“Suppressor factor” of neutrophils: A short story of a long-term misconception

A large body of evidence obtained during the last decade has demonstrated that neutrophils suppress T cell proliferation in different models of inflammation and cell interaction. The commonly used method for assessing cell proliferation and proliferation inhibition is measuring [³H]thymidine incorporation into cells. Earlier, we observed inhibition of [³H]thymidine uptake in experiments on neutrophil-mediated regulation of T cell response in tuberculosis immunity. Here, we used different types of proliferating cells to analyze the nature of the soluble “neutrophil factor” by a variety of methods (dialysis, HPLC, mass spectrometry, and NMR) and unambiguously demonstrated that neutrophils do not synthesize a specific factor inhibiting cell proliferation, but secrete high concentrations of extracellular thymidine that competitively inhibit [³H]thymidine incorporation. Although the physiological significance of thymidine secretion by neutrophils remains unknown, this phenomenon should be carefully considered when designing test systems for studying cell–cell interactions.     

Effective Specific Activity Dilution in Labeled DNA of Cultured Mammalian Cells (L5178Y)

Upon labeling the DNA of cultured mammalian cells with radio-active thymidine it was found that there was a large reduction in the specific activity of radioactive thymidine in DNA when this specific activity was compared to the specific activity of the exogenous supply. This reduction in specific activity may be determined once the effective dilution factor for thymidine in mammalian cells is known. The average effective dilution factor was found to be 2 × 10^-4 M for L5178Y cells in log growth.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Calculation of Cell Production from [3H]Thymidine Incorporation with Freshwater Bacteria

The conversion factor for the calculation of bacterial production from rates of [³H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20°C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 × 10¹⁸ cells mol^-1 of thymidine incorporated into the trichloroacetic acid (TCA) precipitate (standard error = 0.29 × 10¹⁸; n = 54), when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 × 10¹⁸ cells mol^-1 of thymidine incorporated into TCA precipitate (standard error = 1.72 × 10¹⁸; n = 47). The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20°C than at 15 and 10°C. A detailed examination of the [³H]thymidine concentrations that were needed to achieve maximum labeling in DNA was carried out 6 times during a complete growth cycle. During periods with low generation times and high conversion factors, 15 nM [³H]thymidine was enough for the maximum labeling of the TCA precipitate. This suggests that incorporation of [³H]thymidine into DNA is probably limited by uptake during periods with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 × 10¹⁸ cells mol^-1 of thymidine incorporated is used.     

The proliferative effects of retinoic acid on primary cultures of adult rat type II pneumocytes depend upon cell density

Retinoic acid (RA) is important for maintaining integrity of alveolar epithelial cells, but the mechanism has not been defined. We cultured type II pneumocytes at confluent, high cell density (10⁴ cells/mm²) and found that RA (10⁻⁶ M) inhibited thymidine incorporation to 60% of control, despite a dose-dependent increase in epidermal growth factor receptor (EGFR) levels. However, at lower, subconfluent density (10² cells/mm²), RA stimulated thymidine incorporation to 280% of control. EGF increased thymidine incorporation at concentrations as low as 0.1 ng/mL, but no further increase was observed at higher concentrations up to 100 ng/mL. In subconfluent cells co-treated with EGF (100 ng/mL) and increasing concentrations of RA (10⁻⁸ M–10⁻⁵ M RA), thymidine incorporation was significantly greater at all concentrations than RA alone, with greatest increases observed at 10⁻⁷ (422% of control) and 10⁻⁶ (470% of control) M RA. In summary, the effects of RA on thymidine incorporation are sensitive to changes in cell density. RA inhibits thymidine incorporation at high cell density and stimulates thymidine incorporation at low density. RA increases EGFRs in cultured type II pneumocytes, and EGF stimulates thymidine incorporation independent of the cultured cell density. These data may help to explain how RA mediates lung repair in vivo.     

Bacterial metabolism in the River Danube: parameters influencing bacterial production

1. Microbial parameters were determined at five sampling sites in the River Danube up-and downstream of Vienna, Austria, twice monthly over an annual cycle. Bacterial production (BP) was estimated from thymidine and leucine incorporations; additionally, the effect of turbulence on BP and the conversion factors for converting incorporation rates into bacterial cell production were determined using the cumulative approach. 2. BP under turbulent conditions was not significantly different from that under stagnant conditions. For thymidine, a mean annual conversion factor of 3.2 ± 10¹⁸ cells mol^?1 thymidine incorporated was calculated. For leucine, the corresponding factor was 0.07 ± 10¹⁸ cells mol^?1 leucine. Average annual BP calculated by thymidine incorporation was significantly higher than BP calculated from leucine incorporation and ranged from 47.2 to 77.5 μg C 1-^?1 day^?1 depending on the tracer and the conversion factor used. 3. Bacterial growth rates ranged from 0.1 day^?1 during winter to 1.7 day^?1 in the summer. A strong correlation was found between temperature as well as chlorophyll a and bacterial growth when temperature was greater than 5 °C; a major spring phytoplankton bloom at a temperature below 5 °C did not increase BP. 4. Dissolved organic carbon (DOC) concentrations varied between 2 and 7.2 mg C 1-^?1 and comprised between 50 and 92% of the total organic carbon pool in the River Danube, Based on the DOC concentration and an assumed bacterial growth yield of 20% we calculated mean DOC turnover times of around 60 days in the winter and less than 8 days during the summer.     

Comparison of Rates of Flagellate Bacterivory and Bacterial Production in a Marine Coastal System

Protozoan predation on bacteria and bacterioplankton secondary production were simultaneously determined in La Salvaje Beach water during 1990. Protozoan grazing on bacterioplankton was measured from fluorescently labeled bacterium uptake rates; estimates of bacterial secondary production were obtained from [³H]thymidine incorporation rates. Two different conversion factors were used to transform thymidine incorporation rates into bacterial production rates; both of them were specific for La Salvaje Beach and were calculated by using empirical and semitheoretical approaches. The average flagellate predation rate was 14.0 bacteria flagellate^-1 h^-1; the average population predation rate was 7.35 x 10⁶ bacteria liter^-1 h^-1. The estimates of bacterial production differed greatly depending on the conversion factor used, and so did the percentages of bacterial production consumed by flagellated protozoa (4.6% when the empirical conversion factor for La Salvaje Beach was used and 113% when the semitheoretical conversion factor specific for this system was used). The ecological implications of each of these values are discussed.     

Protozoan Grazing and Bacterial Production in Stratified Lake Vechten Estimated with Fluorescently Labeled Bacteria and by Thymidine Incorporation

In stratified Lake Vechten, The Netherlands, protozoan grazing was estimated on the basis of uptake of fluorescently labeled bacteria and compared with bacterial production estimated on the basis of thymidine incorporation. By using a grazer-free mixed bacterial population from the lake in continuous culture, an empirical relationship between cell production and thymidine incorporation was established. Thymidine incorporation into total cold-trichloroacetic-acid-insoluble macromolecules yielded a relatively constant empirical conversion factor of ca. 10¹⁸ (range, 0.38 × 10¹⁸ to 1.42 × 10¹⁸) bacteria mol of thymidine⁻¹ at specific growth rates (μ) ranging from 0.007 to 0.116 h⁻¹. Although thymidine incorporation has been assumed to measure DNA synthesis thymidine incorporation appeared to underestimate the independently measured bacterial DNA synthesis by at least 1.5- to 13-fold, even if all incorporated label was assumed to be in DNA. However, incorporation into DNA was found to be insignificant as measured by conventional acid-base hydrolysis. Methodological problems of the thymidine technique are discussed. Like the cultures, Lake Vechten bacteria showed considerable thymidine incorporation into total macromolecules, but no significant incorporation into DNA was found by acid-base hydrolysis. This applied not only to the low-oxygen hypo- and metalimnion but also to the aerobic epilimnion. Thus, the established empirical conversion factor for thymidine incorporation into total macromolecules was used to estimate bacterial production. Maximum production rates (141 × 10⁶ bacteria liter⁻¹ h⁻¹; μ, 0.012 h⁻¹) were found in the metalimnion and were 1 order of magnitude higher than in the epi- and hypolimnion. In all three strata, the estimated bacterial production was roughly balanced by the estimated protozoan grazing. Heterotrophic nanoflagellates were the major consumers of the bacterial production and showed maximum numbers (up to 40 × 10⁶ heterotrophic nanoflagellates liter⁻¹) in the microaerobic metalimnion.     

Activity of an Attached and Free-Living Vibrio sp. as Measured by Thymidine Incorporation, p-Iodonitrotetrazolium Reduction, and ATP/DNA Ratios

Three independent techniques, [³H]thymidine incorporation, the reduction rate of p-iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA, were adapted to measure the activity of attached and unattached estuarine bacteria. In experiments employing the estuarine isolate Vibrio proteolytica, nutrient concentrations were manipulated by varying the concentration of peptone-yeast extract. In the presence of exogenous nutrients, the activity of free-living cells was greater than that of attached cells as measured by [³H]thymidine incorporation and ATP/DNA ratios. In the absence of peptone-yeast extract, however, the activity of attached cells surpassed that of free-living cells as determined by [³H]thymidine incorporation and INT formazan normalized to DNA. Of the three techniques, [³H]thymidine incorporation was deemed most sensitive for detecting changes in activity resulting from slight differences in nutrient concentration. By this technique, attached cells were much less sensitive to changing nutrient concentrations than were free-living cells. Below a threshold concentration, attached cell activity remained constant, while the activity of unattached cells decreased as a function of decreasing nutrient concentration. The results suggest that loss of cell surface area available for substrate uptake due to attachment may be an important factor in determining the relative activities of attached and free-living cells.     

Direct cell culture assay of thin-layer-chromatographed material without prior substance elution

A method enabling the direct assay in a cell culture system of material separated by thinlayer chromatography (TLC) without prior elution of active material from the TLC sheet is described. After chromatographic development on a plastic cellulose sheet, small disks were punched out from the sheet and transferred to microwells used for cell culture. Lymphoid cells in culture medium were incubated with the disks and the DNA synthesis was measured by [³H]thymidine incorporation. The reliability of the method was tested with l-alanine and thymidine, which are known to enhance the DNA synthesis of lymphoid cells and to interfere with the uptake of labeled thymidine, respectively. The incorporation of [³H]thymidine was stimulated with disks containing l-alanine and inhibited with disks containing unlabeled thymidine, completely in agreement with the expected results. Elution experiments showed that more than 75% of l-alanine and thymidine on the TLC disks was eluted into the culture medium within 1 h. Elution of material of higher molecular weight was also demonstrated. The method was used for the separation and assay of a thymocyte-specific growth factor isolated from calf thymus and greatly facilitated the detection of the active material after TLC.     

Thymidine Metabolism and the Measurement of the Rate of DNA Synthesis in Carrot Suspension Cultures: EVIDENCE FOR A DEGRADATION PATHWAY FOR THYMIDINE

The kinetics of [³H]thymidine incorporation into the DNA of carrot suspension cultures were investigated. At a thymidine concentration of 0.1 micromolar, incorporation into DNA is not quantitative but ceases after only 14% of the thymidine has been incorporated. Thymidine incorporation into DNA is resumed following addition of a second aliquot of thymidine, which is consistent with substrate depletion. In vivo tracer experiments indicate that this may be due to a catabolic route for converting thymidine to β-aminoisobutyric acid. Bearing these observations in mind, conditions for determining the rate of DNA synthesis using [³H]thymidine incorporation have been investigated. It is concluded that by increasing the thymidine concentration to 10 micromolar the assay period may be increased, by reducing the influence of the degradative pathway, and that cell density and incubation time are critical factors in establishing a valid measure of the rate of DNA synthesis using this method.     

In vitro stimulation of human endothelial cells by derivatized dextrans

Summary Derivatized dextrans exert a stimulatory effect on the in vitro growth of human umbilical vein endothelial cells (HUVEC). Measurements of growth were monitored by [³H]thymidine uptake and cell numbers. Our results show that some derivatized dextrans at 4 μg/ml (88 nM) increase the [³H]thymidine incorporation, whereas starting dextran (40 000 Da), dextran sulfate, and carboxymethyl dextran have no effect. In addition, heparin under similar experimental conditions shows a slight inhibitory effect on the HUVEC growth. The stimulatory effect of derivatized dextrans was also found when HUVEC grew during 7 days in medium containing 2% fetal bovine serum. We also observed that derivatized dextrans had no effect on the mitogenic activity of acidic fibroblast growth factor, a mitogenic factor for several cell types including HUVEC. By assessment of [³H]thymidine uptake at 48 h without serum, we concluded that the exogenous growth factors were not involved in the proliferative activity of these components. The stimulatory effects are related to the chemical nature and the proportion of substituents on the synthetic polysaccharides. The data indicate that benzylamide sulfonated groups play a key role in the stimulation of HUVEC growth. Neither carboxyl nor sulfate groups alone exhibit this effect. Thus, the stimulatory capacity of dextran derivatives depends strongly on the respective ratios of the functional groups.     

Heart Mitochondrial TTP Synthesis and the Compartmentalization of TMP

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [³H]thymidine or [³H]TMP as precursors of [³H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [³H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [³H]TMP produced far less [³H]TTP than the amount observed with [³H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [³H]TTP from [³H]TMP was effectively blocked, demonstrating that synthesis of [³H]TTP from [³H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [³H]TMP to [³H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [³H]thymidine was readily converted to [³H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.     

Insulin-like Growth Factor I Regulation of Swarm Rat Chondrosarcoma Chondrocytes in Culture

Insulin-like growth factor I (IGF-I) is anabolic for chondrocytes and is thought to be important in regulating such normal cartilaginous tissues as the epiphyseal growth plate. In the present studies, we have investigated the role of IGF-I in the regulation of neoplastic cartilage. Chondrocytes cultured from a transplantable rat chondrosarcoma were analyzed for responsiveness to IGF-I with respect to DNA and glycosaminoglycan synthesis as determined by labeling with radioactive thymidine and sulfate, respectively. Stimulation of [³H]thymidine and [³⁵S]sulfate incorporation by IGF-I was two to four times that in serum-free controls, with half-maximal stimulation at 1 × 10^-9M. The efficacy of IGF-I was approximately one-half of that of serum in stimulating [³H]thymidine incorporation and was comparable to that of serum for [³⁵S]sulfate incorporation. When Swarm rat chondrosarcoma chondrocytes were cultured in the presence of IGF-I and exposed to graded concentrations of anti-IGF-I antibody, [³H]thymidine incorporation and [³⁵S]sulfate incorporation were attenuated in a dose-dependent fashion to 29 and 25% of antibody-free controls, respectively. Nonspecific antibody not raised against IGF-I was not inhibitory. These observations suggest that the majority of IGF-I action on these cells is susceptible to immunoinhibition. To estimate the contribution of IGF-I to the regulation of these cells by serum, Swarm rat chondrosarcoma chondrocytes were cultured with graded concentrations of either calf serum or fetal calf serum in the presence of anti-IGF-I antibody, nonspecific antibody, or no other additives. Specific antibody attenuated the effect of calf serum on both [³H]thymidine and [³⁵S]sulfate incorporation with overall inhibition of 52% (P < 0.01) and 48% (P < 0.001), respectively. Nonspecific antibody superimposed small, variably stimulatory or inhibitory effects on those of calf serum. When chondrosarcoma chondrocytes were incubated with fetal calf serum, anti-IGF-I antibody exerted a minimal inhibitory effect, reducing both [³H]thymidine and [³⁵S]sulfate incorporation by less than 25%. The immunoinhibition of both pre- and postnatal serum could be overcome in a dose-dependent fashion by increasing serum concentrations. These results suggest that the factors influencing Swarm rat chondrosarcoma chondrocytes may be developmentally regulated and that the contribution of IGF-I to the action of serum increases between fetal and postnatal life. These data support the hypothesis that chondrosarcoma is a somatomedin-responsive neoplasm and suggest that this tumor may be susceptible to interventions directed toward mechanisms that block insulin-like growth factor action.     

Sites of transcription of the Müllerian inhibiting substance gene in mouse testis

The factors involved in the inhibition of ovarian follicular cellular growth after the luteinizing hormone (LH) surge are poorly established. The aim of this study was to investigate the production of an inhibitory growth factor by human ovarian cells. Luteinized granulosa cells were obtained from an assisted fertilization program and were cultured in the presence or absence of follicle-stimulating hormone (FSH) and estradiol. Data obtained by cell counting showed that the number of human luteinized granulosa cells cultured in the presence of fetal bovine serum (10%) increased 1.8-fold within a 2-day period. In serum-free medium, human luteinized granulosa cells were able to incorporate ³H-thymidine, measured during consecutive 48 h periods. During all the periods tested (up to 7 days), low basal levels of thymidine incorporation were measured and were further reduced in the presence of FSH (200 ng/ml) and estradiol (500 ng/ml). To elucidate the possible production of an inhibitory growth factor, ³H-thymidine incorporation by rat granulosa cell cultures was measured in the presence of conditioned media (CM; from human granulosa cell cultures). In this system, FSH and estradiol elicited a tenfold increase in thymidine incorporation. The addition of CM (10% v/v collected on day 2) to FSH- and estradiol-treated granulosa cell cultures produced an inhibition (61%) of thymidine incorporation. The active factor in CM withstood freeze-thawing, was stable for several weeks at – 20°C, became unstable at 4°C, and was heat labile and sensitive to proteolysis. Ultrafiltration using membranes with different molecular weight cutoffs suggested that the factor had a molecular weight >30,000 dalton. We suggest that an inhibitory growth factor produced by human luteinized granulosa cells could be involved in the differentiation of growing follicles to corpus luteum. © 1993 Wiley-Liss, Inc.     

Comparison of effects of epidermal and insulin-like growth factors,gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10 ⁻⁹ M. At concentration 10 ⁻⁸ M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Measurement of bacterial growth rates on the rhizoplane using 3H-thymidine incorporation into DNA

Bacterial growth rates on the rhizoplane of rape seedlings grown in sand were determined using ³H-thymidine incorporation into DNA. Axenic roots incorporated thymidine into DNA, which had to be subtracted from values for roots with associated bacteria. Thymidine incorporation into rhizoplane bacterial DNA ranged between 0.6 and 1.4 pmol thymidine h^–1 root^–1 for 6 to 26-day-old plants. Using a conversion factor, the turnover time of bacteria was calculated to decrease from 9.2 h for 6-day-old plants to 160h for 26-day-old plants. A similar value was found for rhizosphere bacteria of plants grown for 26 days in natural soil.     

DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [³H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [³H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [³H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10⁶ cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.     

DNA Synthesis and Tritiated Thymidine Incorporation by Heterotrophic Freshwater Bacteria in Continuous Culture

Continuous cultivation of heterotrophic freshwater bacteria was used to assess the relationship between DNA synthesis and tritiated thymidine incorporation. The bacteria were grown on a yeast extract medium with generation times of 0.25 to 3.7 days. In six different continuous cultures, each inoculated with a grazer-free mixed bacterial sample from Lake Vechten (The Netherlands), tritiated thymidine incorporation into a cold trichloroacetic acid precipitate and bacterial cell production were measured simultaneously. Empirical conversion factors were determined by division of both parameters. They ranged from 0.25 × 10¹⁸ to 1.31 × 10¹⁸ cells mol of tritiated thymidine^-1 (mean, 0.60 × 10¹⁸ cells mol of tritiated thymidine^-1). In addition, DNA concentrations were measured by fluorometry with Hoechst 33258. The validity of this technique was confirmed. Down to a generation time of 0.67 day, bacterial DNA content showed little variation, with values of 3.8 to 4.9 fg of DNA cell^-1. Theoretical conversion factors, which can be derived from DNA content under several assumptions, were between 0.26 × 10¹⁸ and 0.34 × 10¹⁸ cells mol of thymidine^-1 (mean, 0.30 × 10¹⁸ cells mol of thymidine^-1). Isotope dilution was considered the main factor in the observed discrepancy between the conversion factors. In all experiments, a tritiated thymidine concentration of 20 nM was used. Control experiments indicated maximum incorporation at this concentration. It was therefore concluded that the observed difference resulted from intracellular isotope dilution which cannot be detected by current techniques for isotope dilution analysis.