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H Tsubouchi

Publications and source records attributed to H Tsubouchi.

At least 145 records · Page 8Linked to original sources
PubMed1990

Biological and immunological properties of human hepatocyte growth factor from plasma of patients with fulminant hepatic failure.

We have recently purified human hepatocyte growth factor (hHGF), a heterodimer with molecular weight of about 83,000, from plasma of patients with fulminant hepatic failure (Gohda, E. et al., J. Clin. Invest. 81, 414-419, 1988). Biological and immunological properties of hHGF were examined. Out of the well-known growth factors tested, only epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) stimulated DNA synthesis of adult rat hepatocytes in primary culture. hHGF enhanced the DNA synthesis at less than one-tenth of the molar concentrations of EGF and TGF-alpha. Half-maximal stimulations by hHGF, EGF and TGF-alpha were observed at 30, 400 and 900 pM, respectively. Maximal stimulation by TGF-alpha, however, was greater than those caused by hHGF and EGF. The effect of hHGF was additive with the maximal effects of EGF and TGF-alpha. Anti-hHGF antiserum was prepared in a rabbit by injecting with purified hHGF. This antiserum recognized nonreduced hHGF, but not reduced hHGF. The antiserum for hHGF did not inhibit growth-promoting activity of EGF, that was neutralized by incubation with anti-EGF antiserum. The activity of hHGF was completely inhibited by anti-hHGF antiserum, but not by anti-EGF antiserum. hHGF did not show any cross-reactivity to anti-EGF antiserum as measured by enzyme immunoassay for EGF. Thus, biological and immunological properties of hHGF are different from those of EGF and TGF-alpha.

Animals↗
PubMed1990

Hepatotrophic growth factor in blood of mice treated with carbon tetrachloride.

The presence of a human hepatocyte growth factor (hHGF)-like DNA-synthesis promoter in platelet-poor serum of mice with liver injury was examined. Activity of the serum for stimulating DNA synthesis in cultured rat hepatocytes was low in untreated or vehicle-treated mice, but markedly increased 24 h after carbon tetrachloride administration and then dropped to normal levels prior to the peak of liver DNA synthesis. The effect of the serum was additive with the maximal effects of mouse and human epidermal growth factors, but not with that of hHGF. The growth-stimulating factor in the mouse serum, like hHGF, had affinity for heparin and was heat-labile. These results indicate that the level of a serum hHGF-like hepatocyte growth factor increased in mice treated with carbon tetrachloride prior to liver regeneration.

Alanine Transaminase↗
PubMed1989

Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor.

Amino acid sequences of four peptide fragments of human hepatocyte growth factor purified from the plasma of patients with fulminant hepatic failure were determined. Based on the amino acid sequence of one of the fragments, two oligodeoxyribonucleotide mixtures were synthesized and used to screen a human placenta cDNA library. On the screening, two overlapping cDNA clones for human hepatocyte growth factor were isolated and the nucleotide sequence of the cDNA was determined. The entire primary structure of the protein was deduced from the sequence. The protein consists of 728 amino acid residues, including a possible signal peptide at the N-terminus. The sequence revealed that the heavy and light chains which comprise the protein are encoded by the same mRNA and are produced from a common translation product by proteolytic processing.

Amino Acid Sequence↗
PubMed1989

Clinical significance of human hepatocyte growth factor in blood from patients with fulminant hepatic failure.

We have recently found the presence of human hepatocyte growth factor in sera of patients with fulminant hepatic failure and have purified human hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. In this paper, we report the clinical significance of human hepatocyte growth factor in blood from patients with fulminant hepatic failure. The effect of sera or plasma from 17 patients with fulminant hepatic failure on liver cell growth was examined by use of adult rat hepatocytes in primary cultures. Sera or plasma from 16 of the 17 patients with fulminant hepatic failure stimulated DNA synthesis in hepatocytes more effectively than normal human serum. The mean growth-promoting activity for the 17 patients with fulminant hepatic failure was about 16 times higher than that obtained for normal human serum. This growth-promoting activity of the patients' blood was not related to sex, age, clinical outcome of the patients or type of fulminant hepatic failure, but was intimately related to the clinical grade of hepatic coma. Sera or plasma with Grade III and IV coma showed stimulatory activity on DNA synthesis more markedly than sera or plasma from patients with coma of less than Grade II. In the surviving group, this activity decreased as the hepatic coma of patients improved. In fact, this activity of sera from patients at the recovery stage showed no significant increase compared with that of normal human serum. In the group of terminal patients, this activity increased as the coma developed.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure.

Human hepatocyte growth factor (hHGF) has been purified approximately 209,000-fold with 18% yield from plasma of a patient with fulminant hepatic failure. The purification involves heat treatment of plasma, ammonium sulfate precipitation, and chromatography on Affi-Gel Blue, heparin-Sepharose, and hydroxylapatite. Purified hHGF shows several bands with molecular weights between 76,000 and 92,000. Each band shows growth-stimulating activity on cultured hepatocytes which is proportional to the intensity of the band. After reduction of the sample with 2-mercaptoethanol, SDS-PAGE yields two chains with molecular weights of 31,500-34,500 and 54,000-65,000. The effect of hHGF on DNA synthesis by hepatocytes is half-maximal at 3.5 ng/ml. hHGF stimulates proliferation of cultured hepatocytes more effectively than human epidermal growth factor (hEGF) or insulin, and the effect of hHGF is additive or synergistic with the maximal effects of hEGF and insulin. These results suggest that hHGF is a new growth factor which is different from hEGF.

Cell Division↗
PubMed1987

Effect of roasting on ochratoxin A level in green coffee beans inoculated with Aspergillus ochraceus.

The heat stability of ochratoxin A in green coffee beans inoculated with Aspergillus ochraceus was studied. Heat treatment (roasting) at 200 degrees C for 10 or 20 min reduced the levels of ochratoxin A by only 0-12% in the dried whole beans. Almost all of the ochratoxin A was infused into the coffee decoction when the roasted samples were ground and extracted with boiling water. Therefore, the reduction of ochratoxin A concentration of contaminated coffee beans by roasting under these conditions is ineffective.

Aspergillus↗
PubMed1987

Effect of histamine H2-receptor antagonists on DNA synthesis in adult rat hepatocytes in primary culture. Cimetidine enhanced hepatocytes proliferation stimulated with insulin and epidermal growth factor.

The effects of three of the most widely used histamine H2-receptor antagonists, cimetidine, ranitidine and famotidine, on liver cell growth were studied in vitro using adult rat hepatocytes in primary culture, because these antagonists are commonly given to patients with hepatic cirrhosis or fulminant hepatic failure for protection against peptic ulcers and gastrointestinal hemorrhage. At their clinically effective concentrations in the blood (0.5-5 micrograms/ml cimetidine, 0.25-2.5 micrograms/ml ranitidine and 0.05-0.5 microgram/ml famotidine), these three antagonists did not have any effect on replicative DNA synthesis either in the presence or absence of insulin plus epidermal growth factor (EGF). However, unexpectedly DNA synthesis stimulated by insulin and EGF was found to be enhanced by 0.05-0.5 mg/ml cimetidine, although it was unaffected or inhibited by ranitidine and famotidine at the concentrations tested. Cimetidine caused maximal enhancement of 1.5-2 times the control level of DNA synthesis at a concentration of 0.25 mg/ml. Cimetidine also had an enhancing effect at submaximal concentrations of insulin and EGF, but neither cimetidine nor the other antagonists had any stimulatory effect on DNA synthesis in the absence of insulin plus EGF. This enhancement of DNA synthesis by cimetidine resulted in significant increase in the total DNA content of the hepatocytes in culture. Under the conditions used, cimetidine had the lowest toxicity of these three antagonists and ranitidine the highest, as judged from data on DNA synthesis and the total protein content of cultured hepatocytes, leakage of aminotransferases from the cells and morphological observations.

Animals↗
PubMed1986

Human hepatocyte growth factor in plasma from patients with fulminant hepatic failure.

Plasma from patients with fulminant hepatic failure obtained during plasma exchange therapy, like their serum, demonstrated marked stimulatory activity on DNA synthesis in cultured rat hepatocytes. Heat treatment at 56 degrees C for 30 min did not affect this activity of the plasma, but reduced that of the serum. This growth-promoting activity was confirmed by showing that the patients' serum and plasma increased the labeling index with [3H]thymidine and the total number of nuclei in hepatocyte cultures. The activity of pooled active fractions obtained by gel filtration of the heated plasma was lost completely on heat treatment at 80 degrees C for 10 min or on treatment with trypsin or chymotrypsin, which suggests that it was due to a protein. The human hepatocyte growth factor was purified about 600-fold from heated plasma of a patient by ammonium sulfate precipitation and chromatographies on Affi-Gel Blue and hydroxylapatite. The maximum effect of this partially purified factor on DNA synthesis in cultured hepatocytes was greater than that of epidermal growth factor. The molecular weight of the hepatocyte growth factor was about 85,000 as determined by SDS-PAGE.

Animals↗
PubMed1986

Degradation of [125I]iodoglucagon by normal rat plasma in radioimmunoassay mixture containing aprotinin and its prevention by p-chloromercuriphenyl sulfonate and leupeptin.

Degradation of [125I]iodoglucagon during RIA of glucagon would result in erroneously high values for immunoreactive glucagon (IRG). Normal rat plasma was found to have high activity for glucagon degradation that was not suppressed by the aprotinin and EDTA routinely added to the RIA system. About 30% of the added radioactive glucagon was degraded during RIA in assay mixture at pH 7.4 containing 0.2 ml normal rat plasma, and the IRG value was calculated to be 150-180 pg/ml plasma. The degradation was completely inhibited by addition of 2 mM p-chloromercuriphenyl sulfonate (PCMS) plus 0.25 mM leupeptin as protease inhibitors, and in their presence the IRG value was about 80 pg/ml. The glucagon-degrading activity was about half as much in assay mixture at pH 8.8 as in that at pH 7.4, but the degradation still affected the accuracy of IRG values. When rat plasma was incubated with [125I]iodoglucagon in the assay conditions used for RIA and then subjected to Bio-Gel P-6 filtration, three new peaks of radioactivity were found in low mol wt fractions, with decrease in the peak corresponding to [125I]iodoglucagon, whereas on similar treatment in the presence of PCMS and leupeptin all the radioactivity was recovered in the glucagon fraction. The average recoveries of authentic glucagon as IRG in the absence and presence of the inhibitors were less than 60% and more than 90%, respectively. These findings indicate that determination of plasma IRG in rats by RIA with assay mixture containing aprotinin gives spuriously high values owing to degradation of the radiotracer, and that PCMS and leupeptin should be added to the sample and assay mixture to prevent this degradation.

4-Chloromercuribenzenesulfonate↗
PubMed1985

Caffeine degradation and increased ochratoxin A production by toxigenic strains of Aspergillus ochraceus isolated from green coffee beans.

The growth and ochratoxin A production of Aspergillus ochraceus strains S-235-100 and IFM 0458, which were isolated from green coffee beans and glutinous rice, respectively, were examined in yeast extract-sucrose (YES) medium containing 0.1 to 1.0% caffeine. The mycelial growth and ochratoxin A formation of strain IFM 0458 was inhibited by caffeine at concentrations over 0.1%, and ochratoxin A was not produced at caffeine levels of 0.5% and 1.0%. Contrary to this, A. ochraceus strain S-235-100 produced a larger amount of ochratoxin A in the presence of 0.5% and 1.0% caffeine when grown on YES medium, reaching a maximum after 15 to 20 days of incubation. The formation of ochratoxin A by nine additional strains of A. ochraceus, three strains of A. elegans and one strain of A. sclerotiorum isolated from green coffee beans was determined on rice and ground green coffee media. A significant degree of degradation of caffeine in the green coffee medium was demonstrated with cultures of nine A. ochraceus isolates from green coffee beans. Most of these isolates showed the potential to grow on moist green coffee beans and to produce a significant amount of ochratoxins.

Aspergillus↗
PubMed1984

Increased activities of liver cathepsins T and D in carbon tetrachloride-treated rats.

Changes in activities of a new proteinase cathepsin T as well as some other lysosomal acid proteinases and hydrolases were examined in liver homogenate from rats treated with a single hepatotoxic dose of carbon tetrachloride. The most striking changes were several-fold increases of liver cathepsin T and D activities over their levels in untreated rats 3 days after administration of the agent to rats. Increase of cathepsin T was greater than that of cathepsin D at all doses of the hepatotoxin examined. The activities of N alpha-benzoyl-DL-arginine 2-naphthylamide hydrolase, acid phosphatase, beta-galactosidase and beta-glucuronidase in poisoned rat liver were unchanged or only slightly increased. Cathepsin T and D activities were less enhanced in mitochondrial lysosomal fractions than in the homogenate, and were greatly elevated in the supernatant fractions of liver from the treated rats. As judged from the molecular weights, the elevated activities of cathepsins T and D in the treated rat liver could be attributable to the two cathepsins themselves and not to other proteinases. Administration to rats of other hepatotoxic agents, thioacetamide and dimethylnitrosamine, also induced the elevation of the two cathepsin activities in liver, but on partial hepatectomy the activities of liver cathepsins T and D did not show such marked increases. Nonparenchymal liver cell fractions were responsible for almost all the increased activities of liver cathepsins T and D. It is possible that cathepsins T and D play a role in the heterolytic breakdown of hepatocyte molecules following CCl4 poisoning.

Animals↗
PubMed1983

Immunoreactive glucagon in the vascular walls of the rat.

We detected immunohistochemically immunoreactive glucagon (IRG) in the smooth muscle of blood vessels and the myoepithelial cell of sweat glands of rats using two antisera against pancreatic glucagon; OAL-123 and 30K. The content of IRG in the blood vessels was found to be 320-1, 270 pg per g wet tissue weight. Filtration of the extracted IRG through a Bio Gel P-30 column yielded a single peak of IRG at 3,500 daltons, the same elution volume of pancreatic glucagon. These findings suggest that the blood vessels of the rats is one of the extrapancreatic sources of IRG in plasma, although physiological role of the IRG is not known.

Animals↗
PubMed1983

Glucagon degradation in fraction of big plasma glucagon in rats treated with carbon tetrachloride.

When whole plasma obtained from rats given carbon tetrachloride was applied to a column of Bio Gel P-30, P-150 or P-300, a large molecular form of immunoreactive glucagon (IRG), so called "big plasma glucagon" (BPG), was eluted in the void volume. Glucagon degrading activity, measured under the same conditions as for radioimmunoassay of IRG, was eluted in parallel with IRG. Since standard assay mixture containing aprotinin was used for the measurement of IRG, this degradation of glucagon might be due to an enzyme(s) other than serine proteinases. p-Hydroxymercuribenzoate depressed glucagon degradation and IRG in the BPG fractions to 20% and 30% of the control values, respectively. Other inhibitors of thiol proteinases, such as N-ethylmaleimide and leupeptin, also decreased the values for both glucagon degradation and IRG to a similar extent. However, phenylmethylsulfonyl fluoride and pepstatin had little or no effect on these values. On filtration of plasma from carbon tetrachloride-treated rats on a column of Bio Gel A-1.5m, IRG peaks between 600k and 1,200k daltons were obtained and glucagon degrading activity was again eluted with IRG. When p-chloromercuriphenyl sulfonate and N-ethylmaleimide were added to the elution buffer and assay mixtures, the glucagon degrading activity was almost completely inhibited and the values for IRG between 600k and 1,200k daltons were decreased to less than 10% of those of the control. These results indicate that more than 90% of the IRG in the fractions of BPG obtained from carbon tetrachloride-treated rats could be accounted for entirely by the degradation of immunoreactive 125I-glucagon by plasma proteinases, probably thiol proteinases. Thus we conclude that some proteinases are still active in the radioimmunoassay system of glucagon which is widely used at present, and lead apparently higher glucagon values owing to degradation of 125I-glucagon.

Animals↗