An investigation into the binding of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one to DNA in vitro.
After metabolic activation the carcinogen 15,16-dihydro-11-[3H]methylcyclopenta[a]phenanthren-17-one binds to DNA in vitro, and this binding is prevented by 7,8-benzoflavone. Radioactivity cannot be removed from the DNA with organic solvents or by chromatography on Sephadex G-50, even after heat denaturation of the DNA. Enzymatic hydrolysis yields radioactive fractions, which elute from a column of Sephadex LH-20 immediately after the natural nucleosides. At least two species of reactive metabolites are involved in this bending, those with a half-life of a few hr and others with greater stability. After extraction from the aqueous incubation mixture, they could be detected in discrete polar fractions from separations of the complex metabolite mixture by high-pressure liquid chromatography. Their ability to bind to DNA decreased with time at ambient temperature, and they were rapidly deactivated by acid. 7,8-Benzolflavone acted by suppressing the formation of polar metabolites derived from enzymatic oxidation of the aromatic double bonds. The inhibitor had no effect on the enzymes hydroxylating saturated carbon; hence it is unlikely that metabolism of the methyl group is important in conversion of this carcinogen to its proximate form, although the presence of the 11-methyl group is essential for carcinogenic activity in this series. (+info)
The direct spectrophotometric observation of benzo(a)pyrene phenol formation by liver microsomes.
Optical spectral repetitive scan analysis during the oxidative metabolism of benzo(a)pyrene by liver microsomal suspensions reveals the time-dependent formation of an intermediate(s) of which the visible spectra resemble those of several benzo(a)pyrene phenols. Liver microsomes from 3-methylcholanthrene-treated rats showed a greater rate of formation of the phenols than did microsomes from control animals; the rate of formation catalyzed by liver microsomes from phenobarbital-pretreated rats was intermediate. When 3-hydroxybenzo(a)pyrene was used as a standard for comparison of activity, the rates of formation of phenols were compared when measured by fluorometric, spectrophotometric, or high-pressure liquid chromatographic analytical techniques. An epoxide hydrase inhibitor, 1,1,1-trichloropropene-2,3-oxide, enhanced phenol formation regardless of the source of liver microsomes, and 7,8-benzoflavone inhibited control and 3-methylcholanthrene-induced microsomal metabolism of benzo(a)pyrene, 7,8-Benzoflavone did not effect benzo(a)pyrene metabolism by liver microsomes from phenobarbital-pretreated rats. The effect of inhibitors on the spectrophotometric assay correlates well with the results obtained from benzo(a)pyrene metabolite analysis using high-pressure liquid chromatography. (+info)
A novel H2A/H4 nucleosomal histone acetyltransferase in Tetrahymena thermophila.
Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be approximately 80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena. (+info)
Isolation and complete covalent structure of liver microsomal paraoxonase.
Paraoxonase (PON1) is a serum esterase exclusively associated with high-density lipoproteins; it might confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids in oxidized low-density lipoproteins. Here I show that rabbit liver microsomes contain a PON analogue (MsPON) and report the isolation and complete covalent structure of MsPON. In detergent-solubilized microsomes, MsPON co-purifies with the microsomal triacylglycerol transfer protein (MTP) complex. MsPON was separated from the complex and purified to homogeneity under non-denaturing conditions. Automated sequence analysis of intact MsPON and peptides obtained from enzymic and chemical cleavages led to the elucidation of the complete covalent structure of MsPON. The protein is a single polypeptide consisting of 350 residues. The sequence of rabbit liver microsomal MsPON is 60% identical with that of rabbit serum PON1, and 84% identical with the sequence predicted by a human cDNA of unknown function, designated PON3. MsPON has a hydrophobic segment at the N-terminus that might serve to anchor the protein to the microsomal membrane or to the MTP complex. Unlike in the serum enzyme, two potential N-glycan acceptor sites in MsPON are not glycosylated. An absence of N-glycans was also indicated in the rabbit liver MTP. MsPON has a single free cysteine residue at position 38 and a disulphide bond between Cys-279 and Cys-348. The microsomal enzyme lacks three residues at the N-terminus that are present in the serum protein. MsPON lacks four residues at the C-terminus that are present in the rabbit serum protein but absent from human serum PON1. On the basis of the observation that MsPON displays a high degree of similarity with serum PON1, it is proposed that MsPON might have a function related to that of PON1 in serum high-density lipoprotein complexes. (+info)
Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin.
A chimaeric antisense construct was used to reduce the activities of the two major starch-synthase isoforms in potato tubers simultaneously. A range of reductions in total starch-synthase activities were found in the resulting transgenic plants, up to a maximum of 90% inhibition. The reduction in starch-synthase activity had a profound effect on the starch granules, which became extremely distorted in appearance compared with the control lines. Analysis of the starch indicated that the amounts produced in the tubers, and the amylose content of the starch, were not affected by the reduction in activity. In order to understand why the starch granules were distorted, amylopectin was isolated and the constituent chain lengths analysed. This indicated that the amylopectin was very different to that of the control. It contained more chains of fewer than 15 glucose units in length, and fewer of between 15 and 80 glucose units. In addition, the amylopectin contained more very long chains. Amylopectin from plants repressed in just one of the activities of the two starch-synthase isoforms, which we have reported upon previously, were also analysed. Using a technique different to that used previously we show that both isoforms also affect the amylopectin, but in a way that is different to when both isoforms are repressed together. (+info)
Purinogen is not an endogenous substrate used in endothelial cells during substrate deprivation.
Porcine aortic endothelial cells (PAEC) are known to be metabolically robust. They are capable of surviving extended periods of complete lack of exogenous substrate, and purine release has been shown to be significantly up-regulated. The endogenous substrates used during substrate deprivation, as well as the sources responsible for the increased purine release, have not been completely identified. We tested the possibility that a phosphoglyceroyl-ATP-containing polymer, purinogen, might support PAEC hibernation induced by lack of exogenous substrate. This involved isolation of the acid-insoluble fraction of PAEC, which was presumed to contain purinogen, and analysis by HPLC and 31P NMR. No evidence supporting the presence of triphosphate-containing compounds (purinogen) was found. Similar results were obtained in the rat heart. The majority of the products in the acid-insoluble, alkaline-treated fraction were identified as RNA degradation products (2'- and 3'-nucleoside monophosphates). A [14C]adenosine labelling experiment showed that incorporation of adenosine into the acid-insoluble fraction was almost completely prevented after inhibition of RNA synthesis with actinomycin D. Furthermore, RNA isolated from PAEC and subsequently treated with alkali showed a profile that was almost identical with the HPLC profile of the acid-insoluble fraction. Finally, substrate-free incubation of the cells did not quantitatively or qualitatively influence the distribution of acid-insoluble derivatives. We conclude that PAEC survival during the absence of exogenous substrate is not supported by purinogen but rather by some other, yet-to-be-identified, endogenous substrate. (+info)
Accumulation of astaxanthin all-E, 9Z and 13Z geometrical isomers and 3 and 3' RS optical isomers in rainbow trout (Oncorhynchus mykiss) is selective.
Concentrations of all-E-, 9Z- and 13Z- geometrical and (3R,3'R), (3R, 3'S) and (3S,3'S) optical isomers of astaxanthin were determined in rainbow trout liver, gut tissues, kidney, skin and blood plasma to evaluate their body distribution. Two cold-pelleted diets containing predominantly all-E-astaxanthin (36.9 mg/kg astaxanthin, 97% all-E-, 0.4% 9Z-, 1.5% 13Z-astaxanthin, and 1.1% other isomers, respectively) or a mixture of all-E- and Z-astaxanthins (35.4 mg/kg astaxanthin, 64% all-E-, 18.7% 9Z-, 12.3% 13Z-astaxanthin, and 2.0% other isomers, respectively), were fed to duplicate groups of trout for 69 d. Individual E/Z isomers were identified by VIS- and 1H-NMR-spectrometry, and quantified by high-performance liquid chromatography. Significantly higher total carotenoid concentration was observed in plasma of trout fed diets with all-E-astaxanthin (P < 0.05). The relative E/Z-isomer concentrations of plasma, skin and kidney were not significantly different among groups, whereas all-E-astaxanthin was higher in intestinal tissues and 13Z-astaxanthin was lower in liver of trout fed all-E-astaxanthin (P < 0.05). The relative amount of hepatic 13Z-astaxanthin (39-49% of total astaxanthin) was higher than in all other samples (P < 0.05). Synthetic, optically inactive astaxanthin was used in all experiments, and the determined dietary ratio between the 3R,3'R:3R, 3'S (meso):3S,3'S optical isomers was 25.3:49.6:25.1. The distribution of R/S-astaxanthin isomers in feces, blood, liver and fillet was similar to that in the diets. The ratio between (3S,3'S)- and (3R,3'R)-astaxanthin in the skin and posterior kidney was ca. 2:1 and 3:1, respectively, regardless of dietary E/Z-astaxanthin composition. The results show that geometrical and optical isomers of astaxanthin are distributed selectively in different tissues of rainbow trout. (+info)
Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritionally relevant concentrations.
Daidzein and genistein glucuronides (DG and GG), major isoflavone metabolites, may be partly responsible for biological effects of isoflavones, such as estrogen receptor binding and natural killer cell (NK) activation or inhibition. DG and GG were synthesized using 3-methylcholanthrene-induced rat liver microsomes. The Km and Vmax for daidzein and genistein were 9.0 and 7.7 micromol/L, and 0.7 and 1.6 micromol/(mg protein. min), respectively. The absence of ultraviolet absorbance maxima shifts in the presence of sodium acetate confirmed that the synthesized products were 7-O-glucuronides. DG and GG were further purified by a Sephadex LH-20 column. DG and GG competed with the binding of 17beta-(3H) estradiol to estrogen receptors of B6D2F1 mouse uterine cytosol. The concentrations required for 50% displacement of 17beta-(3H) estradiol (CB50) were: 17beta-estradiol, 1.34 nmol/L; diethylstilbestrol, 1.46 nmol/L; daidzein, 1.6 micromol/L; DG, 14.7 micromol/L; genistein, 0.154 micromol/L; GG, 7.27 micromol/L. In human peripheral blood NK cells, genistein at <0.5 micromol/L and DG and GG at 0.1-10 micromol/L enhanced NK cell-mediated K562 cancer cell killing significantly (P < 0.05). At > 0.5 micromol/L, genistein inhibited NK cytotoxicity significantly (P < 0.05). The glucuronides only inhibited NK cytotoxicity at 50 micromol/L. Isoflavones, and especially the isoflavone glucuronides, enhanced activation of NK cells by interleukin-2 (IL-2), additively. At physiological concentrations, DG and GG were weakly estrogenic, and they activated human NK cells in nutritionally relevant concentrations in vitro, probably at a site different from IL-2 action. (+info)