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Biomedical subjects

Y C Cheng

Publications and source records attributed to Y C Cheng.

At least 289 records · Page 16Linked to original sources
PubMed1981

Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethyl)guanine triphosphate. Effects on primer-template function.

The inhibition of highly purified herpes simplex virus (HSV)-induced and host cell DNA polymerases by the triphosphate form of 9-(2-hydroxyethoxymethyl)guanine (acyclovir; acycloguanosine) was examined. Acyclovir triphosphate (acyclo-GTP) competitively inhibited the incorporation of dGMP into DNA, catalyzed by HSV DNA polymerase; apparent Km and Ki values of dGTP and acyclo-GTP were 0.15 microM and 0.003 microM, respectively. HeLa DNA polymerase alpha was also competitively inhibited; Km and Ki values of dGTP and acyclo-GTP were 1.2 microM and 0.18 microM, respectively. In contrast, HeLa DNA polymerase beta was insensitive to the analogue. The "limited" DNA synthesis observed when dGTP was omitted from HSV or alpha DNA polymerase reactions was inhibited by acyclo-GTP in a concentration-dependent manner. Prior incubation of activated DNA, acyclo-GTP, and DNA polymerase (alpha or HSV resulted in a marked decrease in the utilization of the primer-template in subsequent DNA polymerase reactions. This decreased ability of preincubated primer-templates to support DNA synthesis was dependent on acyclo-GTP, enzyme concentration, and the time of prior incubation. Acyclo-GMP-terminated DNA was found to inhibit HSV DNA polymerase-catalyzed DNA synthesis. Kinetic experiments with variable concentrations of activated DNA and fixed concentrations of acyclo-GMP-terminated DNA revealed a noncompetitive inhibition of HSV-1 DNA polymerase. The apparent Km of 3'-hydroxyl termini was 1.1 X 10(-7) M, the Kii and Kis of acyclo-GMP termini in activated DNA were 8.8 X 10(-8) M and 2.1 X 10(-9) M, respectively. Finally, 14C-labeled acyclo-GMP residues incorporated into activated DNA by HSV-1 DNA polymerase could not be excised by the polymerase-associated 3',5'-exonuclease activity.

Acyclovir↗
PubMed1981

Induction of uracil-DNA glycosylase and dUTP nucleotidohydrolase activity in herpes simplex virus-infected human cells.

HeLa BU cells infected with either the type 1 or the type 2 forms of herpes simplex virus show an increase in the activities of uracil-DNA glycosylase and dUTP nucleotidohydrolase. Under optimal conditions, uracil-DNA glycosylase activity increases approximately 40-fold in HSV type 2-infected cells. In herpes simplex virus (HSV) type 1-infected cells, uracil-DNA glycosylase activity increases only 6-fold. At a KCl concentration of 100 mM, uracil-DNA glycosylase derived from HSV type 2-infected cells is activated 2-fold, while the glycosylase extracted from mock infected HeLa BU cells is inhibited almost 90% at 100 mM KCl. dUTP nucleotidohydrolase activity increases 4-fold and 3-fold, respectively, in HSV type 1- and HSV type 2-infected HeLa BU cells. Nondenaturing polyacrylamide gel electrophoresis of extracts derived from the type 1- and type 2-infected cells indicates distinct electrophoretic mobilities from the host cell enzyme. dUTP nucleotidohydrolase RF values for the mock infected cells, HSV type 1, and HSV type 2 are 0.5, 0.25, and 0.33, respectively. Serum from rabbits immunized against cells infected with herpes simplex virus type 1 or type 2 specifically neutralizes the dUTPase and uracil-DNA glycosylase activities extracted from herpes simplex virus-infected cells. This serum does not neutralize dUTPase or uracil-DNA glycosylase activity derived from mock infected cells.

Cell Transformation, Viral↗
PubMed1981

Herpes simplex virus type I DNA polymerase. Kinetic properties of the associated 3'-5' exonuclease activity and its role in araAMP incorporation.

An exonuclease activity copurified with herpes simplex virus type I (HSV-1) DNA polymerase through DNA-cellulose column chromatography and comigrated with DNA polymerase activity on nondenaturing gel electrophoresis at varied polyacrylamide concentrations. A gapped duplex DNA was the preferred substrate for this exonuclease activity since the hydrolytic activity on this type of DNA was much greater than the hydrolysis of either native or heat-denatured DNA. Using 3'-terminally labeled activated calf thymus DNA as substrate, the exonuclease activity was found to be activated by salt and spermidine in a manner identical with HSV-1 DNA polymerase. This activation was accompanied by increases in apparent Km and Vmax values of the activated DNA substrate. Phosphonoformic acid inhibited both DNA polymerase and exonuclease activities uncompetitively with respect to activated DNA and had a Ki of 2.4 microM at an ionic strength of 0.25 mu. Of the nucleoside 5'-monophosphates tested only the purine ribonucleotides inhibited the exonuclease activity. The inhibition was noncompetitive with respect to DNA, and GMP was about twice as potent as AMP or IMP. 9-beta-D-arabinosyladenine 5'-monophosphate (araAMP) could be incorporated into DNA by HSV-1 DNA polymerase; however, 9-beta-D-arabinosyladenine 5'-triphosphate would not replace dATP in supporting in vitro HSV-1 DNA synthesis. AraAMP incorporated into primer termini caused a significant decrease in the rate of subsequent primer elongation. These 3'-terminal araAMP residues could be removed by the HSV-1 DNA polymerase-associated exonuclease activity in a manner dependent on GMP concentration.

Arabinonucleotides↗
PubMed1981

Mode of action of phosphonoformate as an anti-herpes simplex virus agent.

Phosphonoformate inhibited the replication of Herpes simplex virus (HSV) type 1 and type 2 in culture. The concentration required to inhibit the replication of both types of virus by 2 logs at 28 h post-infection was approximately 150 microM. It was more potent than phosphonoacetate against the growth of both virus types. A virus mutant which is resistant to phosphonoacetate was cross-resistant to phosphonoformate. Arsonoacetate, at 300 microM, had no antivirus activity. Phosphonoformate also inhibited HeLa and KB cell growth; at a concentration of about 500 microM, cell growth was inhibited by 50%. The anti-cell growth effects of the drug were completely reversible. The antivirus effect of phosphonoformate was partially reversible, depending on the time and duration of exposure of infected cultures to the drug. To obtain the maximum antivirus effect, phosphonoformate had to be added within the first 3 h post-virus-infection and be continuously present for at least 18 h. Phosphonoformate, added at 0 h post-infection, suppressed the induction of virus-specific DNA polymerase and DNAase activities. dTMP incorporation into DNA was preferentially inhibited in nuclei isolated from infected cells compared to uninfected cells, and the degree of inhibition varied with the ionic strength of the assay. Phosphonoformate was a potent inhibitor of the purified HSV-1 and HSV-2 DNA polymerases, inhibiting DNA polymerase activity by 50% at a concentration of 3 microM and ionic strength of 0.2.

Antiviral Agents↗
PubMed1981

Synthesis and antiviral activity of 1-(2-deoxy-beta-D-ribofuranosyl)-5-(methylmercapto)-2-pyrimidinone.

1-(2-Deoxy-beta-D-ribofuranosyl)-5-(methylmercapto)-2-pyrimidinone (1b) was synthesized via modification of the silyl method. 1b inhibits the Herpes simplex virus type 1 (98%) and type 2 (97%) at a concentration which is nontoxic to human HeLa cells. The compound shows 50 times greater binding affinity (lower Ki) to the virus-specific thymidine kinase than to the thymidine kinase of uninfected HeLa cells.

Antiviral Agents↗
PubMed1981

Differential activity of potential antiviral nucleoside analogs on herpes simplex virus-induced and human cellular thymidine kinases.

Potential antiviral nucleoside analogs 1-beta-D-arabinofuranosylthymine, the 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-nucleosides of -5-methyluracil, -5-iodouracil, -5-methylcytosine, -5-iodocytosine, and -E-5-(2-bromovinyl)uracil, E-5-(2-bromovinyl)-2'-deoxyuridine, E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil, and 9-(2-hydroxyethyoxymethyl)guanine were studied to compare their phosphorylation rates relative to thymidine by purified thymidine kinases from human and herpes simplex virus sources. Most of these analogs are capable of being phosphorylated by both human and viral enzymes. On the assumption that inhibition constants (Ki) reflect binding affinity, Ki values were determined for these analogs with the same thymidine kinases. In general, these analogs have a greater affinity for the viral enzymes. The amount of the analogs phosphorylated to the monophosphate form, which is presumably necessary to produce cytotoxic effects, was determined by the combined effects of phosphorylation rates and binding affinities. All of these analogs act as preferential substrates for the viral thymidine kinases at low concentrations, which may be one of the main reasons for their selective antiviral action.

Antiviral Agents↗
PubMed1981

Biological and biochemical effects of 2'-azido-2'-deoxyarabinofuranosylcytosine on human tumor cells in vitro.

2-azido-2'-deoxyarabinofuranosylcytosine (Cytarazid), recently synthesized, was found to inhibit the in vitro growth of several human cell lines by 50% at concentrations ranging from 0.06 to 0.2 microM and to prevent the replication of herpes simplex virus types 1 and 2 by 98% at 50 microM. As determined with HeLa cells, the inhibition of cell growth was partially prevented by 2'-deoxycytidine (dCyd) and cytidine but not by uridine or thymidine. Cytarazid proved resistant to deamination by human cytidine/dCyd deaminases purified from acute myelocytic leukemia blast cells and from liver, a property reflected in the inability of tetrahydrouridine to enhance the cytotoxicity of the compound. Cytarazid served as a substrate for cytoplasmic dCyd kinase partially purified from human peripheral chronic lymphocytic leukemia blast cells. At a concentration of 0.4 mM, the nucleoside analog was phosphorylated 2.6 times more effectively by this enzyme than was dCyd, the Km for Cytarazid being 250 microM. In intact HeLa cells, the triphosphate derivative of Cytarazid was the major drug metabolite formed. In these cells, the analog interfered with the incorporation of radiolabeled thymidine into DNA at a concentration and a time interval at which the incorporation of uridine into RNA and amino acids into protein was not inhibited, suggesting that interference with DNA synthesis is a primary drug effect. Further analysis showed that Cytarazid triphosphate interferes with DNA synthesis in intact HeLa cell nuclei and that it inhibits both the alpha- and beta-DNA polymerases purified from HeLa cells in a manner competitive with deoxycytidine triphosphate, with Ki's of 0.6 and 0.7 microM, respectively. Cytarazid triphosphate was not able to replace deoxycytidine triphosphate for the synthesis of DNA in either intact nuclei or in cell-free preparations; but, in the cell-free assay system, the compound was found to interfere with primer-template activity.

Biotransformation↗
PubMed1980

Properties of herpes simplex virus type 1 and type 2 DNA polymerase.

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA polymerases were highly purified from infected HeLa BU cells by DEAE cellulose, phosphocellulose and DNA cellulose column chromatography. DNA exonuclease activity but not endonuclease activity was found associated with both types of DNA polymerase. Both DNA polymerase activities could be activated by salt in a similar fashion with the optimal activity in the range of ionic strength between 0.22 and 0.29 alpha. At an ionic strength of 0.14, spermidine and putrescine in the concentration range (0--5 mM) studied could mimic the action of KCI in stimulating DNA polymerase activity. Spermine, in the same concentration range, had a biphasic effect. At an ionic strength of 0.29 all three polyamines were inhibitory. HSV-1 and HSV-2 DNA polymerase are similar in their column chromatographic behavior, sedimentation rate in sucrose gradient centrifugation, and activation energy, but they differ in their heat stability at 45 degrees C with the HSV-2 enzyme more stable than the HSV-1 enzyme. Kinetic behavior of both enzymes is similar, with Km values for deoxyribonucleoside triphosphates in the range of 5 . 10(-7) to 1.8 . 10(-8) M. IdUTP and dUTP served as apparent competitive inhibitors with respect to dTTP, and AraATP acted as an apparent competitive inhibitor with respect to dATP. AraATP could not replace dATP in the DNA polymerization reaction; in contrast, IdUTP could replace TTP. Phosphonoformic acid behaved as an uncompetitive inhibitor with respect to DNA. The ID(50) value estimated was foind to be dependent on the purity of the DNA polymerase used and the ionic strength of the assay condition. Each DNA-polymerase associated DNA exonuclease had the same stability at 45 degrees C as its DNA polymerase. The associated DNAase activity was inhibited by phosphonoformic acid and high ionic strength of the assay condition.

Cellulose↗
PubMed1980

Uracil DNA-glycosylase. Purification and properties of this enzyme isolated from blast cells of acute myelocytic leukemia patients.

The enzyme uracil DNA-glycosylase has been purified from blast cells of patients with acute myelocytic leukemia. A 1000-fold purification has been achieved and the enzyme appears highly enriched for the uracil glycosylase activity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of the purified enzyme is 30,000. Uracil DNA-glycosylase exhibits activity in the absence of any added metal and the addition of MgCl2, MnCl2, CaCl2, NaCl, or KCl causes inhibition. EDTA as well as EGTA can inhibit enzyme activity. An interesting finding is the biphasic effect of spermine. At a concentration of 25 microM, spermine will cause a 2.5-fold activation of enzyme activity, whereas at concentrations of 100 microM and higher, spermine will inhibit enzyme activity. An Arrhenius plot of glycosylase activity in the presence of 25 microM spermine shows a biphasic curve with the transition temperature being 36 degrees C. Initial velocity studies in the presence of varying concentrations of spermine indicate a change in both the apparent Km and Vmax of the enzyme. Various uracil analogs were tested to establish a structure-activity relationship for this enzyme. It appears from this data that uracil DNA-glycosylase is very specific for uracil moieties. Uracil, acting as a product inhibitor, gives a Ki value of 220 microM.

Blastomeres↗
PubMed1980

Synthesis, biologic effects, and biochemical properties of some 2'-azido- and 2'-amino-2'-deoxyarabinofuranosyl pyrimidines and purines.

Various 2'-azido- and 2'-aminoarabinofuranosyl purine and pyrimidine nucleosides have been synthesized. Among these, the derivatives of cytosine and of adenine inhibit the growth of some tumor cell lines in vitro and in vivo. 2'-Azidoarabinofuranosyl cytosine also interferes with the replication of herpes simplex virus types I and II. Whereas 2'-azidoara-C is resistant to deamination by a partially purified CdR deaminase from KB cells, the adenine derivatives are substrates for aminohydrolases partially purified from calf and mouse intestines. Both azido- and aminoara-C are phosphorylated by partially purified CdR kinases from leukemia L1210 and from human AML blast cells. The accumulated data encourage exploration of the clinical utility of the more potent of these analogues.

Animals↗
PubMed1980

Frequency and levels of antibodies to Epstein-Barr virus-specific DNase are elevated in patients with nasopharyngeal carcinoma.

Sera from healthy individuals and patients with infectious mononucleosis, Burkitt lymphoma, nasopharyngeal carcinoma, or other malignancies were examined for their capacity to neutralize Epstein-Barr virus (EBV)-induced DNase activity. Sera were found that neutralized the EBV DNase but not herpes simplex virus type 1 or type 2 DNases, and vice versa. Sera from 46 of the 49 patients with nasopharyngeal carcinoma examined (94%) neutralized > 6 units of EBV DNase per ml of serum. In contrast, only 19% of 47 patients with Burkitt lymphoma, 12% of 183 patient with other malignancies, 4% of 58 patients with infectious mononucleosis, and none of 101 healthy individuals had such levels of neutralizing activity. The neutralizing factor was found in the IgG fraction derived from nasopharyngeal carcinoma sera. There was no correlation between the concentration of these antibodie and the titers of IgG ad IgA antibodies to the EBV capsid antigen, the early antigen complex, or the EBV-associated nuclear antigen.

Antibodies, Viral↗
PubMed1980

Anti-herpes simplex virus and anti-human cell growth activity of E-5-propenyl-2'-deoxyuridine and the concept of selective protection in antivirus chemotherapy.

E-5-Propenyl-2'-deoxyuridine (E-5-propenyl-dUrd) inhibited the growth of herpes simplex virus (HSV) types 1 (HSV-1) and 2 in culture. The concentration of drug required to give a 2-log reduction in virus titer was 5 microM for HSV-1 and 23 microM for HSV-2. The anti-HSV-1 activity of this agent was more potent than 5-propyl-dUrd, equivalent to E-5(3,3,3-trifluoropropenyl)-dUrd, and less potent than E-5-bromovinyl-dUrd. The HSV-1 mutant (B2006) lacking the ability to induce virus-specific thymidine kinase could not be inhibited by E-5-propenyl-dUrd. The binding constants of E-5-propenyl-dUrd to HSV-1, HSV-2, varicella-zoster virus, and human mitochondrial thymidine kinases were established to be 0.2, 6.2, 0.3, and 0.8 microM, respectively. Thymidine phosphorylation catalyzed by human cytosol thymidine kinase could not be inhibited by E-5-propenyl-dUrd at a concentration 10-fold higher than the thymidine in the assay. When thymidine and E-5-propenyl-dUrd were added concomitantly at equal concentrations to virus-infected cells, the antiviral activity was not reversed in HSV-1 and only partially reversed in HSV-2. E-5-Propenyl-dUrd also inhibited the growth of human cells in culture with 50% inhibitory dose of 50 microM. Since this inhibition could be readily reversed by a lower concentration of thymidine, the idea of selective protection is proposed. This approach could avoid the cytotoxic effect of an antiviral agent with properties similar to E-5-propenyl-dUrd without sacrificing antiviral activity.

Antiviral Agents↗
PubMed1980

Effects of deoxyadenosine triphosphate and 9-beta-D-arabinofuranosyl-adenine 5'-triphosphate on human ribonucleotide reductase from Molt-4F cells and the concept of "self-potentiation".

Deoxyadenosine triphosphate (dATP) acted as a noncompetitive inhibitor with respect to the specific nucleoside triphosphate activator for the reduction of all four common ribonucleoside diphosphates catalyzed by the reductase derived from human Molt-4F (T-type lymphoblast) cells. The inhibition constant of dATP for different ribonucleotide reduction reactions was different, indicating that the binding of the nucleoside triphosphate activator or substrate could modify the binding affinity of dATP to the enzyme. dATP also acted as a noncompetitive inhibitor with respect to cytidine diphosphate (CDP) for reductase-catalyzed CDP reduction. 9-beta-D-Arabinofuranosyl-adenine 5'-triphosphate acted as a competitive inhibitor with respect to either adenosine triphosphate or guanosine triphosphate for CDP or for adenosine diphosphate reduction, respectively. The inhibition constant was 15 microM for CDP reduction and 4 microM for adenosine diphosphate reduction. 1-beta-D-Arabinofuranosyladenine 5'-triphosphate could not substitute for adenosine triphosphate or guanosine triphosphate as the activator for CDP or adenosine diphosphate reduction, respectively. The effects of 9-beta-D-arabinofuranosylcytosine 5'-triphosphate and 5-iodo-2'-deoxyuridine 5'-triphosphate on ribonucleotide reductase were also included for comparison. The "self-potentiation" mechanism of the action of 9-beta-D-arabinofuranosyladenine and 5-iodo-2'-deoxyuridine is discussed.

Adenosine Diphosphate↗