Search PubMed⌕ Search

Biomedical subjects

H Takikawa

Publications and source records attributed to H Takikawa.

At least 109 records · Page 6Linked to original sources
PubMed1988

Dualistic effects of thyroid hormone on a human hepatoma cell line: inhibition of thyroxine-binding globulin synthesis and stimulation of alpha 1-acid glycoprotein synthesis.

In order to examine the action of thyroid hormone on the secretory proteins of the liver, we investigated the effects of thyroid hormones on the synthesis of T4-binding globulin (TBG), alpha 1-acid glycoprotein (AGP), and albumin in a human hepatoblastoma cell line, Hep G2. Hep G2 cells grown to be confluent in medium with 10% fetal calf serum were further cultured in serum-free medium for 4 days, and followed by treatment with hormones for 2 days changing the medium every 24 h. On day 2 (the second 24 h of hormone treatment), about 30% of TBG accumulation was inhibited by 10(-12) M T3 and 50% was inhibited by 10(-8) M T3, although no change was observed on day 1 (the first 24 h of hormone treatment). This inhibitory effect of T3 closely resembled the effect of T3 on [35S]methionine-labeled TBG synthesis by the cells incubated for 3 h after 42 h of pretreatment. About 30-55% of the newly synthesized [35S]TBG immunoprecipitated with anti-TBG serum was inhibited by 10(-8) M T3. These results showed that thyroid hormone inhibited TBG synthesis in Hep G2 cells. On the other hand, T3 stimulated the accumulation of AGP in the media on day 1 (140% of control by 10(-8) M T3), and the effect increased drastically on day 2 (250% of control by 10(-8) M T3). No effect of T3 on albumin accumulation or total protein synthesis was seen. The concentrations of T4 which had significant effects on TBG and AGP accumulation were 10 and 10(3) times higher than those of T3, respectively. In conclusion, thyroid hormone has dualistic effects on the secretory proteins synthesized by a human hepatoblastoma cell line: physiological concentrations of thyroid hormones decrease the synthesis of TBG, but increase the synthesis of AGP.

Carcinoma, Hepatocellular↗
PubMed1988

Comparison of the binding sites of GSH S-transferases of the Ya- and Yb-subunit classes: effect of glutathione on the binding of bile acids.

We have previously observed that the Ya subunit-containing glutathione (GSH) S-transferases from rat liver exhibit a common high affinity binding site for lithocholic acid, bilirubin, and sulfobromophthalein (BSP) (1984. J. Lipid Res. 25: 1177-1183). Subsequently we found that cholic acid and its amidates bound to a site on the Ya subunit separate for the lithocholic acid/bilirubin site (1986. J. Lipid Res. 27: 955-966). We now have extended this work by showing that amidates of lithocholic acid as well as chenodeoxycholic acid and its amidates competitively displace [14C]lithocholic acid from the Ya subunit. GSH did not inhibit binding of any of the ligands to the high affinity Ya site, but did inhibit binding to the cholic acid site on the Ya subunit. We have also defined the binding sites and effects of GSH on the Yb class of subunits. Lithocholic, chenodeoxycholic, and cholic acids (and amidates) shared a common site on the Yb or Y'b subunit, whereas BSP and bilirubin were bound at a different site. Both the bile acid and organic anion sites on the Yb subunit were inhibited by GSH. The inhibition by GSH in all cases (Ya cholic acid site or Yb bile acid or bilirubin sites) was saturable, of the competitive type, and incomplete at maximal GSH concentrations, suggesting that when GSH binds to its distinct substrate site, it induces a conformational change in the proteins affecting the other binding sites.

Animals↗
PubMed1987

A novel binding site for bile acids on human serum albumin.

Binding sites of bile acids on human serum albumin were studied using various probes: dansylsarcosine (site I probe), 7-anilinocoumarin-4-acetic acid (ACAA, site II probe), 5-dimethylaminonaphthalene-1-sulfonamide (DNSA, site III probe), cis-parinaric acid (probe for fatty acid binding site) and bilirubin. Bile acids competitively inhibited the binding of dansylsarcosine to human serum albumin whereas bile acids enhanced the binding of ACAA, DNSA, cis-parinaric acid and bilirubin. Considering the concentrations of bile acids required to inhibit the binding of dansylsarcosine to human serum albumin, the secondary binding site of bile acids may correspond to site I. Dissociation constants (Kd) of the primary binding sites of lithocholic and chenodeoxycholic acid to human serum albumin were approximately 0.2 and 4 microM, respectively, which was measured by equilibrium dialysis at 37 degrees C. All the bile acids and their sulfates and glucuronides inhibited the binding of chenodeoxycholic acid to human serum albumin. Lithocholic and chenodeoxycholic acid and their sulfates and glucuronides exhibited more inhibition than cholic acid and its conjugates. In conclusion, bile acids may bind to a novel binding site on human serum albumin.

Bile Acids and Salts↗
PubMed1987

Sequence of membrane-associated thyroid hormone binding protein from bovine liver: its identity with protein disulphide isomerase.

The complementary DNAs of the bovine liver membrane-associated 3,5,3'-triiodo-L-thyronine binding protein with 55 k-dalton (T3BP) were cloned and the nucleotide sequences were determined. Monospecific antibodies against T3BP were used to screen a bovine liver cDNA library in lambda gtll. We analyzed the sequences of two cloned T3BP cDNAs. The clones encoded a polypeptide of 510 amino acid residues, including a signal peptide of 20 amino acid. Northern blot analysis of bovine and human RNA showed that the mRNAs encoding T3BP are 2.7 kilobase in length. Amino acid sequence of N-terminal and other three peptides isolated from purified T3BP were found in the predicted amino acid sequence from the cDNA sequence. The predicted amino acid sequence is closely homologous (93%) with that of rat protein disulphide isomerase (EC 5.3.4.1), which catalyzes the isomerization of the protein disulphide bonds and has been shown to play an important role in post-translational regulation. The results suggest that T3BP and protein disulphide isomerase should be the same protein.

Amino Acid Sequence↗
PubMed1987

Subunit heterogeneity of cationic human hepatic glutathione S-transferases.

We have purified the major reduced glutathione (GSH) S-transferases from 3 apparently normal human livers: two obtained at surgery and one at autopsy. Purification was by sequential gel filtration. GSH-affinity chromatography, and chromatofocusing. All three livers exhibited the same two major transferase peaks from chromatofocusing at pH 9.0 and 8.7 (designated C1 and C2, respectively) and several (2-4) minor peaks. Another major form (designated A1) from two livers eluted from chromatofocusing at pH 5.4, whereas the major form from the third liver (designated N1) eluted near neutral (pH 6.8). The transferase from erythrocytes eluted at pH 4.6. Isoelectric focusing revealed that the true pI of A1 was pH 7.1 indicating that C1, C2 and A1 are all cationic. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, C1, C2 and A1 exhibited the same single subunit (25,000) whereas N1 was different (26,000). The erythrocyte enzyme had a smaller subunit (23,500). Urea/SDS-polyacrylamide gel electrophoresis resolved the apparent single subunit of A1, C1 and C2 into two distinct subunits. C1 from all 3 livers was a homodimer of the faster migrating subunit (designated subunit I); C2 was a heterodimer (designated I-II); and A1 was a homodimer of the slower migrating subunit (designated subunit II). Hybridization experiments demonstrated that by mixing C1 and A1 we could produce C2 whereas dissociation and reassociation of the subunits of C2 generated C1 and A1 as well as C2. Rabbit antiserum to C1 recognized C1 and C2, but not A1. Thus, the cationic human hepatic transferases are dimers of two distinct subunits.

Amino Acids↗
PubMed1987

3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.

Rat Y' bile acid binders (33 kD) have been previously recognized as cytosolic bile acid binding proteins (Sugiyama, Y., T. Yamada, and N. Kaplowitz, 1983, J. Biol. Chem., 258:3602-3607). We have now determined that these Y' binders are 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSD), bile acid-metabolizing enzymes. 3 alpha-HSD activity copurified with lithocholic acid-binding activity after sequential gel filtration, chromatofocusing, and affinity chromatography. Three peaks of 3 alpha-HSD activity (I, II, III) were observed in chromatofocusing and all were identified on Western blot by a specific Y' binder antiserum. 3 alpha-HSD-I, the predominant form, was purified and functioned best as a reductase at pH 7.0 with a marked preference for NADPH. Michaelis constant values for mono- and dihydroxy bile acids were 1-2 microM, and cholic acid competitively inhibited the reduction of 3-oxo-cholic acid. Under normal redox conditions, partially purified 3 alpha-HSD-I and freshly isolated hepatocytes catalyzed the rapid reduction of 3-oxo-cholic to cholic acid without formation of isocholic acid, whereas the reverse reaction was negligible. The Y' bile acid binders are therefore 3 alpha-HSD, which preferentially and stereospecifically catalyze the reduction of 3-oxo-bile acids to 3 alpha-hydroxy bile acids.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Cyclical oxidation-reduction of the C3 position on bile acids catalyzed by rat hepatic 3 alpha-hydroxysteroid dehydrogenase. I. Studies with the purified enzyme, isolated rat hepatocytes, and inhibition by indomethacin.

We recently identified that the Y' bile acid binders are 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSD). In the present studies, purified 3 alpha-HSD catalyzed rapid 3H loss from [3 beta-3H, C24-14C]lithocholic and chenodeoxycholic acids without net conversion to 3-oxo bile acids under physiologic pH and redox conditions. [3 beta-3H]Cholic acid was a poor substrate. The Y' fraction of hepatic cytosol was exclusively responsible for this activity and 3H was transferred selectively to NADP+. Time-dependent 3H loss was also seen in isolated hepatocytes. Further hydroxylation products of lithocholic and chenodeoxycholic acids lost 3H at the same rate, whereas 3H loss from lithocholic acid rapidly ceased, which suggests compartmentation of this bile acid in hepatocytes. Indomethacin inhibited 3H loss from bile acids either in incubations with the pure enzyme or in isolated hepatocytes. Indomethacin did not alter the initial uptake rate of bile acids by hepatocytes, but caused a redistribution of unconjugated bile acids into the medium at early time points (2.5 and 5.0 min) and that of conjugated bile acids at later time intervals (30 min). 3H loss from the 3 beta position therefore can be used to probe the interaction between bile acids and cytosolic 3 alpha-HSD in intact cells, and indomethacin is capable of inhibiting this interaction.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Cyclical oxidation-reduction of the C3 position on bile acids catalyzed by 3 alpha-hydroxysteroid dehydrogenase. II. Studies in the prograde and retrograde single-pass, perfused rat liver and inhibition by indomethacin.

[3 beta-3H, 24-14C]Lithocholic, chenodeoxycholic, and cholic acids were administered in tracer bolus doses either prograde or retrograde in the isolated perfused rat liver. Little 3H loss from cholic acid was observed, whereas with the other bile acids, 20-40% of the administered 3H was lost in a single pass from perfusate to bile. Most of the 3H loss occurred rapidly (5 min) and was recovered as [3H]water in perfusate. Excretion of bile acids was delayed with retrograde administration, and 3H loss was more extensive. In both prograde and retrograde studies, indomethacin markedly inhibited the excretion of the bolus of bile acid into bile. Indomethacin inhibited the extraction of glycocholate (50 microM) during steady state perfusion without affecting transport maximum for excretion. At lower glycocholate concentration (5 microM), indomethacin inhibited both extraction and excretion. A greater effect was seen on excretion in the latter case, which suggests that displacement of bile acid from the cytosolic protein lead to redistribution in the hepatocyte as well as reflux into the sinusoid. These data suggest that binding of bile acids to cytosolic 3 alpha-hydroxysteroid dehydrogenases occurs extensively during hepatic transit and is important in mediating the translocation of bile acids from the sinusoidal to canalicular pole of the cell.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Thyroxine-induced molting and gonadal function of laying hens.

Twenty laying hens, 238 days of age, were divided into four groups which received daily intramuscular injections of L-thyroxine (T4) (0, 20, 100, or 500 micrograms/kg body weight per day) for 4 weeks. There was no change in body weight or egg production rate of the control (0 microgram T4) or of 20 and 100-micrograms T4 groups over time. Body weight and egg production of the 500 micrograms T4 group decreased markedly and molting started 10 days after T4 injection. Circulating iodothyronine (T4), triiodothyronine (T3), and reverse triiodothyronine (rT3) of the 500-micrograms T4 group increased markedly (30 to 150 times those of the control group) whereas serum luteinizing hormone and progesterone declined after one week and estradiol after two weeks. We concluded that a large dose of T4 (500 micrograms T4/kg, body weight per day) induced an increase in circulating iodothyronine levels, decreased secretion of gonadotropin and sex steroid hormones, and induced molting.

Animals↗
PubMed1986

Binding of bile acids, oleic acid, and organic anions by rat and human hepatic Z protein.

Binding affinities of purified Z proteins from rat and human liver for bile acids, oleic acid, and organic anions were studied. Purification of Z protein from both rat and human hepatic cytosol was performed by gel filtration, chromatofocusing, and hydroxyapatite chromatography. Both purified proteins showed the same molecular weight (Mr = 14,000) and isoelectric points were 6.9 and 6.5 for rat and human proteins, respectively. Binding studies were performed by the competitive displacement of 1-anilino-8-naphthalene sulfonate. Rat and human Z proteins exhibited similar binding affinities for bile acids, oleic acid, and organic anions. Among various bile acids, both proteins bound monohydroxy bile acids with high affinity and trihydroxy bile acids with low affinity; sulfates were bound with higher and glucuronides with lower affinity than their parent bile acids. In comparison with GSH S-transferases, rat Z protein had lower affinity for bile acids than rat GSH S-transferase B and human Z protein had higher affinity for bile acids than human cationic GSH S-transferase. The role for Z protein in the intracellular binding of bile acids may be particularly important in human liver.

Animals↗
PubMed1986

Purification of a 32.5 kDa monomeric sulfotransferase from rat liver with activity for bile acids and phenolic steroids.

Both bile acid and phenolic steroid sulfotransferase activities in rat liver cytosol have previously been identified in fractions corresponding to apparent molecular masses of 60-70 and 30-35 kDa. We purified the latter activity corresponding to a monomeric protein. Activity for bile acids and phenolic steroids co-eluted on sequential chromatography on Sephadex G-75 sf, Affigel blue, chromatofocusing and hydroxyapatite. The protein was homogeneous on SDS-PAGE (32.5 kDa).

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Glucuronidated and sulfated bile acids in serum of patients with acute hepatitis.

The concentrations of glucuronidated and sulfated bile acids in the serum of 15 patients with acute hepatitis were determined by mass fragmentography. Total serum bile acid levels were 13.79-444.10 mumol/liter, and the percentages of glucuronidated and sulfated bile acids were in the wide ranges of 1.7-33% and 2.4-49%, respectively. In four of the five patients for whom serum bile acids were analyzed serially from the acute to the recovery stages of acute hepatitis, the decrease of the glucuronidated and sulfated bile acids was slower than that of nonglucuronidated, nonsulfated bile acids. Thus, the relative proportion of the glucuronides and sulfates in total bile acids apparently increased during the recovery phase. The mechanism for the relative predominance of bile acid esters in serum during recovery is unknown but might reflect an improved excretion of the nonesterified bile acids into bile after the rapid recovery of intrahepatic cholestasis.

Acute Disease↗
PubMed1986

Superoxide anion generating capacity and lysosomal enzyme activities of Kupffer cells in galactosamine induced hepatitis.

To elucidate the function of the reticuloendothelial system of liver in hepatic injury, we investigated the effect of endotoxins on superoxide anion (O-2) generating capacity and lysosomal enzyme activities of Kupffer cells isolated from rats treated with galactosamine (Gal N), with Gal N supplemented with polymyxin B (Polymyxin B-Gal N), with lipopolysaccharide (LPS) and from control rats. After collagenase digestion of the liver and centrifugation over metrizamide gradient, Kupffer cells were prepared by the dish adherence procedure. O-2 production by the cells was examined as chemiluminescence during phagocytosis of latex particles and beta-glucuronidase activities were analyzed. High titers of endotoxemia were detected in LPS and Gal N rats by limulus test, while a low endotoxemia titer was found in Polymyxin B-Gal N rats. Hepatocyte damage was found in Gal N rats, but little was recognized in LPS and Polymyxin B-Gal N rats. In the latter groups, Kupffer cells, activated by endotoxins, showed the enhancement of chemiluminescence and a release of lysosomal enzyme. Though lysosomal enzyme was released from Kupffer cells in Gal N rats, chemiluminescence was slightly suppressed in spite of the high titer of endotoxemia. These results appear to be related to the consumption of O-2 during liver injury. The functional state of Kupffer cells was thus changed by the grade of endotoxemia and hepatic injury.

Animals↗
PubMed1986

[Combined therapies with medroxyprogesterone acetate (MPA) in DMBA-induced rat mammary cancer].

Sixty-one female SD rats with 7, 12-dimethylbenz(a) anthracene (DMBA)-induced mammary cancer were divided into six groups according to the antitumor agent administered: MPA (20 mg/kg, Im), FT 207 (40 mg/kg, Ip), MPA (20 mg/kg, Im) + FT 207 (40 mg/kg, Ip), E2 priming (3 micrograms/body, Im, three days) + MPA (20 mg/kg, Im), E2 (3 mg/body, Im, three days) + MPA (20 mg/kg, Im) and control. The sizes of the tumors in all DMBA rats were measured at 1, 2, 3 and 4 weeks after the start of the administration, and the antitumor effects were compared among the groups. The antitumor effect of MPA + FT 207 was better than that when they were administered separately. It was shown that chemo-endocrine therapy with MPA was very effective. The antitumor effect was in the order of E2 priming + MPA, E2 + MPA and MPA among these three groups. E2 priming before MPA administration was thought to be very promising. The progesterone receptor (PgR) level tended to increase by E2 priming in DMBA tumors. Furthermore, in the E2 priming + MPA group, the tumors with high PgR levels before therapy tended to be more responsive than those with low PgR levels.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Comparison of the affinities of newly identified human bile acid binder and cationic glutathione S-transferase for bile acids.

The bile acid binding properties of the newly identified bile acid binder (Mr = 36,000) (FEBS Lett. 1984. 177: 31-35) and the major cationic glutathione (GSH) S-transferase (Mr = 50,000) in human liver cytosol were compared. Binding affinities were measured by the competitive displacement by bile acids of 1-anilino-8-naphthalene sulfonate (ANS) bound to the proteins and, in some cases, by direct methods of flow dialysis and equilibrium dialysis. The binding affinities for various bile acids by the human bile acid binder were 2-5 orders of magnitude greater than those by human cationic GSH S-transferase. This suggests an important physiologic role for the former protein in intracellular transfer of bile acids in human liver.

Anilino Naphthalenesulfonates↗
PubMed1986

Binding of bile acids by glutathione S-transferases from rat liver.

Binding of bile acids and their sulfates and glucuronides by purified GSH S-transferases from rat liver was studied by 1-anilino-8-naphthalenesulfonate fluorescence inhibition, flow dialysis, and equilibrium dialysis. In addition, corticosterone and sulfobromophthalein (BSP) binding were studied by equilibrium and flow dialysis. Transferases YaYa and YaYc had comparable affinity for lithocholic (Kd approximately 0.2 microM), glycochenodeoxycholic (Kd approximately to 60 microM), and cholic acid (Kd approximately equal 60 microM), and BSP (Kd approximately 0.09 microM). YaYc had one and YaYa had two high affinity binding sites for these ligands. Transferases containing the Yb subunit had two binding sites for these bile acids, although binding affinity for lithocholic acid (Kd approximately 4 microM) was lower than that of transferases with Ya subunit, and binding affinities for the other bile acids were comparable to the Ya family. Sulfated bile acids were bound with higher affinity and glucuronidated bile acids with lower affinity by YaYa and YaYc than the respective parent bile acids. In the presence of GSH, binding of lithocholate by YaYc was unchanged and binding by YbYb' was inhibited. Conversely, GSH inhibited the binding of cholic acid by YaYc but had less effect on binding by YbYb'. Cholic acid did not inhibit the binding of lithocholic acid by YaYa.

Anilino Naphthalenesulfonates↗
PubMed1985

Serum concentrations of glucuronidated and sulfated bile acids in children with cholestasis.

Serum concentrations of nonglucuronidated-nonsulfated, glucuronidated, and sulfated bile acids in 9 control children and 16 children with cholestasis were quantitated by mass fragmentography. Total bile acid levels in control children were 19.55 +/- 2.78 mumol/liter (mean +/- SEM), and glucuronidated and sulfated bile acids comprised 2.6 +/- 0.5 and 17 +/- 3.1%, respectively. In 9 patients with congenital biliary atrasia, total bile acid levels were 167.34 +/- 11.18 mumole/liter of which 2.1 +/- 0.3% were glucuronidated and 15 +/- 1.4% were sulfated. Lithocholic and 3 beta-hydroxy-5-cholenoic acids, which have hepatotoxic effects, were presented in only small amounts in cholestatic children, and they were almost all glucuronidated or sulfated. The percentages of glucuronidated bile acids in control and cholestatic children were lower than in healthy and cholestatic adults, which may be explained by the lower activity of UDP-glucuronyltransferase in neonatal liver.

Adolescent↗