We investigated an in vitro metabolic test using rat liver biopsy samples by TLC-autoradioluminography (ARLG), with a view to developing a method to rapidly assess the drug metabolizing activities of individual patients.Drug metabolizing activity was measured in liver biopsy samples collected from rats in four groups : a female control group, male control group, phenobarbital (PB)-administered male group and cimetidine (CM)-administered male group.The productivity of metabolites of 7-ethoxycoumarin (7-EC), debrisoquine (DB) and diazepam (DZ), respectively, was lower in the female control group than in the male control group, but there were no differences in the productivity of metabolites of 5-fluorouracil (5-FU) and tolbutamide (TB), respectively, between the male and female control groups. Those of 7-EC, TB and DZ were higher in the PB-administered group than in the male control group, but those of DB did not differ between these two groups. Those of 5-FU, 7-EC, TB, DB and DZ were lower in the CM-administered group than in the male control group.Using TLC-ARLG, we could detect drug metabolites in rat liver biopsy samples in a relatively short time span at low concentrations similar to those in vivo. We could also measure drug metabolizing activity in cases with and without the involvement of cytochrome P450. When applied in clinical metabolic tests, TLC-ARLG is expected to be useful for assessing the drug metabolizing activities of patients.
Cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) was photoaffinity-labeled with a specific ligand, 3β, 5α-dihydroxycholestan-6-one (YS-64). Analysis by ODS-HPLC of peptide fragments obtained from the labeled CN-TPBP by trypsinization indicated the existence of a single specifically labeled site. Affinity gels for the purification of CN-TPBP were then prepared. As ligands for the affinity gels, 12-O-tetradecanoylphorbol 13-acetate (TPA), a typical phorbol-type tumor promoter, and benzolactam-V8-310 (BL-V8-310), a structural/biological mimic of teleocidin-class tumor promoters, were adopted. The use of these gels afforded a protein that showed a single band of 58 kDa on SDS-PAGE.
MSSP (c-myc gene single strand binding proteins) were identified as protein factors binding to a putative replication origin/transcriptional enhancer sequence present upstream from the human c-myc gene, and two cDNAs encoding highly homologous proteins, MSSP-1 and MSSP-2, have been cloned. Scr2, independently cloned as a factor which complements the cdc2 defective mutant of Schizosaccharomyces pombe, has turned out to be identical to MSSP-1. MSSP-1/Scr2 and MSSP-2 similarly stimulated the initiation of SV40 DNA replication, and thus were suggested to be involved in regulation of cell cycle movement, especially from the G1 to S phase. Here, we examined the functions of MSSP in apoptosis. MSSP expression plasmids were transfected to human HeLa cells together with a β-galactosidase expression vector. After incubation in the presence of 2% calf serum, cells were stained with X-gal and morphologically apoptotic cells among the β-galactosidase-positive cells were counted. Both MSSP-1 and 2 induced apoptosis in a dose-dependent manner as in the control experiments with c-myc or adenovirus E1A. DNA fragmentation, a hallmark of apoptosis, was also observed in cells transfected with MSSP expression plasmids. The results of experiments using various deletion mutants of MSSP indicated that the region containing one of the two RNP consensus motifs, RNP1-B, is required for induction of apoptosis as well as specific DNA binding activity.
15-Hydroperoxyeicosatetraenoic acid (15-HPETE), an arachidonate lipoxygenase product, is reported to induce severe endothelial injury. In this study, we examined the effect of 15-HPETE on the release of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) from cultured human umbilical vein endothelial cells (HUVEC). The addition of 15-HIPETE to the serum-free medium reduced the release of t-PA antigen from HUVEC, while the release of PAI-1 antigen was significantly enhanced. However, treatment of the cultured HUVEC with α-tocopherol or nordihyroguaiaretic acid completely suppressed the 15-HPETE-induced change in t-PA and PAI-1 antigen release. 15-Hydroxyeicosatetraenoic acid (15-HETE) had no effect on the release of either antigen from cultured HUVEC. The HUVEC surfaces exposed to 15-HPETE decreased the potency for binding antithrombin III. In a reconstituted system with heparin and phosphatidylcholine, 15-HPETE decreased the ability of heparin to inactivate thrombin activity. These results suggest that the fibrinolytic factor release and the antithrombin binding of vascular endothelial cells are impaired by the attack of 15-HPETE, and that the presence of antioxidants prevents the injurious action of lipid hydroperoxide.
Two different forms of oxytocinase (L-cystine aminopeptidase, CAP; EC 220.127.116.11) were purified from the 9000 g and 105000 g precipitate fractions of human placenta homogenate by sequential chromatography on columns of hydroxyapatite, DE-32, nickel ion affinity, and Sephadex G-200. One species (CAP-I) purified from the mitochondrial/lysosomal fraction migrated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with an apparent molecular mass of 61 kDa; the other (CAP-II) from the microsomal fraction was composed of two subunits with molecular masses of 56 and 40 kDa. The molecular masses of CAP-I and CAP-II estimated by gel filtration were 64 and 97 kDa, respectively. The specific activities of the two species for S-benzyl-L-cysteine p-nitroanilide increased by 357- (for CAP-I) and 139-fold (for CAP-II) compared with the starting preparations. The optimal pH values toward the artificial substrate were approx. 7.4-8.0 for CAP-I and 6.8-8.0 for CAP-II. The Km and Vmax values toward oxytocin were 5.6 μM and 23.4 μmol/h/mg protein for CAP-I, and 38μM and 15.6 μmol/h/mg protein for CAP-II. Both enzymes were inhibited by the metal-chelating agents, EDTA and o-phenanthroline, whereas they were specifically activated by addition of Co2+ : CAP-I was more sensitive to these reagents than CAP-II. L-Methionine strongly inhibited CAP-I, while CAP-II activity was only slightly affected. CAP-II was more sensitive to amastatin than CAP-I. Thus, the two enzymes are quite distinct in their molecular nature and biochemical properties. They may play a regulatory role in the metabolism of oxytocin and other biologically active peptides in intact placenta.
Bleomycin (BLM) cytotoxicity was greatly enhanced by vortex-stirring BLM and mammalian cells for a few seconds together with 1 to 10 μg/ml high molecular weight polyacrylic acid (A-119). When suspensions of murine leukemia P388 cells were injeced together with BLM and A-119 into the tail vein of CDF1 mice, cell viability was reduced to 1/1000, the reduction being similar to that obtained by vortex-stirring the cells with BLM and A-119 in vitro. This was corroborated by an increase in the survival time of these mice. The reduction in cell viability was noted only when the cells, BLM, and A-119 were simultaneously injected. There was absolutely no effect when there was a time-lag between cell inoculation and injection of BLM/A-119. These findings suggest that the conditions created by the blood stream may simulate those of vortex-stirring and that, in both cases, rapid uni-directional movement of cells with high molecular weight polyacrylic acid may affect the plasma membrane facilitating internalization of non-permeant materials into cells.
We investigated the effects of a new antiulcer agent, SWR-215([[(1, 2-dihydro-2-oxo-4-quinolinyl)methyl]thio]-N-[[[(4-(1-piperidinylmethyl)-2-pyridinyl]oxy]-Z-2-butenyl]acetamide), on histamine H2-receptors, gastric acid secretion and various acute experimental gastric lesions. SWR-215 showed unsurmountable histamine H2-antagonism on isolated guinea-pig atrium. In gastric secretion studies, SWR-215 exhibited potent and durable inhibitory effects, and the antisecretory activities were much stronger than that of roxatidine acetate hydrochloride (roxatidine) : 5 times stronger on basal acid secretion in pylorus ligated rats, 11 times stronger on histamine-stimulated acid secretion in acute fistula rats, and 2 times stronger on histamine stimulated acid secretion in Heidenhain-pouch dogs, respectively. In various experimental acute gastric lesion studies, SWR-215 potentially inhibited almost all acute gastric and duodenal lesions compared with roxatidine, especially indomethacin-induced and HCl-ethanol-induced gastric lesions, and the inhibitory effects were exhibited at the same or lower doses than those which caused the antisecretory effect.Furthermore, it was considered that the mucosal protective effect of SWR-215 was probably unrelated to the endogenous prostaglandin system in gastric mucosa.These results suggest that SWR-215 possesses both durable antisecretory and mucosal protective effects, and is expected to be a useful drug for the treatment of patients with peptic ulcers.
Anti-ataxic effects of TA-0910, a novel thyrotropin-releasing hormone analog, in mice of the ataxic mutant mouse strain Rolling mouse Nagoya (RMN) are sustained beyond its 2-week oral administration period (Kinoshita et al., Eur. J. Pharmacol., 274, 65-72, 1995). We examined the concentration of TA-0910 in the central nervous system (CNS) of RMN after repeated administration in an attempt to clarify the mechanism of the sustained effect of the drug. Repeated administration of TA-0910 (3 mg/kg/d, i.p.) for 2 weeks produced a long-lasting ameliorating effect on ataxia in RMN, and this effect was maintained until 3 weeks after drug withdrawal. The concentrations of TA-0910 in the cerebrum and brain stem 24 h after the final administration were twice the concentration observed 24 h after single administration. The cerebellum cencentration of TA-0910 was more than 4 times that observed 24 h after final administration. After repeated administrations, the drug concentrtions in the brain tissues gradually decreased, but the drug was still detectable in the cerebrum and brain stem 3 weeks after withdrawal. However, these concentrations of TA-0910 3 weeks after withdrawal were as low those observed 24 h after single administration when there were no anti-ataxic effects. These observations suggest that the long-lasting ameliorating effect on the ataxia during and after repeated administration of TA-0910 is not ascribable to the drug remaining in the CNS of RMN.
The potent mutagen, 5-fluoroquinoline (5-FQ), and non-mutagenic 3-fluoroquinoline (3-FQ) were tested for hepatocarcinogenicity using a medium-term assay system employing quinoline, a moderately mutagenic hepatocarcinogen, as a reference. F344 male rats were given a single i.p. injection of a submanifestational dose of diethylnitrosamine (DEN, 200 mg/kg). Then, quinoline, 3-FQ, or 5-FQ at two doses (0.1% and 0.05%) was added to their diet for a period of 6 weeks, starting from 2 weeks after the DEN injection. Control groups were administered DEN alone. All rats were subjected to a partial (two-thirds) hepatectomy at the end of week 3 and sacrificed at the end of week 8. The number and areas of GST-P (placental glutathione S-transferase)-positive foci induced in the liver increased significantly as a result of treatment with 0.1% quinoline, and this increase was dramatic with 5-FQ at both doses, whereas no increases were noted with 3-FQ at either dose. Thus, the results of the medium-term carcinogenicity assay predicted that quinoline, a hepatocarcinogen, would be deprived of carcinogenicity by fluorine atom substitution at position 3, and would conversely be endowed with a higher carcinogenic capacity by substitution at position 5. A semi-quantitative relationship was demonstrated between carcinogenic and mutagenic potencies.
The hypoglycemic effect of the rhizomes of Smilax glabra ROXBURGH (Liliaceae) was investigated in normal and KK-Ay mice, one of the animal models of non-insulin dependent diabetes mellitus (NIDDM) with hyperinsulinemia.The methanol extract of rhizomes of Smilax glabra ROXBURGH (SM, 100 mg/kg body weight) reduced the blood glucose of normal mice 4 h after intraperitoneal administration (p<0.05), and also significantly lowered the blood glucose of KK-Ay mice under similar conditions (p<0.001). However, SM did not affect the blood glucose in streptozotocin-induced diabetic mice, one of the animal models of insulin-dependent diabetes mellitus (IDDM) with hypoinsulinemia. SM also suppressed epinephrine-induced hyperglycemia in mice. SM-treated KK-Ay mice significantly decreased the blood glucose in an insulin tolerance test. We concluded that the hypoglycemic effect of SM raised insulin sensitivity.
We constructed the co-expression vector, pFK4, in which two cDNAs encoding serine acetyltransferase (SATase) and β-(pyrazol-1-yl)-L-alanine/L-cysteine synthase (β-PA/CSase) from Citrullus vulgaris (watermelon) were over-expressed under the transcriptional control of T7 promoter in Escherichia coli. Accumulation of both SATase and β-PA/CSase in soluble extracts of E. coli was confirmed by immunoblotting. The high enzymatic activities of SATase and L-cysteine synthase (CSase) were detected in cell-free extracts of E. coli carrying pFK4. The activities of the formation of β-PA and L-minosine, plant non-protein amino acids, from O-acetyl-L-serine (OAS) and the precursor heterocyclic compounds, pyrazole and 3, 4-dihydroxypyridine, were also found in the extracts. β-PA was also produced in vivo from L-serine and pyrazole as precursors by E. coli cells transformed with pFK4. β-PA was accumulated mainly in the extra-cellular culture medium. The pronounced accumulation of L-cysteine and L-methionine was observed in the cells transformed with pFK4. Additionally, we also constructed vectors which carried chimeric genes encoding fusion proteins of SATase and β-PA/CSase. However, the fusion proteins tended to form insoluble inclusion bodies and thus to exhibit only weak enzymatic activities. The successful results of pFK4 shows the way to create a new sequential biosynthetic pathway of plant specific amino acids in bacterial cells by means of recombinant DNA technology.
The work described in this paper was designed to evaluate the relevance of in vitro skin penetration studies of peptides across rat skin. The apparent penetration of three peptides, enkephalin, elcatonin and insulin, in the presence of enhancers was not seen in the in vitro method using Franz diffusion cells. However, when a protease inhibitor was mixed in the receptor fluid, the penetration of enkephalin and insulin was observed. Although insulin penetrated in the presence of enhancers, the penetration was extremely small in quantity and the cumulative amount did not increase with time. When the degradation of peptides in the receptor fluid of Franz cell was estimated, these peptides, especially enkephalin and insulin, were rapidly hydrolyzed and were almost completely lost within 3 h in the absence of an inhibitor, while elcatonin was slowly degraded. The addition of protease inhibitors, such as gabexate (20 mM), camostat (20 mM) or bile salt (taurocholate and deoxycholate, 10 mM), to the receptor fluid inhibited the degradation to a considerable extent, with the first-order rate constants decreased to one-tenth compared with the contants without inhibitors. From the inhibitory study using specific inhibitors, it was clarified that enkephalin and elcatonin were mainly hydrolyzed by aminopeptidases, endopeptidases and serine proteases in the viable skin. Consequently, the results obtained from the in vitro penetration studies without inhibitors did not reflect reliable penetration data.Thus, effective protease inhibitor(s) should be used to obtain the data corresponding to the in vivo transdermal experiment. This methodology will provide a means to eliminate the confounding effect of metabolism in permeation experiments.
Enantiospecific acetyl conjugation was examined in the rat liver 105000×g supernatant (cytosol) system using racemic 1-amino-3-(1-naphthyloxy)-2-propanol (NDP), a N-desisopropyl metabolite of propranolol. From the results of chiral separative determination of the samples by HPLC using a Chiralcel OD-R column, more remarkable enantiospecificity was observed in the R(+)-enantiomer on NDP elimination and N-acetyl conjugate (AcNDP) formation.Next, the strength of β-adrenoceptor antagonistic actions and mutagenicities was compared between R(+)- and S(-)-enantiomers of NDP and AcNDP, respectively. In the case of NDP, both enantiomers possessed weak β1-adrenoceptor antagonistic effects on isoproterenol-induced positive inotropic and chronotropic actions in the left and right atria isolated from a guinea pig. These actions of R(+)- and S(-)-NDP were 1700-times and 100-times less potent, respectively, than those of propranolol. β2-Adrenoceptor antagonistic actions of R(+)- and S(-)-NDP in the trachea were 1600-times and 200-times less potent, respectively, than those of propranolol. Enantiospecificity was observed in the β-adrenoceptor antagonistic action of S(-)-NDP, while R(+)-NDP and both enantiomers of AcNDP appeared to be negligible in this action.On the other hand, the mutagenicities of each enantiomer were examined by the Ames method using 13 kinds of Salmonella typhimurium strains. In the case of AcNDP, the numbers of colonies increased according to the substrate concentration only when rat liver 9000×g supernatant fraction (S-9 mixture) was added to the plates containing TA100, YG1029, TA104 and YG3003, and then enantiospecificity was observed in the mutagenicity of S(-)-AcNDP. Thus, the ultimate mutagen might be an active metabolite formed mainly from S(-)-AcNDP.Despite of the addition of rat liver S-9 mixture, R(+)-AcNDP and both enantiomers of NDP did not indicate mutagenicity.
Triacetyl-β-cyclodextrin (TA-β-CyD), a hydrophobic cyclodextrin derivative that is insoluble in water, was used to form a complex with flufenamic acid (FA). Complexes of FA with TA-β-CyD (FA-TA-β-CyD) at various molar ratios (1 : 1, 1 : 2, 1 : 3) were prepared by a kneading method, using ethanol as a solvent. FA-TA-β-CyD complex formation was demonstrated by differential scanning calorimetry and powder X-ray diffractometry. The release rate of FA from the FA-TA-β-CyD complexes was measured in both the Japanese Pharmacopoeia XII 1st fluid pH 1.2 and isotonic phosphate buffer pH 6.8. The release rate of FA from the FA-TA-β-CyD complexes in the isotonic phosphate buffer pH 6.8 was significantly retarded compared to the release rate of FA from the FA-glucose mixture. After 1 h, 100% of the drug was released from the FA-glucose mixture and 10-25% was released from the complexes. When either the powder of the FA-glucose mixture or the FA-TA-β-CyD mixture was administered directly into the intraduodenal lumen in rats, the plasma concentration of FA reached a maximum level within 40 min after administration. On the other hand, when the FA-TA-β-CyD complexes were administered into the intraduodenal lumen, the plasma concentration of FA did not show a sharp peak, but remained at a plateau level (10-18 μg/ml) for 6-8 h. An increased mean residence time of FA following FA-TA-β-CyD complexes administration was observed; however, the AUC0-10 for the FA-TA-β-CyD complexes showed no significant difference from that for the FA-TA-β-CyD mixture. These results indicate that TA-β-CyD may serve as a hydrophobic carrier in sustained-release preparations of FA.The drug-TA-β-CyD complexes may therefore be useful in oral administration to achieve prolonged action and reduced side effects.
Two carboxylesterases with pI 6.0 and 6.2 derived from rat liver microsomes were purified. The two isozymes were remarkably different in substrate specificity, but they had equal enzymatic activity for α-naphthyl acetate and were inhibited equally by phenylmethylsulfonyl fluoride (PMSF) and bis-(4-nitrophenyl) phosphate (BNPP).Carboxylesterases pI 6.0 and 6.2 are identical to the enzymes referred to as hydrolase A and B, respectively, from the results of amino acid sequence analyses. Pranlukast was effectively hydrolyzed by carboxylesterase pI 6.2 but not by the pI 6.0 enzyme, and the difference in the pranlukast metabolism between the human and the rat could be explained by the substrate specificity of carboxylesterase. Furthermore, prodrugs of angiotensin converting enzyme inhibitors were found to be converted to the active drugs after hydrolysis by the carboxylesterases pI 6.0 and 6.2.Carboxylesterases generally catalyze the hydrolysis of ester-type drugs preferentially rather than amide-type drugs.
We have established a method for quantifying serum 16-dehydropregnenolone (3β-hydroxy-5, 16-pregnadien-20-one) sulfate (16-DHP S) by GC-MS. The levels of 16-DHP S at birth were compared in infants grouped as extremely immature (gestational age : 22-27 weeks), pre-term (gestational age : 28-36 weeks) and full-term (gestational age : 37-41 weeks). The average of the serum concentration of 16-DHP S in full-term infants was 0.172±0.104 μmol/l (n=10, mean±S.D.) which was significantly higher than the levels of the extremely immature (0.106±0.054 μmol/l, n=14, p<0.05) and pre-term infants (0.088±0.066 μmol/l, n=33, p<0.01). However, 16-DHP S in sera from normal adults (age 22-73 years, n=40) was not detected.We investigated chronological changes in serum levels of 16-DHP S during the early neonatal period. In extremely immature and pre-term infants, these levels were significantly higher at 2-7 d than those of 16-DHP S at day 0 (p<0.001). The levels at 8-18 d were still significantly higher than those at day 0 (p<0.05), but in full-term infants, these levels did not change at days 0 and 2-7.These results indicate that 16-DHP S is a steroid specific to fetuses and neonates and the involution of the fetal adrenal gland does not affect its serum levels in the early neonatal period.
A synthetic peptide (ANFLVWEIVRKKP) designed from the platelet-derived growth factor (PDGF) B-chain, which is known to act as a PDGF antagonist, induced histamine release from rat peritoneal mast cells. Maximal release by the peptide reached about 50% of the total histamine content in mast cells and half-maximal release occurred at 15 μM. The histamine release induced by the PDGF antagonist was required for the presence of Ca2+ in the medium. Treatment of kinase inhibitors (staurosporine and genistein) with mast cells before exposure to the PDGF antagonist inhibited the release to some extent, while calmodulin antagonists (W-7 and R24571) had little effect. The PDGF antagonist induced the secretion of actin from mast cells concurrently with histamine release, though it had no effect on the distribution of tubulin.These results suggest the possibility that PDGF and its agonists may stimulate and induce exocytosis of peritoneal mast cells.
The present study was undertaken to examine the electrophysiologic effects of nitrous oxide in the dog heart after inducing myocardial infarction, and to compare these with those of other anesthetics. Myocardial infarction was produced by two-stage ligation of the left anterior descending coronary artery in dogs. Seven days after ligation, bipolar electrodes were sutured on the ventricular surface of the infarcted and normal regions for applying electrical stimulation or recording ventricular activation. Ventricular activation time and QT interval on the bipolar electro-cardiogram and PQ interval from the standard limb lead II were measured during atrial pacing. Nitrous oxide 80% did not significantly prolong ventricular activation time, PQ interval or QT interval. However, halothane 1 minimum alveolar concentration (MAC), thiopental 5 and 10 mg/kg and fentanyl 30 μg/kg did prolong ventricular activation time; thiopental and fentanyl prolonged the QT interval. Nitrous oxide did not potentiate the effects of fentanyl. Therefore, electrophysiologic effects of nitrous oxide are much weaker compared with those of thiopental, fentanyl or halothane.
Thyrotropin-releasing hormone (TRH) and its analog, TA-0910, ameliorate the ataxia of the mutant mouse, rolling mouse Nagoya, by metabolic normalization in the ventral tegmental field (VTF). We here investigated the distribution of cerebral TRH receptors in the rolling mouse to clarify the sites of action of these drugs. TRH receptors were widely distributed in multiple brain areas, including in the VTF and the cuneiform nucleus (CnF) which terminates in the VTF. These results suggest that TRH and TA-0910 directly activate the VTF by acting on TRH receptors in the VTF and indirectly activate it through the receptors in the CnF.
We have previously demonstrated that the systemic administration of pilocarpine stimulates striatal acetylcholine (ACh) release in rats using a brain microdialysis technique. In the present study, we investigated whether a nigro-striatal dopaminergic system is involved in the pilocarpine-induced increase in striatal ACh release using dopamine-depleted rats under urethane anesthesia. The application of pilocarpine (0.1-10 mM) via the microdialysis tube increased striatal ACh release in normal rats in a concentration-dependent manner, but it had no effect on the release of glutamate or γ-aminobutyric acid (GABA) from the striatum. The increase in striatal ACh release caused by pilocarpine (1 mM) was enhanced by reserpine and α-methyl-p-tyrosine treatment, which completely depleted dopamine in the striatum. These results suggest that pilocarpine selectively increases striatal ACh release by acting at the striatum, and that the nigro-striatal dopaminergic neurons play an inhibitory role in the pilocarpine-induced ACh release.
Reduction in the blood free carnitine (FC) level as a side effect of sodium valproate (VPA) given epiletic patients was pharmacokinetically studied in connection with changes in the VPA disposition. The serum FC level in patients taking at least one of phenobarbital (PB), phenytoin (PHT) and/or carbamazepine (CBZ) in addition to VPA was significantly lower than that in the controls given only these other anti-epileptic drugs (AEDs). Patients medicated only with VPA also tended to have a lower serum FC level than the controls, although the difference was not significant. Among all the patients taking VPA with or without other AED(s), a significantly positive correlation was observed between the serum FC level and the value of dose and level ratio (L/D) of VPA, indicating that both the serum FC concentration and the L/D value of VPA were remarkably reduced in those patients receiving both medications. These results suggested that reduction in the blood FC level as a side effect of VPA reflected FC deficiency associated with the accelerated degradation of VPA in liver; such a condition appears to result from medication with VPA and other AED(s) which induce(s) enzyme(s) for the VPA metabolism.
The relationship between metabolic chiral inversion and chemical structure of various 4-phenyl-4-oxobutanoic acids (4-OBA), derivatives of anti-rheumatic agent KE-298 [2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid], was investigated in rats. Chiral inversion occurred with the thio-alkyl group, whereas the thio-acyl group played no role in the inversion of 4-OBA. A 2-methylene moiety was required for the inversion. When the 4-carbonyl moiety was removed, chiral inversion was significantly decreased, which provided an affinity for the intramitochondrial medium chain fatty acid CoA ligase. In addition, the distance between the chiral center and the carbonyl moiety was also an important factor for chiral inversion. While a sulfur atom was not indispensable for the chiral inversion, the existence of the sulfur atom influenced its affinity to the long chain fatty acid CoA ligase.
The polyphenolic substance(s) in the hot water extract of Bupleurum chinese (PSF) showed strong mitogenic activity. In this paper, we analyzed PSF by using ESR spectroscopy, and found that i) PSF showed a strong ESR signal on g=2.005 which was similar to the commercially available lignin; ii) Sho-saiko-to, which contains an extract of B. chinense, also showed similar signals on ESR; iii) Powdered B. chinense also showed similar signals on ESR; iii) Powdered B. chinense also showed similar signals on g=2.005. Peroxidase activity, essential for producing polyphenolic substances, was detected in the cold water extract of B. chinense. In addition, the signal intensity of the ESR spectrum of B. chinense was increased after boiling. The data of the ESR spectra of the model reactions using lignin, arginine, proline and maltose also strongly suggested that a certain chemical modification proceeded during the hot water extraction to increase the percentage of the stable free radical. These facts strongly suggested that the mitogenic substance in B. chinense is a polyphenolic substance extracted by hot water, and the structure was modified during the extraction to increase the stable free radical components.
Prostaglandin E2 (PGE2) was converted into prostaglandin B2 (PGB2) by alkaline treatment and quantitated by a novel and specific competitive enzyme immunoassay using an anti-PGB2 antibody and a biotin-PGB2 conjugate as a tracer. This assay was relatively specific for PGE1 and PGE2 (n-6 type); the reactivity for PGE3 (n-3 type) was below 2% of that for n-6 type, and was applicable to the quantitation of PGE2 synthesized in lipopolysaccharide-stimulated peritoneal macrophages from mice fed either a high linoleate or a high α-linolenate diet.
The enhanced effect of urethane anesthesia on the serum creatine kinase (CPK) level following administration of hypolipidemic agents was examined to develop a convenient experimental screening method for drug-induced myopathy. After oral administration of a hypolipidemic agent to rats, 25% urethane solution was infused intravenously at a very low rate using a microinfusion pump. Blood samples were collected 7 h after drug administration and the risk of myopathy was evaluated based on the CPK level. When bezafibrate (BF), simvastatin (SV), or pravastatin (PV) (50-500 mg/kg) was orally administered under urethane infusion, enhanced elevation of the serum CPK level was observed dose dependently for BF and SV, but not for PV. The elevation of serum CPK was much higher with BF than with SV or PV. In addition, when SV or PV (50-500 mg/kg) was coadministered with 50 mg/kg of BF, there was a striking increase in the serum CPK level as compared with the drug alone. Without urethane infusion, no significant elevation in serum CPK level was observed even at a high dose of these hypolipidemic agents. These phenomena suggest that the urethane anesthesia enhanced the elevation of the serum CPK level following administration of hypolipidemic agents. We propose that this method is a simple and speedy screening test for drug-induced myopathy.
The merB-merA-deleted plasmid pMRD141 which contains the intact merT-merP genes of pMRA17 conferred bacterial hypersensitivity not only to Hg2+ but also to C6H5Hg+. The bacterium with pMRD141 took up significantly more C6H5Hg+ than its isogenic strain with the cloning vector BluescriptII. The hypersensitivity to C6H5Hg+ seems to be based on hyperaccumulation of toxic C6H5Hg+ in the absence of detoxifying enzymes encoded by merB and merA. Our results show that bacterial transport of C6H5Hg+ into the cytoplasm is regulated by merT-merP genes.
Using the gene mapping membrane technique, we identified a gene (nemA) that encodes N-ethylmaleimide reductace in Escherichia coli. The open reading frame encodes a polypeptide of 365 amino acids with a molecular mass of 39, 514 Da. The deduced amino acid sequence showed a high degree of homology (87% identical) with the pentaerythritol tetranitrate reductase of Enterobacter cloacae and the morphinone reductase of Pseudomonas putida (52% identical).