Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

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.     

Cryoprotection of murine lymphocyte subpopulations using a microprocessor-controlled cooling system

Single-cell suspensions of splenic lymphocytes from 5- to 6-month-old C57BL/6 mice were cryopreserved using cooling rates ranging from ?0.25 to ?10.0 °C/min with the microprocessor-controlled cooling system developed in our laboratory. The cells (30 × 10⁶ cells/ml) were suspended in RPMI 1640 containing 10% FCS and 10% DMSO, and a total volume of 1.75 ml was frozen. Fluorescein-diacetate staining identified viable cells in unfrozen controls and frozen-thawed suspensions. Functional capacity was assessed in vitro by the incorporation of [³H]thymidine by dividing cells activated with graded concentrations of the T-lymphocyte mitogens, PHA-P and Con A, and the B-lymphocyte mitogen, LPS. High percentages of viable cells were recovered after cooling at rates ranging from ?0.5 to ?5.0 °C/min, as compared with those of unfrozen control suspensions. Incorporation of [³H]thymidine by T and B cells reached similar levels after cooling at rates ranging from ?0.25 to ?5.0 °C/min, and the percentage incorporation of [³H]thymidine as compared with that of unfrozen cells was 80–100%, except for T lymphocytes activated with PHA-P after cooling at ?5.0 °C/min. The relative response of cell suspensions to T- and B-cell mitogens, the relative mitogenic index, was unchanged from that of unfrozen controls in suspensions cooled at all rates including two (?0.25 and ?10.0 °C/min), which permitted recovery of only 55% of unfrozen cells. The importance of the constant cooling rates and rapid compensation for heat released at the phase change using the microprocessor-controlled system and of precise determinations of cellular viability and function are discussed and related to the apparent protection conferred on subpopulations of murine lymphocytes using cooling rates ranging from ? 0.25 to ?10.0 °C/min.     

Sensitivity of early mouse embryos to [H]thymidine

Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to [³H]thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of [³H]thymidine, concentrations as low as 0.1 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs was reduced, and at 10 nCi/ml the number of embryos developing to all three post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highest concentration tested (100 nCi/ml). When embryos were cultured from the 2-cell stage for 3 days in the presence of [³H]thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to [³H]thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages; the lowest concentration that interfered with development was 10 nCi/ml, and exposure at the morula stage was most detrimental to the subsequent development of embryos, particularly that of ICMs. The 24-h exposure of immunosurgically isolated ICMs to [³H]thymidine revealed that the high sensitivity of the ICM to [³H]thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate [³H]thymidine into the DNA.     

Increased uptake of thymidine in the activation of sea urchin eggs. III. Effects of aphidicolin

Uptake and phosphorylation of externally supplied [3H]-thymidine are fully stimulated in fertilized sea urchin eggs exposed to 5.0 micrograms/ml aphidicolin. As in untreated controls, the rate of uptake in aphidicolin-treated eggs increases greater than 50-fold shortly after fertilization, and greater than 85% of the transported thymidine is immediately phosphorylated to the triphosphate. The intracellular levels of [3H]-thymidine triphosphate (3H-dTTP) resulting from an external supply of [3H]-thymidine is therefore equal in aphidicolin-treated and untreated fertilized eggs. Under the same experimental conditions, the incorporation of externally supplied [3H]-thymidine into newly synthesized DNA of fertilized eggs is 90% inhibited by exposure to aphidicolin. The full availability of 3H-dTTP in these eggs further suggests that aphidicolin inhibits specifically at the level of DNA synthesis. This inhibitory effect is proportional to the concentration of aphidicolin between 0 and 5.0 micrograms/ml. In the continuous presence of 5.0 micrograms/ml aphidicolin, fertilized eggs fail to undergo mitotic chromosome condensation, nuclear envelope breakdown, and cytokinesis, suggesting a dependent link between these processes and the completion of nuclear DNA synthesis.     

Pulse-Labeling of Kinetoplast DNA: Localization of 2 Sites of Synthesis Within the Networks and Kinetics of Labeling of Closed Minicircles*

SYNOPSIS. Short pulse-labeling of log phase Crithidia fasciculata cells with [³H]thymidine allowed the autoradiographic visualization of 2 sites of replication of kinetoplast DNA situated at the periphery of the networks and separated by 180°. Longer pulse-labeling led to the previously reported total peripheral labeling pattern. Pulse-labeled networks possess an intermediate density in ethidium bromide-CsCl equilibrium gradients between the densities characteristic of closed networks and open or linear DNA. Removal of ethidium bromide by several methods and treatment of intermediate band networks with RNase and pronase had no effect on the equilibrium rebanding pattern. Closed minicircles of Leishmania tarentolae are not labeled by a short pulse of intact cells with [³H]thymidine. A chase of ～ 3–4 hr is required for the appearance of radioactivity in closed minicircles, a time delay which implies the existence of intermediate events between replication and eventual covalent closure of the minicircles.     

Transport of thymidine during the cell cycle in mitotically synchronized CHO cells

The kinetics of total uptake of thymidine into the cell were determined for cells which had been mitotically synchronized, plated into scintillation vials and pulsed with five concentrations of [³H]-thymidine at various times during the cell cycle. From Lineweaver-Burk plots of these rates, V_max and K_m values were determined for the transport of thymidine. The V_max values ranged from a low of 2.0 pmoles/ min/10⁶ cells in mid-G1 to a high of 99.7 in mid-S before a decline in late S and G2. K_m values displayed only a 5-fold range in values.     

Genistein abolishes nucleoside uptake by cardiac fibroblasts

Genistein, a soy isoflavone, is reported to exert significant beneficial action in several human disorders, which has generated immense interest in the mechanisms underlying its effects on diverse cellular processes. It has anti-proliferative action on many cell types, an effect generally attributed to tyrosine kinase inhibition. In this study, genistein was found to cause total inhibition of [³H]-thymidine incorporation into DNA and a modest reduction in [³H]-proline incorporation into protein in primary cultures of cardiac fibroblasts. The decrease in [³H]-thymidine incorporation was not associated with a decrease in cell proliferation but correlated exactly with low intracellular levels of [³H]-thymidine. Genistein dramatically reduced [³H]-thymidine but not [³H]-proline uptake by these cells in which the equilibrative nucleoside transporter may be the major route of nucleoside uptake. The effect was irreversible and was demonstrable in pulmonary fibroblasts as well. The findings suggest that nucleoside uptake mechanisms may be a novel target of genistein action in cardiac fibroblasts and point to serious limitations in using genistein to assess the role of tyrosine kinase in cell proliferation by the standard technique of [³H]-thymidine incorporation.     

Incorporation of tritiated thymidine by marine bacterial isolates when undergoing a starvation survival response

Bacterial DNA synthesis, as measured by the incorporation of [methyl-³H] thymidine, was examined during conditions of decreasing biomass and non-growth of three heterotrophic marine bacteria. High rates of [³H] thymidine incorporation were recorded during the initial phase of starvation and two strains exhibited a net increase in DNA during the first few hours of starvation. The decreased rate of [³H] thymidine incorporation with the time of starvation, was in agreement with the decrease in the percentage of the total population that showed uptake of labelled thymidine, as seen by a combined autoradiography-epifluorescence technique. It is suggested that new rounds of replications were initiated after cells had been starved for times that well exceeded the time for replication of genomes during growing The initial increase in cell numbers upon transfer of growing cells to a starvation regime was inhibited by nalidixic acid, suggesting that DNA synthesis, rather than an excess of nuclear bodies, allow for the fragmentation process in these strains.     

The effects of cortisol and amino acids on the metabolic activity of rat skin stored in dimethyl sulfoxide buffer at −196 °C

Addition of amino acids to the DMSO buffer reduces the intracellular amino acid depletion of rat skin tissue frozen and stored at ?196 °C.Although prolonged exposure to DMSO progressively inhibits the [2-¹⁴C]glycine and l-[U-¹⁴C]leucine incorporation into the proteins, cortisol and amino acid additions to the buffer medium protect the protein-synthesizing activity. These factors also stimulate the incorporation of [6-³H]-thymidine into DNA. The stimulatory characteristics of cortisol and of amino acids separately are enhanced when both components are added together to the preserving buffer. These effects are noticeable in tissue only exposed to the DMSO buffer without freezing as well as in skin frozen and stored at ?196 °C and subsequently thawed at 40 °C.A stimulatory effect of cortisol and of a free amino acid, supplement to the medium on the α-amino-[1-¹⁴C]isobutyric acid uptake by the cells is only observed in skin exposed for a short period of time to the DMSO buffer, but it is not detectable after longer exposure and after freezing.     

Utility of the modified ATP III defined metabolic syndrome and severe obesity as predictors of insulin resistance in overweight children and adolescents: a cross-sectional study

Background

Inhibition of vascular smooth muscle cell (vSMC) proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs) inhibit vSMC proliferation but it has been reported that they anomalously stimulate [³H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [³H]-thymidine incorporation.

Methods

VSMC proliferation was investigated by [³H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [³H]-thymidine uptake. Cell cycle events were analyzed by FACS.

Results

VSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose) and high (25 mM glucose) increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively). We confirmed an earlier report that troglitazone (at low concentrations) causes enhanced incorporation of [³H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [³H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations. The sulfonylureas chlorpropamide and gliclazide had no inhibitory effect on vSMC proliferation assessed by either [³H]-thymidine incorporation or cell numbers.

Conclusion

TZDs but not sulfonylureas nor biguanides (except phenformin at high concentrations) show favorable vascular actions assessed as inhibition of vSMC proliferation. The activity of rosiglitazone and pioglitazone is enhanced under high glucose conditions. These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases. However, the plethora of potentially beneficial actions of TZDs in cell and animal models have not been reflected in the results of major clinical trials and a greater understanding of these complex drugs is required to delineate their ultimate clinical utility in preventing macrovascular disease in diabetes.     

Differential effects of glucocorticoids on insulin-like growth factor I action in cultured human fibroblasts

Glucocorticoids act synergistically with insulin-like growth factor I (IGF-I) to stimulate DNA synthesis and replication of cultured human fibroblasts. In the present study, we further define glucocorticoid and IGF-I interactive effects on human fibroblast metabolism and growth. IGF-I stimulated dose-dependent increases in early metabolic events. Half-maximal effectiveness was seen at 5–8 ng/ml IGF-I, with mean maximal responses of 1.5-, 2-, and 6-fold for [³H]2-deoxyglucose uptake, [¹⁴C]glucose incorporation, and [¹⁴C]aminoisobutyric acid (AIB) uptake, respectively. A 48-hour preincubation with 10^?7 M dexamethasone markedly enhanced both the sensitivity and maximal effectiveness of IGF-I stimulation of AIB uptake. In contrast, dexamethasone had no effect on IGF-I-stimulated glucose uptake and utilization. Maximum specific binding of [125I]IGF-I to fibroblast monolayers was identical in ethanol control and glucocorticoid-treated cells, with 50% displacement at ～5 ng/ml IGF-I. In addition to its synergism with IGF-I, preincubation with dexamethasone augmented insulin and epidermal growth factor (EGF) stimulation of [³H]thymidine incorporation; dexamethasone had no effect on platelet-derived growth factor or fibroblast growth factor action. Two-dimensional gel electrophoresis identified two specific glucocorticoid-induced proteins in human fibroblast cell extracts with molecular weights of 45K and 53K and pls of 6.8 and 6.3, respectively. These data indicate that IGF-I receptor-mediated actions in human fibroblasts are differentially modulated by glucocorticoids. Glucocorticoids are synergistic with IGF-I in stimulating mitogenesis and amino acid uptake, without having any apparent effect on IGF-I-stimulated glucose metabolism. Glucocorticoid enhancement of growth factor bioactivity may involve modulation of a regulatory event in the mitogenic signaling pathway subsequent to cell surface receptor activation. © 1995 Wiley-Liss, Inc.     

Estimating Bacterioplankton Production by Measuring [3H]thymidine Incorporation in a Eutrophic Swedish Lake

Bacterioplankton abundance, [³H]thymidine incorporation, ¹⁴CO₂ uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 × 10⁹ to 2.4 × 10⁹ cells liter⁻¹ and 7 to 47 μg of C liter⁻¹, respectively. The average bacterial cell volume was 0.185 μm³. [³H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 × 10⁻¹² to 200 × 10⁻¹² mol liter⁻¹ h⁻¹. Bacterial carbon production rates were estimated to be 0.2 to 7.1 μg of C liter⁻¹ h⁻¹. Bacterial production estimates from [³H]thymidine incorporation and ¹⁴CO₂ uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [³H]thymidine in the >3-μm fraction during a chrysophycean bloom in early June. We found that >50% of the ³H activity was in the >3-μm fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the ¹⁴CO₂ uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production.     

Explant culture of rat esophagus in a chemically defined medium

Summary Esophagus from adult male CDF rats was cultured for a period of 28 d in CMRL-1066 medium supplemented with pyruvic acid, HEPES buffer, β-retinyl acetate, and antibiotics. Morphological, radioautographic, and biochemical studies indicated that the survival of the tissue in serum-free medium was equivalent to that in medium containing 5% heat-inactivated fetal bovine serum. There was a relatively constant uptake of [³H]thymidine into DNA and [³H]leucine into protein of the esophageal explants during the incubation. Only the basal cells of the epithelium incorporated [³H]thymidine into their nuclei. The normal morphology of the tissue was preserved when the explants were maintained at both 37 and 30° C, and in either 50 or 20% O₂. Ninety-five percent O₂ was highly toxic to the cells of the explants. This culture system should be suitable for a variety of investigations in esophageal cell differentiation and carcinogenesis.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Effects of [3H]Udr On the Cell-Cycle Progression of L1210 Cells

Tritium-labelled uridine ([³H]UdR) perturbs progression of L1210 cells through the mitotic cycle. the main effect manifests as a slowdown or arrest of a portion of cells in G₂ and is already observed 2 hr after addition of 0.5–5.0 μCi/ml of [³H]UdR into cultures. At 2.5–5.0 μCi/ml of [³H]UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of [³H]UdR for 8–24 hr. A pulse of [³H]UdR of 2 hr duration labels predominantly (95%) cellular RNA. the first cell-cycle effects (G₂ slowdown) are observed when the amount of the incorporated [³H]UdR is such that, on average there are fewer than thirty-six [³H] decays per cell which corresponds to approximately 12–19 rads of radiation. the S-phase slowdown is seen at a dose of incorporated [³H]UdR twice as high as that inducing G₂ effects. the specific localization of [³H]UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed in the light of the differences between the effects of [³H]UdR and [³H]thymidine. Mathematical modelling of the cell-cycle effects of [³H]UdR is provided.     

Brugia pahangi: uptake and incorporation of adenosine and thymidine.

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

Nucleoside transport in choroid plexus: Mechanism and specificity

In vitro the transport into and release of [³H]thymidine, [³H]deoxyuridine, and [³H]nitrobenzylthioinosine (NBTI) from the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, were studied separately. Using the ability of NBTI to inhibit nucleoside efflux from the choroid plexus, the transport of [³H]thymidine and [³H]deoxyuridine into the choroid plexus at 37 °C was measured. Like thymidine, deoxyuridine was transported into the choroid plexus against a concentration gradient by a saturable process that depended on intracellular energy production but not intracellular binding or metabolism. The Michaelis-Menten constants (K_T) for the active transport of thymidine and deoxyuridine into the choroid plexus were 13.6 and 7.2 μM, respectively. Deoxyuridine and adenosine were competitive inhibitors of thymidine transport into the choroid plexus with inhibitor constants (K_I) of 6.8 and 14.5 μM, respectively. [³H]NBTI was also transported into the choroid plexus at 37 °C; unlike [³H]thymidine and [³H]deoxyuridine, the release of [³H]NBTI was not inhibited by NBTI itself. These studies provide evidence that the choroid plexus contains an active nucleoside transport system of low specificity for nucleosides, and a separate, saturable efflux system for nucleosides that is very sensitive to inhibition by NBTI. In vivo these systems transport nucleosides from blood into cerebrospinal fluid.     

Evidence for a 5' leads to 3' direction of hydrolysis by a 5' mononucleotide-producing exoribonuclease from Saccharomyces cerevisiae.

[³H]rRNA labeled at the 5′ terminus with ³²P and [³H]rRNA labeled at the 3′ end with [¹⁴C] (pA)_n have been degraded at 0° with a highly purified exoribonuclease from $\text{Saccharomyces cerevisiae}$. The results show that with the [³²P, ³H] substrate, the ³²P label is rendered acid-soluble at a much faster rate than the ³H label. Both acid-soluble labels are found in 5′ mononucleotide. With the [¹⁴C, ³H]rRNA, the ³H label is hydrolyzed at a faster rate than the ¹⁴C label. The exoribonuclease hydrolyzes in the 5′ → 3′ direction.     

Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.