Thymidine Incorporation Method Research Articles

Stimulation by interleukin-7 of mononuclear cells in peripheral blood, synovial fluid and synovial tissue from patients with rheumatoid arthritis.

To determine how interleukin-7 (IL-7) affects the proliferation of T cells in patients with rheumatoid arthritis (RA), we evaluated the response of mononuclear cells (MNC) obtained from their peripheral blood (PB), synovial fluid (SF) and synovial tissue (ST) to stimulation by recombinant IL-7 and interleukin-2 (IL-2). Each cytokine was administered alone or combined with phytohemagglutinin (PHA). Cellular DNA synthesis was assayed by the [3H]-thymidine incorporation method. The stimulatory effect of 500 u/ml IL-7 on PBMNC obtained from 19 patients with RA was significantly lower than on PBMNC from 19 healthy controls. However, the same degree of stimulatory activity of 500 u/ml IL-2 was observed on the PBMNC from both RA patients and control subjects. The response of PBMNC to a suboptimal dose of PHA (0.2 micrograms/ml) was enhanced by adding either IL-7 or IL-2 (100 or 500 u/ml) to the cultures. The enhanced synthesis of DNA by both RA and control PBMNC on exposure to IL-7 following stimulation by a suboptimal dose of PHA was higher than that of IL-2. The effect of IL-7 on RA PBMNC was significantly greater than that of IL-2 at the concentration of 100 u/ml on PBMNC from the same RA patients. The stimulatory activity of IL-2 at the concentrations of 100 and 500 u/ml on SF MNC and ST MNC exceeded that of IL-7. In particular, an IL-2 dose of 500 u/ml had a marked effect on SF MNC. The PHA response of SF MNC was the lowest seen among the MNC from three different compartments.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors controlling bacterial production in marine and freshwater sediments

We collected benthic bacterial production data measured by (3)H thymidine incorporation (TTI) (25 studies), frequency of dividing cells (FDC) (3 studies), dark-C02 assimilation (1 study) and (3)H-adenine uptake (2 studies) from the literature, which included 18 marine, 6 river, and 2 lake studies. In all of the studies that used the TTI method, (3)H-DNA was isolated and incubations were carried out at in situ temperatures. Most of the researchers also determined (3)H-DNA extraction efficiencies and isotope dilution, thus interpretable estimates of bacterial production were used in the analysis. In marine sediments, bacterial production rates were linked to bacterial biomass, bacterial abundance, sediment organic matter, temperature, and sediment chlorophyll a, with these variables explaining between 40% and 68% of the variation in production rates. Simple relationships between production and bacterial biomass or bacterial abundance, or between production and sediment organic matter, were improved by also including temperature in the analysis of marine sediments. Sediment organic matter explained an appreciable fraction (58%) of the observed production in freshwater sediments. Temperature was the most powerful predictor of the observed variability in specific growth rates (r (2) = 0.48 and r (2) = 0.58) in marine and freshwater sediments, respectively. Thus, bacterial production and specific growth rates are most closely linked to substrate supply and temperature in marine and freshwater sediments.

3 H]Thymidine Incorporation To Estimate Growth Rates of Anaerobic Bacterial Strains

The incorporation of [H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [H]thymidine during growth. It is concluded that the [H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.

Cell-growth quantitation methods for the evaluation of antiestrogens in human breast cancer cells in culture

The antiproliferative activity of the antiestrogen, tamoxifen, on the growth of MCF-7 human breast cancer cells was evaluated using the hemocytometric trypan blue exclusion method, [ 3H]-thymidine incorporation, and a total protein determination. Tamoxifen was evaluated over a concentration range from 10 −9 to 10 −6 M. The hemocytometric trypan blue exclusion method and [ 3H]-thymidine incorporation were sensitive enough to demonstrate the inhibitory influence of tamoxifen on the proliferation of MCF-7 cells at a concentration as low as 10 −9 M. A very good correlation of these two methods was observed in the submicromolar concentration range of tamoxifen. The total protein determination method was only sensitive enough to detect the antiproliferative influence of tamoxifen at concentrations above 10 −6 M. In conclusion, the [ 3H]-thymidine incorporation method was found to be effective and much less time consuming than the hemocytometric trypan blue exclusion method for evaluating the antiproliferative effects of antiestrogens in cultured MCF-7 cells. However, when evaluating antiestrogens, which are cell-cycle specific, the results of the [ 3H]-thymidine incorporation method should be interpreted with caution.

Dentin as inhibitor of bacterial toxicity on pulpal cells in vitro

Many studies have shown that the major cause of pulpal disease is the presence of bacteria or their by-products in the dentinal tubules. The purpose of this investigation was to develop an in vitro model, simulating the pulp chamber, that would permit the study of the transport of bacterial by-products through dentin and their effect on pulpal cells. Human pulpal cells were cultured in a modified Sykes-Moore chamber and exposed through dentin to sonicated extracts of Porphyromonas gingivalis ATCC 33277. The cell response was evaluated with the thymidine incorporation method. The results were compared with the cell response obtained after direct exposure to the same irritant. It was found that dentin significantly restricts the diffusion of bacterial proteins in a 24-h experimental period. The time needed for the first bacterial protein molecule to cross the dentin barrier was 6 h. The "diffusion velocity" of the bacterial proteins was 0.023 microns/s. The proposed model has further applications in biocompatibility and microleakage research.

Comparison of two different cell culture methods in evaluation of biocompatibility of latex urinary catheters

International biocompatibility standards for urinary catheters have not yet been set. The used in vivo animal tests earlier have been shown to be insensitive and expensive. The present study compared the sensitivity and other properties of two in vitro cell culture methods, the reference method of the British Standards Institution (BSI) and the thymidine incorporation method (DNA synthesis inhibition test. The cell culture toxicities of thirty-seven latex catheters were measured and 84.8% of the catheters tested passed the BSI reference test while 94.6% passed the thymidine incorporation test. The overall batch-to-batch correlation was poor (p greater than 0.05), but within brands the correlation was better. This is obviously to be explained by the differences the characteristics of the tests and/or the chemical composition of the catheters. The thymidine incorporation test is easier to perform, requires less manpower, and is therefore less liable to subjective interpretations than the BSI test. The biocompatibility toxicity limits for latex urinary catheters need to be tightened. Our experience in this study indicates that the thymidine incorporation test or tests similar to it, which can be highly automated can be recommended for biocompatibility screening in large series, and BSI reference test can be used additionally in unclear cases.

Decreased cell replication and polyamine content in insulin-producing cells after exposure to human interleukin 1 beta.

Interleukin 1 (IL-1) has been suggested to cause the islet B cell destruction occurring during the development of insulin-dependent diabetes mellitus. One mechanism by which B cell loss can be compensated for is via de novo formation of new cells through replication. In the present study the replicatory activity of cells in isolated rat pancreatic islets and in the insulin-producing cell line RINm5F has been assessed by [3H]thymidine incorporation methods after exposure to 1-25 U/ml of human recombinant IL-1 beta (rIL-1 beta). In the rat islets [3H]thymidine incorporation was decreased by 20% 5 h after exposure to 25 U/ml rIL-1 beta. A similar inhibition was also observed in islets exposed to 2.5 and 12.5 U/ml rIL-1 beta. In the RINm5F cells there was a dose-dependent inhibition of the cell replication to approximately 50% of the controls in cells exposed to 25 U/ml rIL-1 beta for 48 h. This was also accompanied by an increased cell death, as measured by trypan blue inclusion (controls 13% and rIL-1 beta treated cells 25%). The insulin content of the RINm5F cells was reduced by about 40% after a 48-h exposure to 25 U/ml rIL-1 beta. When the exposure of the RINm5F cells to rIL-1 beta was decreased to 24 h there was no increased cell death, but a reduced replicatory activity was still observed. rIL-1 beta decreased the cellular content of the polyamines spermidine and spermine.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and significance of β‐glucosidase activity in lake water

Kinetics of β‐glucosidase activity (βGlcA) and bacterial activity (glucose uptake, thymidine incorporation) and cell numbers were measured in the euphotic zone and in the water column of Pluβsee during spring phytoplankton bloom development and after its breakdown. Heterotrophic bacteria were the major producers of the enzyme. Activity of free β‐glucosidase, unassociated with microbial cells, was negligible. βGlcA displayed a distinct temporal and spatial distribution pattern in lake water. βGlcA was low when algal populations grew actively, but during the algal bloom breakdown βGlcA increased rapidly. The increase in βGlcA was proportional to the abundance of bacteria and to their heterotrophic uptake of glucose, as well as to bacterial production, measured by the thymidine incorporation method. In contrast with its response to pH, β‐glucosidase exhibited no obvious adaptation to ambient temperature of lake water. βGlcA produced by aquatic bacteria was under control of a repression‐induction mechanism, and synthesis was derepressed when the level of directly assimilable hexoses (glucose or galactose) fell below a critical level. The tight relationship between the rates of βGlcA and glucose uptake indicated the existence of a specific, coupled hydrolysis‐uptake system in lacustrine bacteria.

Estimates of bacterial production and mortality in the Ebro river (Spain)

In the terminal part of the Ebro river, bacterial production and mortality have been studied. Bacterial production was estimated by the thymidine incorporation method and from estimates of growth rate. Bacterial mortality and grazing were studied using differential filtration of a 3H‐labelled population. Production was estimated as about 0.15 times 109 bacteria/l/h and total mortality was about 0.016/h. Grazing accounted for about 56% of the total mortality processes.

Assessing Phytoplankton and Bacterioplankton Production During Early Spring in Lake Erken, Sweden

The spring development of both phytoplankton and bacterioplankton was investigated between 18 April and 7 May 1983 in mesotrophic Lake Erken, Sweden. By using the lake as a batch culture, our aim was to estimate, via different methods, the production of phytoplankton and bacterioplankton in the lake and to compare these production estimates with the actual increase in phytoplankton and bacterioplankton biomass. The average water temperature was 3.5 degrees C. Of the phytoplankton biomass, >90% was the diatom Stephanodiscus hantzchii var. pusillus, by the peak of the bloom. The C and O(2) methods of estimating primary production gave equivalent results (r = 0.999) with a photosynthetic quotient of 1.63. The theoretical photosynthetic quotient predicted from the C/NO(3) N assimilation ratio was 1.57. The total integrated incorporation of [C]bicarbonate into particulate material (>1 mum) was similar to the increase in phytoplankton carbon determined from cell counts. Bacterioplankton increased from 0.5 x 10 to 1.52 x 10 cells liter ( approximately 0.5 mug of C liter day). Estimates of bacterioplankton production from rates of [H]thymidine incorporation were ca. 1.2 to 1.7 mug of C liter day. Bacterial respiration, measured by a high-precision Winkler technique, was estimated as 4.8 mug of C liter day, indicating a bacterial growth yield of 25%. The bulk of the bacterioplankton production was accounted for by algal extracellular products. Gross bacterioplankton production (production plus respiration) was 20% of gross primary production, per square meter of surface area. We found no indication that bacterioplankton production was underestimated by the [H]thymidine incorporation method.

The radioactive thymidine incorporation method for the determination of antibiotic susceptibility of gram-negative bacilli.

The thymidine incorporation method was compared with the disc diffusion and broth dilution methods for determining the antibiotic susceptibility of Escherichia coli (233 strains), Proteus mirabilis (72 strains) and Klebsiella species (189 strains). The method measures changes in the rate of DNA synthesis of bacterial strains exposed to an antibiotic by comparing the amount of radioactive thymidine incorporated by these strains in relation to control strains. Good agreement was obtained between the results of the thymidine incorporation and disc diffusion methods for all antibiotics tested except streptomycin and tetracycline. Minimal inhibitory concentrations of antibiotics determined by the thymidine incorporation method after 3 and 5 hours were generally lower than those obtained after 24 hours by the conventional broth dilution procedure. The thymidine incorporation method may be potentially useful for determining the antibiotic susceptibility of bacteria a few hours after inoculation of a clinical specimen.

Bacterial productivity in the water column and sediments of the Georgia (USA) coastal zone: Estimates via direct counting and parallel measurement of thymidine incorporation

Three methods of estimating bacterial productivity were compared using parallel samples of Atlantic Ocean water (within 0.25-15 km of the Georgia coast). The frequency-of-dividing cells (FDC) method and the [(3)H]thymidine incorporation method gave results which were strongly correlated (r=0.97), but the FDC estimates were always higher (X2 to X7) than the [(3)H]thymidine estimates. Estimates of bacterial productivity ranged from 2-4×10(8) cells·l(-1)·h(-1) at 0.25 km from shore to 1-9×10(7)cells·l(-1)·h(-1) at 15 km. A method involving incubation of 3-μm filtrates and direct counting gave results that could not be easily translated into estimates of bacterial productivity. Application of the FDC method to sediment samples gave high productivity estimates, which could be not reconciled with productivity estimates based on sediment oxygen uptake.

Response of sheep lymphocytes to PHA: quantitation by nuclear volume measurement and cell counts.

Phytohemagglutinin response of peripheral blood lymphocytes (PBL) of sheep was studied. Assessment of proliferative response was performed by determination of nuclear volumes and cell counts in cultures from 14 sheep and by incorporation of tritiated thymidine in cultures in four additional sheep. PBL of sheep were found to transform and proliferate with PHA similarly to human peripheral blood lymphocytes with minor differences. Quantitation of the proliferative response by determining the cell count and nuclear volumes provided more information on cell kinetics in culture than the commonly used isotope-labeled thymidine incorporation method.