF (or thapsigargin) caused a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) in cultured porcine aortic endothelial cells, and subsequent application of thapsigargin (or NaF) failed to increase [Ca2+]i. 5. In arterial rings precontracted with phenylephrine, NaF produced endothelium-dependent relaxation followed by sustained contraction even in the presence of L-NOARG and indomethacin. The relaxant response was abolished by high K+ or cyclopiazonic acid. 6. These results indicate that NaF causes endothelium-dependent hyperpolarization, thereby leading to smooth muscle relaxation of rat mesenteric artery. This action appears to be mediated by the promotion of Ca2+ influx into endothelial cells that can be triggered by the emptying of intracellular Ca2+ stores, as proposed for those of thapsigargin and cyclopiazonic acid.

Animals↗
PubMed1999

Production of monoclonal antibody and development of enzyme-linked immunosorbent assay for kanamycin in biological matrices.

Monoclonal antibodies (MAbs) against kanamycin were prepared by using a kanamycin-bovine gamma-globulin conjugate for the immunization of mice. Splenocytes from BALB/c immunized mice were fused with P3X63Ag8U.1 myeloma cells. This resulted in two hybridoma cell lines. Fifty per cent inhibition concentrations (IC50) for the MAbs were 2 and 5 ng ml-1. One MAb (IC50 = 2 ng ml-1) was named #22 and was used to develop quantitative assays for kanamycin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 0.2 ng ml-1 and the standard deviations were 0.2-4.4% for intra-assay and 0.6-4.7% for inter-assay, respectively. The detection limits using peroxidase were 4 ppb in cattle milk, cattle plasma, cattle urine, swine plasma, swine urine and chicken plasma. Using the MAb #22 produced, a rapid test kit based on an immunochromatographic method was developed. The detection limits using the kit were 50 ppb in cattle milk, cattle plasma, cattle urine and chicken plasma.

Animals↗
PubMed1999

Mice with early onset of death (EOD) due to lupus glomerulonephritis.

Both MRL-lpr/lpr (lpr) and BXSB mice fall victim to autoimmune disease as a function of age. To combine their properties, brother-sister mating of (female lpr x male BXSB)F1 mice was done. Mice for mating were selected according to indicators of early onset of glomerulonephritis and subsequent early death (i.e., EOD). This mating was continued for more than 16 generations. The EOD mice thus established had homozygous H-2k/k, lpr/lpr, and possible yaa/- (in the case of males). The average life span of males was 83 days while that of females was 126 days. After 12 weeks of age, the majority (> 80%) of male EOD mice were characterized by the abnormality of urine due to glomerulonephritis. We then characterized how glomerulonephritis was evoked, especially in terms of expanding lymphocyte subsets in various immune organs. Similar to the case of parental lpr mice, the major expanding cells were CD4-8-B220+ TCRint cells in the immune organs and kidney. In addition, myeloid cells were found to infiltrate the kidney. This massive infiltration of both TCRint cells and myeloid cells might be responsible for the onset of acute glomerulonephritis. Even after more than 50 generations, these EOD mice still carry both lpr and yaa genes. These results suggest that EOD mice might be a very useful tool for the study of acute lupus glomerulonephritis which is evoked by the genetic abnormalities.

Acute Disease↗
PubMed1999

Quick recovery in the generation of self-reactive CD4low natural killer (NK) T cells by an alternative intrathymic pathway when restored from acute thymic atrophy.

The thymus comprises the mainstream of T cell differentiation which produces conventional T cells and an alternative pathway which produces primordial T cells with intermediate density of T cell receptor (TCR)-CD3 complex on the surface (i.e. intermediate TCR cells or TCRint cells). We induced acute thymic atrophy in mice by an administration of hydrocortisone (10 mg) or irradiation (6.5 Gy). It was demonstrated that CD3intCD4lowNK1.1+ T cells were immediately generated by an alternative intrathymic pathway without passing through the double-positive CD4+8+ stage, when restored from thymic atrophy (days 3-14). These CD3intCD4lowNK1.1+ T cells mediated self-reactivity and appeared even in the periphery. mRNA of an invariant chain of TCR Valpha14Jalpha281 gene product was detected in these CD4low T cells, but not remaining CD4high T cells. The mainstream of T cell differentiation in the thymus was not restored up to day 14 and there was no leakage of self-reactive clones into the population generated through the mainstream. These results reveal that an alternative intrathymic pathway is associated with the generation of self-reactive T cells, in an early restoration phase after thymic atrophy.

Acute Disease↗
PubMed1999

Enrichment of c-kit+ Lin- haemopoietic progenitor cells that commit themselves to extrathymic T cells in in vitro culture of appendix mononuclear cells.

The appendix as well as the small intestine have recently been found to carry c-kit+ stem cells which give rise to extrathymic T cells. In this study, the properties of c-kit+ stem cells in the appendix of mice were further characterized. When appendix mononuclear cells (MNC) were cultured in the presence of stem cell factor, interleukin-3, interleukin-6 and erythropoietin on a methylcellulose culture plate, the population of c-kitdull Lin- and that of c-kithi Lin- cells expanded. Morphological study revealed that these c-kithi Lin- cells were basophilic granular cells (possibly mast cells). Both populations of cultured appendix MNC were then injected into severe combined immunodeficient mice or cultured with Tst-4 thymic stroma cells. These in vivo and in vitro studies demonstrated that c-kitdull Lin- cells were oligopotent haemopoietic progenitor cells which gave rise to extrathymic T cells, while c-kithi Lin- cells lacked haemopoietic progenitor cell activity. In contrast to c-kit+ stem cells in the bone marrow, those in the appendix did not give rise to myeloid cells and conventional thymic T cells under any of the conditions tested. The present results suggest that the appendix primarily comprises c-kit+ cells which give rise to basophilic granular cells and extrathymic T cells and that such c-kit+ cells have the ability to replicate themselves in culture in vitro.

Animals↗
PubMed1999

TNF-alpha, nitric oxide and IFN-gamma are all critical for development of necrosis in the small intestine and early mortality in genetically susceptible mice infected perorally with Toxoplasma gondii.

We previously reported that genetic susceptibility of mice to peroral infection with T. gondii is associated with CD4+ T cell-dependent, interferon (IFN)-gamma-mediated necrosis of their small intestine. We examined the role of tumour necrosis factor (TNF)-alpha and nitric oxide (NO), in addition to IFN-gamma. At 7 days after infection, a marked increase in CD4+ T cells was observed in lamina propria mononuclear cells (LPC) of the small intestine as compared with normal mice, and significantly greater amounts of mRNA for IFN-gamma, TNF-alpha, and inducible NO synthase (iNOS) were detected in LPC of the small intestine of infected than uninfected animals. Treatment of infected mice with anti-TNF-alpha monoclonal antibody (mAb) or the iNOS inhibitor, aminoguanidine, prevented necrosis and prolonged time to death. Infected iNOS-targeted mutant mice did not develop the disease whereas infected, control mice did. Treatment with anti-TNF-alpha mAb did not affect the expression of IFN-gamma in the LPC but inhibited expression of iNOS in the infected mice, indicating the role of TNF-alpha in the induction of iNOS. These results suggest that NO induced by a combination of IFN-gamma and TNF-alpha through activation of iNOS is a critical mediator of intestinal pathology and contributes to early mortality in genetically susceptible mice.

Administration, Oral↗
PubMed1999

Purification and cDNA cloning of a protein derived from Flammulina velutipes that increases the permeability of the intestinal Caco-2 cell monolayer.

A new protein that decreases transepithelial electrical resistance (TEER) in the human intestinal Caco-2 cell monolayer was found in a water-soluble fraction of the mushroom Flammulina velutipes. This protein, termed TEER-decreasing protein (TDP), is not cytotoxic and does not induce cell detachment, but rapidly increases the tight junctional permeability for water-soluble marker substances such as Lucifer Yellow CH (Mr 457) through the paracellular pathway. TDP was isolated and purified from the aqueous extract of F. velutipes by chromatographic means. Purified TDP was found to be a simple, nonglycosylated protein without intermolecular disulfide bonds, and the apparent molecular mass as estimated by SDS/PAGE and gel filtration is 30 kDa. It was revealed that the N-terminal amino-acid sequence of purified TDP is identical to the recently reported N-terminal sequence of flammutoxin, a membrane-perturbing hemolytic protein, for which the complete primary structure has not yet been reported [Tomita, T., Ishikawa, D., Noguchi, T., Katayama, E., and Hashimoto, Y. (1998) Biochem. J. 333, 24794-24799]. The cDNA coding for TDP was cloned by 5' and 3' rapid amplification of cDNA ends. The ORF encodes a protein with 272 amino-acid residues showing no homology to known proteins. Relevant studies using TDP cDNA will provide insight into the structure-function relationships of membrane pore-forming toxins.

Amino Acid Sequence↗
PubMed1999

Systematic treatment for nocturnal urinary frequency following a sleep-micturition chart.

Seven patients complaining of nocturnal urinary frequency were treated following a sleep-micturition chart. By clinical analysis of the data, the causes of nocturnal urinary frequency were divided into three diagnostic categories: a small bladder capacity group, a sleep disorder group and a large nocturnal urinary volume group. The three groups were treated by anticholinergic agents, sleeping pills and restriction of water intake at night respectively. With the administration of anti-cholinergic agents or sleeping pills nocturnal bladder capacity increased. By restriction of water intake at night nocturnal urinary volume decreased. In all patients nocturnal urinary frequency decreased and sleep efficiency was improved.

Aged↗
PubMed1999

K-ras codon 12 and p53 mutations in biopsy specimens and bile from biliary tract cancers.

To evaluate whether it is useful for diagnosis to detect K-ras and p53 mutations in biopsy specimens and bile of biliary tract lesions, 12 cholangiocarcinomas (CC), eight cases of cholangitis, seven gallbladder carcinomas (GBC), seven gallbladder cholesterol polyps, four cases of adenomyomatosis of the gallbladder and five cases of cholecystitis were examined. K-ras and p53 mutations in bile were detected by a two-step polymerase chain reaction (PCR) and nested PCR-single-strand conformation polymorphism (SSCP) analysis. In addition, p53 protein expression in biopsy specimens from CC were examined by immunostaining. K-ras mutations at codon 12 were detected in 50% of CC and 57.1% of GBC in both biopsy specimens and bile. The incidence of p53 mutations was 33.3% in CC and 42.9% in GBC. p53 protein overexpression was observed in 60% CC biopsy specimens. In contrast, K-ras and p53 abnormalities were not detected in any non-neoplastic biliary tract lesion. K-ras and p53 mutations in biliary tract cancers showed the same mutation patterns in spite of differences in the collection methods used between bile and biopsy specimens or surgically resected tissue. Genetic analysis of K-ras and p53 mutations in biopsy specimens and bile may be useful for the diagnosis of biliary tract cancers, although it may be effectively limited to patients with advanced disease.

Base Sequence↗
PubMed1999

Esophageal adenocarcinoma that probably originated in the esophageal gland duct: a case report.

A case of primary esophageal adenocarcinoma in a 64-year-old man is reported. An ulcerating tumor was located in the middle intrathoracic esophagus. Histopathological examination revealed a moderately differentiated adenocarcinoma, which had invaded down to the adventitia. The cancerous tubuli were lined by flattened cuboidal cells with eosinophilic cytoplasm, which were analogous with the esophageal gland ducts and syringoma of the skin. The carcinoma was spread widely in the lamina propria mucosae without intraepithelial neoplastic elements. An immunohistochemical profile of individual cytokeratins and other epithelial markers in the carcinoma was similar to that of the esophageal gland ducts. Barrett's metaplastic epithelium or ectopic gastric mucosa was not found around the tumor. It is strongly suggested that this unique carcinoma is derived from the esophageal gland ducts.

Adenocarcinoma↗
PubMed1999

Heterotopic sebaceous glands in the esophagus: histopathological and immunohistochemical study of a resected esophagus.

A resected esophagus with numerous heterotopic sebaceous glands was examined in an attempt to determine whether esophageal heterotopic sebaceous glands are the result of a metaplastic process or a congenital anomaly. The present case concerns a 79-year-old Japanese man with numerous esophageal heterotopic sebaceous glands accompanied by superficial esophageal cancer. The resected esophagus possessed numerous heterotopic sebaceous glands, which could be seen clearly as slightly elevated, yellowish lesions. Histological examination of these glands, all of which were located in the lamina propria, revealed lobules of cells that showed characteristic sebaceous differentiation. Bulbous nests of proliferating basal cells showing sebaceous differentiation were occasionally observed in the esophageal epithelium. Of the antibodies against six different keratins used, only anti-keratin 14 labeled both the heterotopic sebaceous glands and the bulbous nests. Acquired metaplastic change of the esophageal epithelium is probably the pathogenetic mechanism involved in these unusual lesions.

Aged↗
PubMed1999

Giant cell interstitial pneumonia in two hard metal workers: the role of bronchoalveolar lavage in diagnosis.

Two cases of hard metal lung disease and pathological findings of giant cell interstitial pneumonia are reported. The cases worked in different factories manufacturing hard metal parts from tungsten carbide and cobalt. Pathological specimens were obtained by percutaneous thoracoscopy and transbronchial lung biopsy. X-ray microanalysis detected only tungsten carbide in the lung specimen of one case. Bronchoalveolar lavage showed diagnostic bizarre macrophages in the lavage fluid.

Adult↗
PubMed1999

Molecular analysis of enterohemorrhagic Escherichia coli isolates in Japan and its application to epidemiological investigation.

Many outbreaks of enterohemorrhagic Escherichia coli (EHEC) O157 infections occurred in Japan in 1996. Molecular epidemiological analyses of the isolates by methods such as XbaI-digested, pulsed-field gel electrophoresis (PFGE), revealed that a variety of PFGE type strains have spread all over Japan. They also showed that such molecular methods are very useful for confirming the epidemiologically related isolates and in assisting the epidemiological investigation. Recent characteristic features of the infection are that the incidence of patients experiencing symptoms such as abdominal cramps, diarrhea and hemorrhagic colitis is higher in children younger than 9 years old; however, that of asymptomatic carriers seems to be higher in adults; and that EHEC O157:H7-serotyped strains are still the main isolates, while the isolation frequency of other serotype strains, such as O26 and O111, is increasing.

Adolescent↗