Biomedical subjects
Publications and source records attributed to R Kato.
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A hamster acetyltransferase, AT-I, has high activities for N-acetylation of arylamines, O-acetylation of N-hydroxyarylamines and N,O-acetyltransfer of N-hydroxyarylacetamides. In the present study, the cDNA was expressed in Salmonella typhimurium TA1538. The new SAT138 strain expressing high levels of AT-I showed remarkably high sensitivity (> 10,000 fold) for a carcinogenic intermediate, N-hydroxy-2-acetylaminofluorene, in an Ames mutagenesis test as compared to the parental TA1538 strain. SAT138 had 650-1,600-fold higher sensitivities for mutagenesis induced by 2-acetylaminofluorene and benzidine in the presence of S9 mix. Higher sensitivities (32-560-fold) were also observed with N-hydroxy-2-aminofluorene, N-hydroxy-4-aminobiphenyl, N-hydroxy-4-acetylaminobiphenyl, N-hydroxy-4-propionylaminobiphenyl and N-hydroxy-phenacetin in the absence of S9 mix. These high sensitivities to arylamines and the related chemicals are accounted for by the efficient expression of AT-I in the cytosol of this bacterium. The unique characteristics of SAT138 having high N-hydroxyarylacetamide N,O-transacetylating activity, which is defective in Salmonella acetyltransferase, provide broadened and high sensitivities for the detection of mutagenic N-substituted chemicals in the Salmonella mutagenesis test.
1. Nicotine, high K+ and maitotoxin caused the inositol polyphosphate accumulation concomitant with 45Ca2+ uptake. 2. Angiotensin II (Ang II) and ATP induced the inositol polyphosphate accumulation without 45Ca2+ uptake. 3. Nifedipine-treatment and Ca(2+)-deprivation inhibited the high K(+)-induced inositol polyphosphate accumulation but failed to inhibit the Ang II-induced inositol polyphosphate accumulation. 4. 12-O-tetradecanoylphorbol-13-acetate inhibited the Ang II-induced inositol polyphosphate accumulation but failed to inhibit the high K(+)-induced one. 5. These results suggest that the formation of inositol polyphosphates may be regulated by two mechanisms, i.e. Ca2+ uptake-dependent mechanisms represented by high K+, and Ca2+ uptake-independent mechanisms represented by Ang II.
The effect of FK506, a potent immunosuppressive agent, on 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13- acetate (TPA)-promoted skin papilloma formation was examined in CD-1 mice. A topical application of FK506 to mouse skin 15 min before each TPA treatment resulted in a dose-related inhibition of tumor formation. FK506 (1 mumol) almost completely inhibited tumor formation. This inhibitory effect of FK506 was not due to any damage inflicted on the initiated cells but due to its anti-tumor-promoting action. A topical application of FK506 also inhibited epidermal ornithine decarboxylase induction and skin inflammation caused by TPA in a dose-related manner. Significant inhibition by FK506 of TPA-induced endogenous protein phosphorylation in intact epidermal cells was not detected. These results indicate that FK506 inhibits TPA-induced tumor promotion at a step distal to the endogenous protein phosphorylation by TPA.
The recA gene of a thermophilic eubacterial strain, Thermus thermophilus (T.th.) HB8, was cloned from a genomic DNA library by Southern hybridization using a gene-internal fragment amplified by the polymerase chain reaction (PCR) method as the probe. The gene encoded a 36 kDa polypeptide whose amino acid sequence showed 61% identity with that of the Escherichia coli RecA protein. Characteristic amino acid changes between the two RecA proteins were found. In the amino acid composition of the T.th. RecA protein, the number of Pro residues was increased, the number of Cys residues was decreased, and Lys residues were replaced by Arg, Asp by Glu, Thr by Val, and Ile by Val or Leu. These changes are supposed to stabilize the native protein conformation against heat denaturation. The amino acid residues in the nucleotide binding site of the protein and in the protein-protein interaction site responsible for the oligomer formation were well conserved. The T.th. recA gene has the ability to complement the ultraviolet light (UV) sensitivity of a E. coli recA deletion mutant. Thus, the thermophilic bacterium has a RecA protein whose function will be common to the E. coli RecA protein.
OBJECTIVE: The present study was designed to investigate the effect of L-arginine administration on patients with essential and secondary hypertension by measuring haemodynamic parameters, neuroendocrine hormones and indicators of nitric oxide (NO) release. DESIGN: Ten patients with essential hypertension and six with secondary hypertension (three with renovascular hypertension and three with primary aldosteronism) were enrolled in the study. METHODS: L-Arginine was administered intravenously to the hypertensive patients. During L-arginine administration, blood pressure, heart rate, cardiac output and neuroendocrine hormones such as catecholamines, plasma renin activity and plasma aldosterone were measured. To examine whether L-arginine administration increases NO production, indicators of NO release in vivo such as plasma cyclic GMP, plasma citrulline and urinary excretion of nitrite and nitrate were measured simultaneously. RESULTS: During administration, mean arterial pressure decreased, heart rate increased, cardiac output increased and total peripheral resistance decreased. The indicators of NO release increased simultaneously during administration. Catecholamine and plasma renin activity, rather than increasing in response to L-arginine-induced hypotension as expected, showed no significant changes except in patients with renovascular hypertension. In all patients plasma aldosterone levels decreased significantly in response to L-arginine administration, regardless of basal plasma renin activity and aldosterone levels. CONCLUSIONS: These results suggest that exogenous L-arginine produces a vasodilatory effect by increasing NO production and that L-arginine, or released NO, modulates the release of neuroendocrine hormones in hypertensive subjects.
Microsomal hydroxylation of tolbutamide in Japanese livers was studied in vitro to ascertain the enzyme catalysing this reaction. Rates of tolbutamide hydroxylation differed individually 33-fold and 42-fold at 0.1 mM and 2.4 mM tolbutamide concentrations, respectively, and were segregated into two groups, rapid and slow metabolizers. An antibody raised against P450 human-2 (a form of CYP2C9) strongly inhibited the hydroxylation in livers of rapid metabolizers but only weakly inhibited in the slow metabolizer. Kinetic experiments further demonstrated a clear distinction in tolbutamide hydroxylation between two groups; the mean of apparent Km values for tolbutamide was 0.25 mM (n = 3) in the rapid group and 2.58 mM (n = 2) in the slow, respectively. These data suggest that different enzymes are involved in the hydroxylation in both metabolizer groups. Furthermore, CYP2C9 produced by cDNA expression in yeasts, catalysed tolbutamide hydroxylation at rates similar to the rapid metabolizer group at both the 0.1 mM and 2.4 mM concentrations. The apparent Km value of the expressed protein for tolbutamide, 0.26 mM, was similar to that determined for the rapid group of microsomal samples. Clear correlations were observed between the rate of microsomal tolbutamide hydroxylation at 0.1 mM and CYP2C9 protein content or the rate of S-mephenytoin 4'-hydroxylation in human liver. These results indicate that considerable portions of microsomal tolbutamide hydroxylation are catalysed by CYP2C9 or the closely related form in the rapid metabolizers.
Metabolism of diazepam (DZP) was studied in vitro to clarify the involvement of different forms of hepatic cytochrome P450 (CYP) in rats, and humans of Japanese and Caucasian origin. Microsomal 3-hydroxylation was the major pathway of DZP metabolism in rats and was inhibited by anti-CYP3A antibodies. Purified CYP3As and CYP2C11 catalysed 3-hydroxylation and N-demethylation, respectively, in the reconstituted systems. The rates of both reactions in human liver microsomes depended on the substrate concentration: the rate of 3-hydroxylation was 3-4 times higher than N-demethylation at 0.2 mM; the two activities were essentially the same at a lower substrate concentration (0.02 mM). Inhibitions of the N-demethylation by anti-CYP2C antibody and S-mephenytoin also depended on the substrate concentration and was detectable only at a low substrate concentration. Kinetic studies revealed the presence of two distinct catalytic activities for the N-demethylation; low Km and low Vmax, and high Km and high Vmax. The former activity seems to be mediated by a CYP2C P450 form. On the other hand, DZP 3-hydroxylation was rather selectively catalysed by a CYP3A P450 at the low and high substrate concentrations. These results were consistent with the observation in vivo that DZP N-demethylation and S-mephenytoin 4'-hydroxylation are closely correlated in humans. These results also suggest that the apparent discrepancy on the role of CYP forms in DZP metabolism in vitro and in vivo may reside in the difference in substrate concentration.
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To study the phosphorylation state of tau in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for tau protein kinase II. Immunoblot experiments using these antisera demonstrated that tau in microtubule-associated proteins is phosphorylated at Ser144 and at Ser315. Almost all tau variants separated on two-dimensional gel electrophoresis were phosphorylated at Ser144 and nearly one-half of them at Ser315. Phosphorylation at Ser144 and at Thr147 of tau isolated from heat-stable brain extracts was shown to be developmentally regulated, with the highest level of phosphorylation found at postnatal week 1. In vitro phosphorylation of tau by tau protein kinase I, a kinase responsible for abnormal phosphorylation of tau found in paired helical filaments of patients with Alzheimer's disease, was enhanced by prior phosphorylation of tau by tau protein kinase II. Thus, we suggest that tau protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of tau in neuronal cells.
A case of epithelial myoepithelial tumour originating from the tracheal gland in a 57 year old woman is described. The tumour was removed by segmental tracheal resection and end-to-end anastomosis. Histologically, the tumour comprised clear cells and presented a monophasic pattern. Immunohistochemical analysis showed that the tumour cells were positive for both S-100 protein and smooth muscle actin. suggesting that this tumour resembles a subtype of epithelial-myoepithelial carcinoma described in the 1990 WHO international classification of salivary glands. Although some reports describe a clear cell dominant epithelial myoepithelial carcinoma, in this case local invasiveness or regional lymphnode metastasis was not proved through investigation. It is therefore concluded that this was an epithelial myoepithelial tumour rather than a carcinoma.
To understand the effects of lipid-lowering agents on the ethanol-induction of hepatic CYP2E1, clofibrate and L-carnitine were administered to adult male rats. The administration of ethanol in the diet (containing 21% calories as ethanol, given for 3 weeks) increased levels of hepatic CYP2E1 protein (1.9-fold that of untreated controls) and mRNA (2.1-fold). In contrast, the administration of clofibrate (0.1%v/v) in an ethanol-containing diet did not significantly increase either CYP2E1 protein (1.1-fold) or mRNA (0.8-fold), in spite of the significant increases in blood ketone bodies. Administration of L-carnitine alone had no clear effect on CYP2E1 and blood ketone body levels. Co-administration of L-carnitine, however, increased liver microsomal CYP2E1 protein (2.5-fold) in rats given an ethanol-containing diet. No difference was observed in the mRNA levels in rats receiving ethanol with and without L-carnitine. These results indicate that clofibrate and L-carnitine modulate ethanol-mediated induction of hepatic CYP2E1 independent of blood levels of ketone bodies. It is also suggested that these lipid-lowering agents affected hepatic CYP2E1 through particular mechanisms, suppression of the specific mRNA and post-translation stabilization.
The efficacy and toxicity of drugs are closely related to the kinetics and metabolism of drugs. The metabolism of drugs is variable depending on individual, age, sex and species differences. Therefore, in this paper, the following strategies of drug metabolism studies on each phase of development of new drugs is discussed: 1. Early selection of drug candidates for the development of new drugs by a preliminary drug metabolism study. 2. Selection of a drug candidate for the repeated administration of drug toxicity study. 3. Drug metabolism studies on sex- and species-related differences in experimental animals. 4. Drug toxicity and metabolic activation of drug candidates. 5. In vitro and in vivo drug metabolism studies in humans. 6. Arrangement of drug metabolism and pharmacokinetic profile of drug candidate for the phase I study. 7. Role of pharmacokinetics and drug metabolism studies in the phase I study. 8. Pharmacokinetic and metabolic studies for the development of chiral drugs. 9. Approach to drug design from the viewpoint of drug metabolism studies. In conclusion, performance of drug metabolism studies starting from the early stage of drug development is a powerful and useful tool to increase the efficiency and to minimize the period and cost for the development of new drugs.
A cDNA (PST-1) isolated from a rat liver cDNA library was expressed in COS-1 cells and found to encode a form of arylsulfotransferase (termed ST1A1), which sulfated p-nitrophenol, alpha-naphthol, minoxidil, beta-estradiol and dopamine, but not dehydroepiandrosterone and cortisol. ST1A1 showed a mobility identical with a major immunodetectable sulfotransferase contained in the livers of male and female rats in Western blots. In addition, ST1A1 mRNA was detected in the liver and extrahepatic tissues by Northern blots.
Regulation of nitric oxide synthase mRNA by interferon-gamma, tumor necrosis factor-alpha, bacterial lipopolysaccharide (LPS) and dexamethasone in rat aortic endothelial cells was examined. The combination of interferon-gamma (100 U/ml) and tumor necrosis factor-alpha (5000 U/ml) evoked a time-dependent increase in nitric oxide synthase mRNA and nitrite/nitrate production, both of which were inhibited by dexamethasone. Neither interferon-gamma (100 U/ml), tumor necrosis factor-alpha (5000 U/ml) nor LPS (100 ng/ml) alone was capable of increasing nitric oxide synthase mRNA and nitrite/nitrate production in these cells. However, combinations of two of the three agents synergistically increased both nitric oxide synthase mRNA and nitrite/nitrate production. When the three agents were applied simultaneously, nitric oxide synthase mRNA and nitrite/nitrate production were both markedly increased. LPS contamination, which may affect the induction of nitric oxide synthase, was below 20 pg/ml in all experiments unless LPS was added exogenously, namely, the effects observed were those of the cytokines themselves. Our results suggest that in endothelial cells, these cytokines regulate the production of nitric oxide at the level of nitric oxide synthase mRNA induction.
The effects of interleukin-1 alpha, tumor necrosis factor-alpha and dexamethasone on the induction of nitric oxide synthase mRNA in rat aortic smooth muscle cells were studied. Neither interleukin-1 alpha (up to 100 U/ml) nor tumor necrosis factor-alpha (up to 5000 U/ml) was capable of inducing nitrite/nitrate production and nitric oxide synthase mRNA in smooth muscle cells. In contrast, treatment for 12 hr or longer with a combination of the two synergistically induced nitrite/nitrate and cyclic GMP production in cell culture media and nitric oxide synthase mRNA, both of which were prevented by dexamethasone. Contamination with bacterial lipopolysaccharide, which may affect the induction of nitric oxide synthase, was below 30 pg/ml in all experiments. Our findings show that dexamethasone and these cytokines regulate the induction of nitric oxide synthase at the mRNA level in vascular smooth muscle cells.
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