Biomedical subjects

H Kuwahara

Publications and source records attributed to H Kuwahara.

At least 19 recordsLinked to original sources

PubMed2000

NE-dlg, a mammalian homolog of Drosophila dlg tumor suppressor, induces growth suppression and impairment of cell adhesion: possible involvement of down-regulation of beta-catenin by NE-dlg expression.

Membrane-associated guanylate kinases (MAGUKs) are known to function as scaffolds for forming multiprotein complexes at the synaptic junctions of neuronal cells and at sites of epithelial cell-cell contact. In Drosophila, mutations of the lethal (1)-discs large (dlg) gene, which encodes a MAGUK protein, leads to post-synaptic structure defects in neuronal cells and neoplastic overgrowth of epithelial cells. We previously showed that NE-dlg (neuronal and endocrine dlg), a human homolog of the dlg, plays a crucial role in formation of synaptic structure in human neuronal cells. Here we demonstrate that NE-dlg, similar to Drosophila dlg, is involved in regulation of cell cycle progression and adhesive ability of non-neuronal cells. Overexpression of NE-dlg in proliferating cells including various cancer cell lines induced growth suppression and impairment of cell adhesive ability. Furthermore, NE-dlg overexpression caused the down-regulation of beta-catenin in cancer cells regardless of mutations in the APC (adenomatous polyposis coli) gene. The PDZ domains of NE-dlg were found to be essential for the growth suppression, loss of adhesive property and down-regulation of beta-catenin. We propose that NE-dlg regulates the cell growth and adhesive ability by controlling the level of beta-catenin through an APC-independent pathway. Inactivation of NE-dlg may therefore contribute to development and/or progression of human neoplasms.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Intraarticular osteoid osteoma associated with synovitis: a possible role of cyclooxygenase-2 expression by osteoblasts in the nidus.

To clarify the condition of development of synovitis associated with intraarticular osteoid osteoma (OO), expressions of cyclooxygenase-2 (COX-2) protein and its messenger ribonucleic acid were investigated both in the nidus and the synovial tissue using immunohistochemical and reverse transcription-polymerase chain reaction analyses. Diffuse and strong COX-2 immunoreactivity was found in osteoblast-like tumor cells in the nidus of all six cases of OO (three of six cases were intraarticular OO associated with synovitis) and one case of osteoblastoma associated with synovitis. Expression of COX-2 messenger ribonucleic acid was demonstrated in one case of OO associated with synovitis, and was higher in the nidus than that in the inflamed synovial tissue. However, there were no significant difference between the nidus and synovium in the expression of cytosolic phospholipase A2, one of the enzymes involved in arachidonic acid metabolism, and inflammatory cytokines such as interleukin-1beta and tumor necrosis factor alpha. Finally, as there was only one case in which the examinations of gene expression were performed, no definitive overall conclusions could be reached; rather it is suggested that COX-2 expressed primarily by osteoblasts in the nidus of intraarticular OO may play a role in activating the pathway of arachidonic acid metabolism, resulting in synovitis of the involved joint.

Adolescent↗

PubMed2000

Helicobacter pylori urease suppresses bactericidal activity of peroxynitrite via carbon dioxide production.

Helicobacter pylori can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced. H. pylori may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of H. pylori against host-derived peroxynitrite (ONOO(-)), a bactericidal metabolite of nitric oxide, focusing on the role of H. pylori urease, which produces CO(2) and NH(3) from urea and is known to be an essential factor for colonization. The viability of H. pylori decreased in a time-dependent manner with continuous exposure to 1 microM ONOO(-), i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO(-) against H. pylori was significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO(-)-induced killing of a urease-deficient mutant of H. pylori or Campylobacter jejuni, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 microM flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO(-) was also suppressed by the addition of 20 mM NaHCO(3) but not by the addition of 20 mM NH(3). In addition, the nitration of L-tyrosine of H. pylori after treatment with ONOO(-) was significantly reduced by the addition of urea or NaHCO(3), as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that H. pylori-associated urease functions to produce a potent ONOO(-) scavenger, CO(2)/HCO(3)(-), that defends the bacteria from ONOO(-) cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa.

Ammonia↗

PubMed2000

Effects of chemical modification of carboxyl groups in the hemolytic lectin CEL-III on its hemolytic and carbohydrate-binding activities.

Effects of chemical modification of carboxyl groups in the hemolytic lectin CEL-III on its activities were investigated. When carboxyl groups were modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine methyl ester, hemolytic activity of CEL-III decreased as the EDC concentration increased, accompanied by reduction of oligomerization ability and hemagglutinating activity. However, binding ability of CEL-III for immobilized lactose was retained fairly well after modification, suggesting that one of two carbohydrate-binding sites might be responsible for such inactivation of CEL-III.

Animals↗

PubMed1999

A novel NE-dlg/SAP102-associated protein, p51-nedasin, related to the amidohydrolase superfamily, interferes with the association between NE-dlg/SAP102 and N-methyl-D-aspartate receptor.

The membrane-associated guanylate kinase proteins have been known to interact various membrane receptors with their N-terminal segments designated the PDZ domains and to cluster these receptors at the target site of the cell membrane. NE-dlg/SAP102, a neuronal and endocrine tissue-specific MAGUK family protein, was found to be expressed in both dendrites and cell bodies in neuronal cells. Although NE-dlg/SAP102 localized at dendrites was shown to interact with N-methyl-D-aspartate receptor 2B via the PDZ domains to compose postsynaptic density, the binding proteins existing in the cell body of the neuron are still unknown. Here we report the isolation of a novel NE-dlg/SAP102-associated protein, p51-nedasin. Nedasin has a significant homology with amidohydrolase superfamily proteins and shows identical sequences to a recently identified protein that has guanine aminohydrolase activity. Nedasin has four alternative splice variants (S, V1, V2, and V3) that exhibited different C-terminal structures. NE-dlg/SAP102 is shown to interact with only the S form of nedasin which is predominantly expressed in brain. The expression of nedasin in neuronal cells increases in parallel with the progress of synaptogenesis and is mainly detected in cell bodies where it co-localizes with NE-dlg/SAP102. Furthermore, nedasin interferes with the association between NE-dlg/SAP102 and NMDA receptor 2B in vitro. These findings suggest that alternative splicing of nedasin may play a role in the formation and/or structural change in synapses during neuronal development by modifying clustering of neurotransmitter receptors at the synaptic sites.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90. A possible regulatory role in molecular clustering at synaptic sites.

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.

Amino Acid Sequence↗

PubMed1999

Superiority of end-to-side anastomosis with the internal jugular vein: the experience of 80 cases in head and neck microsurgical reconstruction.

The authors report their experience with 80 head and neck reconstructions using free-tissue transfer in which end-to-side anastomosis with the internal jugular vein was carried out. An end-to-side anastomosis with the internal jugular vein has the following advantages. Firstly, the technique overcomes the problems of vessel size discrepancy. It is effectively applied for free jejunal transfer or combined flap transfer based on a single vascular pedicle, of which the size of the proximal end of the drainage vein is very large. Secondly, the internal jugular vein has wide capacity to be the recipient of two or more end-to-side anastomoses. It is effectively used for free radial forearm or rectus abdominis myocutaneous flaps in which two or more drainage veins can be included. Thirdly, the respiratory venous pump effect may act directly on the venous drainage of the transferred flap through the internal jugular vein. In our institution, these advantages have made it the technique of choice in head and neck reconstructive microsurgery.

Adult↗

PubMed1999

Characterization of the interaction of hemolytic lectin CEL-III from the marine invertebrate, Cucumaria echinata, with artificial lipid membranes: involvement of neutral sphingoglycolipids in the pore-forming process.

The hemolytic lectin, CEL-III, is a Ca2+-dependent, galactose/N-acetylgalactosamine-specific lectin purified from the marine invertebrate, Cucumaria echinata (Holothuroidea). After binding to specific carbohydrates on the erythrocyte surface, CEL-III forms ion-permeable pores by oligomerizing in the membrane, which leads to colloid osmotic rupture of the cells. When incubated with liposomes composed of total lipids from the human erythrocyte membrane, CEL-III efficiently induced the leakage of carboxyfluorescein (CF) trapped in the vesicles, suggesting the presence of its receptor in the membrane lipids. The rate of CF-leakage increased with increasing temperature, although the hemolytic activity of CEL-III had been found to be much higher at lower temperatures (around 10 degrees C). Identification of the receptor for CEL-III was performed by examining the ability of individual lipids from human erythrocytes to induce CF-leakage from DOPC-liposomes. As a result, the most effective receptor was found to be lactosyl ceramide (LacCer), while globoside (Gb4Cer) also showed slight induction of CF-leakage. On the other hand, a binding assay involving CEL-III-horseradish peroxidase conjugate indicated that CEL-III exhibits similar affinity for LacCer and Gb4Cer, suggesting that the structure or length of the carbohydrate portion of sphingoglycolipids is also relevant as to their ability to induce CF-leakage in addition to their affinity. Electron micrographs of CEL-III-treated liposomes revealed that CEL-III induced considerable morphological changes in the vesicles, while a clearly distinguishable oligomeric structure of the protein was not observed.

Animals↗

PubMed1999

Carbohydrate-dependent hemolytic activity of the conjugate composed of a C-type lectin, CEL-I, and an amphiphilic alpha-helical peptide, 4(3)-beta Ala2.

A lectin-cationic peptide conjugate, 4(3)-CEL-I, was prepared from an invertebrate C-type lectin, CEL-I, and an amphiphilic alpha-helical peptide, 4(3)-beta Ala2 [Ac-(Leu-Ala-Arg-Leu)3-beta Ala2]. When 4(3)-CEL-I was incubated with rabbit erythrocytes, hemolysis was observed, especially at basic pH. Inhibition experiment using some carbohydrates suggested that hemolytic activity of 4(3)-CEL-I was caused by the interaction between 4(3)-beta Ala2 portion in the conjugate and the lipid bilayer after binding to the carbohydrate chains on the cell surface by the lectin activity of CEL-I.

Animals↗

PubMed1998

The ultrastructural characteristics of eccrine sweat glands in a Fabry disease patient with hypohidrosis.

As an investigation of the pathogenetic mechanism of diminished sweating in Fabry disease, an electron microscopy ultrastructural study was conducted on specimens of eccrine sweat glands from a typical patient with Fabry disease who had hypohidrosis, a low skin moisture content, and diminished thermoregulation ability. Numerous characteristic cytoplasmic inclusions were observed in the eccrine sweat glands, the lamellar pattern of which was considerably variable in various types of gland cells. Large vacuolar inclusions predominated in clear cells of secretory coil; lesser vacuoles were also seen in the coiled duct, and the basal cells of the straight duct toward the coiled duct displayed mulberry-like figures. There were some clear cells showing cell damage and necrosis in the secretory coil. Lamellated inclusions were noted in the unmyelinated axons innervating the eccrine sweat glands. The small blood vessels around the eccrine glands were narrowed by swollen endothelial cells with heavy inclusions. These intracytoplasmic deposits may be responsible for the decreased sweating ability in Fabry disease. The factors related to hypohidrosis are also discussed.

Eccrine Glands↗

PubMed1998

Chemical modification of the hemolytic lectin CEL-III by succinic anhydride: involvement of amino groups in the oligomerization process.

CEL-III is a Ca(2+)-dependent lectin from a marine invertebrate, Cucumaria echinata, which shows strong hemolytic activity toward human and rabbit erythrocytes. After binding to carbohydrate receptors, CEL-III oligomerizes in the erythrocyte membrane to form ion-permeable pores, leading to the colloid osmotic rupture of the cells. Since hemolysis was greatly increased in the alkaline pH, especially above pH 9, involvement of amino groups of CEL-III in its hemolytic activity was evaluated using chemical modification by succinic anhydride. After modification of 7 amino groups per protein molecule, the hemolytic activity of CEL-III was reduced to 23% of the native protein, but hemagglutinating and carbohydrate-binding activities were only slightly affected even after modification of 14 amino groups. A circular dichroism spectrum of modified CEL-III showed almost no change in the secondary structure from that of the native protein, indicating that the decrease of hemolytic activity was not caused by partial unfolding of the protein. Immunoblotting analysis of the erythrocyte membrane treated with modified CEL-III showed a decrease in the formation of CEL-III oligomer in the membrane in parallel with the decrease in hemolytic activity. These results suggest that amino groups of CEL-III are involved in its oligomerization in the cell membrane, and their modification leads to inactivation of the protein without much influence on the carbohydrate-binding activity.

Amino Acids↗

PubMed1998

[A study of Article 32 of the Mental Health Act analysis of those who made applications under Article 32].

Article 32 of the Mental Health Act was enacted in 1965 and entitles psychiatric outpatients to financial aid towards payment of psychiatric care costs. Psychiatric outpatients can make applications under Article 32 voluntarily at local public health centers. These centers are operated by the prefectural governments. Therefore, records of the applications are kept by the local public health centers. In order to study the effects of Article 32 on psychiatric care, we analyzed the records of those who applied for such benefits in I. County of Shiga Prefecture, at the K. local Public Health Center, and performed a comparative analysis of applicants vs. non-applicants. We also tried to find out which psychiatric illnesses were more significantly represented among those who applied than among those who did not. Our study showed that about 43.5 percent of all psychiatric outpatients in I. County had made applications for Article 32 benefits. This was significantly higher than any former percentages that had been anticipated. We also discovered that a significantly higher number of people with schizophrenia, atypical psychosis and epilepsy could be identified among those who had applied for benefits than those who had not applied. On the other hand, the number of people with neurosis and emotional disorders seemed higher among those who did not apply for the benefits. In addition, the study indicated that a large proportion of those who applied belonged to the 40 s and 50 s age group and to the group with chronic psychiatric problems. All of the above make it clear that Article 32 has been effective in making psychiatric care accessible to those people who suffer from chronic mental diseases over quite a long period time.

Adult↗

PubMed1997

Small-angle X-ray scattering study on CEL-III, a hemolytic lectin from Holothuroidea Cucumaria echinata, and its oligomer induced by the binding of specific carbohydrate.

Hemolytic lectin CEL-III from a marine invertebrate Cucumaria echinata forms an oligomer upon binding of specific carbohydrate such as lactose at high pH values and in the presence of high concentrations of salt. In this study, using small-angle X-ray scattering, we characterized CEL-III and its oligomer induced by the binding of lactose. The molecular mass of the oligomer was determined as 1019 kDa from its forward scattering value, compared with 47,490 Da for the monomer. This oligomer size is much larger than that estimated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 270 kDa). The monomer has a 24.6 A radius of gyration and can be approximated by a rod which has a 20 A radius and a height of 75 A, while the oligomer has a 101.4 A radius of gyration. Together with the comparison of the radii of gyration and the forward scattering of the cross-section of the monomer and oligomer, it is suggested that in aqueous solution the oligomer comprises three or four molecules of a smaller unit which was observed by SDS-PAGE (270 kDa), held by a relatively weak interaction. The scattering profile also suggests that the oligomer has a hole in its central axis which might be associated with the formation of ion-permeable pores in the erythrocyte membrane by CEL-III during the hemolytic process.

Animals↗

PubMed1997

Cloning and characterization of NE-dlg: a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein interacts with the APC protein.

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dig) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dig). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.

Adaptor Proteins, Signal Transducing↗

PubMed1997

[Analysis of contact dermatitis by cytokines. I].

The study of the participation of T cells and the kinetic studies of cytokines by the RT-PCR method in the challenge phase of contact hypersensitivity reaction (CHR) were performed using skin tissues collected at regular intervals from mice sensitized with 2,4-dinitro-1-fluorobenzene (DNFB). The results obtained from these studies and the further analyses using anti-CD4 and CD8 antibodies showed that the revelation of phenotype of lymph node cells at the reaction site and T lymphocytes at the CHR site were found to play important roles. Furthermore, we examined cytokines formed at the CHR site on the mRNA level. Consequently, it was found that 24 h after the challenge IFN-gamma, IL-6 and IL-1 beta were expressed and 48 h after the challenge IL-2, IL-4 and TNF-alpha were expressed in addition to these cytokines. The results of the expression of IL-12 and those of the examination using IFN-gamma knock out mice suggested that the expression of IFN-gamma among several Th1 types of cytokine is especially important for the formation of CHR.

Animals↗

PubMed1997

[Analysis of contact dermatitis by cytokines. II].

We examined the mechanism of the onset of contact hypersensitivity reaction (CHR) induced by several kinds of chemical substances in mice, focusing on cytokines. Before doing this experiment, we investigated the involvement of T cells in the formation of CHR using each primer of CD4 and CD8 (surface markers of lymphocytes) positive cells. It was suggested that both cells participate in the formation of CHR. On the basis of these results we analyzed cytokines produced in the reaction site on the mRNA level in with or without the presensitization of two kinds of strong contactants. Consequently, there was a tendency that the expression of mRNA of Th1 types of cytokines was commonly observed around 24 h after the reaction irrespective of the presence or the absence of presensitization. These date suggested that CHR can be presumed on the genetic level by the only one priming of strong contactants.

Animals↗

PubMed1997

[Cisplatin/5-fluorouracil intraperitoneal administration superior to intravenous administration for liver metastases of colonic carcinoma].

A 28-year-old female underwent sigmoidectomy for sigmoid colon cancer with peritoneal seeding. One month later, a solitary metastasis was found in S3 of the liver. After CDDP/5-FU intravenous chemotherapy, another metastasis appeared in S7. Intravenous administration showed PD. But the metastatic tumors shrank and became inobservable by CT after the 1st round of CDDP/5-FU intraperitoneal chemotherapy, and S7 tumor could not be identified after the 2nd round. Many previous reports demonstrated the concentration of cytotoxic drug in intraperitoneal administration was much higher than in intravenous administration. Theoretically, intraperitoneal chemotherapy is superior to intravenous chemotherapy for the prevention and treatment of liver metastases. This case demonstrated this hypothesis was right. We think adjuvant intraperitoneal chemotherapy should be re-considered for the prevention of the liver metastases of gastrointestinal cancers.

Adenocarcinoma↗

PubMed1996

Interplane Tunneling Magnetoresistance in a Layered Manganite Crystal

The current-perpendicular-to-plane magnetoresistance (CPP-MR) has been investigated for the layered manganite, La2-2xSr1+2xMn2O7 (x = 0.3), which is composed of the ferromagnetic-metallic MnO2 bilayers separated by nonmagnetic insulating block layers. The CPP-MR is extremely large (10(4) percent at 50 kilo-oersted) at temperatures near above the three-dimensional ordering temperature (Tc approximately 90 kelvin) because of the field-induced coherent motion between planes of the spin-polarized electrons. Below Tc, the interplane magnetic domain boundary on the insulating block layer serves as the charge-transport barrier, but it can be removed by a low saturation field, which gives rise to the low-field tunneling MR as large as 240 percent.

Journal Article↗