Uridine, incorporation

Other model studies related to DNA photo damage include the photocyclisation of 5 and 6-benzyluracils 66, 67 <06OL681> and the related cyclisation of 5-phenylthio uridine, incorporated into a dinucleotide 68 <06OL2527>. 

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 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. 

The second consideration is the chemical nature of the radioactive material. The material presented to the experimental organism must be taken up or quickly metabolized to something else that can. For example, some strains of Saccharomyces cerevisiae are unable to accumulate exogenously provided uridine but can take up uracil quite normally. If, on the other hand, uridine is added to a culture of E. coli, 23% of it is extracellularly converted to uracil in the first 3 minutes of incubation. Figure 3-32 depicts the incorporation of uridine and uracil into TCA precipitable material in E. coli and S. cerevisiae, respectively. In both instances a lag is observed prior to linear incorporation. This lag may vary from 17 seconds for E. coli uridine incorporation to several hours for... 

A recent study shows that adrenals of 8 hour hypopbysectomized (hypoxed) rats secrete less steroids but have unchanged uridine incorporating ability3o. Actinomysin D inhibits uridine and amino acid incorporation in the absence of ACTH but has no effect on steroid secretion in the... 

Incorporation of precursors into DNA, proteins, and lipids is affected less rapidly or to a lesser extent. Respiration is not inhibited. Since the phenylamides at low concentrations do not affect the uptake of precursors or the conversion of nucleosides into nucleotides, inhibition of RNA synthesis must be responsible for the observed effects on uridine incorporation. However, at phenylamide concentrations that are fully inhibitory to growth, a complete inhibition of uridine Incorporation does not occur. Depending on the fungal species used incorporation is reduced to 20-60 X of the control value (12 ). This indicates that only part of the cellular RNA synthesis is sensitive to phenylamides. 

In phenylamide-resistant strains, phenylamides are not able to inhibit ribosomal RNA synthesis, as is indicated by the inability of metalaxyl to interfere with uridine incorporation in a phenylamide-resistant strain of P. megasperma f. sp. megasperma obtained in the laboratory after mutagenic treatment (14) and a phenylamide-resistant strain of Phytophthora infestans (Table III) originating from a potato field (15). 

This indicates that the apparent dissociation constants for benalaxyl and metalaxyl are similar. The binding site has a lower affinity for cyprofuram and oxadixyl, which is in agreement with the lower activity of these compounds against RNA synthesis measured as [ H]uridine incorporation by mycelium (121. 

Strategy for Kunkel oligonucleotide-directed mutagenesis using uridine incorporation as a means to eliminate unmutated plasmid DNA from the pool. 

Duval D, Durant S, Homo-Delarche F. Effect of antiglucocorticoids on dexamethasone-induced inhibition of uridine incorporation and cell lysis in isolated mouse thymocytes. J Steroid Biochem 1984 20 283-287. 

The leaves of this species have also been of interest in tests for radioprotection in a comparative study with the increase in phagocitosis of the reticular endothelial system [113]. More recently, May terms species have attracted a certain interest because of the particular properties, such as antitumoral and contraceptive ones of the extracts. For example, antitumor activity of a number of phenoldienone triterpenes isolated from Maytenus sp. has been shown [114] and it was demonstrated that they inhibit the synthesis of proteins and of uridine incorporation into DNA [115]. 

In P. infestans [17], cymoxanil when applied at concentrations up to 100 pg mL did not inhibit the uptake of radiolabeled precursors of DNA (thymidine), RNA (uridine) or proteins (phenylalanine). However, after less than 2-hours treatment, although uptake was not affected thymidine incorporation was considerably reduced while uridine incorporation was slightly affected and phenylalanine incorporation was insensitive. In contrast, cymoxanil at 10 pg mL did inhibit thymidine or uridine incorporation weakly while inhibition of mycelial growth was complete, suggesting that DNA and RNA synthesis inhibition is a secondary effect. RNA polymerase activity in isolated nuclei was not inhibited by cymoxanil. 

There is no net increase in RNA content, per embryo, however, until 72 hours postcoitum at which time there is a marked acceleration of uridine incorporation accompanying the transformation of the morula into a blastocyst as the embryo leaves the oviduct and enters the uterus (Manes, 1969). The qualitative aspects of RNA synthesis in preimplantation rabbit embryos have been investigated by the electrophoretic separation of tritium-labeled embryonic RNA on polyacrylamide gels (Manes, 1971). Labeling of RNA species having migratory properties identical with those of tRNA are detectable from the 2-cell stage onward. 

Fig. 3. Colony formation after treatment with 10 mAf 3AAB for different times Fig. 4. Inhibition of uridine incorporation by 0-5 mM 3AAB...

Fig. 7A,B. Inhibition of uridine incorporation by 10 rM benzamides vs (A) inhibition of poly-(ADP-ribose)synthetase or (B) Hammet constant...

When [ H]-thymidine incorporation was measured in the presence and absence of 10 mM SAAB, no significant difference was observed. If, however, cells were preincubated in the presence of 10 mM SAAB for increasing periods of time, a distinctly biphasic time course was observed (Fig. 9). At present we have no simple explanation for the initial slow decrease in incorporation. Blockage of cell cycle progress in Gi at approx. 2 h before S-phase could account for the second portion of the curve. The observation that S-nitrobenzamide at 6 mM (which gave an approx, equivalent inhibition of uridine incorporation) did not give this effect suggests such a block could be poly(ADP-ribose) synthetase mediated. To test whether this was indeed a Gi block, we treated cells with 10 mM AAB for 8 h, a treatment which caused minimal toxicity (Fig. S), and then released the block and monitored the recovery of [ H]-thymidine... 

It has clearly been shown that inhibition of poly(ADP-ribose) synthetase was responsible for the cytotoxicity exhibited by five of the six benzamides tested. 3-Nitrobenz-amide was markedly more cytotoxic than would be expected on the basis of its K,. Nitro-compounds have been shown to be cytotoxic and genotoxic. This is thought to arise by reduction to reactive intermediates which can then modify DNA [3]. When uridine incorporation was correlated with the Hammet constant, 3-nitrobenzamide fitted very well. The shorter exposure time in this assay minimises any metabolite effects. 

Hyperthermia is known to result in the appearance of a number of heat shock proteins whose synthesis is controlled at the level of both transcription and translation [3]. Thus, we have examined for effects of cycloheximide and actinomycin D on the alteration of poly(ADP-ribose) metabolism by hyperthermia. The results of Table 2 show that the presence of cyclohexamide resulted in over 80% inhibition of accumulation of poly(ADP-ribose) following hyperthermia, but did not affect accumulation in control cells. In contrast, the presence of actinomycin D, which inhibited uridine incorporation by over 90%, had no appreciable effect (data not shown). 

An analysis of the effects of benzamides on [ H]uridine incorporation into RNA was also performed. The cells were pretreated with various concentrations of the inhibitors for 20 min, then pulse labeled for 30 min with 2 /xCi ml" of [ H]uridine. The incorporation into RNA was performed by standard techniques. When the effects of the... 

Assuming that the synthesis of viral RNA is reduced by the interferon pre-treatment of the cell but the pattern of uridine incorporation per RRA molecule remained unchanged, the amount of virus-induced dsRRA in 1 x 10 interferon pre-treated,mengo virus-infected L cells was estimated to be 2.5 ixg (Table 7) This amount is sufficient to explain the enhanced phosphorylation of P67 reported in the preceeding paragraphs. 

A8 Andersson, J. and Darzynkiewicz, Z. Nucleoprotein changes and uridine incorporation in rat thymus lymphocytes. I. Cellular characterization of age-dependent thymus involution. II. Effect of treatment with anti-thymocyte serum in vitro. Exp. Cell Res., 75, 410-416 417-423 (1972)... 

Uridine incorporation is unchanged by oxipurinol. A slight increase is seen in the presence of purines which is probably due to an increase of ATP production (fig. 1). This increase also shows that the diminished orotic acid incorporation is not only a toxic effect. 

In a first approach to determine the mode of action of the cytostatically active clavines, incorporation studies were performed (Eich et al., 1984a). They revealed that the incorporation of PH]-thymidine into DNA is reduced in the presence of 1-propylfestuclavine. On the other hand, no influence has been determined for [ H]-uridine incorporation into RNA and PH]-phenylalanine incorporation into protein. The inhibition of the thymidine incorporation rate was only observed after a preincubation period with the clavine derivative for 24 h and was not detectable after a pretreatment period of 2h. The assumption was favoured that clavines interfere with DNA replication processes in L5178y cells rather than an unspecific effect on cell growth. This conclusion was based on the observation that all active clavines caused unbalanced growth , a property which they share with other cytostatic agents acting selectively by... 

Genty, N., 1975, Analysis of uridine incorporation in chicken embryo cells infected with vesicular stomatitis virus and its temperature-sensitive mutants Uridine transport, J. Virol. 15 8. 

Reynolds, A.G., and Russell, D.H., 1973, Stimulation of l C-uridine incorporated into RNA by intracistemal injection of putrescine. Fed. Proc., 32 429. 

Fig. 51. [ H] uridine incorporation into the mouse oocyte at different stages of follicle growth expressed as the mean number of grains over the nucleus. Open circles, grains over the nucleolus closed circles, grains over the whole of the nucleus. Vertical lines represent the standard error of the mean. The broken line depicts the change in area of the oocyte nucleus during growth. (After Moore et al., 1974)...

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