Thymidine VOLUME

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. The mixture was incubated at 37°C for 18 hours. After this time, enzyme action was stopped by the addition of four volumes of acetone and one volume of peroxide-free diethyl ether. The precipitated solids were removed by filtration, and the filtrate was evaporated under nitrogen at reduced pressure until substantially all volatile organic solvent had been removed. About 20 ml of aqueous solution, essentially free of organic solvent, remained. This solution was diluted to 100 ml with distilled water. 

Fig. 7.3. Electronic cell volume plotter. Mouse L929 cells were harvested from stationary phase cultures at zero time and subcultured into medium to which thymidine (5 mM) was added at 8 h. At 24 h the thymidine was removed. At various times the cells were harvested by trypsinisation and their volumes measured using a...

Fig. 12.2. Relationship between the concentration of extracellular thymidine and the exogenously derived nuclear pool of dTTP. Mouse L929 cells were incubated for 30 min with [3H]thymidine at the concentrations indicated and the acid-soluble pool extracted (see Fig. 12.1). The concentration of dTTP is calculated on the basis that the radioactive acid soluble pool is completely [3H]dTTP and that it is located in the nuclei of S-phase cells. The volume of a nucleus is taken as 500/xm3. (Reproduced from Adams, 1969a with kind permission of the publisher.)...

The mixed lymphocyte reaction (MLR) test was performed in 96-well flat-bottomed microtiter plates. Selected experimental agents were prepared as 10 mM stock solution in DMSO and a 50-fold dilution of this was prepared in RPMI. Serial dilutions were prepared from this subsequent solution and 10 xl of the diluted stock was added to the well to give concentrations in the assay starting at 9.5 xm. In each well was placed 1.5 x 105 donor cells to give a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum, 2mM L-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide for 120 hours. 3H-Thymidine (0.5 xCi) was added in the final 6 hours of incubation and cell radioactivity levels determined, which were indicative of T-cell proliferation. 

In another study by Wang et al., i.v. injection of liposomes carrying rat insulin promoter (RIK)-thymidine kinase (TK) was found to be less toxic to the liver than the i.p. injection of the same formulation to severe combined immunodeficient mice (SCID) [192], The direct injection of the liposomes to abdominal cavity probably leads to higher local absorption and, thus, higher liver toxicity. In contrast, the i.v. injected volume is smaller (70 pL to 100pL of i.p. injection), which is diluted fast as soon as it enters the body. 

The reaction mixture consists of 100 mM Tris HCI, pH 8.0, 10 mM nicotinamide, 10 mM thymidine, 10 mM dithiothreitol, 5mM MgCb, 10 nM pP]-NAD (900 cpm/pmol) (ADP-ribosylation buffer), purified C3 exoenzyme, and recombinant Rho or a crude homogenate in a total volume of 100 jil. The mixture is incubated at 30 C. To measure the amount of Rho, the ADP-ribosylation reaction should reach a plateau. When 50 ng of C3 exoenzyme... 

After 36 h of culture, add 1 pCi of 3H-thymidine in a small volume of complete growth medium to each replicate in the 96-well plate. 

Fig. 3-93. Separation of nucleosides using anion exchange chromatography. — Separator columns 2 IonPac AS4A eluent see Fig. 3-92 flow rate 1.5 mL/min detection suppressed conductivity injection volume 50 pL solute concentrations 10 ppm cytidine (1), 5 ppm adenosine (2), 10 ppm thymidine (3) and uridine (4), 13 ppm 2 -deoxyguanosine (5), 15 ppm guanosine (6), and inos-ine (7).

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. 

Hagai Ginsburg and Ioav Cabantchik at the Hebrew University of Jerusalem and Kiaran Kirk of the Australian National University (ANU, Canberra, ACT, Australia) have characterized the NPP as a poorly selective anion channel with many characteristics of volume-sensitive anion channels. Kirk et al. (1994) postulated the NPP to be a single channel type. However, a recent analysis of tracer flux and iso-osmotic lysis suggests the NPP may consist of two kinds of channels one present in low copy number per cell (around 4 copies/cell) is selective for charge and size whereas the other is more abundant (around 400 copies /cell) and allows the movement of anions (Cl- and lactate) and nucleosides (thymidine and adenosine) (Ginsburg and Stein, 2004). 

VHLio4 i23 Inhibition of thymidine incorporation into RCC cells, inhibition of MAP kinase pathway, inhibition of RCC proliferation in vitro, growth delay of RCC xenografts, regression of tumor volume, inhibition of invasion of RCC tumor Tat 15... 

A confluent flask should be washed twice in sterile PBS and the cells trypsinized in 10 ml of trypsin/EDTA solution. Transfer the cells into the roller bottle at a density of 1.5 x lO /cm and in a final volume of 400 ml complete medium. Charge the bottle with sterile filtered 5% C02/95% air and place at 37°C on a roller apparatus at a rotation speed of 0.5 rpm. After 2 days the bottle should be confluent. At this point replace the medium with 200 ml complete medium containing 2 vciM thymidine and incubate for a further 11 hr in order to accumulate cells in S phase. This 11-hr thymidine block is most conveniently carried out overnight. 

This method is also used for the analysis of the drug in tablets after employing a resolution solution prepared by dissolving accurately weighed quantities of thymine and thymidine in water, diluted with water to obtain a solution having a known concentration of 0.1 JLg/ml of each component. The resolution, R, between thymine and thymidine is not less than 2.0, and thymine is resolved from the void volume... 

Fig. 3-135. Gradient elution of various nucleosides and nucleotides on a DVB polymer coated with D2.2.2. - Eluant (A) water, (B) 0.1 mol/L KCl gradient 100% A isocratically for 5 min, then linearly to 5% B in 15 min, then linearly to 100% B in 130 min flow rate 1 mL/min detection UV (254 nm) injection volume 20 pL solute concentrations lOpmol/Leach ofcytidine (1), deoxycytidine (2), thymidine (3),...

The known role of vitamin B12 and folic acid in the formation of labile methyl groups (339) helps to explain the replaceability of folic acid (330) or vitamin B12 (331) by thymidine in certain deficient oi anisms. Also of interest is the role of folic acid compounds in the pathway leading to formation of the thymine methyl group. Low concentrations of a folic acid antimetabolite, aminopterin, blocked the utilization of deoxyiuidine for thymidine synthesis (333). The antimetabolite effects of aminopterin on the utilization of one-carbon donors have been known for some time (333, 334). The details of thymine biosynthetis will be discassed elsewhere in this volume (Chapter 24). 

---