Autoradiographic Studies of [methyl-3H]Thymidine Incorporation in a Cyanobacterium (Microcystis wesenbergii)-Bacterium Association and in Selected Algae and Bacteria

The present investigation showed by means of autoradiography that the cyanobacterium Microcystis wesenbergii did not incorporate [³H]thymidine at nanomolar concentrations, whereas its associated heterotrophic bacteria appearing in the gelatinous cover of the cyanobacterium became labeled. Several other tested cyaobacteria and algae did not incorporate [³H]thymidine.     

Changes in fibroblast-derived trypomastigotes of Trypanosoma cruzi during long-term culture.

Fibroblast-derived trypomastigotes (FDTs) of Trypanosoma cruzi that had been in culture for extended periods of time were found to differ in their ability to proliferate in culture when compared to blood-form trypomastigotes (BFTs) and FDTs that had been recently established from blood-forms. "Old" FDTs transform into amastigotes/spheromastigotes and epimastigotes and readily incorporate [3H]thymidine in medium alone or in the presence of mouse spleen cells, whereas "new" FDTs and BFTs did not incorporate [3H]thymidine although they did transform in culture. These differences should be considered when FDTs are used for physiologic and immunologic studies of T. cruzi.     

The relationship between cell division and melanocyte differentiation in epidermal cultures from mouse embryos

Cultures of 14-day embryonic mouse epidermis that include melanoblasts initiate melanin synthesis 30 hr after plating, a schedule that is 2.5 days earlier than in vivo. In order to determine if the accelerated differentiation of melanoblasts is related to a cessation of cell proliferation in the cultures, a study of [³H]thymidine incorporation by melanoblasts and melanocytes was made. Autoradiograms of 14-day epidermal cultures grown for 48 hr in medium containing [³H]thymidine revealed that melanoblasts continue to proliferate during this time period. A second population of melanoblasts that did not incorporate [³H]thymidine was also present in these cultures. The relative numbers of dividing and nondividing melanoblasts change with the age of the epidermis cultured. Ninety-one percent of the melanoblasts in 13-day epidermis take up [³H]thymidine, 63% incorporate [³H]thymidine in 14-day cultures, and only 29% take up label in cultures of 15-day epidermis. It appears from these results that melanoblasts during their migration from the neural crest are proliferative cells and that during the early invasion of the epidermis a nonproliferative population of melanoblasts is established. Both populations coexist in the epidermis and subsequently undergo differentiation on the same time schedule.     

Electron microscopic autoradiographic and electron microscopic immunohistochemical studies on the anti-HPO antibody-producing cells

The secondary immune responses in mouse popliteal lymph nodes to horseradish peroxidase (HPO) were studied by a combination of electron microscopic autoradiography and electron microscopic immunohistochemistry in order to clarify the relationship between antibody-producing and DNA-synthesizing capacities of the plasmacytic series. The anti-HPO antibody-containing cells, which increased in number 72 h after the secondary antigenic stimulation, were mainly immunoblasts and immature plasma cells. Immunoblasts containing anti-HPO antibody incorporated [³H]thymidine more actively than did immature plasma cells containing anti-HPO antibody. In 144 h after the secondary antigenic stimulation, antibody containing cells consisted mainly of mature plasma cells and immature plasma cells. Immature plasma cells containing the anti-HPO antibody incorporated a little [³H]thymidine, but mature plasma cells containing anti-HPO antibody did not incorporate any [³H]thymidine.     

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.     

Selective incorporation of thymidine in the organelle DNA of Polytoma obtusum

Polytoma obtusum was found to selectively incorporate exogenous thymidine into its leukoplast DNA. The nuclear DNA was unable to incorporate [3H]thymidine, although both DNA species could be labeled with radioactive adenine with similar efficiencies. The mitochondrial DNA (mitDNA), which had a buoyant density of 1.714 g/ml and was banded slightly on the heavier side of the nuclear DNA peak, was also found to incorporate a small amount of [3H]thymidine. These observations suggest that P. obtusum lack cytoplasmic thymidine kinase, whereas the enzyme is present in both leukoplast and mitochondria.     

Retention of bone cell viability in mouse calvarial explants after cryopreservation

Newborn mouse calvaria, cyropreserved at -196 degrees C in serum-free medium containing dimethyl-sulfoxide, were compared to unpreserved explants for bone cell viability by [3H]thymidine uptake. Other explants were studied using autoradiography to compare the histological appearance of the cryopreserved and control unpreserved explant sites of cellular localization of [3H]thymidine. After short-term cryopreservation, calvarial bone cells, including less differentiated osteoprogenitor cells, survived as indicated by their incorporation of the DNA precursor. With culture continuing for up to 24 hr after thawing and in the continuous presence of [3H]thymidine, additional labeled thymidine was incorporated, indicating that the proliferative ability of explant cells persists after cryopreservation. Cryopreserved bone explants did not, however, incorporate the same amount of labeled thymidine as did controls at each time point studied. These events, as measured quantitatively and observed by autoradiography of the tissue, indicate that newborn calvarial bone cell proliferation in vitro continues after cryopreservation. The large surface:mass ratio of the tissue and its proportionate volume of calcified matrix apparently permits it to behave as an isolated cell population with regard to the diffusion of the cryoprotectant and thermal conductivity, thus permitting the retention of explant viability.     

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

The incorporation of [³H]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 [³H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [³H]thymidine during growth. It is concluded that the [³H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.     

A Study on the lack of [methyl-3H] thymidine uptake and incorporation by chemolithotrophic bacteria

Five chemolithotrophic bacteria were tested for their ability to incorporate [methyl-³H] thymidine. None of the bacteria incorporated the label, even after incubation for 24 hours. The inability of these bacteria to incorporate thymidine appears to be due to an absence of transport mechanisms for exogenous nucleosides. As a result of these findings, it is concluded that activities deduced from labeled thymidine incorporation measurements probably do not include the activity of chemolithotrophic bacteria.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates.

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

Effect of 5-fluoro-2'-deoxyuridine on [h]thymidine incorporation by bacterioplankton in the waters of southwest Florida

The effect of 5-fluoro-2'-deoxyuridine (FdUrd) on [methyl-H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-H]thymidine or [6-H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [H]thymidine labeling of protein and RNA, but caused some inhibition of [H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [H]thymidine incorporation.     

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos

Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 microCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 microCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of < or = 1 h, 22% had a G1-phase of < or = 2 h, 22% had a G1-phase of < or = 3 h and 13% had a G1-phase of < or = 4 h. The G1-phase in 85% of the 4-cell embryos was < or = 3 h, that in 8% of embryos was < or = 4 h and that in 7% of embryos was < or = 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 microM, and the appropriate concentrations which did not impair development were 0.05-0.5 microM.     

Effect of estradiol on the early incorporation of (3H) thymidine into uterine DNA on immature mouse.

The incorporation of [3H]thymidine into uterine DNA was markedly depressed within 10 to 30 minutes after intraperitoneal administration of 17beta-estradiol to immature mouse. Maximum inhibition occurred about 6 hours after the hormone was administered. Uterine DNA content and the amount of [3H]thymidine incorporated into the acid-soluble fraction was not affected during the period of hormone-induced inhibition. Moreover, the in vitro incorporation of [3H]thymidine by isolated estradiol-treated mouse uterus was blocked. In contrast to the uterus, 17beta-estradiol did not influence the incorporation of thymidine into mouse liver DNA. Evidence is presented to show that the incorporation of thymidine into uterine DNA was blocked initially by 17beta-estradiol.     

Incorporation of [methyl-3H]thymidine by obligate and facultative anaerobic bacteria when grown under defined culture conditions

Abstract Incorporation of [ methyl -³H]thymidine into bacterial DNA was determined for a range of axenic anaerobic bacterial cultures: fermentative heterotrophs, sulphate-reducing bacteria, purple sulphur bacteria, acetogens and methanogens. Anaerobically growing Bacillus sp. and the obligate aerobe Thiobacillus ferrooxidans were also investigated. Actively growing cultures of sulphate-reducing bacteria belonging to the genera Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfobotulus and Desulfobulbus , purple sulphur bacteria ( Chromatium vinosum OP2 and Thiocapsa roseopersicina OP1), methanogens ( Methanococcus GS16 and Methanosarcina barkeri ) and an acetogen ( Acetobacterium woodii ) did not incorporate [ methyl -³H]thymidine into DNA. The only obligate anaerobes in which thymidine incorporation into DNA could be unequivocally demonstrated were members of the genus Clostridium . Anaerobically growing Bacillus sp. also incorporated thymidine. These data demonstrate that pure culture representatives of major groups of anaerobic bacteria involved in the terminal oxidation of organic carbon and anoxygenic phototrophs within sediments are unable to incorporate [ methyl -³H]thymidine into DNA, although some obligate and facultative anaerobes can. Variability in thymidine incorporation amongst pure culture isolates indicates that unless existing techniques can be calibrated to take this into consideration then productivity estimates in both aerobic and anaerobic environments may be greatly underestimated using the [ methyl -³H]thymidine technique.     

The structural and functional properties of light and dark mouse hepatocytes]

The morphofunctional analysis of the light and dark hepatocytes of mouse C3HA line has shown that the dark cells have increased electronic density of hyaloplasm in contrast to the light ones. The light hepatocytes predominate among tetra- and octaploid cells. Both cell types are able to incorporate [3H] uridine and [3H] thymidine. Mitosis are met among the light hepatocytes only.     

Effect of 5-Fluoro-2′-Deoxyuridine on [3H]Thymidine Incorporation by Bacterioplankton in the Waters of Southwest Florida

The effect of 5-fluoro-2′-deoxyuridine (FdUrd) on [methyl-³H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-³H]thymidine or [6-³H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [³H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [³H]thymidine labeling of protein and RNA, but caused some inhibition of [³H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [³H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [³H]thymidine 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.     

Specific modifications of hepatoma cell-surface glycoproteins with enzymes : Effects on in vitro growth as investigated by the use of lectins

The effects of enzymic treatment on the interactions between Zajdela's tumor cells and various lectins. Concanavalin A (ConA); Wheat Germ Agglutinin (WGA); Robinia lectin; have been studied. (1) The number of lectin-binding sites and the affinity constants were investigated. (2) The effects of the lectins on cell growth and [3H]thymidine incorporation were studied on untreated and enzyme-treated cells. It was observed that treatment of tumor cells with neuraminidase resulted in a change in the binding characteristics of each lectin. However, additional treatment of the cells with galactose oxidase had no further effect on lectin binding. ConA and Robinia lectin induced a decrease of the untreated tumor cell growth and a stimulation of the [3H]thymidine incorporation. This paradoxal result may be explained as a consequence of the stimulation of the [3H]thymidine uptake observed in the presence of lectins. The enzymatic treatments themselves did not change the cell growth although they did induce a change in the effect of ConA and Robinia lectin on cell growth and [3H]thymidine incorporation. As a result of neuraminidase treatment, the effects of ConA were totally suppressed but those of Robinia lectin only partially. Although WGA interacted with untreated and enzyme-treated cell surfaces, it had no effect on tumor cell growth nor [3H]thymidine incorporation. The results are discussed in terms of lectin transport.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.