Extensive assignments of resonances in the 600 MHz 1H-NMR spectra of cerebrospinal fluid are reported. These have been achieved by the measurement of a combination of two-dimensional experiments ...comprising homonuclear J-resolved, COSY45, and double-quantum filtered COSY (DQCOSY) spectra. By these means the previous total of 18 endogenous metabolites, of which in general only selected resonances have been assigned, has been augmented to 46 molecules including all of the resonances of both alpha- and beta-anomers of glucose. With only a few exceptions all resonances have been assigned for all of the metabolites. In addition, the effect of freeze-drying on the 600 MHz 1H-NMR spectrum of human cerebrospinal fluid (CSF) is presented using both lyophilization with reconstitution into either H2O or D2O. Freeze-drying and reconstitution into H2O causes a significant sharpening of many small molecule resonances, including notably those of glutamate and glutamine as well as other amino acids and in addition causes the loss of volatile components, principally acetone. Further exchange of the H2O solvent by D2O causes no additional changes in the spectra.
This study has investigated the interactions of a number of methyl-enedioxyphenyl compounds with hepatic cytochrome P-450. The administration of isosafrole to rats resuited in the formation of an in ...vivo isosafrole metabolite-cytochrome P-450 complex which caused inhibition of the hepatic monooxygenase activities. In the oxidized state this in vivo metabolite complex readily dissociated in vitro following the addition of certain lipophilic Type I binding compounds, many of which were substrates for the monooxygenase. The time-dependent dissociation of the in vivo metabolite complex resulted in an apparent increase in the cytochrome P-450-mediated monooxygenase activities. The enzyme activities which increased following displacement of the complex were those generally associated with induction by 3-methylcholanthrene. Induction by isosafrole resulted in an increase in the monooxygenase activities of hepatic microsomes from both C57BL/10 and DBA/2 mice, strains which are respectively "responsive" and "non-responsive" to induction by polycyclic aromatic hydrocarbons. The monooxygenase activities which showed the largest increases in activity following dissociation of the isosafrole metabolite-cytochrome P-450 complex were those which were generally increased by 3-methylcholanthrene induction. The induction profile of the hepatic microsomes from both C57BL/10 and DBA/2 mice was compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Isosafrole induced a pattern of proteins similar to those induced by both phenobarbitone and 3-methylcholanthrene. Spectral investigations in isolated hepatocytes demonstrated Type I and Type II binding spectra but not Type RI spectra. The addition of methylenedioxyphenyl compounds to a suspension of isolated hepatocytes in vitro resulted in the formation of absorption maxima at 427 nm and 460 nm although the 460 nm peak was small. However, the rate of formation of the 427 nm and 455 nm absorption maxima during oxidative metabolism of isosafrole in hepatic microsomes was shown to be pH-dependent, with formation of the 427 nm maximum favoured at low pH whilst a higher pH favoured formation of the 455 nm maximum. Isosafrole-related material was also found to be apparently irreversibly bound to the cellular components of isolated rat hepatocytes incubated 14 with o - 14C isosafrole in vitro. This binding was increased by prior induction of the hepatic monooxygenase with phenobarbitone, 3-methylcholanthrene and isosafrole.
An extensive set of computed molecular properties, both steric and electronic, have been calculated using molecular orbital and empirical methods for benzoic acid (1) and a congeneric series of ...substituted benzoic acids, i.e. 2-, 3- and 4-fluorobenzoic acids (2-4), 2-, 3- and 4-trifluoromethyl benzoic acids (5-7), 2-, 3- and 4-methylbenzoic acids (8-10), 4-amino benzoic acid (11), 2-fluoro-4-trifluoromethyl benzoic acid (12), 4-fluoro-2-trifluoromethyl benzoic acid (13), 3-trifluoromethyl-4-fluorobenzoic acid (14). We have monitored the urinary excretion profiles and determined the metabolic fate of compounds 2-7, 12-14 in the rat using high resolution 1H and 19F NMR spectroscopy. Corresponding data for compounds 1,8-11 are taken from the literature. In all cases phase II glucuronidation or glycine conjugation reactions dominated the metabolism of these compounds. Compounds 5, 7, 12, 13 have ester glucuronides as their major metabolites; the rest primarily form glycine conjugates. Compounds (1-12) have been classified according to their calculated physicochemical properties using pattern recognition methods and principal components maps have been used as a novel type of structure-metabolism diagram. The maps of compounds in the physicochemical property space served to separate the compounds into the two major classes which related to their principal metabolic fate in vivo, namely glucuronidation versus glycine conjugation. Compounds 13 and 14 were used as further probes of the property space, and dominant metabolic fates of glucuronidation and glycine conjugation, respectively, were predicted from the previous "training set map". The metabolic fate of compounds 1-14 can thus be classified according to a simple set of physicochemical rules. Investigation of the physicochemical properties which are important in distinguishing the metabolic fate of the compounds may give insight into key features of the drug-metabolizing enzyme active sites and hence provide information on basic mechanisms of benzoate metabolism.
The assignment of the
1H,
19F, and
13C NMR chemical shifts and coupling constants of 2-deoxy-2-fluoro-
d-ribose, an important intermediate in the synthesis of antiviral nucleoside drugs, is reported ...and the NMR spectra are used to determine the proportions of the pyranose and furanose forms together with the anomeric ratios in acetone-
d
6 solution. The β-pyranose isomer is shown to exist at equilibrium with both
4
C
1 and
1
C
4 conformations in approximately equal proportions in fast exchange. The α-pyranose isomer at equilibrium is predominantly in the
4
C
1 form but the
1C
4 conformer is also present in solution, the two forms being in intermediate exchange on the
19F NMR timescale but in fast exchange on the
1H and
13C NMR timescales. For both the pyranose and furanose forms, the β-anomer predominates. The results are similar to those for
d-ribose.
This study has investigated the interactions of a number of methyl-enedioxyphenyl compounds with hepatic cytochrome P-450. The administration of isosafrole to rats resuited in the formation of an in ...vivo isosafrole metabolite-cytochrome P-450 complex which caused inhibition of the hepatic monooxygenase activities. In the oxidized state this in vivo metabolite complex readily dissociated in vitro following the addition of certain lipophilic Type I binding compounds, many of which were substrates for the monooxygenase. The time-dependent dissociation of the in vivo metabolite complex resulted in an apparent increase in the cytochrome P-450-mediated monooxygenase activities. The enzyme activities which increased following displacement of the complex were those generally associated with induction by 3-methylcholanthrene. Induction by isosafrole resulted in an increase in the monooxygenase activities of hepatic microsomes from both C57BL/10 and DBA/2 mice, strains which are respectively "responsive" and "non-responsive" to induction by polycyclic aromatic hydrocarbons. The monooxygenase activities which showed the largest increases in activity following dissociation of the isosafrole metabolite-cytochrome P-450 complex were those which were generally increased by 3-methylcholanthrene induction. The induction profile of the hepatic microsomes from both C57BL/10 and DBA/2 mice was compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Isosafrole induced a pattern of proteins similar to those induced by both phenobarbitone and 3-methylcholanthrene. Spectral investigations in isolated hepatocytes demonstrated Type I and Type II binding spectra but not Type RI spectra. The addition of methylenedioxyphenyl compounds to a suspension of isolated hepatocytes in vitro resulted in the formation of absorption maxima at 427 nm and 460 nm although the 460 nm peak was small. However, the rate of formation of the 427 nm and 455 nm absorption maxima during oxidative metabolism of isosafrole in hepatic microsomes was shown to be pH-dependent, with formation of the 427 nm maximum favoured at low pH whilst a higher pH favoured formation of the 455 nm maximum. Isosafrole-related material was also found to be apparently irreversibly bound to the cellular components of isolated rat hepatocytes incubated 14 with ω - 14C isosafrole in vitro. This binding was increased by prior induction of the hepatic monooxygenase with phenobarbitone, 3-methylcholanthrene and isosafrole.
Cytochrome P450scc metabolizes vitamin D3 to 20-hydroxyvitamin D3 (20(OH)D3) and 20,23(OH)(2)D3, as well as 1-hydroxyvitamin D3 to 1alpha,20-dihydroxyvitamin D3 (1,20(OH)(2)D3). It also cleaves the ...side chain of 7-dehydrocholesterol producing 7-dehydropregnenolone (7DHP), which can be transformed to 20(OH)7DHP. UVB induces transformation of the steroidal 5,7-dienes to pregnacalciferol (pD) and a lumisterol-like compounds (pL).
To define the biological significance of these P450scc-initiated pathways, we tested the effects of their 5,7-diene precursors and secosteroidal products on leukemia cell differentiation and proliferation in comparison to 1alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D3). These secosteroids inhibited proliferation and induced erythroid differentiation of K562 human chronic myeloid and MEL mouse leukemia cells with 20(OH)D3 and 20,23(OH)(2)D3 being either equipotent or slightly less potent than 1,25(OH)(2)D3, while 1,20(OH)(2)D3, pD and pL compounds were slightly or moderately less potent. The compounds also inhibited proliferation and induced monocytic differentiation of HL-60 promyelocytic and U937 promonocytic human leukemia cells. Among them 1,25(OH)(2)D3 was the most potent, 20(OH)D3, 20,23(OH)(2)D3 and 1,20(OH)(2)D3 were less active, and pD and pL compounds were the least potent. Since it had been previously proven that secosteroids without the side chain (pD) have no effect on systemic calcium levels we performed additional testing in rats and found that 20(OH)D3 had no calcemic activity at concentration as high as 1 microg/kg, whereas, 1,20(OH)(2)D3 was slightly to moderately calcemic and 1,25(OH)(2)D3 had strong calcemic activity.
We identified novel secosteroids that are excellent candidates for anti-leukemia therapy with 20(OH)D3 deserving special attention because of its relatively high potency and lack of calcemic activity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We have successfully achieved selective gene expression in human nasopharyngeal carcinoma (NPC) by exploiting the presence of the Epstein–Barr virus (EBV), utilizing a transcriptional targeting ...strategy (J. H. Li et al., 2002, Cancer Res.62: 171). Building on this platform, we have generated a novel ΔE1 adenoviral vector mediating the expression of a mutant noncleavable form of the FasL gene (HUGO-approved symbol TNFSF6) (ad5oriP.ncFasL). We observe that this therapy induces significant cytotoxicity in the EBV-positive NPC cell line C666-1, mediated by the induction of caspase-dependent apoptosis. The addition of ionizing radiation therapy (RT) causes additional cytotoxicity. Ex vivo infection of C666-1 cells with adv.oriP.ncFasL completely prevents tumor formation in SCID mice followed for up to 100 days. The combination of intratumoral adv.oriP.ncFasL with RT causes regression of established nasopharyngeal xenograft tumors for 2 weeks' duration. Systemic delivery of this targeted strategy achieves 50-fold higher gene expression in nasopharyngeal tumors than in normal organs. Intravenously injected adv.oriP.ncFasL results in mild perturbation of liver function that returns to normal 2 weeks after initial therapy. These results demonstrate the efficacy of our EBV-specific targeting strategy, which allows the potentially safe and effective utilization of a highly potent membrane-based apoptotic gene.
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