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Biomedical subjects

I Wada

Publications and source records attributed to I Wada.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effects of cyclosporin A on water-immersion stress-induced gastric lesion and gastric secretion in rats.

Cyclosporin A is an immunosuppressive agent which is well known as a specific inhibitor of calcineurin (protein phosphatase 2B). In this study, we investigated the effects of cyclosporin A on water-immersion stress-induced gastric ulcer formation and gastric acid secretion in rats. We also examined the localization of calcineurin immunohistochemically. Calcineurin was specifically expressed in gastric parietal cells and chief cells of the gastric mucosa. The intraperitoneal administration of cyclosporin A dose-dependently suppressed the development of gastric mucosal lesions induced by water-immersion stress and inhibited gastric acid secretion, as assessed by pylorus ligation. These results indicated that calcineurin may play an important role in gastric acid secretion.

Animals↗
PubMed2000

Corticosteroid pretreatment prevents small intestinal mucosal lesion induced by acetic acid-perfusion model in rats.

One of the important problems in experimentally induced small intestinal lesions is that there is no reproducible model of diffuse and stable mucosal lesion. In this paper, we studied in detail the effects of continuous perfusion of various concentrations of acetic acid on the rat small intestinal mucosa. In order to evaluate its applicability for screening of the preventive effect of drugs on gut damage, we also evaluated the efficacy of corticosteroid pretreatment in preventing acetic acid-induced mucosal lesion. Male Sprague-Dawley rats were fasted for 12 hr, and the small intestinal lumen was perfused with 1%, 1.5%, 2.5%, 3%, 3.75% (pH 2.4-2.6) acetic acid or saline (control) at 1 ml/min for 15 min. In separate experiments, the effect of preadministration of budesonide (0.5 or 0.75 mg/kg/day) and prednisone (0.75 mg/kg/day) on 1.5% acetic acid-induced mucosal damage was investigated. Macroscopic and microscopic lesions occurred diffusely in a concentration-dependent fashion. Histological findings revealed signs of transmural inflammation characterized by mucosal-submucosal edema, ulceration, and neutrophil infiltration. Mucosal-submucosal height had an inverse relation with the acetic acid concentrations perfused. Myeloperoxidase activity levels increased several-fold in the acetic acid-perfused groups. Corticosteroid pretreatment prevented microscopic damage and was associated with reduction of MPO activity levels in 1.5% acetic acid-perfused rats. We conclude that this simple and reproducible model could be applied for the screening of new drugs in the gastrointestinal tract in which large numbers of animals are taken into account.

Acetic Acid↗
PubMed2000

Cell surface activities of the human type 2b phosphatidic acid phosphatase.

Several isozymes of mammalian type 2, Mg(2+)-independent phosphatidic acid phosphatase (PAP-2) have recently been cloned, and they are predicted to have their catalytic sites exposed at the cell surface membranes. We investigated the mode of utilization of extracellular lipid substrates by the human PAP-2b expressed in HEK293 cells as a green fluorescent fusion protein. We first confirmed the plasma membrane localization of the expressed PAP-2b. PAP-2b actively hydrolyzed exogenously added lysophosphatidic acid and short-chain phosphatidic acid. In the case of dephosphorylation of lysophosphatidic acid, the reaction products, including inorganic phosphate and monoacylglycerol, were recovered exclusively in the extracellular medium. Interestingly, PAP-2b exhibited negligible activities toward long-chain phosphatidic acid either exogenously when added or generated within the membranes by treating the cells with bacterial phospholipase D. These findings indicate that PAP-2b acts at the outer leaflet of cell surface bilayers and can account for the ecto-PAP activities previously described for various types of cells.

Cell Line↗
PubMed2000

[Nasal foreign bodies in 299 cases].

Although nasal foreign bodies very commonplace in daily clinical practice, their simplicity in pathology and diagnosis is so unique that case reports based upon a large number have been rather scarce. We report 299 verified cases of nasal foreign bodies seen during the past 13 years from January 1986 to December 1998, at our institute, together with clinical and statistical analysis. The total number of first visits during the surveyed period was 55,312 and nasal foreign bodies comprised 0.54%. Of these 217 subjects (72.6%) appeared in emergencies. Man comprised 172 (58%) and woman 127 (42%), with the right side comprising 166 cases (57%), the left side 123 (42%) and bilateral 3 cases (1%). In monthly distribution, cases are more often seen in November and December and less often seen in January, with the monthly average being 25. In age distribution, the majority were seen in those under 10 years old, excluding 6 cases. The average age was 4.0 and the range 1 month to 81 years. In the majority of cases, foreign bodies remained only a short time (with in 24 hour) but in 15 cases stayed rather a long time (over 24 hour). In specificity, the majority of foreign bodies were toys, with plastic bullets used with air guns most frequent, in 46 cases (15.3%), followed by beads in 36 cases (12.0%). Other foreign bodies seen comparatively often were pieces of tissue paper, buttons, and plastic toys components.

Adolescent↗
PubMed2000

Postnatal development of synovial capillaries of rats with special reference to permeability.

The size of a substance is a major factor determining whether it can permeate the wall of synovial capillaries. The maximum diameter of particles that can move across the synovial capillary wall has generally been thought to be 50 nm. We studied the permeability of the synovial capillaries of the rat between day 20 and 30 after birth using a polystyrene particle whose diameter was 240 nm. In addition using lecithin-coated polystyrene particles, we studied the maturation of the barrier function supported by endothelial and peripheral cells against foreign bodies. Lecithin-coated particles were found within the fibroblast-like synovial cells near the capillary in the 20 day-old rats, while non-coated particles remained in the endothelial wall and in the peripheral cells of capillaries. In the 30 day-old rats, lecithin-coated particles were present in the peripheral cells and the neighboring synovial cells; however, the non-coated particles were never found in the synovial or perisynovial cells. The present study shows that the size of the transportable substance by transcytosis may be larger than previously thought. Furthermore, the synovial capillaries functionally changed between day 20 and 30 suggesting that active movement of the joint led to the functional maturation of the synovial capillaries.

Animals↗
PubMed1999

Failure to detect genetic alteration of the mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) gene in hepatocellular carcinomas in Japan.

The mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) suppresses cell growth through binding to the insulin-like growth factor 2 (IGF2) and latent complex of the transforming growth factor-beta (TGF-beta). Recently, it was reported in the United States that loss of heterozygosity (LOH) and mutations in exons 27, 28, and 31 of the M6P/IGF2R gene are frequent in hepatocellular carcinomas (HCCs) and adenomas. In view of the possible importance of this finding, especially for differential diagnosis of small hepatic lesions, we analyzed 43 primary HCCs, 2 adenomatous hyperplasias (AHs), and 3 regenerative nodules (RNs) developing in 42 Japanese patients in Japan for LOH using the polymorphic locus and for mutations by both single strand conformation polymorphism (SSCP) and direct sequencing methods. In the LOH study, 21 out of 22 informative HCCs and all of the informative AHs and RNs showed no allelic loss. In mutational studies of exons 27, 28, and 31, no mutations were detected either by SSCP or direct sequencing analysis in any of the 48 lesions. Thus inactivation of the M6P/IGF2R gene because of genetic alteration does not appear to be essential for hepatocarcinogenesis in Japan.

Adenoma↗
PubMed1999

Molecular characterization of the type 2 phosphatidic acid phosphatase.

Phosphatidic acid phosphatase (PAP) converts phosphatidic acid to diacylglycerol, thus regulating the de novo synthesis of glycerolipids and also signal transduction mediated by phospholipase D. We initially succeeded in the cDNA cloning of the mouse 35 kDa PAP bound to plasma membranes (type 2 enzyme). This work subsequently led us to the identification of two human PAP isozymes designated 2a and 2b. A third human PAP isozyme (2c) has also been described. The cloned enzymes are, in common, N-glycosylated and possess six transmembrane domains. The transmembrane dispositions of these enzymes are predicted and the catalytic sites are tentatively located in the 2nd and 3rd extracellular loops, thus suggesting that the type 2 PAPs may act as ecto-enzymes dephosphorylating exogenous substrates. Furthermore, the type 2 PAPs have been proposed to belong to a novel phosphatase superfamily consisting of a number of soluble and membrane-bound enzymes. In vitro enzyme assays show that the type 2 PAPs can dephosphorylate lyso-phosphatidate, ceramide-1-phosphate, sphingosine-1-phosphate and diacylglycerol pyrophosphate. Although the physiological implications of such a broad substrate specificity need to be further investigated, the type 2 PAPs appear to metabolize a wide range of lipid mediators derived from both glycero- and sphingolipids.

Amino Acid Sequence↗
PubMed1999

Association of 72-kDa heat shock protein expression with adaptation to aspirin in rat gastric mucosa.

It is well documented that gastric mucosa can increase its resistance to mucosal damage caused by aspirin during repeated long-term administration of aspirin. However, the underlying mechanism of this adaptation is not well established. In the present study, we investigated the effect of long-term (chronic) administration of aspirin on expression of heat shock proteins (HSPs), which are known as endogenous cytoprotectants, in rat gastric mucosa. Rats were administered aspirin (100 mg/kg) daily for up to 20 days. After various periods of aspirin administration, a high dose of aspirin (250 mg/kg) was administered, and the mucosal damage was assessed. Expression of heat shock proteins (HSPs) in gastric mucosa was evaluated by Western blot. Intracellular localization of each HSP was studied immunohistochemically. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels were also investigated. Long-term aspirin administration resulted in development of resistance to aspirin-induced mucosal damage, and the increase of HSP72 expression correlated with mucosal resistance to aspirin. No significant increase was observed in HSP60 and HSP90 levels. Immunohistochemical study showed an increase of HSP72 in the cytoplasm of mucosal surface cells. The PGE2 level was suppressed and no change in the level of LTB4 was observed. It is possible that HSP72 could play important roles in gastric mucosal adaptation when the PGE2 level is suppressed by NSAIDs.

Adaptation, Physiological↗
PubMed1999

Early onset of X-linked Emery-Dreifuss muscular dystrophy in a boy with emerin gene deletion.

A boy developed contractures of the Achilles tendons at 3 years and of the postcervical muscles at 7 years, although neither contractures of the elbows nor cardiac abnormality were recognized by the age of 9 years. Muscle computed tomography scanning revealed changes characteristic of muscle involvement. Emerin was not detected in the biopsied muscle, and RT-PCR and PCR-based genomic DNA analyses of the emerin gene demonstrated no amplification product in the patient. These results confirmed the diagnosis of X-linked Emery-Dreifuss muscular dystrophy (EDMD), and reinforce the necessity of molecular genetic diagnosis of the membrane protein emerin in younger patients with possible EDMD before appearance of the typical symptoms, to avoid sudden cardiac death.

Child↗
PubMed1999

Promotion of tyrosinase folding in COS 7 cells by calnexin.

To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.

Animals↗
PubMed1999

Pharmacokinetics of etoposide after intrathoracic instillation to lung cancer patients with pleural effusion.

OBJECTIVE: To examine etoposide (VP16) levels in serum and pleural effusion after intravenous infusion or intrathoracic instillation to lung cancer patients. METHODS: Four patients were administered VP16 by intrathoracic instillation and three patients were administered it intravenously. Serum, urine, and pleural effusion were collected and VP16 levels in the biological fluids were determined by HPLC. Pharmacokinetic parameters were calculated. RESULTS: VP16 distributed rapidly into pleural effusion after intravenous infusion. In two of three patients, VP16 levels in pleural effusion were maintained at constant levels more than 24 hours in spite of the decline in serum VP16 levels. After intrathoracic instillation, VP16 in pleural effusion reached high levels and eliminated slowly. Serum levels of VP16 were relatively low compared with those in pleural effusion. CONCLUSION: It was demonstrated that intrathoracic instillation of VP16 might be useful for managing malignant pleural effusion and reducing systemic side-effects by cutting down the dose.

Adenocarcinoma↗
PubMed1999

High rate of small TP53 mutations and infrequent loss of heterozygosity in malignant liver tumors associated with thorotrast: implications for alpha-particle carcinogenesis.

Epidemiological studies have revealed that malignant tumors occur in the liver approximately 20 years after injection of Thorotrast. We investigated genetic changes in the TP53 gene (formerly known as p53) in malignant liver tumors related to Thorotrast to cast light on the mechanisms of alpha-particle carcinogenesis. A total of 19 autopsy cases of liver malignancies [11 hepatocellular carcinomas (HCC), 5 cholangiocellular carcinomas (CCC) and 3 angiosarcomas (AS)] were analyzed. Using archival tissues, loss of heterozygosity (LOH) at the 17p13 locus was analyzed. Then single-strand conformation polymorphism analysis and sequencing were performed to detect mutations in exons 5 to 8 of the TP53 gene. As a result, 15 cases were informative in terms of polymorphism, and 4 cases showed LOH (3 HCC and 1 AS). Eight cases showed 9 mutations in exons and 2 in introns: 7 transitions (6 HCC and 1 CCC), 2 transversions (1 HCC and 1 AS), and 2 deletions (2 HCC). The direct action of alpha particles is thought to result in relatively large deletions such as those detected by LOH. Therefore, the low frequency of such changes (27%) compared to point mutations (47%) suggests that the genetic changes in the TP53 gene in the liver tumors related to Thorotrast were not caused mainly by direct actions of alpha particles but rather by indirect effects that may have been due to cycles of necrosis and regeneration.

Alpha Particles↗
PubMed1999

Ultrastructural study of cells at the synovium-cartilage junction: response of synovial cells of the rat knee joint to intra-articularly injected latex particles.

To study phagocytosis in synovial cells at the synovium-cartilage junction, we used polystyrene latex spheres which induced no infiltration of inflammatory cells into the synovial tissues and observed them for a long term period. The latex bead suspension was injected into the knee joint cavities of 60 day-old male Wistar rats. The animals were then sacrificed at 1, 4, 7, 14, 28 and 56 days after the injections and their synovial tissues including the patellar cartilage were resected for subsequent examination with the transmission electron microscope. On day 1, particles were phagocytised intensively by both type A (macrophage-like) and type B (fibroblast-like) cells. Particles were more numerous in the cytoplasm of type A rather than type B cells. The number of synovial cells containing particles and the number of particles present in these cells decreased remarkably on day 4. By day 7, particles were also observed in the tendon. On day 14, particles were observed in the cytoplasm of chondrocyte. It was noted that particle density within cells began to increase again after day 28. On day 56, numerous particles were observed in type A and type B cells and moreover within the intercellular matrix. Even tendon cells actively engulfed the particles. The results of the present study suggest that both type A and B synovial cells, chondrocytes and tendon cells possess the ability to phagocytise foreign materials. Moreover, the possibility is proposed that the synovium-cartilage junction is the point where the synovial fluid exits and particles drain via the blood circulation.

Animals↗
PubMed1999

A possible barrier function of the articular surface.

Since MacConaill first reported the existence of a thin additional layer of the articular cartilage and named it the lamina splendens, there have been various opinions as to the role of this layer in the lubrication of the articular surface. We studied the superficial portion of the articular cartilage in the 20 day-old and 30 day-old rats using light and transmission electron microscopy. Furthermore, we studied the articular cartilage of the rat whose "cover layer" had been removed mechanically. Also, intraarticular latex beads injection, intraarticular dye injection using lithium carmine and supravital staining experiments were performed. On day 20, dye injected intraarticularly was clearly observed by light microscopy in chondrocytes situated in the deeper layers. The dye injected in the 30 day-old rats, however, was not seen in the chondrocytes but was found only in the superficial layer. Dye was found in the chondrocytes when supravital staining was performed in the articular cartilage of 30 day-old rats after mechanical removal of the cover layer. By transmission electron microscopy, a superficial layer consisted of fine filamentous structures was observed on the articular surface of the 30 day-old rats. The cover layer was destroyed by intraarticular injected latex beads in 30 day-old rats. These findings strongly support the idea that the cover layer acts as a barrier against substances which invade from the surface of the articular cartilage. The development period of the cover layer coincides with the initiation of weight bearing, and joint cartilage debris and pressure changes might further promote maturation.

Animals↗
PubMed1998

A splice mutation in the human canalicular multispecific organic anion transporter gene causes Dubin-Johnson syndrome.

The human Dubin Johnson syndrome (DJS) is a rare autosomal recessive disorder characterized by chronic conjugated hyperbilirubinemia and impaired hepatobiliary transport of non-bile salt organic anions. A highly homologous phenotype exists in the transport deficient (TR-) Wistar rat, which has a defective canalicular multispecific organic anion transporter (cMOAT). This protein mediates adenosine triphosphate-dependent transport of a broad range of endogenous and xenobiotic compounds across the (apical) canalicular membrane of the hepatocyte. The cDNA encoding rat cMOAT has recently been cloned, and this mutation in the TR- rat has been identified. Subsequently the human homologue of rat cMOAT localized in the liver was found to be the cause of DJS. In an individual with DJS, we have identified a single novel nucleotide substitution in the exon-intron junction of the cMOAT gene which generates liver cDNA with a 67bp exon deletion.

Anion Transport Proteins↗
PubMed1998

Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts.

The p16INK4A cyclin-dependent kinase (Cdk) inhibitor is now recognized as a major tumor suppressor that is inactivated by a variety of mechanisms in a wide range of human cancers. It is also implicated in the mechanisms underlying replicative senescence since p16INK4A RNA and protein accumulate as cells approach their proscribed limit of population doublings in tissue culture. To obtain further evidence of its role in senescence, we have sought ways of overexpressing p16INK4A in primary human diploid fibroblasts (HDF). To circumvent the low transfection efficiency of primary cells we have exploited a recombinant form of the full-length p16INK4A protein fused to a 16 amino acid peptide from the Drosophila antennapedia protein. This peptide has the capacity to cross both cytoplasmic and nuclear membranes allowing the direct introduction of the active protein to primary cells. Here, we show that antennapedia-tagged wild-type p16INK4A protein, but not a functionally compromised tumor-specific variant, causes G1 arrest in early passage HDFs by inhibiting the phosphorylation of the retinoblastoma protein. Significantly, the arrested cells display several phenotypic features that are considered characteristic of senescent cells. These data support a role for p16INK4A in replicative senescence and raise the possibility of using the antennapedia-tagged protein therapeutically.

Antennapedia Homeodomain Protein↗
PubMed1998

Effect of preinduction of heat-shock proteins on acetic acid-induced small intestinal lesions in rats.

Bowel dysfunction such as irritable bowel syndrome caused by stress is well described. Previous reports suggest that stress is known to cause the release of endogenous substances such as catecholamine, beta-endorphine, 5-hydroxytryptamine, corticotropin-releasing factor, and thyrotropin-releasing hormone (TRH). However, the role played by these neurohormonal mediators in bowel dysfunction under stress conditions is not well known. We investigated the influence of water-immersion stress or TRH administration on the expression of 60-kDa, 72-kDa, and 90-kDa heat-shock proteins (HSP60, HSP72, and HSP90, respectively) in rat small intestinal mucosa by Western blot and immunohistochemical analyses. The cytoprotective function of preinduced HSPs on experimentally induced mucosal damage also was studied. In order to investigate the influence of preinduction of HSP60 on small intestinal damage, the small intestinal lumen was perfused with 1.5% acetic acid 1 ml/min for 15 min with or without pretreatment with water-immersion stress or TRH administration. Expression of HSP60 was significantly increased by water-immersion stress or TRH administration in the small intestinal mucosa, whereas HSP72 and HSP90 did not increase. Interestingly, expression of this protein showed the biphasic peak pattern after water-immersion stress or TRH administration. Each peak was observed 3-6 hr and 21-24 hr after the initiation of water-immersion stress or TRH administration. Immunohistochemical study also showed a significant increment of HSP60 in both the cytoplasm and nuclei of the small intestinal mucosal cells. No histopathologic alteration was observed in rat small intestinal mucosa after each treatment. Small intestinal damage caused by 1.5% acetic acid perfusion was not influenced by preinduction of HSP60. We demonstrated that water-immersion stress or TRH administration specifically induced HSP60, although preinduction of this protein did not show a cytoprotective function in the small intestinal mucosa.

Acetic Acid↗