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.     

DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [³H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [³H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [³H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10⁶ cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.     

CHANGES IN THE DNA SYNTHETIC PERIOD DURING THE PHASE OF RAPID GROWTH IN CULTURED DIPLOID FIBROBLASTIC CELLS

The DNA synthetic period in diploid fibroblastic cell populations was studied during the phase of rapid growth in vitro , using the labelled mitotic wave method and quantitative autoradiography.
It was found that the length of the DNA synthetic period increased progressively, from early passage levels, with the number of generations in vitro. the increase was mainly in the time taken to synthesize the first 50%, and especially the first 10%, of DNA.
The change was detectable despite the complication due to a low level of tritium incorporation after the removal of tritiated thymidine from the cultures.     

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.     

THE SYNTHESIS OF PHOSPHOLIPIDS IN THE NUCLEUS AND NUCLEAR MEMBRANE OF SYNCHRONIZED HeLa CELLS

HeLa cells were synchronized by a double thymidine block and pulse labeled at different stages of the cell cycle with ³H-choline. The specific activity of phospholipids extracted from the cell, the nucleus and the nuclear membrane showed a progressive increase from S to G₁; the incorporation of choline into phospholipids of asynchronous cells showed a specific activity intermediate between the values of S and G₁ cells. Similar results were obtained when ³²phosphorus was used as a precursor instead of choline. Thin layer chromatographic analysis of phospholipids extracted from cells in S and from cells in G₁ failed to show any difference in the distribution of radioactivity among the various phospholipid classes. Choline uptake by HeLa cells in different phases of the cell cycle did not show significant variations. However, during the synchronization process, shortly after the addition of excess thymidine, an increased uptake of choline by cells and an increased incorporation of choline into phospholipids were found. The results indicate that some of the changes occurring in phospholipids synthesis may not be cell cycle dependent, but may be the effect of the synchronizing process.     

THE TIME OF SYNTHESIS AND THE CONSERVATION OF MITOSIS-RELATED PROTEINS IN CULTURED HUMAN AMNION CELLS

p-Fluorophenylalanine (PFPA), an analogue of phenylalanine which may be incorporated into proteins, increases the duration of mitosis. In the present experiments, based upon quantitative analyses of time-lapse cinemicrographic films, brief treatments of cells with PFPA are shown to affect the duration of metaphase in only those cells which enter division during or shortly after treatment. The offspring of cells with prolonged metaphases also tend to have prolonged metaphases. Analyses of the kinetics of the appearance of prolonged metaphases indicate that some protein specifically associated with mitosis is synthesized primarily during a period which corresponds closely to G₂. The manner in which the defect is passed on to daughter cells indicates that the protein involved is conserved and reutilized by daughter cells for their subsequent divisions. Comparable experiments performed with low concentrations of puromycin indicate that the major effect of PFPA is due to its incorporation into protein rather than its ability to inhibit protein synthesis. The fact that puromycin-induced effects can also be passed on to daughter cells is interpreted to mean that cells make only specific amounts of some mitosis-associated proteins and that if a cell "inherits" a deficiency in such protein it is not able to compensate for the deficiency.     

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.     

THE PERIOD OF DNA SYNTHESIS PRIOR TO MEIOSIS IN THE MOUSE

Sixteen pregnant female mice were operated on and H³-thymidine was injected into the amniotic cavities of the uterus. The injection was given between the 6th and 14th days of fetal life. Eighty-eight fetuses received thymidine in this way. Another series of 16 pregnant females was injected intraperitoneally with H³-thymidine between the 5th and 14th days of pregnancy. Two of these females were killed 16 days after the observation of the vaginal plug. The remaining 30 females were allowed to give birth to their progeny. The progeny was killed at birth and the ovaries of the newborn females fixed at once. Labeled oocytes at late pachytene and early diplotene were clearly seen in individuals that received the isotope between the 10th and 12th to 13th days of fetal life, but the period of DNA synthesis preceding meiosis is at the 12th to 13th days of fetal life. Since meiosis is recognized by the 14th day, only the oocyte labeling originating from mothers injected at the 12th and 13th days may be considered as representing the DNA synthesis of the premeiotic replication.     

SYNTHESIS OF NUCLEAR RNA IN NERVE AND GLIAL CELLS

—Tritium-labelled RNA precursors were injected at 30 min intervals into the fourth ventricle of rats or rabbits. After 4 h the nuclei from neurones, astrocytes, and other glial cells were isolated and RNA extracted. Investigations were performed in order to establish optimum conditions for RNA extraction from this particular material. The sedimentation patterns obtained in sucrose gradients were similar to those of nuclear RNA from other mammalian tissues and showed the presence of RNA species with high specific activities in the region of the gradient between 10S and 16S and above 28S. All three types of nuclei contained a 45S and a 38S RNA. Moreover, a 32S component could be identified in astrocytic nuclei, a 35S fraction in neuronal nuclei, and both a 32S and 35S RNA in nuclei from glial cells. The nuclei from the various cell types also differ with respect to the rate of incorporation of the label into the nuclear RNA, being four times higher in astrocytic and neuronal nuclei than in those derived from the other glial 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.     

THE PATTERN OF DNA SYNTHESIS IN THE CHROMOSOMES OF HUMAN BLOOD CELLS

The sequence in which various regions of the chromosomes of human blood cells complete DNA synthesis in vitro has been studied through the use of H³-thymidine labeling and autoradiography. Certain of its aspects have been defined, and these may serve as a basis for comparing the pattern of synthesis in cells of other tissues. In general, the long chromosomes continue replication later than the short ones. Variability of the sequence has been prominent. One pair from Group 13–15 and pair No. 17 complete replication early. In certain other chromosomes, replication is very active late in the S period, e.g. one X of the female cell, the Y of the male cell, two of Group 4–5, two of Group 13–15, the Nos. 16, and the Nos. 18. In the normal human female a striking correlation exists between the late replication of one of the X chromosomes, condensation during the intermitotic period, and presumed genetical inactivation. The pattern of replication characterizes certain chromosomes whose structural features alone are non-distinctive, and it may be useful in studies of cells in which a chromosomal aberration occurs.     

THE KINETICS OF CONTACT INHIBITION IN MAMMALIAN CELLS

To elucidate the process of contact inhibition in mammalian cells, we investigated the kinetics of growth arrest in [³H]thymidine labelled embryonic chinese hamster (Cricetulus griseus L.) cells after the addition of various concentrations of unlabelled cells. It was observed that after the contact inhibition concentration had been reached, the cells grew undisturbed for one more generation. In the following 24 hr the concentration fell back to the level at the beginning of the experiment and stayed there.     

DNA SYNTHESIS IN THE GLIAL CELLS OF SCRAPIE-AFFECTED MOUSE BRAIN

Radioautographic techniques have been used to study the incorporation of [³H]-thymidine into the DNA of glial cells in normal and scrapie-affected mouse brain. Some differences were seen in the morphological distribution and also in the size distribution of labelled nuclei in sagittal sections of normal and affected brain. Counts of the total number of labelled nuclei per sagittal section showed a progressive decline from 50 to 15 per section in normal brain over the 4-month experimental period. However, in scrapie-affected brain the number increased markedly to 120 per section 5-6 weeks after inoculation and stayed at this level for the remaining 9-10 weeks of the incubation period. The significance of these results in relation to the pathogenesis of scrapie is discussed.     

THE NUCLEOLI IN MITOTIC DIVISIONS OF MAMMALIAN CELLS IN VITRO

In a number of mammalian cell strains nucleoli persisted through mitosis. This phenomenon was especially pronounced in several cell lines derived from Chinese hamster tissues. All the methods employed, including radioautography with tritiated uridine, cytochemical stains (methyl green-pyronin and azure B), fluorescent microscopy (coriphosphine O), ribonuclease digestion, and electron microscopy, demonstrated that the bodies identified as persistent nucleoli in the mitotic stages had the same characteristics as did the nucleoli in the interphase. Persistent nucleoli may attach to the chromosomes or may be free in the cytoplasm. In cells where no persistent nucleoli as such were noted, nucleolar material was observed to attach to the chromosomes in shapeless masses which moved with the chromosomes during anaphase. At least a portion of the nucleolar material was included in the daughter nuclei, presumably for immediate use for protein synthesis after cell division.     

血管紧张素Ⅱ对培养心肌细胞蛋白质和DNA合成的影响

本文利用体外细胞培养技术,观察了血管紧张素Ⅱ对Ｗｉｓｔａｒ乳鼠的心肌细胞和心肌组织中非心肌细胞的促生长作用。结果表明：随ＡｎｇⅡ浓度的升高,心肌细胞直径、总蛋白含量及＾３Ｈ－Ｌｅｕ掺入率均呈剂量依赖性增加,而各组间＾３Ｈ－ＴｄＲ掺入率及心肌细胞数目则无明显变化。相反,ＡｎｇⅡ在引起心肌组织中非心肌细胞（主要是成纤维细胞）的＾３Ｈ－Ｌｅｕ掺入率增加的同时,也引起其＾３Ｈ－ＴｄＲ掺入率及细胞数目的增加     

NUCLEAR ENVELOPE-ASSOCIATED RESUMPTION OF RNA SYNTHESIS IN LATE MITOSIS OF HELA CELLS

The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G₁) has been investigated in synchronized HeLa S₃ cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G₁ was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope.     

STATIONARY PHASE OF CULTURED MAMMALIAN CELLS (L5178Y)

The stationary phase of the mammalian cells L5178Y in culture can be divided into two stages: (a) an early phase characterized by the decline of mitotic index, followed by a stabilization of the cell number, and (b) a late stage, occurring several hours after the flattening of the growth curve, during which dead or dying cells appear in the cultures. The estimates of rates of cell progress showed that the rates from G₁ to S and from G₂ to M were affected in the early stationary phase. The main cause of cessation of increase in cell number in the early stationary phase is resulted from the decline in mitotic index, which is caused by prolongation of the G₂ period. The importance of the G₂ stage in regulating the cell growth is discussed in relation to other known situations in the literature.