3H uridine

To establish whether rifaximin, like the other members of the rifamycin family [36, 58], specifically inhibits bacterial RNA synthesis the effect of this antibiotic as well as that of rifampicin and chloramphenicol on RNA (via 3H-uridine incorporation), DNA (via 3H-thymidine incorporation) and protein (via 35S-methionine incorporation) synthesis was studied in growing cultures of Escherichia coli [59], While chloramphenicol reduced protein synthesis, both rifaximin and rifampicin inhibited RNA synthesis in a concentration-dependent fashion. In contrast, none of them affected 3H-thymidine incorporation into DNA. These data suggest that rifaximin, like rifampicin, inhibits RNA synthesis by binding the (3 subunit of the bacterial DNA-dependent RNA polymerase [60],... 

The inhibition of RNA synthesis may be followed by the use of [3H]-deoxyuridine or [3H]-uridine in much the same manner as those used for the DNA assays above [183-186]. 

The measurement of incorporation of radioactive metabolites is frequently used as a response to short and intermediate duration cytotoxicity. Measurements of pH]-thymidine incorporation into DNA and [3H]-uridine incorporation into RNA are the two most common methods of quantifying drug cytotoxicity. Furthermore, [125I]-iododesoxyuridine, a specific and stable label for DNA synthesis, is also employed, as well as measurements of [32P]-phosphate release into medium or incorporation into nucleotides, in addition to the incorporation of [14C]-glucose, [3H]-amino acid, and 45calcium. 

Fig. 5.1. Effect of increased NaCl concentration of the incorporation of [3H]uridine and [3H]leucine into CHO cells. Hamster CHO cells were set up as coverslip cultures in a multiwell dish in Eagle s medium (Glasgow modification) containing 10% calf serum (105 cells/0.5 ml). After overnight growth, 10 /x 1 of [3H]uridine (0.5 pCi) or [3H]leucine (2 fxCi) was added per well, and 30 min later 10 fil 5 M NaCl was added to half the wells. Coverslips were harvested at the indicated times and washed twice in ice-cold BSS, 4 times in cold 5% TCA and twice in absolute ethanol. The cells were dissolved in 0.5 ml hyamine hydroxide and the radioactivity counted using a scintillator of 0.5% diphenyloxazole in toluene. , Control, i.e. 120 mM NaCl o, 220 mM...

H]uridine is generally used as a precursor specific for RNA but this is only partially true as significant amounts of radioactivity may enter DNA particularly in the form of cytosine (Adams, 1968 Oldham, 1967). Many of the problems encountered with the use of [3H]thymidine also arise in the use of [3H]uridine but very little has been done in the search for a rigorous solution. 

Fig. 12.5. Effect of length of autoradiographic exposure on grain count. BHK21/C13 cells labelled with [3H]uridine were covered with Ilford L4 emulsion (diluted with an equal volume of water) dried in a horizontal position and exposed in air at room temperature for the indicated times. (Courtesy of K. Shaw and Dr. J.D. Pitts.)...

Autoradiography is a technique for locating radioactive compounds within cells it can be conducted with light or electron microscopy. Living cells are first exposed to the radioactive precursor of some intracellular component. The labeled precursor is a compound with one or more hydrogen ( H) atoms replaced by the radioisotope tritium (3H) e.g., [3H]thymidine is a labeled precursor of DNA, and [3H]uridine is a labeled precursor of RNA (Chap. 7). Various tritiated amino acids are also available. The labeled precursors enter the cells and are incorporated into the appropriate macromolecules. The cells are then fixed, and the samples are embedded in a resin or wax and then sectioned into thin slices. 

Among the variety of methods that have been developed to quantify nucleobase deamination products, including simple HPLC quantification [134], 32P-postlabe-ling of 2 -deoxyribonucleosides released from DNA [135] and uptake and labeling of DNA with [3H]uridine [136] or [3H]2 -deoxyuridine [137], all lack the specificity and sensitivity required to detect background levels of dU in DNA. This problem has been overcome with the development of methods involving gas or liquid... 

Despite the PPG showing a weak activity, it has developed further antimicrobial assays. Pardo and cols, reported [34] the MIC= 400 pg/mL and MBC= 800 pg/mL of verbascoside. Moreover, Avila and cols. [33] studied its mode of action in vitro. There is no evidence of inclusion of [3H]-leueine into the cell, whereas [3H]-thymidine and [3H]-uridine were not observed. That resulted in the conclusion that the mode of action of verbascoside is through the inhibition of protein production, since leucine is an important metabolite in protein synthesis. 

Figure 1. Time course of protein and viral RHA synthesis in HeLa cells infected with EMC. A culture at 4x10 cells/ml in MEM medium, 59 fetal calf serum, 2mM glutamine and 14 mM HEPES-KOH, pH 7 2, was infected with JOOpfu/cell of EMC. To measure protein synthesis, 0.1 ml of the culture were added at the indicated times to 0.9 ml of methionine-free medium containing 10 0 /ml of 35S )-methionine and incubated 20 min. To measure EHA synthesis, 0.2 ml of the culture were added to 0.8 ml of medium containing 6.25 M g /ml of actinomycin D and incubated 5 min 10 ixCi of ( 3H )-uridine were then added for 1 5 min. The incubations were stopped by adding 10 ml of cold spinner salts solution and the cells collected by centrifugation. The pellet was dissolved in 1% Ha-dodecyl sulphate and precipitated with 5% trichloroacetic acid for counting.

P33 Popov, N., Schmidt, S., Schulzeck, S. and Matthies, H. Incorporation of intraventricularly administered (3H)-uridine monophosphate, pH] cytidine monophosphate, and (14C)-orotic acid into brain and liver RNA of the rat. Acta Biol. Med. Ger., 28, 13-20 (1972) (Germ.)... 

Kitts DD, Krishnamurti CR, Kitts WD 1980 Uterine weight changes and 3h-uridine uptake in rats treated with phytoestrogens. Can J Anim Sci 60 531-534... 

Transglycosylation of 2 -deoxy-2 -3H-uridine with 5-halouracils, catalysed by pyrimidine nucleoside phosphorylase, was used to prepare the labelled 2 -... 

These platinum complexes cause a consistent inhibition of DNA synthesis iji vivo.1 1 At very low concentrations, 2 selectively inhibited the incorporation of thymidine-CH3-3H, uridine-5-3H, L-leucine-14c and L-leucine- H into mammalian cells.It may undergo sequential transformations with loss of chloride, and the resultant platinum species may act blfunctionally to crosslink adjacent nuclephilic centers of DNA through covalent binding.183... 

Seyama, Y., Kalckar, H. M. Specific tritium labeling of uridine diphosphogalactose 4-epimerase by d-1-3H Galactose. Biochemistry 11, 36—39 (1972). 

By 1942 it was clear from ultraviolet cytophotome-try developed by Caspersson21 and from cytochemical work of Brachet2223 that RNA had something to do with protein synthesis. Use of radioautography with 3H-containing uridine showed that RNA was synthesized in the nucleus of eukaryotic cells and was transported out into the cytoplasm 24/25... 

Absorption of purines and pyrimidines by cestodes occurs by a combination of passive diffusion and mediated transport. In H. diminuta, purine and pyrimidine uptake is very complex and seems to involve at least three carrier systems (Table 6.9), two of which appear to bind several substrate molecules simultaneously (631). Pyrimidine transport was thought to involve allosteric regulation because the relation between initial uptake and substrate concentration was sigmoidal. However, more recent work (890) has indicated that the sigmoidal kinetics of pyrimidine transport in H. diminuta is an isotope effect, obtained only when 2-14C-labelled pyrimidines were used absorption kinetics of methyl-l4C- and 3H-labelled pyrimidines were hyperbolic. Nucleosides (thymidine, uridine, adenosine and guanosine) are absorbed by H. diminuta, H. citelli and H. microstoma via a specific sodium-dependent, mediated system involving at least two carriers (347). Interestingly, the mechanism displays a diurnal periodicity in H. diminuta (616, 617). 

The benzylidene acetal is cleaved by acidic hydrolysis (e.g., 0.01 N H2SO4, 100°C, 3h, 92% yield 80% AcOH, 25°C, f,/2 for uridine - 60b h, conditions that do not cleave a methylenedioxy group. The rate of acid-catalyzed hydrolysis of benzylidene acetals increases as the size of the substituent R increases. The second-order rate constant k, on going from R — Me to R — f-amyl, increases about 100-fold, indicating that steric effects play a large role in determining hydrolysis rates. ... 

Saladino R, Crestini C, Palamara AT, Danti MC, Manetti F, Corelli F, Garaci E, Botta M. Synthesis, biological evaluation, and pharmacophore generation of uracil, 4(3H)-pyrimidinone, and uridine derivatives as potent and selective inhibitors of parainfluenza 1 (Sendai) vims. J Med Chem 2001 44 4554 1562. 

Fig. 14. Synchronization with seven heat shocks. Three samples (I, II, and III) were treated with methotrexate + uridine (M + U) as indicated a fourth served as the control (C). The four samples received unlabeled and 3H thymidine(T, H-T) as indicated. Subsequently groups I to IV were sampled at intervals for autoradiography. The curves show the percentage of cells with a labeled nucleus. (From Zeuthen. 1970. Exp. Cell Res., 61 311-325.)...

Exchange of tritium for hydrogen by tritiated biomolecules and s >sequent loss of THO into the environment was examined by incubating the labeled biomolecule and scintillator in a sealed film tube at ambient temperature. The sanples (in triplicate) were then counted at timed intervals. Three tritiated nucleosides were compared in this manner, adenosine (2,8-3h) from New England Nuclear (Boston, MA.) and guanosine (8-3h) and uridine (5-3h) both from ICN (Irvine, CA.). 

Figure 4 shows the changes in activity of film tubes containing either THO or tritiated biomolecules. Activities of both adenosine (2-8%) and uridine (5-3h) remain constant... 

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