Studies of the incorporation of [5-3H]uridine during activation and transformation of lymphocytes induced by phytohaemagglutinin. Dependence of the incorporation rate on uridine concentration at certain critical concentrations

1. Partially purified pig blood lymphocytes were stimulated to transform in culture with phytohaemagglutinin. Initial cell activation was assessed by measuring the increase of uridine incorporation into RNA induced by phytohaemagglutinin. The phytohaemagglutinin/serum ratio for this effect was similar to that required for transformation; however, no inhibition at high phytohaemagglutinin/serum ratios was found during cell activation. 2. Without replenishment of medium the pool of competitors with added uridine for incorporation fell to zero during 2 days of culture. At certain critical pool concentrations uridine itself could stimulate the rate of uridine incorporation. 3. Most of the tritium from [5-(3)H]uridine added at the initiation of culture had been incorporated into RNA by the end of the second day of culture; the subsequent loss of radioactivity preceded a fall in the total RNA content of cultures. 4. RNA was qualitatively examined on sucrose density gradients. In the first 30min. after the addition of phytohaemagglutinin, increased labelling occurred predominantly between the 28s and 4s regions of the gradients. On the second day of culture with phytohaemagglutinin mainly RNA sedimenting beyond the 28s region of gradients was labelled in 30min.     

The effect of dibromodulcitol on resting and dividing lymphoid cells

The effect of dibromodulcitol (DBD) on the incorporation of labelled precursors into DNA and RNA fractions of PHA-stimulated human lymphocytes and of P388F lymphoma cells at various stages of their growth was studied. Both cell systems showed sensitivity to the drug within the concentration range of 1–10 μg/ml.When DBD was added before phytohaemagglutinin (PHA), human lymphocytes showed a DNA labelling that was more affected than RNA. In contrast, by adding DBD after PHA, RNA labelling was much more inhibited than DNA. In the latter case, the decrease in DNA labelling occurred only 24 h after drug treatment whereas RNA labelling was decreased 1 h after treatment. Levels of DBD which normally produced 30% inhibition in plating efficiency of P388F lymphoma cells affected uridine-5-T incorporation to a different extent at different stages of growth of the culture. Enhanced RNA labelling occurred in early exponential stage while at later stages of growth, RNA synthesis was depressed.     

Metabolic activity of Trypanosoma lewisi cultured in vitro in the presence of normal or ablastinic rat serum

Trypanosoma lewisi, in cultures supplemented with normal rat serum, synthesized the majority of new RNA during the first 13 hr of the culture, whereas DNA synthesis occurred from the 8th hr onwards and amino acids were incorporated into macromolecules uniformly throughout the 24 hr culture period. Thymidine was taken up by the parasite only between the 7th and 14th hr of culture, unlike uracil and amino acids which were taken up as required. Ablastin, a trypanostatic factor in the serum of infected rats, maximally inhibited DNA synthesis if it was added to the cultures before the 5th hr, partially inhibited synthesis if added between the 5th and 11th hr, but if added after this had no inhibitory effect. Ablastin only partially inhibited RNA synthesis and was without effect on amino acid utilisation.     

The Effect of Radiation Upon Lymphocyte Response to Pha

Abstract. The proliferative response of lymphocytes to phytohaemagglutinin has been studied following incremental doses of ⁶⁰Co gamma radiation delivered at the beginning of culture. A dose-dependent inhibition of ³H-thymidine uptake is apparent in 48 hr and 72 hr cultures throughout the 250–2000 rad range, and the effect appears more marked at the later labelling time. Parallel autoradiographic studies have revealed changes in the cell cycle kinetics of irradiated cultures which underlie the effects observed at the whole-culture level. Irradiation reduces the number of cells reaching the first period of DNA synthesis and causes dose-dependent delays at specific points in the cycle of proliferating cells.     

Human monocyte activation by supernatants from concanavalin A (con A) stimulated lymphocytes

Human peripheral lymphocytes were stimulated with Concanavalin A (Con A) in the absence of serum. Supernatants were collected from control and mitogen stimulated lymphocyte cultures and fractions pooled according to the elution before, together with or after human serum albumin which was added as a marker. Only one fraction derived from Con A stimulated lymphocyte culture Supernatants which eluted immediately after human serum albumin had a significant effect on the metabolism and structure of human monocytes in vitro. Monocytes separated by human serum albumin and incubated with this fraction for 20 hr had an increase in nuclear RNA synthesis. Monocytes attached to cover slips in Leighton tubes showed an increase in the percentage of phagocytizing cells and phagocytic activity. Electron microscopy demonstrated highly phagocytic cells containing numerous Golgi associated granules and strands of nondilated rough surfaced endoplasmic reticulum in presence of the active fraction.     

Effects of insulin, hydrocortisone and prolactin on cell morphology, growth and survival of mammary organoids from mid-pregnant mice cultured on collagen gels.

Mammary epithelial organoids consisting of groups of lobular-alveolar acini were prepared from mid-pregnant mice and cultured for 24, 48, 96 and 192 hr on attached collagen gels in the presence of combinations of insulin, hydrocortisone and prolactin. The organoids rapidly attached to the gels and with all the combinations of hormones used colonies of cells spread out as a monolayer from the organoids within 48 hr. Although colony formation continued for up to 192 hr in culture, the maintenance of parental organoid structure after 96 and 192 hr was strongly favoured when hydrocortisone was present in the culture medium. The presence of hydrocortisone produced a dose-dependent increase in the amount of organoid DNA associated with the collagen substratum but decreased the rate of DNA synthesis by the organoids, as measured by the incorporation of labelled thymidine into DNA, in a dose-dependent manner under these conditions. The results suggest that the presence of hydrocortisone minimised the loss of cells from the collagen matrix in these cultures.     

Increase in activity of glucose 6-phosphate dehydrogenase in mouse mammary tissue cultured with insulin

1. In organ cultures of mammary tissue from C3H mice we observed increases in the activity of glucose 6-phosphate dehydrogenase similar to that occurring at parturition. 2. In 22hr. cultures of tissue from late-pregnant mice insulin was required for the increases, but the further addition of prolactin, corticosterone and certain other hormones had no effect. The rise in activity occurred over the second half of the culture period. 3. Results from culture of adipose tissue, and mammary tissue rich in adipose tissue, strongly suggest that the rise in activity occurs in mammary parenchymal rather than adipose cells. 4. In 45hr. cultures prolactin prevented a fall in enzyme activity between 22hr. and 45hr. If the medium contained serum the activity at 22hr. was unaffected, but it continued to rise up to 45hr., and prolactin then had no effect. 5. The enzyme also increased in activity in cultures of mammary tissue from mid-pregnant mice. Insulin was again required, the activity was higher at 45hr. than at 24hr. and prolactin increased the activities at both these times. 6. Actinomycin D, cycloheximide and puromycin at low concentration in the media of 22hr. cultures all prevented increases in enzyme activity. Hydroxyurea at a concentration that inhibited the incorporation of [(3)H]thymidine into DNA by 92% had little effect. 7. Actinomycin D and cycloheximide largely failed to prevent the rise in enzyme activity if added after 3.5hr. and 12hr. respectively. Hence all essential RNA and protein synthesis appears to be finished by 3.5hr. and 12hr., although most of the increase in enzyme activity occurs gradually between 12hr. and 22hr. 8. We suggest that the increases in enzyme activity, both in culture and in the living animal at parturition, are induced by an influx of glucose that is restrained during pregnancy by the growth-hormone-like action of placental lactogen.     

Changes in lipid pattern of HeLa cells exposed to immunoglobulin G and complement

1. Immunoglobulin G was isolated from sera of non-immunized rabbits or rabbits immunized with whole HeLa cell homogenate. The anti-HeLa immunoglobulin G and its Fab fragment precipitated the particulate 400000g-min. fraction of HeLa cell homogenate. 2. Immunoglobulin G from immunized or non-immunized rabbits and fresh or inactivated complement were added to HeLa cell cultures. Changes in the cell count and cellular contents of DNA, RNA, protein, total and individual phospholipids, cholesterol (and esters) and ganglioside were followed. 3. Addition of immunoglobulin G from non-immunized rabbits and guinea-pig serum (complement) caused a transient increase in DNA followed by a permanent increase in RNA, protein, dry weight and number of cells per culture. 4. Addition of anti-HeLa immunoglobulin G and active complement caused an increase in the cellular content of cholesterol, total phospholipids, lysophosphatidylcholine and lysophosphatidylethanolamine greater than the increase of the controls and a decrease in the molar percentages of phosphatidylcholine and phosphatidylethanolamine as compared with the controls. 5. The cholesterol/phospholipid ratio remained constant. 6. The appearance of lysophosphoglycerides was transient, reaching a maximum 3hr. after addition of anti-HeLa immunoglobulin G. 7. The content of lysophosphoglycerides in HeLa cultures exposed to immunoglobulin G from non-immunized rabbits ranged from 50% to 30% of the values obtained from cultures exposed to the anti-HeLa immunoglobulin G and complement. 8. The changes in the lipid pattern of the HeLa cells were associated with the appearance of juxta-nuclear vacuoles in cells, but were apparently not specifically related to the presence of active complement.     

Increased cytolytic T lymphocyte activity induced by 2-mercaptoethanol in mixed leukocyte cultures: kinetics and possible mechanisms of action.

The 20- to 50-fold increase in cytolytic T lymphocyte (CTL) activity caused by the addition of 50 muM 2-mercaptoethanol (2-ME) at the onset of a one-way murine mixed leukocyte culture (MLC) between C57BL/6 and DBA/2 splenic lymphocytes appears to be unrelated to early events in the culture: if 2-ME was present for the first 24 hr of culture only, there was no increase on day 4, but if addition of 2-ME was delayed until the last 24 hr of culture, the CTL activity was almost as high as that of cultures that were exposed to 2-ME for the entire 4-day culture period. The increase of CTL activity caused by delayed addition of 2-ME ("2-ME rescue") was used to investigate the mechanism by which the thiol induces differentiation of CTL from precursor cells. 2-ME rescue was mimicked by two other thiols, dithiothreitol and cysteamine phosphate, but at higher concentrations. Because the latter compound has no free sulhydryl group until it diffuses into cells and is enzymatically dephosphorylated, we conclude that thiols may increase the differentiation of CTL from precursor cells by an intracellular process involving free sulphydryl groups rather than by interaction with membrane sulfhydryls or destruction of inhibitor cells or their products. Cell separation experiments indicated that 2-ME rescue was independent of the presence of B lymphocytes and of adherent cells (macrophages) and was restricted to a subpopulation of T lymphocytes that developed into large lymphoid precursor cells during the first 3 days in culture even without 2-ME. The development of this subpopulation required DNA synthesis between 24 nad 72 hr after the onset of MLC. When 2-ME was added to day-3 MLC, CTL activity increased slightly as early as 4 hr later, but the major increase occurred during the second half of the 24 hr "rescue"period. Because this increase was inhibited by cytosine arabinoside (ARA-C), it seems likely that DNA synthesis is associated with and may be required for the differentiation of large precursor lymphoid cells into CTL after the addition of 2-ME.     

Pyruvate-Induced changes in hepatoma cell morphology and macromolecular synthesis

Cells maintained in basal growth medium with 0.2–1.0% serum often require citric acid cycle intermediates for optimal viability. We have found that pyruvate added to minimal growth medium causes cellular flattening and formation of external processes accompanied by increaded DNA synthesis in cultured hepatoma cells (HTC cells). Cells were cultured in plalstic T-flasks (0.5, 1.0, or 2.0 × 10⁶ cells/flask) containing 5 ml medium (90% Eagle's Basal Medium (BME) and 10% Swim's S-77) with various concentrations of fetal calf serum (0.2,0.25, 0.5, 1.0, 2.0, 10%) and either pyruvate (50, 100, 250,500, 1,000μg/ml), or one of: dibutyryl cAMP (DBcAMP) or dibutyryl cGMP (DBcGMP) at 10^?3, 10^?4, or 10^?5 M. At 44–48 hr cultures were pulsed with tritiated thymidine, uridine, or lecucine. Cells became attached to the plastic surface within 24hr. Cells in medium with 0.25 to 2.0% serum had a rounded appearance. With added pyruvate, cellular flattening, process formation, and an increased adherence to the substratum was absorbed. By 48 hr, culture without pyruvate grew in rounded clusters; with pyruvate, cells formed extensive interconnecting processes that appeared loosely attached to the monolayer surface. At the cell densities tested, process formation was maximal with 250 to 500 μg/ml pyruvate. Cytochalasin B blocked flattening and process formation; EDTA (1 mg/ml) caused retraction of processes within 3 min, and a slow dissolution of these structures within cells was observed. DBcAMP or DBcGMP did not induce process formation. Flattening and process foormation in pyruvate-enriched cultures were accompanied by marked stimulation of DNA synthesis and smaller increases in RNA and protein synthesis. Cell number was not affected. These pyruvate-induced changes suggest that alterations in energy metabolism, or precursors that enhance viability and macromolecular synthesis in mammalian cell cultures, may exert marked effects on cellular morphology without corresponding changes in growth of neoplastic liver cells.     

Control of the Synthesis of Macromolecules During Amino Acid and Thymine Starvation in Bacillus subtilis

Studies of Maal?e, Lark, and others with amino acid- and thymine-starved cultures revealed successive steps in the biosynthesis of Escherichia coli chromosomes. In this study, the corresponding mechanisms in Bacillus subtilis 168 were examined. Using a strain requiring both thymine and tryptophan, we found that, 3 hr after the start of amino acid starvation, when the deoxyribonucleic acid (DNA) content of the culture had increased 40 to 50%, DNA synthesis ceased. After 4 to 5 hr, 100% of the cells were immune to thymineless death; their chromosomes had presumably been completed. Immune cultures slowly incorporated (3)H-thymine. Thymine incorporation increased 20-fold 30 min after readdition of amino acids, indicating reinitiation of chromosome synthesis. Simultaneous presence of amino acids and thymine was required for reinitiation. If 5-bromouracil (5-BU) was added instead of thymine, newly replicated DNA segments could be separated by centrifugation in CsCl. Analysis of the CsCl fractions by a transformation assay showed that the order in which the markers were synthesized was ade-16, thr-5, leu-8, metB5. Less than half the chromosomes started resynthesis synchronously in 5-BU. Nevertheless, chromosome alignment in the amino acid-starved culture is probably very good: marker frequency analysis of its DNA gives the same normalized frequencies as DNA from "perfectly" aligned spores. Full viability is maintained in the chromosome-arrested culture for 10 hr in thymine-free medium in the absence or presence of amino acids. In the latter condition, protein synthesis proceeds, and the cells filament and become more lysozyme-sensitive. Such cells must be incubated and plated on hypertonic or on slow-growth media; otherwise, they undergo "quasiosmotic" thymineless death. This death is thus apparently not directly attributable to any damage of chromosomal DNA. Further, weakening of the teichoic acid portion of the cell wall is not involved, since (32)P incorporation into teichoic acid is normal. Chloramphenicol prevents quasiosmotic thymineless death and also inhibits (32)P incorporation into teichoic acid. Chromosome-synthesizing cultures suffer thymineless death of two types: quasiosmotic death, and death insusceptible to osmotic rescue.     

Mechanism by Which Fiber Antigen Inhibits Multiplication of Type 5 Adenovirus

Purified fiber antigen of type 5 adenovirus inhibited the multiplication of type 5 adenovirus by 50% when 35 mug of fiber antigen protein was added to 10(6) KB cells in suspension culture. Although the fiber antigen reduced the number of virions adsorbed per cell when a multiplicity of infection of 50,000 plaque-forming units (PFU)/cell was employed, the number of cells infected was not diminished under these conditions. If a low multiplicity of infection (1.1 PFU/cell) was used, viral adsorption was not detectably decreased. The fiber antigen did not reduce the capability of virions to liberate their viral deoxyribonucleic acid (DNA). The biosyntheses of DNA, ribonucleic acid (RNA), and protein were blocked about 20 to 25 hr after the addition of fiber antigen to cultures of uninfected or type 5 adenovirus-infected KB cells. Most of the fiber antigen protein became cell-associated between 22 and 36 hr after it was added to cells. The hexon antigen neither inhibited viral multiplication nor blocked the biosynthesis of DNA, RNA, or protein. Moreover, the hexon did not attach to KB cells. The profound effects of the fiber antigen were not due to the induction of an interferon-like substance, for actinomycin D did not reduce the ability of the fiber to inhibit multiplication of type 1 poliovirus.     

Selective Inhibition of Reovirus Ribonucleic Acid Synthesis by Cycloheximide

Cycloheximide, at a concentration of 10 mug/ml, rapidly blocked protein synthesis in L cells infected with reovirus. When the drug was added before 5 hr postinfection, synthesis of both single- and double-stranded varieties of virus-specific ribonucleic acid (RNA), which normally commences between 6 and 7 hr after infection, was blocked. When the cycloheximide was added at 9 hr after infection, uptake of uridine-H(3) into RNA, for the succeeding 6 hr at least, was similar to that of an infected culture without the drug. This latter uptake of uridine-H(3) in the presence of cycloheximide was largely into single-stranded RNA, since double-stranded RNA synthesis was rapidly and markedly inhibited by the cycloheximide. Single-stranded RNA formed in the presence of cycloheximide was found not to be a precursor of viral progeny, double-stranded RNA. Synthesis of double-stranded RNA in the infected cell probably requires prior synthesis of a new protein, which has a rapid rate of turnover. The possibility that formation of single-stranded RNA is preceded by synthesis of a second new protein is discussed.     

Differentiation in the early chick embryo: effects of bromodeoxyuridine on erythropoiesis

Organ cultures of chick embryos at the definitive streak stage will normally show the first appearance of hemoglobin in erythroid cells after 20 hr of culture. Addition of 5-bromodeoxyuridine (BrdU) will prevent hemoglobin formation when added at the beginning of culture, but the tissue becomes resistant to the inhibitor when addition is delayed for approximately 10 hr. The inhibitory effect of BrdU is canceled or reversed if thymidine replaces BrdU at the beginning of culture or later. Transfer to thymidine containing medium even after 20 hr permits hemoglobin formation to occur at almost the normal time, thus making it unlikely that a complete cell cycle or DNA replication during a complete S period is required for the reversal of inhibition.Even when the appearance of cytologically detectable hemoglobin is inhibited by BrdU, some globin is synthesized and RNA sequences specific for globin are present, but in decreased amount, unless the inhibitor is given very early. BrdU does not affect the synthesis of any particular RNA species or of polyadenylic acid, but it does lower the rate of uptake of adenosine into the ATP pool. While not affecting cell cycle times in cell cultures, BrdU greatly reduces cell numbers.     

Effect of cell physiological state on infection by rat virus

Infection by rat virus has been studied in cultures of rat embryo cells to evaluate the Margolis-Kilham hypothesis that the virus preferentially infects tissues with actively dividing cells. An enhancement of infection was seen in cultures infected 10 hr after fresh medium was added as compared to infection of stationary cultures (infected before addition of fresh medium). Since addition of fresh medium stimulates deoxyribonucleic acid (DNA) synthesis, the number of cells per culture synthesizing DNA at the time of infection was compared with the proportion of cells which synthesized viral protein. Cells were infected before the medium change and 10 or 24 hr after the medium change and were pulse-labeled with ³H-thymidine at the time virus was added. The cells were allowed to initiate viral protein synthesis before they were fixed and stained with fluorescein-conjugated anti-rat virus serum. Fluorescence microscopy permitted both labels to be counted simultaneouly and showed that the greatest proportion of cells synthesizing viral protein were those which had incorporated ³H-thymidine at the time of infection.     

Dissociation of differentiation and proliferation in the primary induction of cytolytic T lymphocytes by alloantigens

The requirement for DNA synthesis in the induction of cytolytic T lymphocytes (CTL) by alloantigens has been investigated. C57BL/6 splenic T cells purified by passage on nylon wool columns were stimulated in vitro in mixed leukocyte culture (MLC) and assayed for cytotoxicity against 51Cr-labeled target cells. With this system, CTL activity was detectable after 24 hr of MLC and reached high levels after 48 hr. Addition of cytosine arabinoside (ARA-C) or hydroxyurea to such cultures at concentrations that were sufficient to inhibit DNA synthesis by greater than 98% did not reduce CTL activity measured after 24 hr; however, the increase in activity that occurred between 24 and 48 hr in control cultures was strongly reduced (or abolished) by these drugs. Velocity sedimentation analysis of MLC cells activated for 48 hr in the presence of ARA-C further revealed that CTL precursor lymphocytes had enlarged into medium- to large-sized CTL under these conditions. These studies provide direct evidence that the primary induction of CTL by alloantigens can be dissociated into a differentiation step, which occurs within 24 hr in the absence of DNA synthesis and is accompanied by blast transformation, and a subsequent proliferation.     

Early stimulation of ATP turnover induced by growth factors : Synergistic effect of EGF and insulin and correlation with DNA synthesis

Addition of a mixture of EGF + insulin to quiescent cell cultures synergistically stimulates the cells to reinitiate DNA synthesis and cell division. We have previously demonstrated that this mixture rapidly increases ATP turnover in quiescent cells. The present work shows that each of the two growth factors, EGF and insulin, when added separately to quiescent cells was able to stimulate the phosphorylation of the organic acid-soluble compounds (Po) pool and ATP turnover. The stimulation of ATP turnover was closely correlated with the increase in phosphorylation of the Po pool which suggests that Po labelling reflects the ATP turnover. In many experiments, the synergy between the two growth factors on the early increase in phosphorylation of the Po pool was clearly shown. Doubling the concentration of EGF (12-24 ng/ml) or insulin (50-100 ng/ml) did not increase early stimulation of phosphorylation of the Po pool, whereas simultaneous addition of the two growth factors induced a greater stimulation than that of each growth factor separately added. The augmentation in Po labelling after addition of EGF or insulin alone was transient. The synergistic effect of the two growth factors was more significant when determined 150 or 300 min after growth-factor addition. In our experimental conditions, each of the two growth factors, EGF and insulin, was able to induce a stimulation of DNA synthesis. However, the best stimulatory effect was observed with the mixture of the two which synergistically increased DNA synthesis determined between 6 and 24 h after growth-factor addition. The comparison between DNA replication and Po labelling suggests a correlation between the increase in DNA replication and in the total ATP synthesized in the first 5 h after cell stimulation by growth factors added separately or in combination.     

Synthesis of reovirus ribonucleic acid in L cells

Kudo, Hajime (The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.), and A. F. Graham. Synthesis of reovirus ribonucleic acid in L cells. J. Bacteriol. 90:936-945. 1965.-There is no inhibition of protein or deoxyribonucleic acid (DNA) synthesis in L cells infected with reovirus until the time that new virus starts to form about 8 hr after infection. At this time, both protein synthesis and DNA synthesis commence to be inhibited. Neither the synthesis of ribosomal ribonucleic acid (RNA) nor that of the rapidly labeled RNA of the cell nucleus is inhibited before 10 hr after infection. Actinomycin at a concentration of 0.5 mug/ml does not inhibit the formation of reovirus, although higher concentrations of the antibiotic do so. Pulse-labeling experiments with uridine-C(14) carried out in the presence of 0.5 mug/ml of actinomycin show that, at 6 to 8 hr after infection, two species of virus-specific RNA begin to form and increase in quantity as time goes on. One species is sensitive to ribonuclease action and the other is very resistant. The latter RNA is probably double-stranded viral progeny RNA, and it constitutes approximately 40% of the RNA formed up to 16 hr after infection. The function of the ribonuclease-sensitive RNA is not yet known. Synthesis of both species of RNA is inhibited by 5 mug/ml of actinomycin added at early times after infection. Added 6 to 8 hr after infection, when virus-specific RNA has already commenced to form, 5 mug/ml of actinomycin no longer inhibit the formation of either species of RNA.     

Retention of Labelled Dna Precursor By Murine Cells After A Single Administration of Tritium Labelled Thymidine

Abstract. The central zone of the rat lens epithelium, extending half way from the centre to the periphery of a whole mount preparation, normally has less than 1% of the cells in the cell cycle at any given time. Mechanical wounding initiates a burst of proliferation in the central zone. DNA synthesis begins 14 hr after wounding followed by mitosis 10 hr later. When [³H]TdR was applied at 2 hr prior to S phase, some moderately heavy and some light labelling was observed after the onset of S phase. When [³H]TdR was applied 5 hr before S phase (9 hr after wounding), all the cells were lightly labelled. Only small amounts of the label were available to these cells 5 hr after application. It is significant that there was labelling in this group because it indicates the persistence of relatively small intracellular pools of [³H]TdR for several hours after the initial 'pulse' labelling of cells. Determinations of the duration of S phase were based on the assumption that pulse labelling may be affected by the persistence of the pools of [³H]TdR and consequent light labelling of the cells.