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Biomedical subjects

Y Shintani

Publications and source records attributed to Y Shintani.

At least 55 records · Page 3Linked to original sources
PubMed1999

Urotensin II is the endogenous ligand of a G-protein-coupled orphan receptor, SENR (GPR14).

Two molecular species of urotensin II (UII) were isolated from porcine spinal cords and identified as the endogenous ligands of a G-protein-coupled orphan receptor, SENR (sensory epithelium neuropeptide-like receptor), which is identical to GPR14. We established a CHO cell line stably expressing the rat SENR and investigated several tissue extracts to evoke the response mediated by the SENR. Extract from porcine spinal cords showed an activity of arachidonic acid metabolites release from SENR-expressing cells and was purified using HPLC. Two active substances were isolated and their sequences were determined as GPTSECFWKYCV and GPPSECFWKYCV, which were revealed to be porcine UII. Synthetic UII peptides caused arachidonic acid metabolites release activity in the rat SENR-expressing cells with an EC(50) value of 1 nM. Three cDNAs encoding the precursor proteins of porcine UII were cloned from a porcine spinal cord cDNA library; 2 consist of 121 amino acid residues and the other, which seemed to be a splicing variant, consist of 85 residues.

Amino Acid Sequence↗
PubMed1998

Compensatory apoptosis in preneoplastic liver of a transgenic mouse model for viral hepatocarcinogenesis.

The HBx protein of hepatitis B virus has been shown to induce hepatocellular carcinoma in transgenic mice as direct evidence for its involvement in hepatocarcinogenesis. In these transgenic mice, however, it is not clear why hepatocytes do not acquire a neoplastic phenotype by 13 months old despite the continuous growth stimulation by the HBx protein from 2 months old. In this study, we show that the accelerated proliferation of hepatocytes is counterbalanced by apoptosis, which maintains liver homeostasis. A decrease in the extent of apoptosis seems to precede the emergence of neoplasia in the transgenic mouse liver. The disappearance or block of apoptotic signals, which may be the result of additional genetic or epigenetic aberrations, may result in the preneoplastic hepatocytes becoming neoplastic.

Aging↗
PubMed1998

Protein phosphatase 2B-dependent pathway of insulin action on single Cl- channel conductance in renal epithelium.

The apical membrane of distal nephron epithelium (A6) has a Ca(2+)-dependent outwardly rectifying Cl- channel with single channel conductances of 3 pS for outward current and 1 pS for inward current under the basal condition. The single channel conductance for inward currents increased as cytosolic Ca2+ concentration ([Ca2+]c) was elevated, while the single channel conductance for outward currents did not change at the range of [Ca2+]c from 10 nM to 1 mM. Insulin (100 nM) increased the single channel conductance for the inward current by increasing the sensitivity to cytosolic Ca2+ by 400-fold, but did not affect the single channel conductance for the outward current. Further, insulin increased the open probability of the channel. These effects of insulin were completely blocked by cyclosporin-A, an inhibitor of protein phosphatase type 2B (PP2B) which dephosphorylates phospho-tyrosine in addition to phosphoserine/threonine, but not by okadaic acid, an inhibitor of protein phosphatase type 1 and 2A. Further, these effects of insulin were also completely blocked by W7, an antagonist of calmodulin which is required for activation of PP2B. Lavendustin A, an inhibitor of protein tyrosine kinase (PTK), mimicked these effects of insulin; this action of lavendustin A required 1 hr after its application, while within 30 min after its application lavendustin A had no significant effects on the single channel conductance. On the other hand, lavendustin A blocked the insulin action for a relatively short time period (i.e., within 30 min after their application). However, H89 (an inhibitor of protein kinase A) or H7 (an inhibitor of protein kinases A, C and G) did not mimic the insulin action. Application of PP2B or protein tyrosine phosphatase to the cytosolic surface of the inside-out patch membrane increased the single channel conductance and the open probability as did insulin in cell-attached patches. The insulin-induced increases in single channel conductance and open probability were reversibly decreased by application of PTK catalytic subunit in the presence of ATP through a decrease in the sensitivity to cytosolic Ca2+, but not by protein kinase A. These observations suggest that as intracellular signalling of insulin action, PP2B-mediated dephosphorylation of phospho-tyrosine of the channel protein (or channel-associated protein) is a novel mechanism for regulation of single channel conductance, and that at least two different types of PTKs regulate the channel characteristics.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Persistent viremia after recovery from self-limited acute hepatitis B.

To define the duration of viremia in the course of acute hepatitis B, we semiquantitatively determined the levels of hepatitis B virus (HBV) DNA in the sera, using polymerase chain reaction (PCR) coupled with Southern blotting, of non-immunocompromised patients with self-limited acute hepatitis B. In the sera of 10 of 11 patients, HBV DNA, which was presumably coated with viral proteins, was detected for a long period after recovery, even at the final observation times, which ranged from 6 to 19 months after disease onset. To characterize the mode of HBV that was present in serum, we immunoprecipitated immune complexes in sera by the addition of anti-human immunoglobulin G (IgG) and determined the levels of HBV DNA separately in the supernatants and pellets. In the acute phase of hepatitis B, high levels of HBV DNA were detected both in the supernatants and pellets at comparative levels. After the convalescent phase, the amount of HBV DNA in the supernatant decreased with respect to that in the pellets. It is notable that, in most cases, serum HBV persisted as a form of immune complex even after the seroconversion to antibody to hepatitis B surface antigen (anti-HBs). These data suggest that the replication of HBV may persist in some organs, most likely in the liver or peripheral blood cells, for a long period after recovery from acute hepatitis B, and the data indicate the possible transmission of HBV from organ transplantation donors who exhibit serological markers of past infection only.

Acute Disease↗
PubMed1998

Regulation of Fas gene expression in HeLa cells as determined by modified RT-PCR.

We determined human Fas messenger RNA (mRNA) levels in HeLa cells using a 'mutagenic' reverse transcription-polymerase chain reaction, which quantitates mRNA levels using the corresponding genomic DNA as an internal control. The expression level of Fas mRNA was very low in serum-deprived quiescent HeLa cells. In conjunction with the start of cell-cycle progression upon the addition of serum to culture medium, the Fas mRNA level gradually increased, reached its peak at 36 h and returned to the basal level after 48 h. HeLa cells at 36 h exhibiting a high level of Fas mRNA expression were more susceptible to the anti-Fas antibody apoptotic signal. Thus, the regulation of Fas expression is associated with cell-cycle progression, and this method for Fas mRNA detection may be useful, particularly for the analysis of small amounts of samples.

Antibodies↗
PubMed1998

The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice.

Hepatitis C virus (HCV) is the main cause of chronic hepatitis worldwide. Chronic hepatitis ultimately results in the development of hepatocellular carcinoma (HCC). However, the mechanism of hepatocarcinogenesis in chronic HCV infection is still unclear. The ability of the core protein of HCV to modulate gene transcription, cell proliferation and cell death may be involved in the pathogenesis of HCC. Here, we report the development of HCC in two independent lines of mice transgenic for the HCV core gene, which develop hepatic steatosis early in life as a histological feature characteristic of chronic hepatitis C. After the age of 16 months, mice of both lines developed hepatic tumors that first appeared as adenomas containing fat droplets in the cytoplasm. Then HCC, a more poorly-differentiated neoplasia, developed from within the adenomas, presenting in a 'nodule-in-nodule' manner without cytoplasmic fat droplets; this closely resembled the histopathological characteristics of the early stage of HCC in patients with chronic hepatitis C. These results indicate that the HCV core protein has a chief role in the development of HCC, and that these transgenic mice provide good animal models for determining the molecular events in hepatocarcinogenesis with HCV infection.

Animals↗
PubMed1998

Serum follistatin concentrations are increased in patients with septicaemia.

OBJECTIVE: Follistatin (FS) is the specific binding protein of activin, a growth and differentiation factor of many cell types. Both factors have almost ubiquitous tissue distributions. In vitro, FS is secreted by vascular endothelial cells and this can be stimulated by bacterial compounds. For this reason, serum FS levels were examined in patients with septicaemia. PATIENTS: Five male and four female patients of different age with various forms of septicaemia and different clinical outcome. MEASUREMENTS: Serum concentrations of FS, C-reactive protein (CRP) and blood leucocyte counts were determined repeatedly in all nine patients; samples from age- and sex-matched healthy volunteers served as controls. RESULTS: The median of the maximum FS concentrations of septicaemic patients was threefold higher than in healthy controls (P = 0.008). The highest increase observed was approximately 40 times normal. Serum FS levels in patients with septicaemia showed large variations between individuals. Serum FS levels parallelled those of CRP but were not correlated to the leucocyte counts. CONCLUSION: Serum follistatin concentrations in humans are elevated during septicaemia and appear to parallel serum C-reactive protein levels.

Adult↗
PubMed1998

Macrolipasemia in Crohn's disease.

A 38-year-old male patient who had been treated for Crohn's disease was found to have serum lipase activity that was persistently increased approximately 10-fold above the normal upper limit. He was diagnosed with chronic pancreatitis based on slightly elevated elastase-1 level and retrograde pancreatography showing slight dilatation of the main pancreatic duct. Therefore, the hyperlipasemia was thought to be due to pancreatitis. However, the serum amylase and trypsin was not increased at any time, and no serious findings suggestive of pancreatitis were detected on morphologic examination. Thus, there were discrepancies between the serum lipase activity and other laboratory and clinical findings. Exclusion chromatography of the patient's serum suggested macromolecular lipase, and further immunologic testing including affinity chromatography, enzyme-linked immunosorbent assay, and immunoprecipitation assay showed that serum lipase was bound to immunoglobulin Gkappa. Therefore, the hyperlipasemia was caused by immunoglobulin-linked lipase, termed "macrolipasemia." Macrolipasemia has rarely been reported, and this is the first reported case of macrolipasemia accompanied by Crohn's disease.

Adult↗
PubMed1998

Proliferative effect of phospholipase A2 on rat periacinar fibroblastoid cells of the pancreas.

We have shown previously that rat pancreatic periacinar fibroblastoid cells (PFCs) can be cultured from isolated pancreatic acini. In the present study, immunocytochemical examination of the PFC extracellular matrix was performed using antibodies against prolyl hydroxylase alpha and beta subunits, types I, III, and IV collagen, fibronectin, and laminin. The PFC content of alpha-smooth muscle actin and platelet-derived growth factor (PDGF) receptor were studied by immunoblotting. We demonstrated that PFCs synthesized extracellular matrix and expressed alpha-smooth muscle actin and PDGF receptors. These results suggested that PFCs resemble myofibroblasts and may play a critical role in pancreatic fibrosis. Conversely, pancreatic-type phospholipase A2 (P-PLA2), one of the pancreatic digestive enzymes, has been shown to induce DNA synthesis of Swiss 3T3 fibroblasts. To determine whether this enzyme is involved in pancreatic fibrosis, we studied P-PLA2's proliferative and chemotactic effects on PFCs as well as its digestive activity. The proliferative and chemotactic effects were investigated using 3H-thymidine incorporation and a chemotactic assay, respectively. P-PLA2 had both proliferative and chemotactic effects. P-PLA2 is considered a growth factor for PFCs and is implicated in pancreatic fibrosis.

Actins↗
PubMed1998

Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide.

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current (Isc) without affecting total Isc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the Isc. The effect was reversed by the addition of N-monomethyl-L-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.

Amiloride↗
PubMed1998

Identification of a single cytosine base insertion mutation at Arg-597 of the beta subunit of the human epithelial sodium channel in a family with Liddle's disease.

We describe a family with Liddle's disease caused by a novel mutation of the beta subunit of the human epithelial sodium channel (ENaC). A 15-year-old Japanese female was referred to our outclinic because of hypertension. The physical examination showed no abnormal findings except mild hypertension, but the laboratory data revealed low levels of plasma renin activity, plasma aldosterone and serum potassium. A comprehensive analysis of steroid hormones showed only high levels of urinary free cortisol and 17-hydroxycorticosteroids. During loading tests, blood pressure and serum potassium responded well to triamterene and slightly to spironolactone, but did not respond to dexamethasone. In addition, the normal ratio of tetrahydrocortisol plus 5alpha-tetrahydrocortisol to tetrahydrocortisone in a 24 h urinary excretion test strongly suggested a diagnosis of Liddle's disease rather than apparent mineralocorticoid excess syndrome. DNA sequence analysis of members of this family revealed a single cytosine base insertion at Arg-597 of the beta human ENaC in the proband and her mother, leading to a loss of the last 34 amino acids from the normally encoded protein as the result of a frameshift. We conclude that a de novo cytosine insertion into the final exon of the C-terminus of the beta human ENaC is responsible for Liddle's disease in this Japanese family.

Adolescent↗
PubMed1997

Sialadenitis histologically resembling Sjogren syndrome in mice transgenic for hepatitis C virus envelope genes.

Hepatitis C virus (HCV), a major causative agent of non-A, non-B chronic hepatitis, is also suggested to be associated with extrahepatic manifestations such as mixed cryoglobulinemia and glomerulonephritis. Two independent lines of transgenic mice carrying the HCV envelope genes have been shown previously to express the HCV envelope proteins in organs, including the liver and salivary glands, which results in no pathological changes in the liver. Further analysis of these animals now has revealed that they develop an exocrinopathy involving the salivary and lachrymal glands. This pathology resembles Sjogren syndrome, which also is suggested to have a possible association with chronic hepatitis C. These observations suggest that HCV might be involved in the pathogenesis of sialadenitis in humans and that this transgenic mouse system would be a good animal model for the study of HCV infection.

Age Factors↗
PubMed1997

GS-164, a small synthetic compound, stimulates tubulin polymerization by a similar mechanism to that of Taxol.

PURPOSE: During our search for new microtubule effectors as anticancer agents, we have found that a small synthetic molecule designated GS-164 interferes with the assembly of porcine microtubule proteins and has cytotoxic activity against a wide range of human tumor cell lines. In this study, we investigated mode of action of the compound in comparison with Taxol and colcemid. METHODS: To gain an insight into the mode of action of GS-164, we used an in vitro microtubule polymerization assay and a flow-cytometric measurement technique. Microtubule organization and the level of tubulin polymerization in HeLa cells were also examined by immunofluorescence microscopy and cytoskeletal protein analyses, respectively. RESULTS: GS-164 stimulated assembly of microtubule proteins in vitro in a concentration-dependent and a GTP-independent manner. Furthermore, as with Taxol, the microtubule polymerization induced by GS-164 was antagonized by podophyllotoxin, a tubulin polymerization inhibitor, and microtubules formed by GS-164 were resistant to disassembly by calcium or low temperatures. GS-164 in the micromolar range arrested the cell cycle of HeLa cells in the mitotic phase leading to cell death. GS-164 also increased the amounts of cellular microtubules in HeLa cells, resulting in the formation of microtubule bundles. CONCLUSION: These results indicate that GS-164 stimulates microtubule assembly by a similar mechanism to that of Taxol. A comparative conformational analysis of GS-164 and Taxol suggested that the structure of the former mimics the minimum essential sites of Taxol required to exert the Taxol-like activities described above. Although the cytotoxicity of GS-164 against human tumor cells was 1000-fold lower than that of Taxol and GS-164 was one-tenth as active as Taxol in vitro, these findings pave the way for synthesizing clinically useful anticancer agents using GS-164 as a lead compound.

Animals↗
PubMed1997

Activation of Na+-permeant cation channel by stretch and cyclic AMP-dependent phosphorylation in renal epithelial A6 cells.

It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na+ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na+, K+, Li+, and Cs+, but little selectivity for Ca2+ (PCa/PNa < 0.005) or Cl- (PCl/PNa < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability (Po) of the IBMX-activated channel. This channel had one open ("O") and two closed (short "CS" and long "CL") states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the Po of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model "CL <--> CS <--> O" was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for CL from CS, resulting in an increase in Po. Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Hepatitis C virus core protein induces hepatic steatosis in transgenic mice.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide, which finally leads to development of hepatocellular carcinoma. Chronic hepatitis C is characterized by several histological features in the liver which discriminate it from other forms of hepatitis: bile duct damage, lymphoid follicles and steatosis (fatty change). Little is known, however, about the role of HCV or its viral proteins in the pathogenesis of hepatitis. Recently, the core protein of HCV has been suggested to have a transcriptional regulatory function, and thereby to be involved in inducing phenotypic changes in hepatocytes. To clarify whether or not the HCV core protein has an effect on pathological phenotypes in the liver, two independent transgenic mouse lines carrying the HCV core gene were established. These mice developed progressive hepatic steatosis, indicating that the HCV core protein plays a direct role in the development of hepatic steatosis, which characterizes hepatitis C. This transgenic mouse system would be a good animal model for the study of pathogenesis in human HCV infection.

Animals↗
PubMed1997

Regulation of follistatin production by rat granulosa cells in vitro.

The aims of this study were to apply enzyme-linked immunosorbent assays (ELISA) for human follistatins (FS) to measure total immunoreactive (ir-) rat FS and free rat FS, and investigate the regulation of production of total ir-FS and free FS by rat granulosa cells (GC) in vitro. Production of ir-inhibin was monitored as an index of GC function. The ELISAs for total ir-FS, based on an immunoradiometric assay developed recently for human FS, and free FS, based on capture of FS by a monoclonal antibody and detection by activin A binding, had sensitivities of 0.4 and 0.8 ng recombinant human (rh-) FS 288/ml, respectively, and did not cross-react with inhibin A, rLH, or FSH. rh-Activin did not cross react in the total ir-FS ELISA, but interfered with the measurement of free FS. Dilutions of GC-conditioned medium were parallel to the standard curve of rh-FS 288 for each assay. The values obtained in the free FS assay were 10- to 20-fold higher than those in the total ir-FS ELISA, suggesting that rat FS may be recognized by the antibodies differently than the human standard. Both total ir-FS and free FS production by undifferentiated GC from diethylstilbestrol (DES)-treated, immature rats increased with cell number and time in culture and were stimulated dose dependently by FSH, rh-activin A (except free FS, which was not measured because of interference), forskolin, and phorbol 12-myristrate. The effects of FSH and activin on FS production by undifferentiated GC were additive. There were significant effects of degree of differentiation of GC on basal FS production and responsiveness to FSH, LH, and rh-activin A. Both total ir-FS and free basal FS production increased up to 4-fold with the degree of differentiation of GC, produced by treating rats in vivo with DES (undifferentiated), DES plus FSH (partially differentiated), or DES plus FSH plus hCG (fully differentiated). The addition of FSH in vitro increased FS production by undifferentiated and partially differentiated GC, but not by fully differentiated GC. The only detectable effect of LH on FS production was on partially differentiated GC. Activin A stimulated total ir-FS production by undifferentiated and partially differentiated GC, but inhibited total ir-FS production by fully differentiated GC. Ir-inhibin production in these experiments was similar to that of FS with the following exceptions; phorbol 12-myristrate inhibited ir-inhibin production by undifferentiated GC, basal ir-inhibin decreased in fully differentiated GC, FSH stimulated ir-inhibin only in undifferentiated GC, and rh-activin A stimulated ir-inhibin at all stages. It is concluded that 1) FS protein production by cultured undifferentiated rat GC is up-regulated by FSH and activin, possibly via both protein kinase A and C pathways; 2) increasing GC differentiation is associated with a significant increase in basal FS production by rat GC and a change in the hormonal regulation of FS production; and 3) FS and ir-inhibin production by cultured rat GC can be differentially regulated. The results are consistent with the hypothesis that activin tone decreases within follicles as they develop due to increased production of the activin-binding protein FS.

Activins↗