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.     

STUDIES ON RNA SYNTHESIS IN TWO POPULATIONS OF NUCLEI FROM THE MAMMALIAN CEREBRAL CORTEX

A method is presented for the rapid separation of cell nuclei from the rabbit cerebral cortex into two populations. The first of these consists largely of nuclei with the morphological characteristics of neuronal nuclei, the second almost entirely of nuclei with the morphological characteristics of glial cell nuclei. From studies based upon sensitivity to the toxin α-amanitin, the ratio of incorporation of different bases, ionic requirements and differential sensitivity to actinomycin D, it is concluded that under both the classical low and high salt conditions described by other workers, two enzymes are active in RNA synthesis. The presence of a third enzyme of low activity cannot be excluded. No qualitative difference in the number of enzymes involved in RNA synthesis in neuronal and glial cell nuclei has been found, but there are quantitative differences in activity between the two nuclear populations.     

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.     

DURATION OF CELL CYCLES IN CULTURED ROOTS OF CONVOLVULUS

The durations of the cell cycle in physiologically different regions of the meristem of cultured roots of Convolvulus arvensis were determined by the metaphase-accumulation technique involving colchicine. The cell cycle in the root cap increases from 13 hr in the actively dividing initials of the first tier to 155 hr in the slowly dividing initials of tiers 2–4 to an indeterminate value for derivatives of the initials in the root cap columella. The cycle times for the cells of the central cylinder and cortex are 21 and 27 hr, respectively. The cells of the quiescent center have a cycle of an estimated 420 hr. The duration of the cell cycle in these different regions is discussed in relation to the increased duration of G₁ in slowly or non-dividing cells. The possible regulation of cell division by the synthesis of a cell-division factor in the quiescent center is also discussed.     

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.     

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.     

香烟焦油中稳定性自由基等物质对细胞DNA合成的影响

用苯、乙烷及异丙醇(7:2:1)萃取烟焦油得到含有稳定性自由基的萃取物.萃取物能损伤DNA模板,从而强烈抑制细胞DNA合成.萃取物具有强还原力.升温(20,45和85℃)不但使自由基浓度下降,同时使萃取物还原能力下降,把萃取物与细胞温育后,细胞内出现了一个新的稳定自由基信号,它随温育时间延长而增大,g值为2.0008,不同于萃取物本身的ESR信号(g=2.0032).     

A DEMONSTRATION OF SEVERAL DEOXYRIBONUCLEASE ACTIVITIES IN MAMMALIAN CELL MITOCHONDRIA

Extracts of purified mitochondria from adult rabbit liver and kidney have been prepared by lysis with Triton X-100. Such extracts contain deoxyribonuclease activity demonstrable at alkaline pH. Studies utilizing the effects of substrate variation, differing ionic strength, nucleoside di- and triphosphates, and SH-group inhibitors reveal the existence of at least five distinguishable deoxyribonuclease activities in these extracts. Assay of lysosomal and mitochondrial enzyme markers indicates no significant lysosomal contamination of the mitochondrial extracts. Further studies also suggest that the alkaline deoxyribonuclease activity is specifically located in or in association with mitochondria.     

DNA MICROSPECTROPHOTOMETRY OF SHOOT APICAL MERISTEM CELL POPULATIONS IN CERATOPTERIS THALICTROIDES (FILICALES)

A microspectrophotometric analysis of the DNA content of cell populations in the shoot apical meristem of young and adult stages of the filicalean fern Ceratopteris thalictroides was conducted to determine if the previously reported uptake of labeled DNA precursors by the apical cell was a consequence of endomitotic DNA replication or of DNA synthesis preceeding mitosis. Results demonstrate that in both the young and adult plants the estimated DNA content of the apical cell nuclei parallels that of the other cells of the meristem. There was no evidence of an “apical zone” of endopolyploid cells.     

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.     

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.     

SYNTHESIS OF RNA IN MAMMALIAN CELLS DURING MITOSIS AND INTERPHASE

Chinese hamster cells in the mitotic and G₁ phases of the growth cycle were incubated for 30 or 60 min in suspension tissue culture and pulse-labeled with tritiated uridine. After appropriate chases, washes, and extractions, it was found that all incorporation into the nucleic acid may be accounted for by those cells in interphase. An average of 410 counts was found for incorporation into the cell population (approximately 2.0 x 10⁵ cells) of which over 80% of the cells was initially in mitosis. The increasing number of cells leaving mitosis and entering interphase during the 30 min incubation was theoretically able to account for 470 counts. In addition, short-pulse labeling experiments have shown a consistent linear relationship between the percentage of cells in division and the incorporation of the isotope, which strongly suggests that, if 100% of the cells were in mitosis, the counts would be essentially zero. Thus, the entire label may be attributed to those cells in interphase where portions of the chromosomal material are known to be already extended.     

CELL DIVISION AND DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS DEPRIVED OF ESSENTIAL AMINO ACIDS

The question of amino acid requirements for DNA synthesis and cell division has been studied in Tetrahymena pyriformis by depriving cells of histidine and tryptophan at defined stages in the interdivision interval. Deprivation any time before DNA synthesis does not prevent the initiation of such synthesis but completely inhibits the following division and limits the increase in DNA, as measured microspectrophotometrically, to 20 per cent. H³-thymidine added to the medium is not incorporated during the 20 per cent increase. Deprivation after DNA synthesis is initiated does not prevent the continuation (to completion) of DNA synthesis, and cell division ensues. H³-thymidine added to the medium under these conditions is incorporated into macronuclear DNA. The data indicate that some amino acid-dependent event occurs, about the time of the beginning of the DNA synthesis period, which is not essential for initiation of DNA synthesis but which is essential for the maintenance of synthesis once it has begun. These results are further discussed in terms of enzymes required to convert thymidine (and possibly the other three deoxyribonucleosides) to the immediate precursor of DNA synthesis.     

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.