Genotoxicity of ptaquiloside, a bracken carcinogen, in the hepatocyte primary culture/DNA-repair test

The genotoxicity of ptaquiloside (PT), recently isolated from bracken fern and shown to be carcinogenic, was examined by means of the hepatocyte primary culture/DNA-repair test. PT elicited clear unscheduled DNA synthesis with a dose-response effect. The result indicates that PT is a genotoxic carcinogen.     

Fractal property of eye movements in schizophrenia

 On the basis of a temporal model of animal behavior we conducted temporal analysis of eye movements in schizophrenic subjects (n=10) and normal controls (n=10). We found a fractal property in schizophrenic subjects, the fixation time of eye movement during reading ambiguous and difficult sentences showing a clear inverse power law distribution. An exponential distribution of a nonfractal nature was found in normal controls. Received: 21 July 1995/Accepted in revised form: 30 April 1996     

Participation of cytochrome P450 in mutagenic activation of the carcinogen 3'-hydroxymethyl-N,N-dimethyl-4-aminoazobenzene and its N-demethylated compounds by rat liver

Mutagenicity of the hepatocarcinogen 3'-hydroxymethyl-N, N-dimethyl-4-aminoazobenzene (3'-CH2OH-DAB) and its N-demethylated compounds was examined. Rat-liver 9000 X g supernatant (S9) fraction was used together with Salmonella typhimurium TA98 or TA100 as a tester strain. The expression of mutagenicity of 3'-CH2OH-DAB, 3'-hydroxymethyl-N-methyl-4-aminoazobenzene (3'-CH2OH-MAB) and 3'-hydroxymethyl-4-aminoazobenzene (3'-CH2OH-AB) required the presence of both microsomes and cytosol as sources of enzymes as well as NADPH as a cofactor. 3'-CH2OH-AB showed positive mutagenicity on both strains in the presence of liver S9 from untreated rats whereas 3'-CH2OH-DAB and 3'-CH2OH-MAB were negative. The treatment of rats with polychlorinated biphenyls (PCB) or 3-methylcholanthrene (3-MC) resulted in a marked increase in the ability of S9 to activate these three compounds, whereas phenobarbital (PB) induction was not effective, except for the activation of 3'-CH2OH-AB. The mutagenic activities of the three compounds in strain TA98 were considerably decreased by adding cytochrome c to the S9 mixture, but the activation reactions were insensitive to 1-(1-naphthyl)-2-thiourea (NTU) and methimazole, high-affinity flavin-containing monooxygenase (FMO) substrates. Metyrapone and 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A, potent inhibitors of cytochrome P450, had no inhibitory effect on the activation of these compounds by S9 from PCB-treated rat livers. In contrast, 7,8-benzoflavone (BF), a specific inhibitor of cytochrome P448, decreased the activities of 3'-CH2OH-DAB and 3'-CH2OH-MAB by 88 and 78%, respectively, but the inhibition was negligible for 3'-CH2OH-AB.     

Induction and inhibition of estradiol hydroxylase activities in MCF-7 human breast cancer cells in culture

The effects of estradiol, progesterone, and tamoxifen on the activity of estradiol 2- and 16 alpha-hydroxylases were studied in human breast cancer cell cultures using a radiometric assay. After 5 days' exposure to these compounds, incubations in the presence of either [2-3H]estradiol or [16 alpha-3H]estradiol as substrate were carried out. In MCF-7 cells, estradiol (10(-8) M), progesterone (10(-6) M) and tamoxifen (10(-6) M) significantly increased 16 alpha-hydroxylase activity (estradiol; 21% progesterone 10% to 32%; tamoxifen 21% to 31%; P less than 0.01). Synergistic effects were observed when the cells were successively exposed to tamoxifen and progesterone. Simultaneous treatment with tamoxifen plus estradiol or estradiol plus progesterone showed no change from estradiol alone. On the other hand, although estradiol had no direct effects on 2-hydroxylase activity, tamoxifen decreased this enzymatic activity significantly at 10(-6) M (23% to 37%). Progesterone acted synergistically to further decrease this reaction. Treatment with only progesterone caused an increase in 2-hydroxylation. In contrast, a subline of MCF-7 cells with low estrogen receptor levels showed only minimal enzyme-hormone responses. Likewise, treatment of the estrogen receptor-negative MDA-MB-231 human breast cancer cell line with these compounds showed no effects on either 2- or 16 alpha-hydroxylase activity. In the progesterone receptor-rich T47D cell line, estradiol decreased both activities while progesterone increased both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of cDNA for batroxobin, a thrombin-like snake venom enzyme

The cloned cDNA for batroxobin has been expressed in E. coli. Batroxobin could only be obtained as intracellular aggregates of fusion proteins, fused with a small peptide. To obtain the mature batroxobin, the recognition sequence for thrombin was inserted between the peptide and the mature batroxobin. This fusion protein accumulated in an insoluble form and could easily be purified. After site-specific cleavage of the fusion protein with thrombin, recombinant batroxobin was isolated by preparative electrophoresis. Batroxobin with enzymatic activity was obtained by the refolding of recombinant batroxobin.     

Synthesis of N-acetylglucosaminides of unconjugated and conjugated bile acids.

3-N-Acetylglucosaminides of unconjugated, glycine- and taurine-conjugated bile acids have been synthesized. Bile acids appropriately protected were condensed with acetochloroglucosamine through the 3 alpha-hydroxyl group by means of the Koenigs-Knorr reaction using cadmium carbonate as a catalyst. Subsequent borohydride reduction and/or alkaline hydrolysis provided desired 3-N-acetylglucosaminides of unconjugated bile acids. Glycine-conjugates were obtained from N-acetylglucosaminides of unconjugated bile acids and ethyl glycinate by the carbodiimide method. The preparation of N-acetylglucosaminides of taurine-conjugates was attained by the Koenigs-Knorr reaction of bile acid p-nitrophenyl esters followed by condensation with taurine. 7-N-Acetylglucosaminides of ursodeoxycholates were prepared in a similar fashion. The convenient synthesis of 3-N-acetylglucosaminides of unconjugated bile acids is also described.     

Presence of 2-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, novel endogenous amines, in parkinsonian and normal human brains

2-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline were identified for the first time as novel endogenous amines in parkinsonian and normal human brains by gas chromatography-mass spectrometry. It is of interest that these tetrahydroisoquinolines are analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which produces Parkinson's disease.     

Stimulatory effect of basic fibroblast growth factor on induction of heat shock protein 27 in osteoblasts: role of protein kinase C

In a previous study we showed that basic fibroblast growth factor (bFGF) stimulates activation of protein kinase C through phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether bFGF stimulates the induction of heat shock protein (HSP) 27, a low-molecular-weight HSP, and HSP70, a high-molecular-weight HSP, in MC3T3-E1 cells and the mechanism behind the induction. bFGF increased the level of HSP27 while having little effect on HSP70 level. bFGF stimulated the accumulation of HSP27 dose-dependently in the range between 1 and 30 ng/ml. bFGF induced an increase in the level of the mRNA for HSP27. The bFGF-stimulated accumulation of HSP27 was reduced by inhibitors of protein kinase C. The bFGF-induced HSP27 accumulation was reduced in protein kinase C-downregulated MC3T3-E1 cells. U-73122, an inhibitor of phospholipase C, and propranolol, a phosphatidic acid phosphohydrolase inhibitor, suppressed the bFGF-stimulated HSP27 accumulation. These results strongly suggest that bFGF stimulates HSP27 induction through protein kinase C activation in osteoblasts.