Thymidine cultures

The cell fusion mixture is transferred to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Unflised myeloma cells are unable to grow as they lack HGPRT. Unflised normal spleen cells can grow but their proliferahon is limited and they eventually die out. The hybridoma cell can proliferate in the HAT medium as the normal spleen cell supplies the enzyme which enables the hybridoma to utilize extracellular hypoxanthine. 

Production of Mucosal Damage 2.3.1.2.1 Cell culture Stimulated neutrophils are known to be cytotoxic to cells in vitro (Dull et al., 1987 Dallegri et al., 1990 Grisham et al., 1990b). Several in vitro systems have been used to demonstrate oxidative damage to intestinal cells. Xanthine/XO increased Cr release and decreased [ H]thymidine uptake by IEC-18 small intestinal epithelial cell monolayers in a dose-dependent manner (Ma et al., 1991). Rat enterocytes show decreased trypan blue exclusion and increased protein release when incubated with neutrophils stimulated... 

Myhr B, Bowers L, Caspary WJ. 1985. Assays for the induction of gene mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in culture. In Ashby J, de Serres FJ, et al., eds. Progress in mutation research. Vol. 5. Evaluation of short-term tests for carcinogens. Amsterdam, The Netherlands Elsevier Science Publishers, 555-568. 

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],... 

In a study using cultured L5178Y mouse lymphoma cells, Rogers and Back (1981) reported that both 1,1-dimethylhydrazine and 1,2-dimethylhydrazine induced forward mutations at the thymidine kinase level in the absence of an extraneous metabolic activation system. The investigators also noted that the two dimethylhydrazines appeared to have different modes of action under these conditions. 

The mouse lymphoma forward mutation test at the thymidine kinase locus (detects mutations to a nonfunctional thymidine kinase in a line of culture mouse lymphoma cells). 

Microwaves inhibit thymidine incorporation by DNA blockage in cultured cells of the Chinese hamster irradiated cells had a higher frequency of chromosome lesions (Garaj-Vrhovac et al. 1990). Microwaves induce teratogenic effects in mice when the intensity of exposure places a thermal burden on the dams and fetuses, resulting in a reduction in fetal body mass and an increased number of resorptions (O Connor 1990). 

Stock cultures are established from frozen ampoules of cells that have been treated with thymidine, hypoxanthine, methotrexate and glycine for 24 h, which purges the culture of pre-existing TK / mutants. This cell stock is used for a maximum of two months. 

Microbial growth studies also gave an important clue to the intracellular role of vitamin B12 when it was observed that the presence of thymidine overcame the need for B12 in the culture medium of Lactobacillus lactis dorner, suggesting B12 was required for the biosynthesis of thymidine. 

Cell proliferation was addressed by conventional H-thymidine incorporation assays. Briefly, PBMCs were cultured in the presence of IRIV and liposomes, and in absence of any stimuli. On day 5 of culture, cells were pulsed with H-thymidine for 18 hours, then harvested, and cell proliferation was determined by tracer incorporation measurement. 

Figure 1 Continued on next page) Immunopotentiating reconstituted influenza virosomes (IRIV) induced antigen specific proliferation of CD4+CD45RO+ cells. (A) Peripheral blood mononuclear cells (PBMQ from healthy donors n=3) were cultured in the absence of stimuli (Neg), in the presence of IRIV (V), and in the presence of control liposomes (L) at the indicated dilutions. Proliferation was measured on day 6 of culture by H-thymidine incorporation. (B) Cord blood mononuclear cells from two donors were cultured in the absence of stimuli (Neg) or in the presence of phytohaemag-glutinin (PHA), concanavalin A (ConA), IRIV (V) or L at the indicated concentrations. Proliferation was measured on day 3 of culture for PHA and ConA cultures and on day 6 for IRIV and L stimulated cultures. (Q Purified CD4+ or CD8+ cells were cocultured with autologous irradiated PBMC in the absence of stimuli (Neg) and in the presence of IRIV (V) at the indicated concentrations. Proliferation was measured on day 6 of culture by H-thymidine incorporation. (D) Purified CD4/CD45RA+ cells and CD4/CD45RO-I-cells were isolated from PBMC of one healthy donor and cocultured with autologous irradiated PBMC in the presence of IRIV (V) or L at the indicated concentration. Proliferation was measured on day 6 of culture by H-thymidine incorporation. Source From Ref 6.

With the exception of the effect on microtubules described in the foregoing paragraph, the bisindole alkaloids have little or no effect on macro-molecular synthesis at subtoxic concentrations (21,22). In experiments utilizing radiolabeled precursors ([ H]leucine, -uridine, or -thymidine) cells cultured in the presence of vinblastine showed no differential incorporation of radioactivity. Furthermore, there is no indication that treatment of cells with vinblastine or vincristine produces alterations in cellular DNA (23,24). 

Successful fusion (2) is a rare event, but the frequency can be improved by adding polyethylene glycol (PEG). To obtain only successfully fused cells, incubation is required for an extended period in a primary culture with HAT medium (3), which contains hypoxan-thine, aminopterin, and thymidine. Amino-pterin, an analogue of dihydrofolic acid, competitively inhibits dihydrofolate reductase and thus inhibits the synthesis of dTMP (see p. 402). As dTMP is essential for DNA synthesis, myeloma cells cannot survive in the presence of aminopterin. Although spleen cells are able to circumvent the inhibitory effect of aminopterin by using hypoxanthine and thymidine, they have a limited lifespan and die. Only hybridomas survive culture in HAT medium, because they possess both the immortality of the myeloma cells and the spleen cells metabolic side pathway. 

Fig. 13. Phytohemagglutinin (PHA) dose response curves of 10 patients with multiple myelomatosis and 10 controls. Peripheral blood lymphocytes were cultured and stimulated with PHA in increasing concentrations. Transformation was assessed by counting the uptake of radiolabeled thymidine.

Mutagenesis Entacapone was mutagenic and clastogenic in the in vitro mouse lymphoma/thymidine kinase assay in the presence and absence of metabolic activation, and was clastogenic in cultured human lymphocytes in the presence of metabolic activation. 

Cytotoxic effect. Gas phase of mainstream cigarette smoke, in monolayer culture of mouse lung epithelial cells, produced an increase in cytotoxicity in a dose-dependent manner. Cell viability of cultures exposed to gas phase with only the nonorganic components was equivalent to controls. Removal of volatile organic constituents resulted in almost elimination of cytotoxicity of the smoke . Smoke condensate and tobacco extract, at high concentrations in Lewis lung adenocarcinoma cells and mice spleen lymphocytes, were cytotoxic. Smaller doses increased thymidine incorporation in both cell types. Lymphocytes were more susceptible to the toxic effect of tobacco prod-... 

Oberly, T.J., Bewsey, B.J. Probst, G.S. (1985) Tests for the induction of forward mutation at the thymidine kinase locus of L5178Y mouse lymphoma cells in culture. Prog. Mutat. Res., 5, 569-582... 

Fig. 12. The relation between hepatocyte shape ( ), 3H-thymidine uptake ( ) expressed as ratio to hepatocytes cultured on polystyrene dishes coated with 1 pg/ml PVLA solution and hepatic function, bile acid secretion (A), expressed as ratio to bile add secretion of hepatocytes cultured on dishes coated with 1 pg/ml of PVLA solution (Reproduced from New Functionality Materials, Vol. B [Ref. 181] through the courtesy of Elsevier Sdence Publisher B.V.)...

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