H-thymidine

Human tissues can synthesize purines and pyrimidines from amphibolic intermediates. Ingested nucleic acids and nucleotides, which therefore are dietarily nonessential, are degraded in the intestinal tract to mononucleotides, which may be absorbed or converted to purine and pyrimidine bases. The purine bases are then oxidized to uric acid, which may be absorbed and excreted in the urine. While little or no dietary purine or pyrimidine is incorporated into tissue nucleic acids, injected compounds are incorporated. The incorporation of injected [ H] thymidine into newly synthesized DNA thus is used to measure the rate of DNA synthesis. 

DNA synthesis assays. DNA synthesis was monitored by incorporation of H-thymidine into TCA precipitable material as described by McCaffrey and Rosner (32),... 

To ensure that the inhibition of EGF binding by palytoxin was not a consequence of cell toxicity, the effect of palytoxin on DNA synthesis in Swiss 3T3 cells was monitored. When cells were incubated in the presence of palytoxin, 10% fetal calf serum, and H-thymidine for 19.5 hr, no depression in the extent of H-thymidine incorporation into DNA was detected up to 3.7 pM palytoxin (Table I). Although 11 pM palytoxin was toxic when present for a prolonged period, under the conditions of the assays described above no toxicity was detected (Table I). When cells were incubated in the presence of palytoxin, 0.1% fetal calf serum, and H-thymidine, palytoxin did not stimulate significant incorporation of H-thymidine into DNA. Thus, although it can modulate the EGF receptor system under these conditions, palytoxin alone does not appear to be mitogenic for Swiss 3T3 cells. 

Confluent Swiss 3T3 cells were serum-starved by incubation for 48 hr in DME containing 0.1% PCS. Cells were then incubated with the indicated compound at 37 C in the presence of H-thymidine, 0.1 or 10% PCS for 19.5 hr, washed, and then assayed for H-thymidine incorporation into DNA as described in Methods. 

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... 

Both H-thymidine incorporation and radiolabeled leucine incorporation techniques have been recently used to determine bacterial activity and growth in the rhizosphere of barley seedling (28), Bacteria were initially released from the rhizosphere using homogenization and centrifugation before adding the labeled substrates. The cell incorporation rate was twice as high in the rhizosphere than in bulk soil. In addition, both the leucine and thymidine incorporation rates increased with the distances from the root tip (28). 

Pennanen T (2001) Microbial communities in boreal coniferous forest humus exposed to heavy metals and changes in soil pH a summary of the use of phospholipids fatty acids, Biolog and H-thymidine incorporation methods in field studies. Geoderma 100 91-126... 

Heilman B, Brandt I. 1986. Effects of carcinogenic halogenated aliphatic hydrocarbons on [ H] thymidine incorporation into various organs of the mouse A comparison between 1,2-dibromoethane and 1,2-dichloroethane. Mutat Res 163 193-199. 

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.

Verhoef, J., Petersen, P. K and Quie, P. G. (1977) Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes a quantitative assay using [ H]thymidine labeled bacteria. J. Immunol. Methods 14, 303-311. 

Administration of resorcinol (0.25% in the diet) for 20 weeks did not induce hyperplasia or papillomatous lesions in the forestomach in Syrian golden hamsters. Labelling index, after an intraperitoneal dose of /m67/7v7- H]thymidine, was not elevated in the pyloric region, forestomach or urinary bladder (Hirose et al., 1986). 

Effects of Major Human Brain Gangliosides on H-Thymidine Incorporation (CPMxlO ) Into PBMC In Response to Con-A. 

Effects of Minor Human Brain Gangliosides on H-Thymidine... 

Effects of Adding Gangliosides at Various Times After Culture Initiation on Incorporation of H-Thymidine (CPMxlO 4) Into PBMC in Response to Con-A and Poke Weed Mitogen3... 

Comparison of the Effects of Gangliosides. Liposomal-Bound Gangliosides, and Liposomes Added at Various Time Intervals on - H-Thymidine Incorporation (CPMxlO J) Into PBMC in Mixed Leukocyte Cultures... 

Control cultures consisted of PBMC preincubated in RPMI 1640 plus 6% FBS (Media). The PBMC were then removed, washed twice and recultured with or wi hout 18 microgms Con-A in microtiter plates. 96 hours later, H-thymidine incorporation was measured. 

The megaloblastic response to vitamin B12 deficiency seems to be unique to human beings deficient animals develop neuropathy, but have unimpaired hemopoeisis. It may be that human beings are more reliant on the de novo synthesis of TMP and less able to salvage it from DNA breakdown than other species. The normal suppression of the incorporation of [ H] thymidine into DNA by added dUMP (Section 10.3.3.3) is less than 3% in the fruit bat, 23% in the rat, and 65% in humans. 

Cells that have been preincubated with deoxyuridine, then exposed to [ H] thymidine, incorporate little or none of the labeled material into DNA. This is because of both dilution of the labeled material in the larger intracellular pool of newly synthesized TMP and also inhibition of thymidylate kinase by thymidine triphosphate. 

In normal cells, the incorporation of [ H] thymidine into DNA after preincubation with dUMP is 1.4% to 1.8% of that without preincubation. By contrast, cells that are deficient in folate form little or no thymidine from dUMP and incorporate nearly as much of the [ H] thymidine after incubation with dUMP as they do without preincubation. 

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