[en] A simple combination of autoradiography, to determine when a cell synthesized DNA, and sister chromatid differential staining, to determine how many times a cell has divided, was used to follow up the proliferating fate of human lymphocytes in culture. Cells were incubated continuously with 5-bromodeoxyuridine (BrdU) and pulse-labelled with 0.1 μCi/ml [³H]thymidine at various times after stimulation with phytohemagglutinin (PHA). The cells were then harvested at 4 h intervals up to 72 h, and the percentage of labelled mitoses was determined separately in first, second, or third division cells. The data showed that the cycling cells, whether they began cycling at earlier or later times after stimulation, had about the same generation times of 12-14 h. This confirms that the heterogeneity of cell generations seen in short-term lymphocyte cultures is in large part due to the difference in the times when cells began cell cycling in response to PHA. 34 references, 2 figures, 1 table

[en] Root meristematic cells of nine unrelated diploid higher plants with genome sizes that differ 82-fold and with S periods that differ 4-fold have similar replicon sizes and single replication fork rates that average 22 μm/h and 8 μm/h respectively. The average replicon size of 22 μm is near the 18 μm obtained by extrapolation of measurements, taken from DNA fiber autoradiograms, to zero pulse time with [³H]thymidine. The data suggest that the duration of S is determined by the minimal number of replicon families that function sequentially during DNA replication

[en] Serum stimulation of DNA synthesis in quiescent mouse BALB/c 3T3 cells is accompanied by changes in total and intracellular Na⁺, K⁺, Mg₂⁺ and Ca₂⁺ content and in externally bound divalent cations. A sharp increase in the rate of [₃H] thymidine incorporation into DNA occurred after 10 h incubation following stimulation with 20% calf serum. This increase in DNA synthesis was accompanied by a decrease in intracellular Ca₂⁺, little change in K⁺ and an increase in Mg₂⁺ which reached 15% after 17 h. Incubation of confluent cultures for 17 h in media containing various serum concentrations caused the rate of DNA synthesis to increase rectilinearly with concentration, being 30-fold greater at 20 than at 0% serum. Concomitantly, intracellular cation changes occurred, levels at 20% serum as compared with 0%, being Na⁺, 91%; K⁺, 103%; Mg₂⁺, 114%; and Ca₂⁺, 41%. Externally bound divalent cations exhibited substantial serum dependence, bound Mg₂⁺ at 20% serum decreasing to 45%, and Ca₂⁺ to 22% of values at 0% serum. Results suggest that serum exerts its effects on cell growth at least in part by perturbing the plasma membrane, causing displacement of membrane divalent cations with a resultant change in membrane permeability and subsequently in cellular ion content. Although various cations including Na+, K⁺, and Ca₂⁺ may be of significance in the regulation of cellular growth, our data from this and previous studies support the concept that intracellular Mg₂⁺ is of primary importance in the control of cellular metabolism and growth

[en] A single-gelatin expanded film method for double-isotope autoradiography is described. A preliminary classification based upon silver-grain distribution is used to assign labeled cells to ³H only or with ¹⁴C classes. Optical sectioning combined with grain counting is employed to obtain ratios for classifying cells labeled with ¹⁴C into ¹⁴C only and ³H + ¹⁴C classes. The method has been tested with CHO-K1 cells in plateau-phase cultures using two 24-h labeling periods. The experimental design allowed for independent estimation of the expected frequencies of label classes under conditions that provided a wide range of possible label levels and combinations. Previous methods have used time-consuming applications of two emulsion layers and exposures to distinguish between cells labeled with ³H only or with ¹⁴C and do not identify the ³H + ¹⁴C class. A single-gelatin expanded film requires only one exposure and permits all label classes to be determined by an objective grain-counting procedure

[en] DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident

[en] Patterns of histone phosphorylation and histone H2A subfractionation have been compared in cultured cell lines from two species of deer mice, Peromyscus eremicus and Peromyscus boylii, which differ considerably in their content of heterochromatin but which contain essentially the same euchromatin content. DNA measurements by flow microfluorometry indicated that P. eremicus cells contained 34.2% more DNA than P. boylii cells, and C-band chromosome analysis indicated that the extra DNA in P. eremicus was present as constitutive heterochromatin. Subfraction of histone H2A by acid-urea polyacrylamide preparative gel electrophoresis in the presence of non-ionic detergent showed that each cell line contained two H2A subfractions. Incorporation of ³²PO₄ into these histones indicated that the steady state phosphorylation of the two H2A subfractions was not the same, the more hydrophobic H2A being greater than two times more phosphorylated than the less hydrophobic H2A in both cell lines. A comparison of the two cell lines indicated that the cell line with 34.2% greater constitutive heterochromatin contained a similar excess (29%) in its ratio of the more highly phosphorylated, more hydrophobic H2A subfraction to the less hydrophobic H2A subfraction. It is suggested that this enrichment of the more highly phosphorylated, more hydrophobic H2A subfraction may be related to the amount of constitutive heterochromatin present in the genome

[en] The floral organs of Tradescantia clone 4430 were used to investigate, in terms of cell cycle parameters, cellular behaviour during the maturation of a terminally differentiating system. Petals were sampled at different stages of development for (a) cell number; (b) nuclear DNA content by cytophotometry; (c) [³H]thymidine incorporation into nuclei by autoradiography; and (d) pigment production by spectrophotometry. DNA synthesis was confirmed by measurement of [³H]thymidine incorporation into TCA-insoluble material and changes in DNA content by colorimetric estimation of DNA extracts by diphenylamine. The development of the petal involved four sequential steps. First, there was an increase in cell number, an event characterized by mitoses, DNA synthesis, a few cells in G2 and a predominance in G1. Second, there was a cessation of cell division and DNA synthesis when all the cells accumulated in G1. Third, there was a shift of a large proportion of the total cell population from G1 to the G2 stage of the cell cycle and finally, there was pigment production. In addition, cytophotometric analysis of individual tissues in the mature petal revealed tissue specific differences in the proportion of cells in G2

[en] In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from [U-¹⁴C]glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state

[en] Replicon sizes were measured in Simian Virus 40 (SV40)-transformed and untransformed normal human, xeroderma pigmentosum (XP), and mouse 3T3 cells with an x-ray plus bromodeoxyuridine (BUdR) photolysis method. Replicon sizes in SV40-transformed cells were at least twice those in untransformed counterparts, but DNA fork displacement rates were only slightly increased

[en] Isolated rat hepatocytes could be made permeable to small molecules such as [¹⁴C]sucrose (but not to proteins) but subjecting the cells to repeated discharges in a high-voltage field. During subsequent incubation at 37⁰C, the permeability changes were reversed within 15 min, the electron-injected [¹⁴C]sucros remaining trapped inside the re-sealed plasma membrane. Autophagic sequestration of [¹⁴C]sucrose, i.e., the transfer of radioactivity from cytosol to sedimentable vesicles (autophagosomes and lysosomes), could be followed by incubating the [¹⁴C]sucrose-loaded hepatocytes for up to 2 h at 37⁰C. After incubation, the cells were disrupted by a single high-voltage discharge in electrolyte-free medium (sucrose), and sedimentable cell components were separated from the cytosol by centrifugation through metrizamide. By the use of these methods, which are particularly suitable for the analysis of many small cell samples, it could be shown that [¹⁴C]sucros was autophagically sequestered in the hepatocytes at a rate of 4-5%/h. The sequestration was nearly completely inhibited by the specific autophagy inhibitor 3-methyladenine