NUCLEAR MEMBRANES OF CULTURED MAMMALIAN CELLS IN THE PERIOD PRECEDING DNA SYNTHESIS

When kidney cells are cultured directly from the rabbit, the nuclear membranes undergo a change that can be measured as an increase in electrophoretic mobility. The change appears to begin immediately upon culture and is maximal 2 hours later, after which the mobility remains constant at the elevated level. Actinomycin D and p-fluorophenylalanine, but not EDTA or ionizing radiation, suppress the increase in nuclear electrophoretic mobility. With synchronously growing L cells, no change was detected in nuclei from cells taken during various parts of the division cycle.     

RELATION OF PROTEIN SYNTHESIS TO THE DIVISION CYCLE IN MAMMALIAN CELL CULTURES

The distribution of the reaction product of a staining method for adenosine triphosphatase (ATPase) in rat small intestine, kidney, and liver was studied with electron microscopy. Several procedures were tried but the best results were obtained from tissue that had been quenched in liquid nitrogen, sectioned at 25 µ in a cryostat, fixed for 30 to 90 minutes at 4°C in formalin-sucrose buffered to pH 7.2, incubated with substrate, and then osmicated and prepared for electron microscopy in the usual way. This procedure enabled the localization of mitochondrial ATPase to be studied. In tissue fixed in small blocks in osmium tetroxide for 3 minutes prior to incubation with substrate, good preservation was noted, and the reaction product for ATPase was localized on the cell membrane and nuclei. The reaction product was present in abundant amount in the nuclei, and particularly within nucleoli, of all tissues studied. Because the histochemical localization of nuclear enzymes poses numerous interpretative problems at the present time, the significance of this nuclear localization is uncertain. Cell (plasma) membranes were the site of localization, especially at areas where it has been proposed that active transport mechanisms may occur, namely, on the microvilli of intestinal epithelium, endothelial lining of capillaries, glomerular epithelial cell membranes, basal infoldings of the cell membrane of renal tubules, on the microvilli of bile canaliculi, and on the microvilli of proximal convoluted tubular epithelial cells. ATPase localization on the cristae mitochondriales was also demonstrated.     

THE CHARACTERIZATIO OF MONOAMINE OXIDASE IN CULTURED MAMMALIAN CELLS

The expression of monoamine oxidase (MAO) in a variety of mammalian cells has been investigated employing tryptamine as substrate. The enzyme present in those cell lines having sufficient activity for detailed analysis exhibited a monophasic response to the inhibitor clorgyline. On this basis the cell lines examined were found to express only A, or only B, type activity. Hypoxanthineguanine phosphoribosyl transferase (HGPRT) deficient derivatives of both MAO type A, or MAOtype B, expressing cells were examined. The HGPRT status of the cells appeared to have little influence on the expression of the enzyme.     

CELL PROLIFERATION IN COMPLEX TISSUES: THE CONTROL OF THE MITOTIC CYCLE OF CELL POPULATIONS IN THE CULTURED ROOT MERISTEM OF SUNFLOWER (HELIANTHUS)

Experiments were performed with cultured primary root tips of sunflower (Helianthus annuus var. Russian Mammoth) to determine: (1) if progression in the mitotic cycle of meristematic cells was nutritionally controllable by carbohydrate starvation and replenishment; (2) where in the mitotic cycle control was effected; and (3) whether nutritional deprivation could be used to detect phenotypically different subpopulations in a complex tissue. Meristematic cells were rendered stationary by carbohydrate starvation, as indicated by the absence of cell division; this condition was reversed by carbohydrate provision. After 72 or 96 hr of starvation most cells stopped in G1 (80–90%) and G2 (10–20%), and a very few (“leaky” cells) continued to enter S. “Leaky” cells represent a small population with an S period of approximately 4.1 hr that either lack a principal control point in G1 or have an unusual metabolism whereby the control point requirements are met and have a carbohydrate dependence for mitosis. Though phenotypically different, no specific functions can be attributed to “leaky” cells at this time.     

THE DETERMINATION OF PICOMOLE AMOUNTS OF ACETYLCHOLINE IN MAMMALIAN BRAIN

Abstract— In any assay for the determination of acetylcholine based on the conversion of choline to a product, the immediate problem is the removal of endogenous choline. Other published enzymatic assays have taken advantage of electrophoresis to accomplish this goal. In the assay to be described, this is accomplished by the enzymatic phosphorylation of endogenous choline by choline kinase. Once this reaction is complete, endogenous acetylcholine is simultaneously hydrolysed and then phosphorylated with [³²P]ATP. The labelled product [³²P]phosphorylcholine is separated from the labelled substrate by precipitation of the ATP and further separation is accomplished on microcolumns of ion exchange resin. Using this methodology, picomole amounts of acetylcholine, derived from tissue, can be measured.     

MEASUREMENTS OF RATES OF PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The use of tracer concentrations of labelled amino acids to measure incorporation in incubated slices of brain results in wide fluctuations with time in the specific activity of the precursor. Using concentrations of about 1 mm of labelled amino acid facilitates the accurate measurement of rates of synthesis. These higher precursor levels in the medium decrease the fluctuations in free amino acid specific activity due to dilution by endogenous amino acid and the production of amino acid by protein degradation, and decrease the lag in incorporation due to transport phenomena. Concentrations of 1 mm amino acid in the medium did not inhibit protein synthesis; with valine, leucine, phenylalanine, lysine and histidine, incorporation rates were similar when measured at trace concentrations and at 1 mm medium levels. The source of amino acid for protein synthesis appears to be intracellular. No evidence could be found for the preferential use of extracellular medium amino acid. The rate of incorporation of amino acids in incubated slices of rat brain was 0.087 per cent of the protein amino acid/h.     

THE EFFECT OF pH ON GROWTH, PROTEIN SYNTHESIS, AND LIPID-RICH PARTICLES OF CULTURED MAMMALIAN CELLS

A simple procedure has been established for controlling and measuring the pH of media in which the bicarbonate-carbonic acid system is the predominant buffer. The HCO^-₃ concentration was maintained at 22.5 mM and the H₂CO₃ concentration was varied by equilibrating the media with 0.5 to 40 per cent CO₂ in air. The curve relating extracellular pH to 3 day cell growth was similar for glass-attached HeLa and Chang liver cells. Maximum growth occurred over a pH range of 7.38 to 7.87. Cell growth declined precipitously on the alkaline side and more gradually on the acid side of the optimal pH range. Comparable pH growth curves were also obtained with newly isolated cells from rat liver and skeletal muscle. It was shown that the effect of pH on growth was independent of the CO₂ concentration and that the essential nutrients in the medium were stable over the pH range studied. Although alkalosis depressed the 3 day cell population, cells exposed to a pH of 8.0 to 8.2 grew at the maximal rate for the first 12 to 24 hours. Growth then ceased abruptly and the cells entered a steady state with respect to net protein synthesis. This was followed by cytoplasmic retraction and cell death. Increasing the concentrations of calcium or magnesium in the medium failed to prevent the effects of alkalosis. Moreover, the increase in CO^-₃ concentration of the media and the concomitant decrease in Ca⁺⁺ ion concentration that occur at high pH were eliminated as determining factors in the growth failure and death. While acidosis had a less pronounced effect on the 3 day cell population, its effect on the growth rate was immediate. The increase in cell generation time was proportional to the H⁺ ion concentration. In each of the cell lines studied, acidosis was accompanied by a striking increase in the number of cytoplasmic perinuclear granules. These granules which stain supravitally with Janus green are extracted from fixed cells with lipid solvents. They maintain their identity in cell homogenates and may be isolated from the other subcellular structures by differential centrifugation; at 100,000 g they form a distinct layer at the top of the supernatant fraction. On the basis of their physical and chemical properties, these granules have been called lipid-rich particles. The accumulation of lipid-rich particles in acidosis was independent of the growth rate and the CO₂ concentration.     

VARIABILITY OF SHORT-TERM CULTURES OF HeLa S-3 CELLS: CHANGES IN DNA DISTRIBUTIONS AND RATES OF DNA SYNTHESIS

DNA distributions of HeLa S-3 cells in spinner culture exhibit significant time—dependent changes. The major differences appear to occur in the S-phase region. Significant changes in the rates of DNA synthesis in several S-phase subcompartments correlated well with the changes in the DNA distributions. It is proposed that fluctuations in these rates of DNA synthesis are a reflection of the inherent instability of these abnormal, heteroploid cells.     

EFFECT OF EHRLICH ASCITES CELL CHALONE ON NASCENT DNA SYNTHESIS IN ISOLATED NUCLEI

Ehrlich Ascites Tumor (EAT) chalone has been shown to inhibit nascent DNA synthesis by inhibiting DNA polymerase α and β (Nakai, 1976), but one of the problems in studying eurkaryotic DNA replication has been the relative impermeability of the cell membrane to precursors and macromolecules; hence, to circumvent this restriction without sacrificing the integrity of the replication process, a broken cell system utilizing nuclei in aqueous media was investigated. Isolated nuclei appear to continue the process of DNA replication that was proceeding in vivo before their isolation and under optimal conditions are able to initiate new synthesis (Fraser & Huberman, 1977). The effects of partially purified EAT chalone on nascent DNA could be studied directly in this nuclear system, which excluded effects of the cell membrane, nucleotide pools and other cytosol elements. A concentration-related inhibition of [³H]thymidine triphosphate ([³H]-dTTP) incorporation was noted over a chalone range of 50–200 μg/ml. It appears that chalone can inhibit DNA polymerase α directly within the nucleus without an intermediate step such as a cell membrane receptor.     

TURNOVER OF CELL-RENEWING POPULATIONS UNDERGOING CIRCADIAN RHYTHMS IN CELL PROLIFERATION

Evidence is presented in support of the concept of partial synchrony of cells as the cause for circadian rhythms in DNA synthesis and mitotic activity. The nonuniform age distribution of cells in cycle indicated that equations based on total asynchrony were not applicable for calculation of cytokinetic parameters in cellrenewing populations undergoing circadian rhythms. The integration of the circadian mitotic curve is introduced as a simple and accurate method for determination of proliferation rate and turnover time. An approximately linear increase in the labeling index following repeated injections of ³H-thymidine demonstrated that nearly 100% basal cells in hamster cheek pouch epithelia were in cycle during a turnover time. These experiments suggest that if there is a G₂ phase in cellrenewing tissues, this is short with respect to turnover time and that it may be a specific compartment where the control of cell proliferation operates.     

ENZYMES OF ENERGY-CONVERTING SYSTEMS IN INDIVIDUAL MAMMALIAN NERVE CELL BODIES1

Abstract— The activities of 7 enzymes (hexokinase, phosphofructokinase, glucose-6-P dehydrogenase, 6-P-gluconic dehydrogenase, NADP linked isocitric dehydrogenase, malic dehydrogenase and lactic dehydrogenase) were measured in individual nerve cell bodies of 8 different neuronal types: pyramidal cells from cerebral cortex and Amnion's horn, Purkinje cells, giant cells in the reticular formation, Deiters’nucleus cells, facial nucleus cells, anterior horn cells and dorsal root ganglion cells. Samples of similar size were analysed from the molecular layer of cerebellum. The cell bodies were dissected from frozen-dried tissue sections and weighed on quartz fibre balances. The weights ranged from 0–2 ng for the smallest pyramidal cells to 9 ng for the largest giant cells. The specific enzymatic reactions were carried out in small volumes (0–01–5 μl) under mineral oil (‘oil-well technique’). The NADPH₂ or NAD formed was amplified by‘enzymatic cycling’and measured fluorometrically. A new cycling method was used for measuring the NAD formed in three of the enzymatic methods. Double cycling was used to measure glucose-6-P and 6-P-gluconate dehydrogenases in the smallest cell bodies. Each type of neuron exhibited a unique enzyme pattern, but four general patterns could be distinguished. The most variable of the enzymes was glucose-6-P dehydrogenase which was nearly 10-fold higher in anterior horn cells than in pyramidal cells from the cerebral cortex. Malic dehydrogenase was the most constant, with a 3-fold range from the highest (Purkinje cells) to the lowest (dorsal root ganglion cells).     

EFFECT OF PENTOBARBITAL ON MITOCHONDRIAL SYNTHESIS IN CULTURED GLIAL CELLS

Abstract— Primary cultures of glial cells prepared from brains of newborn rats were grown for 1 week and then exposed to 5 × 10⁻⁴ m -pentobarbital (PB) for 4 weeks. Compared with glial cells grown in drug-free medium throughout, exposure to PB significantly increased hexokinase activity, primarily in its mitochondrial form. Furthermore, cellular protein and RNA concentrations were significantly higher in barbiturate-cultivated than in control cells. Pulse labelling with [³H]thymidine after 4 weeks of PB exposure resulted in a significant increase (332%) in ³H incorporation into mitochondrial DNA while ³H incorporation into nuclear DNA was reduced by 58%. In addition, there was a time dependent increase in the size and number of mitochondria as determined in electron micrographs. These results are interpreted to reflect an increased mitochondrial metabolism in glial cells after chronic exposure to the barbiturate and may constitute a compensatory mechanism to the depressant action of this drug.     

CELL DIVISION RATES OF EUCARYOTIC ALGAE MEASURED BY TRITIATED THYMIDINE INCORPORATION INTO DNA: COINCIDENT MEASUREMENTS OF PHOTOSYNTHESIS AND CELL DIVISION OF INDIVIDUAL SPECIES OF PHYTOPLANKTON ISOLATED FROM NATURAL POPULATIONS1

The cell cycle marker event of DNA replication in eucaryotic algae was identified using ³H-Thymidine (³H-TdR) incorporation. The frequency of cells (F) within a population undergoing DNA replication was estimated and the cell division rate (μF) calculated. In laboratory cultures the rates of cell division calculated from changes in cell numbers (μN) and μF were similar. Dual labelling with ³H-TdR and NaH¹⁴CO₃ enabled rates of cell division and photosynthesis to be coincidently measured for individual species of algae. Using these single species radioisotope techniques, several distinct photosynthesis irradiance and cell division irradiance relationships were found for: (1) different species of phytoplankton isolated from the same sample, and (2) the same species isolated from different environments. These techniques allow the coupling between photosynthesis and cell division to be examined with high resolution for algae in situ.     

THE FLUOROMETRIC DETERMINATION OF 5-METHOXYTRYPTAMINE IN MAMMALIAN TISSUES AND FLUIDS

Abstract— Using a sensitive and specific fluorometric procedure involving selective extraction, reaction of the extracts with o -phthalaldehyde (OP), separation of the OP derivatives by TLC, and determination of fluorescent characteristics and intensities, we have detected and measured 5-methoxytryptamine, (5-MT) in various central and peripheral tissues and fluids of the rat, dog, baboon, and man.
Distribution of 5-MT in peripheral tissues of the rat seemed to parallel that of 5-HT, with highest levels being found in the gastrointestinal (GI) tract and Harderian gland, regions that are rich in 5-HT and have been reported to contain systems capable of methylating 5-HT. 5-MT was detected in the lung, plasma, kidney, spleen, and heart of the rat. 5-MT was present in the CNS of all species examined. No marked interspecies differences were observed. In the rat CNS, the regional distribution of 5-MT did not parallel that of 5-HT indicating that the systems for the synthesis, uptake, or transport of 5-MT might be different than for 5-HT. Pretreatment of rats with iproniazid resulted in a 50% increase in whole brain 5-MT. Reserpine pretreatment had no effect, indicating that the storage or release mechanisms for 5-MT are different than for the conventional amine transmitters. 5-MT was detected in human CSF and urine but not in plasma. These data indicate that 5-MT, a compound with potent pharmacological properties, is more widely distributed in the mammalian body than had previously been supposed.     

THE TEMPORAL SCHEDULE OF DNA SYNTHESIS AND CELL DIFFERENTIATION

The temporal schedule of DNA synthesis in cells of developing and adult mice is analysed by means of Feulgen cytofluorometry combined with tritiated thymidine autoradiography. The results obtained with cells taken from liver, esophageal epithelium and mucosae of gastrointestinal tracts seemed to conform to the hypothesis that a cell at a particular state of cytodifferentiation possesses specifically inactivated sets of late replicating genes showing a specific pattern of the temporal schedule of DNA synthesis.     

缺血缺氧对体外培养星形胶质细胞细胞活化和细胞周期的影响

目的观察缺血缺氧损伤对星形胶质细胞细胞活化和细胞周期的影响。方法用流式细胞仪及BrdU掺入法检测缺血缺氧后不同时间点星形胶质细胞细胞周期变化和细胞的增殖活力;用荧光免疫细胞化学技术测定胶质细胞纤维酸性蛋白(GFAP)及细胞周期蛋白cyclinD1的表达水平。结果体外缺血缺氧损伤后星形胶质细胞S期较正常组明显增高,6h达高峰,BrdU掺入法显示损伤后6h星形胶质细胞的增殖活力最高,而随后S期细胞数目及细胞增殖活力都呈下降趋势。在缺血缺氧早期,GFAP阳性染色增强,6h最高;缺血缺氧12h后GFAP阳性染色变弱,而cyclinD1的表达在损伤后逐渐增加,在24h时达高峰。结论缺血缺氧损伤激活星形胶质细胞,使其进入新的细胞周期,出现细胞的增殖反应;cyclinD1参与了损伤后星形胶质细胞的修复和增殖;细胞周期事件与星形胶质细胞的增殖活化密切相关。