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Biomedical subjects

Y Z Xu

Publications and source records attributed to Y Z Xu.

At least 19 recordsLinked to original sources
PubMed1992

Modulation of deoxycytidylate deaminase in intact human leukemia cells. Action of 2',2'-difluorodeoxycytidine.

Cellular metabolism studies had demonstrated previously that low cellular concentrations of 2',2'-difluorodeoxycytidine (dFdC) nucleotides are eliminated by deoxycytidylate deaminase (dCMPD), whereas dCMPD activity is inhibited at high cellular dFdC nucleotide levels (Heinemann et al., Cancer Res 52: 533-539, 1992). An assay for measuring dCMPD activity in intact human leukemia cells has now been developed to permit investigations of the interactions of dFdC nucleotides with dCMPD in intact cells in which the regulated nature of this enzyme was not disrupted. Using [14C]dCyd as the substrate, radioactivity that accumulated in dTTP was quantitated after high-pressure liquid chromotography by a radioactive flow detector. The assay was first characterized using either the dCMPD inhibitor tetrahydrodeoxyuridine (H4dUrd) which directly inhibits dCMPD, or thymidine and 5-fluoro-2'-deoxyuridine (FdUrd) which indirectly inhibit and activate dCMPD, respectively, by affecting the cellular dCTP:dTTP value. Measured by this in situ assay, there was a strong correlation between dCMPD activity and dCTP:dTTP levels. Consistent with previous studies using partially purified enzyme, incubation of cells with dFdC resulted in a concentration-dependent inhibition of dCMPD in situ. The mechanism of modulation of dCMPD by dFdC, however, was clearly different from that of thymidine and FdUrd. In addition to the effect of dFdC on cellular dCTP:dTTP, our findings also suggested an additional inhibitory mechanism, possibly a direct interaction between dCMPD and dFdC 5'-triphosphate. Thus, results obtained using this direct assay of dCMPD in intact cells support the hypothesis that dCMPD is inhibited by nucleotides of dFdC.

Antimetabolites, Antineoplastic↗
PubMed1992

Cellular elimination of 2',2'-difluorodeoxycytidine 5'-triphosphate: a mechanism of self-potentiation.

2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and found they were dependent on cellular concentration after incubation of CCRF-CEM cells with dFdC and washing into drug-free medium. When the drug was washed out at low cellular dFdCTP levels (less than 50 microM), dFdCTP elimination was linear (t1/2 = 3.3 h), but it became biphasic at intracellular dFdCTP levels greater than 100 microM. Although the initial elimination rate was similar at all concentrations, at higher concentrations the terminal elimination rate increased with increasing cellular dFdCTP concentration, with a nearly complete inhibition of dFdCTP elimination at 300 microM. The deamination product 2',2'-difluorodeoxyuridine was the predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the major excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine transformed the monophasic dFdCTP degradation seen at low dFdCTP levels into a biphasic process, whereas the deoxycytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP elimination. An in situ assay indicated that dCMP deaminase activity was inhibited in whole cells, an action that was associated with a decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. We conclude that dFdC-induced inhibition of dCMP deaminase resulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity.

Antimetabolites, Antineoplastic↗
PubMed1992

Chromatographic separation of oligodeoxynucleotides with identical length: application to purification of oligomers containing a modified base.

A general procedure is described for separation and purification of oligodeoxynucleotides of identical length but different base composition, in particular, of oligomers containing modified bases such as 4-substituted thymines and 6-substituted guanines, using an anion-exchange column (either Mono Q or NucleoPac). The modified oligomers can be well separated from the analogous oligomers containing unmodified thymine or guanine under the basic conditions of the chromatography. The effects of oligomer length, base composition, and lipophilicity on the separation are discussed. A general rule which can be used for prediction of the order of elution of different oligomers and for estimation of tautomeric form of a modified base in the oligomer is presented.

Anion Exchange Resins↗
PubMed1992

[Transdermal properties of timolol and factors affecting its permeability in vitro].

V-C horizontal diffusion cell has been used to carry out the initial study of timolol across intact and stripped hairless mouse skin and human cadaver skin. Also, we have systematically studied the effect of some factors such as hydration time, medium pH value as well as various transdermal enhancers on the permeability of timolol in vitro. We investigated the determination method of timolol by RP-HPLC using YWG-C18 column with water-acetonitrile-triethylamine (87:13:1) as the mobile phase. The results indicated the feasibility of transdermal absorption of timolol. The permeabilities of timolol raised significantly with properly extending of hydration time, increasing of medium pH and with presence of various kinds of transdermal enhancers. With 10% propylene glycol and 1% azone were used conjunctly, they showed obviously synergistic effects. All these studies contributed theoretically and practically to the further development and utilization of timolol in the form of transdermal patch.

Administration, Cutaneous↗
PubMed1991

Nitrosamine-induced cancer: O4-alkylthymine produces sites of DNA hyperflexibility.

The carcinogenic properties of N-nitroso compounds are associated with their ability to alkylate DNA, in particular to form O6-alkylguanine and O4-alkylthymine. DNA duplexes containing either O6-alkylguanine or O4-alkylthymine were synthesized, and each duplex was ligated to form a set of DNAs of increasing length with the alkylated base out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of the DNA. The DNA contained the sequence 5' CAA 3', which is the 61st codon of the K-ras gene, because this codon is a preferred site of mutation for a number of carcinogens including the methylating carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK). O4-Methylthymine or O4-ethylthymine replaced thymine in either of the two A.T base-pairs of this codon (normally CAA), and O6-methylguanine replaced the guanine in the G.C pair. All the sequences containing O4-alkylthymine exhibited anomalous, slow, gel migration and ligated to form circles of unusually small diameter. In general, the effect was seen when the alkylated base-pair was out of phase with the helical repeat as well as when it was in phase, suggesting that the alkylated base-pair confers flexibility which is largely isotropic, i.e., has no preferred direction, rather than anisotropic flexibility or bending. However, at pH 8.3 the 21-base-pair set containing O4-alkylT.A had significantly greater anomalous migration than the 16-base-pair set, suggesting that the flexibility produced by this base-pair has a significant anisotropic component and thus resembles true bending.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1991

Anoxic block of GABAergic IPSPs.

In rat hippocampal slices GABAergic IPSPs are very rapidly suppressed by anoxia (in less than 2 min). Both early (GABAA) and late (GABAB) components are affected. After reoxygenation, the IPSPs recover, but only slowly and not always completely. Iontophoretic applications of GABA or baclofen indicated no major depression of responses during anoxia. It is therefore unlikely that the anoxic suppression of IPSPs is caused by desensitizations of GABA receptors. A more probable explanation is a failure of GABAergic neurons to release GABA from inhibitory nerve terminals.

Action Potentials↗
PubMed1990

A simple method for the solid phase synthesis of oligodeoxynucleotides containing O4-alkylthymine.

A route to prepare the cyanoethyl-phosphoramidite monomer of O4-alkylthymine and a method for the routine solid-phase synthesis of oligodeoxynucleotides containing O4-alkylthymine are described. This method has been used to make DNA sequences up to 48 bases in length. The amino function of the adenine and guanine in the sequence were protected with the phenoxyacetyl group, and that of cytosine with the isobutyryl group. The phosphodiesters were protected with the cyanoethyl group. This allowed complete deprotection of the oligomer with alkoxide ions (methanol/1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) for the oligomers containing O4-methylthymine, or ethanol/DBU for those containing O4-ethylthymine) thus avoiding the use of ammonia which is known to attack the O4-alkylthymine to form 5-methylcytosine. There was no detectable loss of the alkyl group to form thymine.

Alkylation↗
PubMed1990

Schistosoma mansoni: purification and characterization of a membrane-associated leucine aminopeptidase.

Membrane-associated leucine aminopeptidase (EC 3.4.11.1, LAP) has been purified to homogeneity from Schistosoma mansoni egg homogenates by a combination of ultracentrifugation, chromatofocusing, and molecular sieve chromatography. A 260-fold increase in specific activity was observed after purification. This is a metalloenzyme, containing carbohydrate moieties. Optimal enzyme activity was found at neutral pH. Enzyme activity was measured using L-leucine-7-amino-4-trifluoromethylcoumarin (L-Leu-AFC); in addition, schistosome egg LAP hydrolyzed a variety of other aminopeptidase substrates. Hydrolysis of L-Leu-AFC was inhibited by a number of aminopeptidase inhibitors, including 1,10-phenanthroline, bestatin, and amastatin.

Animals↗
PubMed1990

Solid-phase synthesis of oligodeoxynucleotides containing O6-alkylguanine.

Practical methods for the routine solid-phase synthesis of oligodeoxynucleotides containing O6-alkylguanine are described. It is shown that if the 2-amino group of the alkylated base is protected with a phenylacetyl group, and a mixture of nitrobenzaldoximate ions and ammonia used to remove the protecting groups from the oligomer at the end of the synthesis, there is negligible formation of 2,6-diaminopurine and that after chromatography pure oligomers are obtained.

Base Sequence↗
PubMed1990

Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2',2'-difluorodeoxycytidine.

The new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) is a specific inhibitor of DNA synthesis that has marked cytotoxicity and therapeutic activity. A 2-hr incubation with 0.1-10 microM dFdC decreased cellular viability 78-97%. This treatment reduced deoxynucleoside triphosphate pools, similar to the action of the ribonucleotide reductase inhibitor hydroxyurea. The most pronounced decrease occurred in the dCTP pool, quantitatively followed by the decrease of dATP, dGTP, and dTTP. In contrast, inhibition of DNA synthesis by arabinosylcytosine did not affect the dCTP level, whereas dATP, dGTP, and dTTP pools increased, but less than 2-fold. The incorporation of [5-3H]cytidine into the dCTP pool, a measure of ribonucleotide reductase activity in whole cells, was reduced to 3% of controls by 0.1 microM dFdC, but to only 40% by 0.1 microM ara-C. Each drug decreased incorporation of [5-3H]cytidine into DNA to a similar extent (greater than 94%), suggesting limitation by a reaction proximal to this step. The cellular concentration of dFdC 5'-diphosphate was 0.3 microM at 50% inhibition of the in situ activity of ribonucleotide reductase. Direct assays of partially purified ribonucleoside diphosphate reductase (EC 1.17.4.1) demonstrated 50% inhibition by 4 microM dFdC 5'-diphosphate; dFdC 5'-triphosphate was much less inhibitory. We conclude that dFdC 5'-diphosphate acts as an inhibitor of ribonucleoside diphosphate reductase.

Cell Line↗
PubMed1989

Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5'-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.

Base Sequence↗
PubMed1989

Dantrolene suppresses the hyperpolarization or outward current observed during anoxia in hippocampal neurons.

Hyperpolarizations, or outward currents, recorded in CA1 pyramidal cells during brief anoxia (2-3 min) (but not postanoxic hyperpolarizations) are markedly reduced (92 +/- 4.8%) by dantrolene sodium, applied by superfusion (10-20 microM). This effect, which is at least partly reversible by prolonged washing, is in keeping with the idea that anoxia activates a Ca2(+)-sensitive K conductance by releasing Ca2+ from internal stores.

Animals↗
PubMed1989

Solid-phase synthesis of oligoribonucleotides.

An efficient method is described for solid-phase synthesis of oligoribonucleotides that involves use of the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection, 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection and a phosphoramidite coupling procedure.

Drug Stability↗
PubMed1989

Schistosoma mansoni: egg morphology and hatchability.

Schistosoma mansoni eggs were isolated and staged microscopically according to size. The eggs were then allowed to hatch and were reexamined; the hatching percentage of mature eggs was calculated. The results show clearly that not only do immature eggs not hatch, but also mature eggs greater than 160 microns fail to hatch.

Animals↗
PubMed1988

Effect of praziquantel on Schistosoma mansoni eggs: leucine aminopeptidase (LAP) activity and anti-LAP antibodies.

High levels of leucine aminopeptidase (LAP) have been detected in Schistosoma mansoni eggs. In this report we demonstrate that this enzyme is immunogenic in mice infected with either S. mansoni or S. japonicum. The anti-schistosomal agent, praziquantel, causes rapid release of this enzyme from schistosome eggs in vitro. Praziquantel treatment of S. japonicum infected mice is associated with a significant decrease in anti-LAP antibodies. Anti-LAP IgM antibody levels decreased more than did anti-LAP IgG antibodies.

Animals↗