Biomedical subjects
Publications and source records attributed to T Satoh.
We cloned and sequenced a 3296-bp cDNA encoding the rat Ras GTPase-activating protein (GAP). Comparison of the nucleotide (nt) and deduced amino acid (aa) sequences to those of previously described GAP cDNAs revealed greater than 90% identity. Homology is highest between rat and mouse GAP both at the nt (96% identity) and deduced aa levels (98% identity).
Tetrafibricin is a novel nonpeptidic fibrinogen receptor inhibitor isolated from Streptomyces neyagawaensis NR0577. Its competitive and selective fibrinogen receptor blockage was demonstrated in this study. Tetrafibricin competitively inhibited (Ki = 9.9 nM) the binding of biotinylated fibrinogen to purified active glycoprotein (GP) IIb/IIIa immobilized on plastic plate. When RGDS and tetrafibricin were added in combination, the inhibition was additive. The binding of other RGD-containing proteins, fibronectin and von Willebrand factor, to active GPIIb/IIIa were also completely inhibited by tetrafibricin. The fact that tetrafibricin did not inhibit the binding of von Willebrand factor to GPIb/IX indicates the specific blockage of tetrafibricin for GPIIb/IIIa. Fibrinogen receptor inhibition of tetrafibricin was also confirmed by its ability to inhibit 125I-fibrinogen binding to platelets stimulated with ADP. Because of its competitiveness and specificity, tetrafibricin can be used in a new structural model for the design of fibrinogen receptor inhibitors.
Tetrafibricin; a nonpeptidic fibrinogen inhibitor from microbial origin, showed potent antiaggregation activities on human platelet aggregation induced by either ADP, thrombin or collagen (IC50s = 5.6, 7.6 and 11 microM, respectively) in platelet rich plasma. The ability to inhibit aggregation in platelets treated with chymotrypsin confirmed the GPIIb/IIIa blockage of tetrafibricin. Tetrafibricin blocked the release of serotonin induced by ADP but it did not block the release reaction induced by thrombin. When added to platelets formerly aggregated with ADP, tetrafibricin caused rapid and complete deaggregation. As for the selectivity among other Arg-Gly-Asp -dependent integrins, tetrafibricin seems to be more specific for glycoprotein (GP) IIb/IIIa than RGDS is. This is because it had no effect on adhesion of bovine aortic endothelial cells to RGD-containing proteins. Tetrafibricin is the first nonpeptidic fibrinogen receptor inhibitor that may be valuable for the study on platelet aggregation inhibitors.
A novel series of 3,4-dihydroxychalcones was synthesized to evaluate their effects against 5-lipoxygenase and cyclooxygenase. Almost all compounds exhibited potent inhibitory effects on 5-lipoxygenase with antioxidative effects, and some also inhibited cyclooxygenase. The 2',5'-disubstituted 3,4-dihydroxychalcones with hydroxy or alkoxy groups exhibited optimal inhibition of cyclooxygenase. We found that 2',5'-dimethoxy-3,4-dihydroxychalcone (37; HX-0836) inhibited cyclooxygenase to the same degree as flufenamic acid and 5-lipoxygenase, more than quercetin. Finally, these active inhibitors of 5-lipoxygenase inhibited arachidonic acid-induced mouse ear edema more than phenidone.
The inducibility of Glutathione S-transferase (GST) in male Sprague-Dawley rats, treated with phenobarbital (PB), 3-methyl-cholanthrene (MC) and ethoxyquin (ETQ), was examined in detail. The subunit compositions of hepatic and renal GST were determined by using a reverse-phase HPLC technique. In liver, PB was found to induce the Yb1, Yb2, Ya1, Ya2 and Yk subunits by about 2.1-, 1.8-, 1.8-, 4.4- and 2-fold, respectively, while MC induced the Yb2, Yc, Ya2 and Yk subunits by about 1.5-, 1.5-, 6- and 1.7-fold, respectively, and ETQ increased the levels of Yb1, Yb2, Yc, Ya2 and Yk subunits by about 2.1-, 1.7-, 1.9-, 14.9- and 1.8-fold, respectively. In contrast, kidney cytosolic GSTs were induced only by treatment with ETQ and PB and MC had little or no effect. The Pi class subunit Yp in the rat kidney was increased about 4-fold and the Mu class Yb2 was induced by about 2-fold, by the ETQ treatment.
We cloned a human thyrotropin-releasing hormone receptor (TRH-R) gene and its pituitary cDNA, and examined whether TRH-R mRNA may be expressed and function in the GH-secreting pituitary adenomas of patients with acromegaly. The human TRH-R consists of 398 amino acids and is a member of the G protein-coupled receptor family. The gene has a single intron in the coding sequence. An addition of TRH caused an obvious increase in intracellular calcium concentration in chinese hamster ovary cells expressing this receptor. Although highly homologous to the rat and mouse TRH-Rs, the human TRH-R has several differences in the carboxyl-terminal structure. The sequence and functioning analyses showed the same expression of TRH-R mRNA in the pituitary adenomas as that in the normal pituitaries, indicating that 1) some adenomas of patients with acromegaly express an intact and functional TRH-R, and that 2) the paradoxical response of GH secretion to TRH found in the patients might be a direct effect of TRH on TRH-R expressed in the adenoma.
Ras is involved in signal transduction of various factors for growth, differentiation, and oncogenesis. Recent studies have revealed several proteins that function upstream and downstream of the Ras signaling pathway. However, its immediate downstream target molecular has not yet been identified. In an effort to identify the Ras-associated downstream proteins, we added recombinant Ha-Ras in a GTP-bound form to cell-free lysates and used several antibodies against Ras to immunoprecipitate Ras complexes. We found that a serine/threonine kinase, Raf-1, was coimmunoprecipitated with Ha-Ras by two anti-Ras antibodies (LA069 and Y13-238), whereas a neutralizing antibody against Ras (Y13-259) could not precipitate Raf-1. The coimmunoprecipitation was observed with a complex of Ras and guanosine 5'-[gamma- thio]triphosphate but not with a complex of Ras and guanosine 5'-[beta-thio]diphosphate. The GTP-dependent association of Ha-Ras with Raf-1 was observed with lysates of various types of cultured cells, including NIH 3T3, pheochromocytoma (PC) 12, Ba/F3, and Jurkat T cells, and also with crude extracts from rat brain. Furthermore, Raf-1 was precipitated with a transforming Ha-Ras mutant ([Val12]Ras) and wild-type Ha-Ras but not with an effector-region mutant ([Leu35,ARg37]Ras) that lacks transforming activity. These results indicate that Ras.GTP physically associates with Raf either directly or through other component(s) and strongly suggest that Raf functions in close downstream proximity to Ras in mammalian cells.
The argU (dnaY) gene product, a minor tRNA(Arg), from Escherichia coli has the anticodon N*CU with an unidentified modified nucleoside N* in position 34 [Kiesewetter, S., Fisher, W. & Sprinzl, M. (1987) Nucleic Acids Res. 15, 3184]. In the present study, argU tRNA was purified from E. coli A19 strain and nucleoside N* was characterized by the TLC and HPLC analyses. Nucleoside N* was found to be different from any naturally occurring modified nucleosides. From unfractionated E. coli tRNA species, nucleoside N* was prepared in an amount sufficient for 1H-NMR experiments. By the analyses of one-dimensional and two-dimensional NMR spectra, nucleoside N* was suggested to be 5-methylaminomethyluridine (mnm5U), which was confirmed by comparison with a chemically synthesized preparation of mnm5U. Thus, the occurrence of mnm5U in mature tRNA was found for the first time. Further, the modification of U(34) to mnm5U in this tRNA was found to contribute to the strict recognition of two degenerate codons terminating in A and G.
Caco-2 cells grown on membrane filters were used as a model to study the effects of NB-598, an inhibitor of squalene epoxidase, on cholesterol absorption from the intestinal epithelia. NB-598 (10 microM) inhibited the synthesis of sterol and sterol ester from [14C]acetate without affecting the synthesis of other lipids such as phospholipids (PL), free fatty acids (FFA) and triacylglycerol (TG). When labeled lipid was apically loaded as a micellar lipid solution into Caco-2 cell cultures, NB-598 reduced basolaterally secreted radioactivity in cholesterol, cholesterol ester, PL and TG. Furthermore, NB-598 suppressed the basolateral secretion of apolipoprotein (apo) B. When microsomes prepared from control Caco-2 cells were incubated with 10 microM NB-598, acyl CoA:cholesterol acyltransferase (ACAT) activity was inhibited slightly. After incubating Caco-2 cells with 10 microM NB-598, a slight reduction in cellular ACAT activity was also observed. These results suggest that suppression of the secretion of particles containing apo B and reduction of cellular ACAT activity in the intestinal epithelia are part of the mechanism of the cholesterol-lowering effect of NB-598.
Differences in the responses to glucocorticoids and pregnenolone 16 alpha-carbonitrile (PCN) of three isozymes of hepatic microsomal carboxylesterase, namely RL1, RL2 and RH1, in male rats were studied. The administration of dexamethasone dose-dependently increased isocarboxazid hydrolase activity, whereas p-nitrophenyl acetate-hydrolyzing activity was decreased dose-dependently. Betamethasone, methylprednisolone and PCN also markedly increased isocarboxazid hydrolase activity. A radial immunodiffusion assay indicated that carboxylesterase reactive with antibodies was induced by these steroids. Carboxylesterase isozyme RL2 was strongly induced by dexamethasone, methylprednisolone and PCN. In contrast, RL1 and RH1 were decreased by dexamethasone, but not by the other steroids. Estradiol benzoate had a synergic effect on the PCN-induced increase of isocarboxazid hydrolase, but the actions of the glucocorticoids were not affected. It is concluded that hepatic microsomal carboxylesterase isozymes in rats differ considerably from each other in their response to various steroids. These data are also indicative of the importance of glucocorticoids in hepatic xenobiotic metabolism.
NB-598, a specific inhibitor of squalene epoxidase, suppressed the secretion of cholesterol and triacylglycerol from HepG2 cells into the medium. L-654,969, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibited the secretion of cholesterol as potently as NB-598, but did not suppress the secretion of triacylglycerol. Both compounds decreased the intracellular cholesterol content almost equally, and neither of the compounds reduced the intracellular triacylglycerol content. The suppression of lipid secretion by NB-598 was associated with a significant reduction in apolipoprotein (apo) B secretion into the medium. Therefore, the suppression of lipid secretion by NB-598 may be caused by a reduction in the number of triacylglycerol-rich lipoprotein particles. In contrast, the suppression of cholesterol secretion by L-654,969 may be due to a modulation of lipoprotein lipid composition, since this agent did not reduce the secretion of apo B or triacylglycerol. The secretion of apo A-I was unaffected by either NB-598 or L-654,969. Pulse chase studies using [35S]methionine showed that the suppression of apo B secretion by NB-598 depended on an enhancement of intracellular degradation of apo B. These results indicate that the secretion of apo B from HepG2 cells is not regulated by the lipid synthesis alone, and suggest that the mechanism of the hypolipidemic effect of NB-598 involves the suppression of triacylglycerol-rich lipoprotein secretion from the liver as well as an inhibition of cholesterol synthesis in the liver.
We have demonstrated previously that musk xylene, a non-mutagenic carcinogen, is a novel and specific inducer of CYP1A2 in rats (Iwata et al., Biochem Biophys Res Commun 184: 149-153, 1992). In the present study, the effects of musk xylene (50, 100 or 200 mg/kg body weight, i.p., for 5 consecutive days) on both Phase I and Phase II metabolizing enzymes in rat liver were investigated further and more completely. Among the mixed-function oxidases monitored, 7-ethoxycoumarin deethylase and 7-pentoxyresorufin depentylase activities were increased at all dose levels from 1.6- to 1.7-fold and 2.6- to 3.1-fold, respectively. Benzo[a]pyrene hydroxylase activity was increased significantly at only the 200 mg/kg dose level of musk xylene (1.5-fold). Regarding Phase II enzymes, activities of both cytosolic DT-diaphorase and glutathione S-transferase (GST) were increased up to 2.0- to 2.4-fold by musk xylene in a dose-dependent manner. Western blot analysis revealed that the changes in these activities were caused by increases in the amounts of DT-diaphorase and GST Ya subunit. Microsomal UDP-glucoronyltransferase (UDPGT) activity assayed with p-nitrophenol as substrate was increased 1.6- to 2.0-fold. These results show that musk xylene induces both Phase I cytochrome P450 mixed-function oxidase (CYP1A2 specific) and Phase II metabolizing enzyme systems (DT-diaphorase, GST Ya subunit and UDPGT) in rat liver.
Interindividual variability of carbonyl reductase levels in human livers (N = 11) was examined by measuring reductase activity toward various substrates and by western blot analysis using anti-rat ovarian carbonyl reductase CR2 antibody. The carbonyl reductase activity toward p-nitrobenzaldehyde (PNBA) (58.1 +/- 5.4 nmol/mg protein/min, mean +/- SE) was highest among the substrates examined, followed by 4-benzoylpyridine (4BP) (14.4 +/- 2.0 nmol/mg protein/min) and p-nitroacetophenone (PNAP) (2.00 +/- 0.37 nmol/mg protein/min). The reductase activity (6.33 +/- 0.56 pmol/mg protein/min) toward 13,14-dihydro-15-keto-prostaglandin F2 alpha (15KD-PGF2 alpha), which is a diagnostic substrate for rat ovarian carbonyl reductases, was relatively high compared to that in other species. Western blot analysis revealed that each human liver contained several immunoreactive proteins to anti-CR2 antibody. The activities toward 15KD-PGF2 alpha (r = 0.85, P < 0.01) and 4BP (r = 0.84, P < 0.01), but not PNBA (r = 0.53, not significant) or PNAP (r = 0.52, not significant), were closely correlated with the relative amounts of the high molecular weight immunoreactive proteins determined with a densitometer. Thus, the major carbonyl reductases in human liver are similar to those of rat ovarian enzymes.
A specific binding of the cholecystokinin (CCK)-releasing peptide (monitor peptide) to isolated rat jejunal mucosal cells was investigated. The 125I-labelled purified monitor peptide bound to the rat jejunal cells, and a large excess amount of the non-labelled monitor peptide inhibited the binding. The binding was completed within 60 min at 37 degrees C. The optimum pH for the binding was 8-9. A Scatchard plot of the specific binding was linear, and the dissociation constant was 50 nM. The density of the monitor-peptide-binding sites was high in duodenum but low in ileal and absent in colonic mucosa. A recombinant monitor peptide and four kinds of point mutants of it were prepared. The binding of the mutant monitor peptides to the cells indicated that only a trypsin inhibitor of the mutants could bind to the mucosal cells. Human pancreatic secretory trypsin inhibitor inhibited the specific binding, but other trypsin inhibitors, i.e. bovine basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, egg-white trypsin inhibitor, leupeptin, antipain and FOY-305, did not affect the specific binding at all. These findings suggested that the specific binding site for the monitor peptide on the jejunal mucosal cells has a trypsin-like specificity, exhibiting a special specificity for the pancreatic-secretory-trypsin-inhibitor family. Autoradiography of an affinity-cross-linked complex of the 125I-labelled intact monitor peptide and the binding site suggested that its molecular mass was 33 kDa or 53 kDa in the presence or absence of 2-mercaptoethanol respectively.
We have investigated the nuclear transport of U1 and U5 snRNPs by microinjection studies in oocytes from Xenopus laevis using snRNP particles prepared by reconstitution in vitro. Competition studies with snRNPs showed that the Sm core domain of U1 snRNPs contains a nuclear location signal that acts independently of the m3G cap. The transport of U1 snRNP can be blocked by saturation with competitor U1 snRNPs or by U5 snRNPs, which indicates that the signals on the respective Sm core domains interact with the same transport receptors. Further, by using a minimal U1 snRNP particle reconstituted in vitro and containing only the Sm core RNP domain and lacking stem-loops I to III of U1 RNA, we show that this is targeted actively to the nucleus, in spite of the absence of the m3G cap. This indicates that under certain conditions the NLS in the Sm core domain not only is an essential, but may also be a sufficient condition for nuclear targeting. We propose that the RNA structure of a given snRNP particle determines at least in part whether the particle's m3G cap is required for nuclear transport or can be dispensed with.
Malignancy-associated hypercalcemia is mainly caused by excessive production of parathyroid hormone-related protein (PTHrP) by the tumor. Using anti-PTHrP-(1-34) monoclonal murine antibody (anti-PTHrP MoAb), we studied whether repeated injection of the homologous antibody would continuously decrease the serum calcium concentration in hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors, leading to prolongation of their survival time. Daily SC injections of anti-PTHrP MoAb decreased the serum calcium concentration almost to within the normal range in nude mice bearing transplanted human PTHrP-producing tumors (T3M-1, EC-GI, PC-3, and FA-6) but not in a nude mouse bearing a transplanted parathyroid carcinoma. The antibody did not affect FA-6 tumor growth either in vitro or in vivo. Pancreatic carcinoma cells (FA-6), which caused the most severe hypercalcemia, were inoculated into 6-week-old nude mice. When severe hypercalcemia (approximately 19 mg/dl) had developed, daily SC injection of anti-PTHrP MoAb was started. Within 18 days of this time point, all untreated tumor-bearing mice (n = 10) died of hypercalcemia and cachexia, whereas all the treated mice (n = 10) showed an increase in body weight and survived for at least 25 days. Histologic examination of the treated mice revealed a marked decrease in osteoclastic bone resorption, without toxicologic findings in the kidney and liver. These results suggest that passive immunization against PTHrP can continuously ameliorate the hypercalcemia and markedly prolong the survival time of severely hypercalcemic, tumor-bearing mice. If a human monoclonal antibody against PTHrP-(1-34) could be developed, then passive immunization would be potentially one of the most effective therapies for patients with malignancy-associated hypercalcemia due to excessive production of PTHrP.
A factory survey on dose-response relationship in toluene toxicity was conducted in 1985-1989 in four cities in China. The examination items consisted of personal diffusive sampling for TWA exposure measurement, questionnaires on subjective symptoms, hematology and serum biochemistry, and clinical examination including simple neurology tests. Hippuric acid was also determined in urine samples collected at the end of the shift. With selection criteria that (1) complete results were available on all study items and (2) valid toluene exposure data (i.e., toluene shared 90% or more of the exposure) were obtained for the exposed, 452 toluene-exposed workers (206 men and 246 women; toluene exposure at 24.7 ppm as GM) and 517 nonexposed controls (246 men and 271 women) were selected. The subjective symptoms increased in close association with the intensity of exposure to toluene; the threshold concentration appeared to exist at 100 ppm in the case of symptoms during work, and it might be at 50-100 ppm when symptoms off work were evaluated. During the work with exposure at higher concentrations, various symptoms possibly related to CNS or local effects (e.g., eyes, nose, and throat) were complained, and dizziness and floating sensations were identified as typical symptoms with significant dose-response relationship. Several symptoms persisted off work, most of which were apparently related but not necessarily limited to CNS effects. Hematology and serum biochemistry were essentially negative.
In order to determine if there are any residual effects of long-term exposure to mercury vapor, neurobehavioral tests were given to ex-mercury miners about 18 years after the cessation of mercury exposure. Seventy-six male ex-mercury miners who had been exposed to relatively high concentrations of mercury vapor (over 1.0 mg/m3) and with a past history of mercury intoxication were compared to age (+/- 3 years)-, sex-, and years of education-matched controls. Although the extent of the workers' symptoms caused by mercury poisoning, termed erethismus merculialis, markedly decreased after the cessation of exposure, the prevalence of neurological symptoms (such as hand tremors, headaches, and slurred speech) and symptoms of senility (such as low-back pain, loss of sexual desire) in the ex-miners was significantly higher than those in the controls. Matched-pair analysis showed that performances of motor coordination, Simple reaction time, and Short-term memory in the ex-miners were significantly deteriorated compared to those of controls. There are slight but persistent effects on neurobehavioral function, especially on motor coordination function, among mercury miners more than 10 years after the cessation of exposure.