Unscheduled incorporation of thymidine in ultraviolet-irradiated human lymphocytes

The nature, degree, and kinetics of unscheduled thymidine incorporation previously shown to occur in 90 % of irradiated lymphocytes was stud-incorporation was sever ely depressed i n t h e presence of 10(-4) M acriflavine and by low temperature, but was unaffected by 10(-3) M hydroxyurea or caffeine. Over a dose range of 25 to 400 ergs/mm2, the uptake of thymidine was increased by a factor of only 1.6, although the survival of lymphocytes, measured 5 days after irradiation, decreased by almost two orders of magnitude. (The survival curve suggests that 90% of the lymphocytes have a D0 of 35 ergs/mm(2) and 10 % have a D0 of 250 ergs/mm(2).) After exposure to 25 ergs/mm(2), over 70 % of the cells survived for 5 days in culture; moreover, cells which had been stimulated by this dose to incorporate thymidine transformed and divided after exposure to phytohema-glutinin. The final uptake of thymidine was significantly greater when a total dose of 75 ergs/mm(2) was fractionated into three doses of 25 ergs/mm(2) given at six hourly intervals than when it was given as a single dose. The degree of thymidine incorporation and the fraction of leukemic cells labeled were not significantly different from those in normal lymphocytes.     

Effect of serum fractions on the incorporation of tritiated thymidine in activated lymphocytes: I. Human serum from normal adult subjects

Fractionation of normal human adult serum using Centrisart or gel filtration at neutral pH shows that serum thymidine activity, measured as the ability to stimulate the 3H-thymidine uptake into lectin-activated lymphocytes, is represented by several molecular forms distinct from the endogenous Sm-C. The main thymidine activity is found in the 50-70 K range.     

Trypsin-treated serum potentiates thymidine incorporation into concanavalin a stimulated thymocytes

When either heat-inactivated rat serum or culture medium supplemented with 5% rat serum is incubated with trypsin at 37° for 1–16 hours, activity is generated which potentiates the incorporation of (³H) thymidine (³H-TdR) into concanavalin A (con A) stimulated rat thymocytes. Evidence is presented that the potentiating activity is not due to residual trypsin following its sequential inactivation with soybean trypsin inhibitor (STI) and phenylmethylsulfonyl fluoride (PMSF).     

Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization

Factor D, a DNA binding protein that enhances the activities of diverse DNA polymerases with a common restricted set of templates, was initially characterized in mouse liver but has resisted extensive purification. In this paper, we report that a similar stimulatory activity can be obtained in highly purified form from nuclei of rabbit hepatocytes. The rabbit liver protein increases the rates at which several DNA polymerases copy sparsely primed natural DNA templates and primed synthetic poly(dT), but it has no effect on the rates of copying of activated DNA or of poly(dG), poly(dA), and poly(dC). Direct binding of the purified stimulatory protein to an oligomer that contains a (dT)16 base stretch is visualized by retardation of the nucleoprotein complex on nondenaturing electrophoretograms. In the presence of the enhancing factor, Michaelis constants, Km, of responsive polymerase for singly primed bacteriophage M13 DNA and for poly(dT), but not for poly(dA), are decreased. Product analysis of M13 DNA primer extension indicates that the rabbit factor augments the apparent processivity of DNA polymerase by decreasing the extent of enzyme pausing at a tract of four consecutive thymidine residues in the template. Gel filtration of the native stimulatory protein yields an apparent relative molecular size of 58 +/- 2 kilodaltons. Stimulatory activity is readily inactivated by heat or by trypsin digestion, but it is resistant to micrococcal nuclease, N-ethyl-maleimide, or calcium ions.     

Effect of serum fractions on the incorporation of tritiated thymidine in activated lymphocytes: II. Comparison between cord blood and the blood of newborn infants

After serum Sephacryl 300 gel filtration, the major circulating forms of TA in serum are macromolecular. However different patterns are found in cord blood and in the blood of newborns at 24 hr of life. The low values found in cord blood are not due to inhibiting factor(s). TA in serum fractions does not parallel immunoreactive Sm-C levels. The pattern of immunoreactive Sm-C are similar in both cord blood and newborn blood.     

Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

Lymphocyte transformation in vitro : V. Thymidine incorporation into unstimulated lymphocytes

In vitro conditions and kinetics of ¹⁴C-thymidine incorporation into unstimulated lymphocytes were studied. Lymphocytes cultured during 3 to 9 days displayed a progressive increase of thymidine uptake with time. The addition of varying numbers of autologous mitomycin-treated lymphocytes to cultures containing a fixed number of untreated lymphocytes markedly enhanced thymidine uptake per non-mitomycin-treated lymphocytes. In the latter cultures a sharp rise of thymidine uptake between the 7th day of culture was seen. Supernatants of non-stimulated lymphocytes, whether mitomycin-treated or not, showed no stimulating effect on autologous normal cells in these experiments. Although the mechanism of the enhancing effect of mitomycin-treated autologous lymphocytes remains unclear, it obviously may interfere in culture experiments in which the antigen specific stimulatory effect of mitomycin-treated cells is measured, which can only be detected by including the appropriate controls.     

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.     

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.     

Design and synthesis of boronic-acid-labeled thymidine triphosphate for incorporation into DNA

The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.     

The N-terminal hexapeptide fragment of IGF II stimulates thymidine incorporation into fibroblasts.

We synthesized the N-terminal hexapeptide fragment of IGF II to study potential binding to NMDA receptors in analogy to the N-terminal tripeptide of IGF I. The amino acid sequence of the hexapeptide is furthermore identical with the C-terminal sequence of the casiragua insulin B chain. The hexapeptide did not bind to the NMDA receptors, but was found to promote [3H]-thymidine incorporation into fibroblasts at concentrations of 10(-8) - 10(-5) M in a dose-dependent manner. Since [125I]-hexapeptide did not bind to IGF receptors, indirect competition studies using either labelled IGFs or insulin had to be used. The competition of hexapeptide at a concentration of 10(-5) M with labelled IGF I or II was about equal to that of 10(-9) M IGF I or II. IGF receptors were apparently up-regulated by the hexapeptide, as has also been described for insulin. When using casiragua insulin as labelled ligand, IGF II and casiragua insulin competed with equal potency, whereas the hexapeptide at 10(-7) M caused an apparent up-regulation of the casiragua insulin binding sites. Our results that the hexapeptide stimulates [3H]-thymidine incorporation and up-regulates IGF II and casiragua insulin binding sites may be connected to one or several of the following findings: the hystricomorph insulins--of which the casiragua insulin is a member--stimulate DNA synthesis to a greater extent than other insulins; the insulin and type 1 IGF receptor binding regions are localized predominantly in the C-terminal region of the insulin B chain; and the "cooperative" site regulating the affinity of the insulin receptor is also located in the C-terminal region of the insulin B chain. Further experiments will be needed to clarify the exact mechanism.     

Stimulating action of human serum on the incorporation of tritiated thymidine in human lymphocytes activated by lectins: demonstration of the GH-dependency and its limitations

Using thymidine uptake by activated human lymphocytes to measure the growth-stimulating activity of human serum, the authors demonstrate that thymidine activity was significantly correlated (p less than 0.001) with GH levels. r = 0.729 when GH values are below 3.5 ng/ml, r = 0.509 when GH values are below 8 ng/ml and r = 0.337 for all of the patients. Low levels of GH are sufficient for the generation of this activity.     

Brugia pahangi: uptake and incorporation of adenosine and thymidine.

The uptake and incorporation of adenosine and thymidine by infective larvae, 10-day-old juvenile, and adult stages of Brugia pahangi were investigated using scintillation counting and autoradiographic techniques. No evidence of thymidine incorporation by the worm was obtained in this study. Scintillation counting methods demonstrated that ¹⁴C-labeled adenosine was incorporated by all three stages of this filarial worm. Autoradiography, performed on worms incubated in [³H] adenosine from 5 min through 2 hr, revealed that following 5–15 min incubation the greatest degree of adenosine incorporation occurred in the hypodermis and somatic cords. Adenosine incorporation into the deeper body tissues, including the gut, increased significantly with longer periods of incubation. The results obtained further support the concept that nutrient uptake in B. pahangi occurs by a transcuticular route.