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Biomedical subjects

A Yamauchi

Publications and source records attributed to A Yamauchi.

At least 19 recordsLinked to original sources
PubMed1995

Phosphatase toward MAP kinase is regulated by osmolarity in Madin-Darby canine kidney (MDCK) cells.

We have reported that MAP kinase and its activator were activated by increase in extracellular osmolarity in Madin-Darby canine kidney (MDCK) cells [J. Clin. Invest. 93 (1994) 2387-2392]. The activation of MAP kinase quickly disappeared when cells in hypertonicity were shifted to isotonicity. Present study was planned to elucidate the mechanism for the inactivation of MAP kinase when osmolarity decreased. Combination of two different phosphatase inhibitors, 10(-6) M okadaic acid and 0.2 mM sodium orthovanadate, blocked the inactivation of MAP kinase after the decrease in osmolarity. We also demonstrated that phosphatase toward MAP kinase was activated in response to the decrease in osmolarity. These results suggest that MAP kinase is inactivated by phosphatase that is activated when osmolarity decreased.

Animals↗
PubMed1995

Na+/myo-inositol cotransport is regulated by tonicity in cultured rat mesangial cells.

Mesangial cells are considered to be faced with osmotic stress under physiological (such as extraglomerular mesangial cells) and pathophysiological (for example, diabetes mellitus) conditions. To see if mesangial cells have an osmoregulatory mechanism, like renal medullary cells, we measured the intracellular contents of organic osmolytes in isotonic and hypertonic conditions. Cultured rat mesangial cells are well tolerant of acute increase in osmolality up to 500 mOsm/kg. The myo-inositol content increased in hypertonic cells more than six-fold the value in isotonic cells. The contents of glycerophosphorylcholine and sorbitol also increased but were less than that of myo-inositol. The Na(+)-dependent myo-inositol uptake in hypertonic cells was a 12-fold uptake in isotonic cells, reaching a maximum 24 hours after the switch to a hypertonic medium. The uptake rate increased as medium osmolality increased from 300 to 500 mOsm/kg. Raffinose is the most effective solute to increase the myo-inositol uptake. NaCl, glucose and mannitol also increased the uptake rate (NaCl > glucose > mannitol). The increased uptake by hypertonicity was the result of an increase in Vmax without change in Km and was dependent on RNA and protein synthesis. These results indicate that mesangial cells respond to extracellular hypertonicity by increasing myo-inositol transport activity and accumulating myo-inositol into the cells, suggesting that myo-inositol functions as an organic osmolyte in mesangial cells.

Animals↗
PubMed1995

Amino acids and glucose differentially increased extracellular 5-hydroxyindoleacetic acid in the rat brain.

To reveal the role of serotonergic neurons in the regulation of feeding, the levels of 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of serotonin, in the striatum and the hypothalamus were continuously monitored by an in vivo microdialysis technique. Intake of 20% casein diet did not induce significant changes in the 5-HIAA level in these regions. When rats were fed on 5% casein diet (83.5% carbohydrate diet) for 2 h, the level of 5-HIAA in the striatum gradually increased and reached a maximum (226 +/- 44% of basal level, M +/- SEM, n = 7) at 4 h after stopping the diet. In the medial hypothalamus, its level also increased to 183 +/- 19% (n = 10) at 2 h after starting the diet. On the other hand, a 60% casein diet increased the level of 5-HIAA in the lateral hypothalamus to 138 +/- 19% (n = 10) at 2 h after starting the diet. The intravenous infusion of each of these nutrients, glucose, amino acid mixture or lipid, produced more rapid elevation of the 5-HIAA level than oral intake of the diets. When rats were infused with glucose, its level in the striatum continued to be elevated. In the medial hypothalamus, glucose infusion increased 5-HIAA to the maximum (189 +/- 38%, n = 7) at 4 h after starting infusion. In contrast, serotonergic neurons in the lateral hypothalamus seemed to respond only to infusion of the amino acid mixture, and the level of 5-HIAA reached 163 +/- 14% (n = 5) of the basal level at 1 h after starting the infusion. These results suggest that rapid elevation of glucose or amino acids may independently stimulate serotonin metabolism in these brain areas, participating in the feedback regulation of nutrient intake.

Amino Acids↗
PubMed1995

[Significance of prostate fluid carcinoembryonic antigen in the diagnosis of prostate cancer].

Clinical studies of carcinoembryonic antigen (CEA) in prostate fluid were performed on 121 men; 29 patients with prostate cancer, 65 with benign prostatic hypertrophy, 10 with prostatitis and 17 without any prostatic diseases. The CEA level in prostate fluid in the prostate cancer group was significantly higher than that in any other group. However, it could not demonstrate any particular advantage when compared with the prostate specific antigen (PSA) in serum. Additional research was done comparing the sensitivity and specificity of CEA level in prostate fluid in the population with slightly elevated PSA level (3.0-14.9 ng/ml). The sensitivity and the specificity were 82% and 83%, respectively. These findings suggest the usefulness of the measurement of prostate fluid CEA as an adjunctive tool in the diagnosis of prostate cancer in the population with a slightly elevated PSA level.

Carcinoembryonic Antigen↗
PubMed1992

Molecular cloning of the cDNA for an MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is regulated by hypertonicity.

Cells in the hypertonic renal medulla maintain their intracellular ion concentration at isotonic levels, despite much higher concentrations of extracellular electrolytes, by accumulating high concentrations of nonperturbing small organic solutes termed osmolytes. Taurine has been identified as a nonperturbing osmolyte in the renal medulla and Madin-Darby canine kidney (MDCK) cells. In hypertonic medium, the increased accumulation of taurine in MDCK cells is the result of increased activity of a Na(+)- and Cl(-)-dependent taurine transporter. We have isolated a cDNA encoding a Na(+)- and Cl(-)-dependent taurine transporter, whose sequence corresponds to a protein of 655 amino acids with significant amino acid sequence similarity to previously cloned Na(+)- and Cl(-)-dependent transporters, including the MDCK cell betaine/gamma-aminobutyric acid transporter and several brain neurotransmitter transporters. Northern hybridization indicates that mRNA for the taurine transporter is present in renal cortex and medulla, ileal mucosa, brain, liver, and heart. The abundance of mRNA for the taurine transporter is increased in MDCK cells cultured in hypertonic medium, suggesting that regulation of transport activity by medium hypertonicity occurs at the level of mRNA accumulation.

Amino Acid Sequence↗
PubMed1992

Molecular genetic and immunological analysis of dystrophin of a young patient with X-linked muscular dystrophy.

We examined the nucleotide sequence of deleted part of dystrophin mRNA and its translational product with immunoblot and immunohistochemical methods in a 6-year-old boy with a deleted DMD/BMD gene. On Southern blot analysis of his genomic DNA, we found a deletion of exons 10 to 37 in the DMD/BMD gene, which was expected to preserve the translational open reading frame (ORF). Dystrophin mRNA from his biopsy sample was amplified by polymerase chain reaction (PCR) and sequenced. The mRNA lacked the sequence corresponding to the gene from exons 10-37, and the translational ORF was preserved. The transcript was expected to code a 260 kDa protein. Dystrophin expressed in this patient was investigated with immunological methods. A 260 kDa protein was detected by immunoblot analysis with antidystrophin antiserum against nondeleted regions. These observations confirmed the preservation of the reading frame and the 260 kDa protein was produced as a mutant dystrophin. All these are compatible with the diagnosis of BMD. However, the immunohistochemical pattern of his muscle cells was peculiar. With deleted-region-directed antiserum, the membrane was not stained at all as in DMD patients. In contrast, with nondeleted-region-directed antiserum, all the muscle cell membrane was stained continuously as in non-DMD/BMD individuals. These are quite different from the staining pattern in most BMD patients where muscles are stained patchily or discontinuously.

Amino Acid Sequence↗
PubMed1992

Expression and growth-promoting effect of adult T-cell leukemia-derived factor. A human thioredoxin homologue in hepatocellular carcinoma.

Adult T-cell leukemia-derived factor (ADF), originally defined as an interleukin-2 receptor inducer, is a human thioredoxin homologue. ADF is detected in many malignant tissues and has a growth-promoting effect on transformed cells. In this study, ADF expression was examined immunohistochemically in human liver cell lines and liver tissues, and its growth-promoting effect was tested on human hepatoma cells. On three liver cell line--PLC/PRF/5, HepG2, and Chang liver cells--ADF stained positively and also was detected by immunoblotting. ADF had strong staining in the fetal liver (n = 8), although it was faint in the normal adult liver (n = 6). In hepatocellular carcinoma (n = 25), ADF expression generally was enhanced and was very strong in 52% (13 of 25) of the cases, although it was moderate in cases of chronic hepatitis or cirrhosis. ADF augmented the growth of PLC/PRF/5 cells and showed an additive effect with epidermal growth factor. These results indicate possible involvement of ADF in cell activation and growth of hepatocytes, as is the case with lymphocytes.

Carcinoma, Hepatocellular↗
PubMed1992

Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein.

Kidney medullary cells in situ, as well as kidney-derived Madin-Darby canine kidney (MDCK) cells accumulate nonperturbing, small organic solutes (osmolytes), including myo-inositol, when bathed in hypertonic media. Accumulation of osmolytes balances the osmolality of extracellular fluid without raising intracellular salts that would perturb cellular functions. In hypertonic media, increased myo-inositol accumulation is the result of increased activity of a Na+/myo-inositol cotransporter. We have isolated a cDNA encoding a Na+/myo-inositol cotransporter from MDCK cells using expression in Xenopus oocytes. The cDNA sequence predicts a protein of 718 amino acids with a significant amino acid sequence similarity to the Na+/D-glucose cotransporters of absorbing epithelia. Transporter mRNA is present in kidney and brain and is markedly induced in MDCK cells by medium hypertonicity, demonstrating that adaptation to hypertonic stress involves up-regulation of transporter mRNA accumulation.

Amino Acid Sequence↗
PubMed1992

Cloning of a Na(+)- and Cl(-)-dependent betaine transporter that is regulated by hypertonicity.

Many hypertonic bacteria, plants, marine animals, and the mammalian renal medulla are protected from the deleterious effects of high intracellular concentrations of electrolytes by accumulating high concentrations of the nonperturbing osmolyte betaine. When kidney-derived Madin-Darby canine kidney (MDCK) cells are cultured in hypertonic medium, they accumulate betaine to 1,000 times its medium concentration. This results from induction by hypertonicity of high rates of betaine transport into cells. We have isolated a cDNA (BGT-1) encoding a renal betaine transporter by screening an MDCK cell cDNA library for expression of a betaine transporter in Xenopus oocytes. The cDNA encodes a single protein of 614 amino acids, with an estimated molecular weight of 69 kDa. The deduced amino acid sequence exhibits highly significant sequence and topographic similarity to brain gamma-amino-n-butyric acid (GABA) and noradrenaline transporters, suggesting that the renal BGT-1 is a member of the brain GABA/noradrenaline transporter gene family. Expression in oocytes indicates that the BGT-1 protein has both betaine and GABA transport activities that are Cl(-)- as well as Na(+)-dependent and functionally similar to betaine and GABA transport in MDCK cells. Northern hybridization indicates that transporter mRNA is localized to the kidney medulla and is induced in MDCK cells by hypertonicity.

Amino Acid Sequence↗
PubMed1992

Lymphocyte transformation and thiol compounds; the role of ADF/thioredoxin as an endogenous reducing agent.

ADF (adult T-cell leukemia-derived factor), an inducer of IL-2R with growth promoting activity, is a homologue of thioredoxin which is involved in many thiol-dependent reducing reactions. ADF is constitutively produced and released by human lymphoid cell lines transformed by lymphocyte-tropic viruses, such as human T-lymphotropic virus type I (HTLV-I) and Epstein-Barr virus (EBV). We found that the viability and growth of these ADF high-producer cell lines (ATL-2, HUT102, MT-2, 3B6 and RPM18866) were highly dependent on L-cystine in the culture. In contrast to the relative cystine independency of ADF low-producer cells (Jurkat, Jijoye, U937 and K562), the growth of ADF high-producer cells was almost completely suppressed in L-cystine-free condition. Their viability and growth in L-cystine-free medium were markedly improved by 5 x 10(-5) M L-cysteine, 5 x 10(-5) M 2-ME or 10(-3) M GSH and partially by 10(-3) M DTT. The results demonstrate the requirement of reducing condition involving thiol compounds for the optimal growth of the virally transformed lymphoid cells. Furthermore, recombinant ADF (rADF) and suboptimal dose of 2-ME additively enhanced the growth of ATL-2 cells in L-cystine-free medium, implying the possible involvement of endogenous reducing agents such as ADF/thioredoxin homologue in the process of lymphocyte transformation/activation.

Cell Line↗
PubMed1992

Oncotic pressure regulates gene transcriptions of albumin and apolipoprotein B in cultured rat hepatoma cells.

The mechanism of the accelerated syntheses of albumin and apolipoprotein B (apo B) in response to decreased oncotic pressure was investigated in cultured rat hepatoma H4-II-E cells. Addition of dextran (mol wt 6-9 x 10(4)) to the culture medium decreased the levels of albumin and apo B mRNAs in an oncotic pressure-dependent manner. The reductions of both mRNAs were attenuated with increase in the molecular weight of dextran, which resulted in a decrease in oncotic pressure. Addition of macromolecule increased the viscosity in medium; however, alteration of viscosity appeared not to correlate with albumin and apo B mRNA levels. Transcriptional run-on assays with isolated nuclei from dextran-treated vs. untreated hepatoma cells indicated that the changes in steady-state mRNA levels were mainly controlled at the transcriptional step. Treatment with cycloheximide increased albumin mRNA to the basal level, which was effectively suppressed by dextran, and resulted in superinduction of apo B mRNA. These changes occurred primarily at the transcriptional step. These results suggest that regulations of the expressions of the albumin and apo B genes for adaptive increases in the mRNAs may require the continued synthesis of a labile protein(s) or a limiting transcription factor(s). We conclude that oncotic pressure plays an important role in regulation of expression of the albumin and apo B genes at the transcriptional step.

Animals↗
PubMed1991

Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells.

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.

Antigens, Surface↗
PubMed1991

Presence of human immunoglobulin G anti serum pancreatic elastase 1 autoantibodies and their influence on elastase 1 radioimmunoassay.

Human immunoglobulin G (IgG) anti human pancreatic elastase 1 autoantibodies were detected in sera of patients with pancreatic disorders. The characteristics of these anti elastase 1 autoantibodies and their influence on radioimmunoassay (RIA) for elastase 1 were investigated. They were placed in the IgG class by the double antibody method, and most were assumed to be of a monoclonal type from their elution profiles in gel filtration analysis. The presence of autoantibodies in serum caused an increase in apparent elastase 1 values and a decrease in the recovery of elastase 1 exogenously added to the serum. These results suggest that elastase 1 immunoassay data for autoantibody positive sera can cause misjudgement of clinical stages of patients.

Autoantibodies↗
PubMed1991

Hepatocyte growth inhibitory factor derived from HTLV-I(+) T cell lines: effect on the epidermal growth factor-dependent proliferation of rat hepatocytes.

A human T cell leukemia virus-I infected T cell line, ATL-2, produces an interleukin-2 receptor inducing factor, adult T cell leukemia (ATL)-derived factor (ADF). In the conditioned medium (CM) of ATL-2, we found an inhibitory activity on the epidermal growth factor (EGF)-dependent proliferation of primary cultured rat hepatocytes, measured by cell number and [3H]thymidine incorporation. ATL-2 CM dose-dependently inhibited hepatocyte proliferation. This activity was fractionated by gel filtration at a molecular size of 15,000 to 40,000 and was tentatively called hepatocyte growth inhibitory factor (HGI). Further fractionation with the ion-exchange column indicated that HGI was separable from ADF. Nevertheless, there was a positive correlation between HGI and ADF production, because the HGI activity was also detected in the CM of another ADF producer cell line (HUT102), while no significant HGI activity was detected in the CM of low ADF producer cell lines, ED and MOLT4.

Animals↗
PubMed1991

Renal Na-myo-inositol cotransporter mRNA expression in Xenopus oocytes: regulation by hypertonicity.

Canine renal cells in culture (MDCK cells) accumulate organic osmolytes, including myo-inositol (MI), in response to hypertonic stress. When medium tonicity is increased, intracellular concentration of MI rises because hypertonicity elicits increased uptake of MI via Na-MI cotransporter(s). To study the mechanism for this increase in cotransporter activity, poly(A)+ RNA isolated from MDCK cells maintained in hypertonic or isotonic medium was injected into Xenopus oocytes, and Na-dependent MI uptake was measured 3-5 days later. Poly(A)+ RNA from hypertonic cells induced clear expression of the cotransporter. In contrast, oocytes injected with poly(A)+ RNA isolated from MDCK cells maintained in isotonic medium exhibited cotransporter activity like oocytes injected with water. Upon size fractionation of RNA, peak activity appeared in a fraction that contained poly(A)+ RNA with median size of approximately 4 kilobases. Na-dependent MI uptake by poly(A)+ RNA-injected oocytes was inhibited by both phlorizin and phloretin. We suggest that hypertonicity-induced upregulation of the Na-MI cotransporter involves an increase in mRNA and synthesis of cotransporter protein(s).

Animals↗
PubMed1991

Myo-inositol and betaine transporters regulated by tonicity are basolateral in MDCK cells.

Myo-inositol and glycinebetaine are compatible osmolytes accumulated in the renal medulla and in MDCK cells cultured in hypertonic media. Both osmolytes are taken up by MDCK cells on Na-coupled transporters. The maximal velocity (Vmax) of both cotransporters is increased by culture in hypertonic medium. When hypertonic MDCK cells are shifted to isotonic medium there is a large transient efflux of osmolytes. To determine the polarity of the cotransporters and the transient efflux, we grew MDCK cells on a porous support to assay transport separately at their apical and basolateral surfaces. In hypertonic cells, basolateral uptake of both osmolytes was 1) more than 10-fold apical uptake, 2) greater than 96% Na dependent, 3) 25- (myo-inositol) and 16-fold (glycinebetaine) uptake in isotonic cells, reaching a maximum 24 h after the switch to hypertonic medium. When medium osmolarity was decreased from hypertonic to isotonic, myo-inositol uptake reversed to the isotonic level within 1 day; glycinebetaine uptake decreased more slowly. When medium osmolarity was decreased from hypertonic to isotonic, there was a large transient increase in basolateral efflux of both osmolytes.

Animals↗