Thymidine kinase concentrations

L D. The conversion of penciclovir to its active form requires initial monophosphorylation by viral thymidine kinases, then conversion to its active triphosphate form by cellular enzymes. Thus, the concentration of penciclovir triphosphate is particularly high in cells infected with its target viruses (e.g., HSV, VZV, HBV). Foscarnet is a pyrophosphate analogue that does not require activation. Oseltamivir is a neuraminidase inhibitor that is con-... 

Famciclovir is the diacetyl ester prodrug of 6-deoxypencidovir, an acyclic guanosine analog (Figure 49-2). After oral administration, famciclovir is rapidly deacetylated and oxidized by first-pass metabolism to penciclovir. It is active in vitro against HSV-1, HSV-2, VZV, EBV, and HBV. As with acyclovir, activation by phosphorylation is catalyzed by the virus-specified thymidine kinase in infected cells, followed by competitive inhibition of the viral DNA polymerase to block DNA synthesis. Unlike acyclovir, however, penciclovir does not cause chain termination. Penciclovir triphosphate has lower affinity for the viral DNA polymerase than acyclovir triphosphate, but it achieves higher intracellular concentrations. The most commonly encountered clinical mutants of HSV are thymidine kinase-deficient these are cross-resistant to acyclovir and famciclovir. 

Penciclovir triphosphate has lower affinity for the viral DNA polymerase than acyclovir triphosphate, but it achieves higher intracellular concentrations and has a more prolonged intracellular effect in experimental systems. The most commonly encountered clinical mutants of HSV are thymidine kinase-deficient and are cross-resistant to acyclovir and famciclovir. 

Although there is obviously a through-put such that [3H]dTTP is being formed and removed by DNA synthesis it appears that the size of the radioactive deoxythymidylate pool is determined by the concentration of extracellular thymidine (Fig. 12.2). This may come about by a combination of forward promotion of thymidine kinase by its substrate and feedback inhibition by dTTP (Ives et al., 1963). The size of the pool remains constant once equilibrium has been reached until extracellular thymidine levels begin to fall. This will happen within a few hours when the concentration of extracellular thymidine is 10-6M and even at 10-5 M 10% may be utilised within... 

Cells lacking thymidine kinase (TK- cells) can be isolated by treating cell cultures with high concentrations (30 jug/ml) of 5-bromodeoxyuridine, which kills cells containing the enzyme thymidine kinase due to incorporation of large amounts of the analogue into the cells DNA. 

By growing cells in the presence of increasing concentrations of aminopterin a number of resistant cells lines have been isolated (Hakala and Ishihara, 1962 Littlefield, 1969). These have been characterised as having either an altered permeability to the drug or an altered folate reductase or an increased rate of synthesis and hence increased amounts of the enzyme (Alt et al., 1976), resulting, at least in part, from a selective amplification of the dihydrofolate reductase gene (Alt et al., 1978 Schimke et al., 1988). The problem is considered in more detail in 11.8.1. The importance of the antifolates lies in their role in the HAT selection technique ( 13.5) devised by Szybalski (1962) (see also Szybalski et al., 1962 and Littlefield, 1964) for the isolation of hybrids between mutant cells defective on the one hand in thymidine kinase and on the other hand... 

Preparation of Crude Extracts The parasite larvae or adult forms were homogenized by grinding with sand in a mortar (placed on ice) with three to five volumes of ice-cold 0.05 M phosphate buffer, pH 7.5, containing 0.1 M KCl and 0.01 M 2-mercaptoethanol, except for assays of thymidine kinase activity, when phosphate buffer was substituted by Tris-HCl buffer at the same concentration and pH. The suspension was sonicated and centrifuged to obtain crude extract. 

Deoxycytidine kinase is an enzyme catalyzing the phosphorylation of deoxycytidine in nucleotide salvage biosythesis. It will also phosphorylate deoxyadenosine and deoxyguanosine when these compounds are present at higher concentrations. Deoxycytidine kinase is inhibited by dCTP. Unlike thymidine kinase, whose activity fluctuates over the course of the cell cycle, the activity of deoxycytidine kinase stays relatively constant. 

Methotrexate toxicity to normal cells is reduced by supplying an alternative cofactor, leukovorin, or by bypassing the folate pathway by adding thymidine. Thymidine is converted directly to thymidylate by thymidine kinase. Administration of these agents after methotrexate is called leukovorin or thymidine "rescue." Leukovorin and th50iudine do not "rescue" tumor cells from methotrexate because they do not reach adequate concentration in tumor cells. 

MBOCA induced gene mutations at the thymidine kinase (TK) locus in mouse lymphoma cells (Caspary et al. 1988 Myhr and Caspary 1988). Unscheduled DNA synthesis (UDS) was induced in HeLa cells (Martin and Mcdermid 1981), in rat primary hepatocytes at >10 pmol (McQueen et al. 1981 Mori et al. 1988 Williams et al. 1982), and in hamster (McQueen et al. 1981) and rabbit (McQueen and Wiliams 1987) hepatocytes. The concentration that tested positive in the mouse was 50 jmol (McQueen et al. 1981). Sensitivity to MBOCA showed species-specific variations rat > mouse > hamster > rabbit (McQueen et al. 1981, 1983). Because hepatocytes have their own metabolic activation systems, no exogenous metabolic activation is needed. In assays using attachment independence as an end point, MBOCA, at concentrations near the LC o, transformed baby hamster kidney (Daniel and Dehnel 1981 Styles 1981), rat embryo (Dunkel et al. 1981 Traul et al. 1981), and Balb/3T3 cells (Dunkel et al. 1981). Transformation assays have not been evaluated... 

HEPT does not compete with pH-A/c]thymidine for phosphorylation by thymidine kinase derived from MT-4 cells. 2) The synthetic triphosphate of HEPT does not inhibit HIV-1 RT (reverse transcriptase) at concentrations much higher than that of its ECso, irrespective of the template-primer used [either poly(rA)-oligo(dT) or poly(c)-oligo(dG)]. Thus, HEPT can be considered a highly unique and specific lead for anti-HIV-1 agents. This prompted us to carry out synthesis of its analogues with a directed aim to improve its activity. 

The pool appears to expand indefinitely as the extracellular concentration of thymidine increases up to 10 mM (Fig. 12.2), which shows that the kinases are present in vast excess (Cleaver and Holford, 1965 Gentry et al., 1965 Cooper et al., 1966 Adams, 1969a Stimac et al., 1977). 

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