Inorganic Phosphorus Stimulation of Bacterioplankton Production in a Meso-Eutrophic Lake

Experiments were conducted to determine whether production of heterotrophic bacterioplankton in a small meso-eutrophic lake was influenced by the dissolved inorganic phosphorus (DIP) supply. DIP may indirectly limit bacterial production by limiting phytoplankton, which in turn may limit the carbon available to bacteria. Direct DIP limitation of bacteria occurs where the availability of DIP for bacteria is insufficient to maintain growth. This work examined direct DIP limitation of bacteria by removing phytoplankton and incubating flasks with or without added P in the dark. Bacterial production was measured via the rate of incorporation of [³H]thymidine ([³H]TdR) into DNA. Bacterial abundance was followed with epifluorescent direct counts. Rates of [³H]TdR incorporation were significantly greater in flasks with added DIP, and changes in cell abundances generally paralleled increases in [³H]TdR incorporation. Even very small additions of P (0.05 μM) were sufficient to stimulate production. DIP addition to whole lakewater also stimulated [³H]TdR incorporation relative to that in zero-addition controls, but there was not a concurrent increase in bacterial cell numbers. The stimulation of [³H]TdR incorporation after DIP addition to whole lakewater was significantly less than the stimulation due to DIP addition to 1-μm-pore-size-filtered lakewater. In this study, addition of DIP caused as much as an eightfold stimulation of [³H]TdR incorporation.     

Preparation, characterization, and functions of rabbit lymph node populations. III. Antigen-induced uptake of thymidine and dibutyryl cyclic AMP: inhibition of thymidine uptake by cholera toxin and dibutyryl cyclic AMP.

Keyhole limpet hemocyanin (KLH)-primed lymph node cell (LNC) populations were incubated with various amounts of KLH and the cellular incorporation of tritiated thymidine ([³H]TdR) or tritiated N⁶, O^2′ dibutyryl cyclic AMP ([³H]DbcAMP) was determined. T LNC responded more vigorously than did complement receptor lymphocytes (CRL), i.e., B cells, at all KLH concentrations, during all time intervals examined, and in the presence or absence of normal rabbit serum (NRS). The depletion of adherent cells from KLH-primed LNC resulted in no significant decrease in KLH-induced incorporation of either [³H]TdR or [³H]DbcAMP in any of the LNC populations. Thus it appeared that variation among LNC populations in the incidence of macrophages did not account for the marked variation in their responses. Cultures containing equal numbers of T and CRL were induced to incorporate more [³H]TdR or [³H]DbcAMP than either population cultured separately or the sum of their individual responses. It was concluded that KLH-induced incorporation of these substances into primed, isolated LNC, was primarily manifested in the T-cell population. The synergism seen in cultures containing mixtures of T and CRL suggested that B cells are induced to incorporate [³H]TdR or [³H]DbcAMP in the presence of antigen and T-cell product(s). KLH-induced incorporation of [³H]TdR into KLH-primed LNC was inhibited by cholera enterotoxin (CT) and DbcAMP as previously reported. However, CT or DbcAMP inhibited this incorporation into T LNC to a greater extent than into CRL or unfractionated LNC.     

Measuring bacterial production via rate of incorporation of [3H]thymidine into DNA

Thymidine (TdR) incorporation into DNA as a measure of bacterial production in environmental samples relies on assumptions about what organisms incorporate exogenous thymidine, extent of dilution of labelled thymidine by internal and external pools, and analytical methods for recovery and purification of bacterial DNA. We have examined these assumptions with regard to the feasibility of using [³H]TdR incorporation in the water column and sediments of a blackwater river. The extent of dilution of added [³H]TdR may be determined with isotope dilution plots (Moriarty and Pollard, 1981 and 1982) and these indicate a wide range of degree of participation of added [³H]TdR. Previously described methods for extracting DNA from sediment bacteria may lead to underestimates and we described a more efficient recovery scheme.     

LOW LEVEL INCORPORATION OF TRITIATED THYMIDINE INTO THE NUCLEAR DNA OF PURKINJE NEURONS OF ADULT MICE

Adult mice were pulse labeled with tritiated thymidine [³H]TdR and killed 9 hr later. A low level incorporation of [³H]TdR into the nuclear DNA of Purkinje neurons was found in autoradiographs. Enzymatic digestions with DNase and with RNase in combination with autoradiographic grain counts indicate that a portion of nuclear DNA is not stable in the Purkinje nucleus. These results are discussed in light of reports of the stable nature of DNA in Purkinje neurons of adult mice.     

Electron microscope analysis of amplifying ribosomal DNA from Xenopus laevis.

The inhibition of human prostatic epithelial cell (MA-160) replication by cAMP and certain analogs was explored in tissue cultures. When untreated fetal bovine serum was used to supplement the culture medium, cyclic AMP (cAMP) markedly inhibited cell growth. The inhibition was reversed by equimolar concentrations of uridine. Inhibition by 8-methyl-thio-cAMP (MES) was somewhat less effective and was not reversed by uridine. After heat treatment of the fetal bovine serum, which inactivated the cAMP phosphodiesterases, cAMP became less effective in cell growth inhibition, whereas the activity of MES remained unaltered. Dibutyryl cAMP (db-cAMP) had no effect on cell growth, however, when combined with the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), significant retardation of cell replication was observed. Cells treated for 24 h with 0.5 mM MES took up and incorporated significantly less [³H]TdR and [³H]uridine than control cells. Treatment of cells with 0.5 mM cAMP for 24 h, on the other hand, resulted in both substantially increased [³H]TdR uptake and increased [³H]uridine incorporation into RNA. The effects of similar treatment with db-cAMP plus MIX closely paralleled those of MES with marked inhibition of the uptake and incorporation of both thymidine and uridine.     

Endogenous Regulation of Endothelial Cell Proliferation

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [³H]TdR incorporation as well as their proliferation. the inhibition is dose- and time-dependent; maximum inhibition of [³H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 μg/ml and reaches a maximum at 600 μg/ml. the blockage of [³H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 μg of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.     

FAILURE TO IDENTIFY A SPERMATOGONIAL CHALONE IN ADULT IRRADIATED TESTES

The adult irradiated rat testis was used as a model system to confirm the existence of a spermatogonial chalone. Rats were given 330 rad whole body ⁶⁰Co irradiation, a dose which selectively destroys most of the spermatogonial population except for the radioresistant A_s stem cells. 11 days after irradiation, when spermatogonial numbers were minimal, the rats were injected with a testicular or liver extract prepared from normal adult rats, or with saline. Each group received a total of four injections given at 4 hr intervals. 2 hr before death, the animals were injected with [³H]TdR. Testicular DNA was isolated and the incorporation of [³H]TdR was determined. The mean ± s.e. ct/min per μg DNA in rats given testicular extract (9·38 ± 0·94) was no different than in those receiving liver extract (10·43 ± 2·01) or saline (7·23 ± 0·69). Autoradiographic studies indicated that variability in counts within or between groups could be attributed to variations in the number of pre-leptotene spermatocytes which incorporated [³H]TdR for the meiotic divisions. Quantitatively, there were no differences between groups in terms of the numbers of A spermatogonia, their labelling indices, or mitotic activity. Therefore, the presence of a spermatogonial chalone could not be demonstrated using crude extracts from normal testes in this irradiated model.     

EFFECT OF TRITIATED THYMIDINE ON THE KINETICS AND VIABILITY OF 9L CELLS IN VITRO

The colony-forming efficiency of 9L rat gliosarcoma cells was unaffected by treatment with 0.1 μCi/ml of [³H]TdR. However, when cells were treated with 1 or 10 μCi/ml of [³H]Tdr, cell growth was reduced and cell survival decreased. When monolayer 9L cells were treated with 1 μCi/ml of [³H]TdR for up to 72 hr, approximately 5% survived, which is closely related to the percentage of non-cycling cells in this system. When cells were treated with 10 μCi/ml of [³H]TdR for 72 hr, less survival was observed. the additional cell kill observed may be induced by [³H]TdR released from doomed cells into petri dishes during the incubation period of the colony-formation assay.     

Underestimation of DNA Synthesis by [3H]Thymidine Incorporation in Marine Bacteria

A direct comparison of [³H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [³H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [³H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [³H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [³H]thymidine incorporation and isotope dilution assays.     

Digestive Tract Cell Proliferation and Food Consumption Patterns of Ha/Icr Mice

The relationship between the daily pattern of food consumption and the proliferation rate of the qesophagus, stomach, forestomach, small intestine and colon of Ha/ICR mice was examined. Proliferative activity was determined by [³H]TdR incorporation on a wet weight tissue basis, along with selective counting of labelled nuclei. Under conditions of ad libitum feeding with a 12 hr light cycle (lights on at 0600) mice eat most of their food during the dark period. A distinct circadian rhythm was observed in the oesophagus, stomach, forestomach and colon with the peak of [³H]TdR incorporation between 0400 and 0600 and the nadir between 1600 and 1800. Although a circadian fluctuation was observed in the small intestine, its amplitude was much less than in other areas. This rhythmic change in proliferation rate could be phase shifted by allowing the mice to feed only between 0800 and 1600 for 14 days. Under these conditions the peak in proliferative activity occurred between 1800 and 2000. Fasting reduced the daily level of proliferative activity in all of the digestive tract sites studied, and for all areas except the oesophagus greatly reduced or eliminated the circadian fluctuation. the forestomach and colon were the most influenced by fasting with 24 hr [³H]TdR incorporation reduced to 30–40% of the control value. Refeeding following a 48 hr fast produced a rapid increase in proliferative activity peaking at levels well above the control value at 16 hr after the onset of refeeding. the major exception to this was the small intestine which slowly returned to the control value during the first 24 hr. Partial refeeding produced a diminished refeeding response. Once the normal pattern of food consumption was re-established following refeeding the normal proliferative fluctuations were again observed.     

Interactions of Phospholipid Vesicles with Murine Lymphocytes

The effect of unilamellar lipid vesicles composed of dioleoyl lecithin (DOL), egg yolk lecithin (EYL), 1:1 EYL:cholesterol (Chol), dipalmitoyl lecithin (DPL), and dimyristoyl lecithin (DML) on the mitogenic response in mouse lymphocytes was tested. Cortisone-resistant thymocytes were briefly treated with lipid vesicles and subsequently stimulated with concanavalin A (con A). All of the lipid vesicles induced an enhanced mitogenic response on day 3 as tested by [³H]TdR incorporation and by counting total cells. The order of enhanced [³H]TdR incorporation (?5.3 times the control) was DML>DPL>1:1 EYL:Chol>EYL?DOL> untreated control cells. These increases were paralleled by increased numbers of total cells. The response of spleen cells to a B-cell mitogen, bacterial lipopolysaccharide, was similarly enhanced by vesicle pretreatments in the same order. Vesicle treatments alone were not mitogenic.

Pretreatment of cells with lipid vesicles modified lectin binding: DML and DPL increased the binding of [¹²⁵I]con A by three to four times the control, whereas 1:1 EYL:Chol, EYL, or DOL had little or no effect. The binding of [125I]phytohemagglutinin-P (PHA-P) to vesicle-treated cells was indistinguishable from untreated cells. The lectin (con A; PHA-P)-induced agglutination of vesicle-treated cells was also modified by different lipid vesicles in the same order as the mitogenic response.

Based on the results presented in the accompanying report [6], we find that the cell surface adsorption properties of the applied lipid vesicles correlate with their ability to enhance the mitogenic response, and that they modify agglutinability and lectin binding. These results are further discussed in terms of the possible alteration of membrane properties and subsequent cellular activity.     

SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([³H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([³H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [³H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G₁ because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.     

Effects of epidermal growth factor on deoxyribonucleic acid synthesis and stomach weight in hypophysectomized adult mice

Epidermal growth factor (EGF) or saline was administered intraperitonally to hypophysectomized adult male CD2F₁ mice or intact controls at 0700 hr. Subgroups of mice were killed at 4, 8, or 12 hr after injection. EGF was shown to stimulate [³H]TdR incorporation into DNA into several organs as previously reported. The response to EGF was found to be enhanced in both hypophysectomized and fasted mice. Differences in [³H]TdR incorporation into DNA, corneal epithelium mitotic index, RNA in pancreas and kidney of hypophysectomized and intact mice are reported. EGF was shown to result in stomach enlargement due to increased luminal contents in both hypophysectomized and intact mice.     

Different Sensitivities of Granulocyte-Macrophage and Erythroid Progenitor Cells of Normal Human Bone Marrow to S-Phase-Specific Agents

The extraordinary sensitivity of early erythroid progenitor cells (BFU-e) of normal human bone marrow to tritiated thymidine ([³H]TdR) was studied. While exposure of bone-marrow cells to [³H]TdR for 1 hr resulted in the death of only 40% of the granulocyte-macrophage progenitor cells (CFU-c), 90% of BFU-e were killed. Experiments in which normal bone-marrow cells were mixed with bone-marrow cells which had been exposed to [³H]TdR demonstrated that the excessive killing of BFU-e by [³H]TdR reflected carry-over of the [³H]TdR by the exposed cells. A carry-over effect was not observed for CFU-c, suggesting the presence of a fundamental difference in the metabolism of TdR between CFU-c and BFU-e. There was a suggestion of a carry-over effect regarding two other S-phase-specific agents, hydroxyurea and 1-β-D-arabinofuranosylcytosine.     

DIFFERENT LABELLING PATTERNS IN MOUSE LYMPHOID TISSUES WITH [3H]DEOXYCYTIDINE AND [3H]THYMIDINE

The percentages of labelled lymphocytes in smear preparations of mouse thymus were higher than those in similar preparations of mesenteric lymph nodes with either generally labelled tritiated deoxycytidine, [³H]CdR, or tritiated thymidine, [³H]TdR. Lymphocytes in the thymus cortex and in germinal centres of mesenteric lymph nodes were intensely labelled with [³H]CdR, whereas with [³H]TdR lymphocytes in the peripheral region of thymus and medullary cords of mesenteric lymph nodes were heavily labelled. The majority of lymphocytes in thymic cortex and germinal centres of mesenteric lymph nodes were labelled weakly with [³H]TdR. Thus, labelling patterns with [³H]CdR differed from those with [³H]TdR in lymphoid tissues of the mouse. Mouse lymphocytes can utilize [³H]CdR as a precursor molecule for cytosine and thymine in DNA. The ratio of radioactivity of thymine to that of cytosine was measured biochemically in DNA extracted from lymphocytes labelled with [³H]CdR. This radioactivity ratio in thymus was higher than that in mesenteric lymph nodes. These results suggest that the metabolic activities of utilizing CdR for DNA synthesis differ within lymphocyte populations in various lymphoid tissues in the mouse.     

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.     

CELL POPULATION KINETIC PROFILE OF THE MOUSE THYMUS, AND THE CHANGES INDUCED BY PREDNISOLONE

The cell population kinetic parameters of the thymus in BALB/c mice have been estimated using stathmokinetic and [³H]TdR techniques in both control animals and animals treated with prednisolone. FLM data were analysed by computer using the Gilbert program. The study showed that prednisolone had an inhibitory effect mainly in the DNA synthesis phase and in G₁. Stathmokinetic data also showed a decrease in the cell birth rate and an increase in the apparent cell cycle time (or potential doubling time) after treatment. The labelling index, the mitotic index and the growth fraction were also decreased. The study also shows a good agreement between the data obtained by stathmokinetic and [³H]TdR techniques.     

Use of Radiolabelled Thymidine and Leucine To Estimate Bacterial Production in Soils from Continental Antarctica

Tritiated thymidine incorporation (TTI) into DNA was used to examine bacterial production in two soil types from the Robertskollen group of nunataks in northwestern Dronning Maud Land, providing the first estimates of bacterial production in soil habitats on the Antarctic continent. Although estimates of bacterial productivity in soils near to bird nests (344 (plusmn) 422 ng of C g [dry weight](sup-1) h(sup-1)) were higher than those for soils from beneath mosses (175 (plusmn) 90 ng of C g [dry weight](sup-1) h(sup-1); measured by TTI at 10(deg)C), these differences were not significant because of patchiness of bacterial activity (P > 0.05). TTI- and [(sup14)C]leucine ([(sup14)C]Leu)-derived estimates of bacterial production were similar when incubations of 3 h were used, although incubations as short as 1 h were sufficient for measurable uptake of radiolabel. Dual-label incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) into DNA and [(sup14)C]Leu into protein indicated that TTI did not reflect bacterial production of in situ assemblages when incubations were longer than 3 h. Isotope dilution analysis indicated that dilution of the specific activity of exogenously supplied [(sup3)H]TdR by de novo synthesis of TdR precursor could be limited by additions of [(sup3)H]TdR at a concentration of 1 nmol per ca. 115 mg of soil. TTI exhibited a psychrotrophic response to variation in temperature, with a temperature optimum of ca. 15(deg)C and a Q(inf10) value for 0 to 10(deg)C of 2.41.     

Effects of Toxic Substances on Natural Bacterial Assemblages Determined by Means of [3H]Thymidine Incorporation

The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were examined by means of [³H]thymidine incorporation into trichloroacetic acid-insoluble material. Results from a large number of coastal marine and freshwater samples suggest the following. (i) The effects of the three toxicants included reductions in the bacterial cell number as well as changes in rates of [³H]thymidine incorporation and in [³H]thymidine incorporation per cell. The concentrations that inhibited [³H]thymidine incorporation by 50% ranged from 3 to 11 mg liter⁻¹ for 3,5-dichlorophenol, 6 to 10 mg liter⁻¹ for 2,4-dinitrophenol, and 21 to 123 mg liter⁻¹ for potassium dichromate, with a tendency to higher values in bacterial assemblages from more eutrophic environments. (ii) The effects of 3,5-dichlorophenol and potassium dichromate determined by [³H]leucine incorporation into bacterial protein were similar or larger than those obtained from [³H]thymidine incorporation. (iii) Two to four hours of exposure to the toxicants was necessary before stable maximum effects were found in [³H]thymidine incorporation. (iv) Storage of natural environmental samples should be avoided, since tests with water stored for 1 to 3 days sometimes produced results different from results obtained from in situ tests. (v) The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were relatively constant during periods with different growth rates in the assemblages, during various periods of the year, and between samples from freshwater and marine localities. With some precautions, [³H]thymidine incorporation can be used as a quick and sensitive method for determining the effects of toxicants on aquatic bacterial assemblages from natural environmental samples.     

A correction: inhibitory activity in the conditioned medium of embryonic chick bones is due to thymidine

Earlier studies from this laboratory suggested that embryonic chick bones in organ culture released into the culture medium a specific inhibitor of bone cell proliferation as defined by inhibition of [3H]TdR incorporation into DNA. Dialysis and membrane ultrafiltration experiments suggested that the inhibitory substance (IS) had a molecular weight between 6000 and 14,000. However, subsequent studies on the purification of IS have revealed that the inhibitory activity in bone-conditioned medium is of lower molecular weight and has several properties in common with thymidine (TdR): (1) IS coeluted with [3H]TdR upon gel filtration chromatography on Sephadex G-10. (2) IS bound to charcoal but not to cation or anion exchange resins. (3) Bone-conditioned medium decreased incorporation of [3H]TdR into the free [3H]TdR pool of cells in monolayer culture. (4) Conditioned medium inhibited [3H]TdR incorporation into [3H]thymidine monophosphate in a reaction catalyzed by thymidine kinase. The equivalent concentration of TdR in conditioned medium as estimated by thymidine kinase assay was sufficient to account for the reduction in [3H]TdR incorporation into bone cell DNA. No evidence was found for a specific inhibitor of bone cell proliferation other than TdR. Hence we conclude that the inhibitory effect of IS is due to dilution of [3H]TdR by nonradioactive TdR. Furthermore, media conditioned by several tumor cell lines also contained a low-molecular-weight component which inhibited [3H]TdR incorporation. The results suggest that organ- and cell-conditioned media can contain significant concentrations of TdR which can artifactually inhibit [3H]TdR incorporation in cell proliferation assays.