The relation between length of the cell cycle duration and RNA content in HeLa S3 cells

The cell cycle of HeLa S3 cells synchronized by hydroxyurea was investigated by flow cytometry. Metachromatic fluorochrome acridine orange was used to strain the DNA and RNA of the cells differentially. Periodic changes in the cellular DNA and RNA contents were observed through five cell cycles. The G1 and S phases of synchronized HeLa S3 cells that contained large amounts of RNA became shorter than those of cells that contained smaller amounts of RNA.     

RNA dependence in the cell cycle of V79 cells

The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.     

SYNCHRONIZATION OF BABY RAT HEPATOCYTES: A NEW TEST FOR THE DETECTION OF FACTORS CONTROLLING DNA SYNTHESIS IN RAT HEPATIC CELLS

The subcutaneous injection of irritating substances to baby rats results in a very reproducible wave of synchronized S phase DNA synthesis in hepatic cell involving 20% of the total population. Use has been made of this reaction to detect factors affecting DNA synthesis in hepatic cells. It enables substances to be tested during precise periods of the cell cycle. Two activities which were detected in normal adult rat serum, could not be found in the serum of the baby rat or of the partially hepatectomized adult rat: an activity inhibiting the progression of hepatocytes through the cell cycle in the late G₁ phase, and an activity inducing the production of binucleate hepatocytes, effective in the late G₁ and in the S phase.     

Fertility Politics as "Social Viagra": Reproducing Boundaries, Social Cohesion, and Modernity in Italy

In this article, we investigate the delicacy of adopting pronatalism as a public position in Italy. Mounting scientific and political knowledge about the demographic "problem" exposes a new hegemonic formation that low fertility is dangerous. Drawing on ethnographic contexts, political debates, media publications, and policy documents, we trace the "demographic emergency" and compare two policies: a monetary baby bonus and a law restricting assisted reproduction. The coexistence of incentives to counter superlow fertility with prohibitions on high-tech baby making reflect the contested governance of "social cohesion." We conclude that scholarly and popular discourses serve as a sort of "social Viagra." Ultimately, both policies sought to rejuvenate family norms. Both aimed to fortify the political terrain of a nation-state struggling to achieve and maintain modernity against a backdrop of immigration and aging. Modernity became a weapon of the state to exert control over Italian fertility practices and of its critics to deploy orientalizing representations of backwardness.     

Primordial germ cells and early stages of oogenesis in Ophryotrocha puerilis (Polychaeta,Dorvillediae)

Summary Each setigerous segment of the protandric polychaete Ophryotrocha puerilis contains two primordial germ cells. A ventral furrow in the gut wall together with the peritoneal lining of the gut forms a genital blood vessel. The gonocytes are located within the peritoneum of this genital blood vessel. At sexual maturity the gonocytes undergo a proliferation cycle, the first division of which gives rise to a cell which is extruded into a forming outpocketing of the coelomic lining. The stem cell remains within the peritoneum. Inside the forming gonad the detached cell goes through a series of four mitotic divisions. The resulting 16 cells are interconnected by cytoplasmic bridges. These bridges are arranged in a very regular pattern which allows the mitotic cycles to be followed. While remaining still within the gonad the 16 cells begin to synthesize yolk and to take up exogenous yolk precursors. At this stage a differentiation into oocytes and nurse cells becomes visible. The oocytes deposit yolk platelets of the definitive size whereas the polyploid nurse cells produce only small yolk bodies that are passed to the adjacent oocytes. In a later stage the cell bridges between adjacent nurse cells are cut and pairs of one oocyte and one nurse cell are released to the coelomic cavity during breakdown of the gonadal sac. Oocyte-nurse cell-complexes then freely float in the coelomic fluid. The proliferation of gonadal cells is well synchronized within one segment. In anterior segments, however, gonadal proliferation usually begins earlier than in posterior segments but smaller oocytes in posterior segments catch up within a few days. Finally a batch of oocytes is produced in which all the oocytes are of the same size (120 m). The origin of the primordial germ cells remains unknown.     

Comparison of synchronized Chinese hamster ovary cells obtained by mitotic shake-off, hydroxyurea, aphidicolin, or methotrexate

Synchronized cell populations are necessary to study many aspects of cell biology. We have developed a method to obtain highly synchronized Chinese hamster ovary cell populations in S phase or G2 phase by utilizing mitotic selection followed by incubation with either hydroxyurea, aphidicolin, or methotrexate for 12 h. Flow cytometry analysis shows that the coefficient of variation in the spread of the cell population in S phase is as low as 6%. Drug toxicity studies compare the effects of the various drugs on G1 and S phase cells. The use of aphidicolin or hydroxyurea results in the most highly synchronized cell populations, but methotrexate yields inadequate synchronization. These results demonstrate that both aphidicolin and hydroxyurea are useful drugs for obtaining highly synchronized cell populations after an initial synchrony in mitosis. Aphidicolin is perhaps the best choice because of less toxicity to S phase cells when used in low concentrations.     

Cyclin A associates with the fusome during germline cyst formation in the Drosophila ovary

Regulated changes in the cell cycle underlie many aspects of growth and differentiation. Prior to meiosis, germ cell cycles in many organisms become accelerated, synchronized, and modified to lack cytokinesis. These changes cause cysts of interconnected germ cells to form that typically contain 2(n) cells. In Drosophila, developing germ cells during this period contain a distinctive organelle, the fusome, that is required for normal cyst formation. We find that the cell cycle regulator Cyclin A transiently associates with the fusome during the cystocyte cell cycles, suggesting that fusome-associated Cyclin A drives the interconnected cells within each cyst synchronously into mitosis. In the presence of a normal fusome, overexpression of Cyclin A forces cysts through an extra round of cell division to produce cysts with 32 germline cells. Female sterile mutations in UbcD1, encoding an E2 ubiquitin-conjugating enzyme, have a similar effect. Our observations suggest that programmed changes in the expression and cytoplasmic localization of key cell cycle regulatory proteins control germline cyst production.     

Cell-cycle research with synchronous cultures: an evaluation

The baby-machine system, which produces new-born Escherichia coli cells from cultures immobilized on a membrane, was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth. In the present article, we put forward a model to describe the behaviour of cells produced by this method, and provide quantitative evaluation of the parameters involved, at each of four different growth rates. Considering the high level of selection achievable with this technique and the natural dispersion in interdivision times, we believe that the output of the baby machine is probably close to optimal in terms of both quality and persistence of synchrony. We show that considerable information on events in the cell cycle can be obtained from populations with age distributions very much broader than those achieved with the baby machine and differing only modestly from steady state. The data presented here, together with the long and fruitful history of findings employing the baby-machine technique, suggest that minimisation of stress on cells is the single most important factor for successful cell-cycle analysis.     

Heterogeneity of Shigella flexneri during selective synchronization

The study of a number of main growth characteristics of S. flexneri (S) cells during the first and second cycles of their cultivation in a solid culture medium in Peshkov's chamber from seed material, nonsynchronized and synchronized by C. E. Helmstetter's method, has revealed that this method does not permit obtaining completely synchronized cultures. Four physiological cell clusters, differing in the time of generation, growth rate, size at the beginning and the end of the cycle and the time of adaptation, have been detected.     

Synchronous growth and synthesis of macromolecules in a naturally wall-less volvocale,Dunaliella bioculata

Summary Dunaliella bioculata, a naturally wall-less unicellular green alga, can be induced to divide synchronously when subjected to a 12 hours light-12 hours dark cycle. This rhythmic cell division will last for at least 15 days under a subsequent constant illumination. Synchronization can be improved when cells are submitted to 8 hours light-16 hours dark cycles under bright white light (10,000 lux). In these conditions the cell division gives rise to two daughter cells: The chronology of DNA, RNA and proteins synthesis has been studied during such a synchronized cell cycle. DNA synthesis begins 4 hours before the outset of cell division and is completed after two hours in the dark; in difference, illumination seems necessary to the synthesis of RNA and proteins.     

Cell-cycle dependent changes in sensitivity to γ-rays in synchronously dividing yeast culture

Changes in survival of yeast cells following γ-irradiation at different stages of the cell cycle were studied using a well synchronized culture. Maximum radioresistance occurs at the end of the S phase. Maximum radiosensitivity is observed just before entry into the S phase. The high degree of synchrony obtained allows more precise measurement of the extent of survival changes than has been achieved until now with partially synchronized cultures. Indeed, after a 60 krad irradiation we find a 100 % survival for cells which have just finished the S phase of the first cell cycle, against a 2 % survival for cells which are ready to enter the S phase of the second cell cycle. As the culture desynchronizes through successive cell cycles we have been able to follow the way in which survival curves are modified. We can extrapolate that with a perfectly synchronized culture the survival of ‘early S’ cells to a 60 krad irradiation would not be 2 % but 0.01 %. The high radioresistance observed at the end of S phase can hardly be explained simply in terms of DNA target or accumulation of radioprotectors. More likely the end of the S phase is a favourable stage for repair processes, at which time two genomes are able to recombine.     

Two-stage, self-cycling process for the production of bacteriophages

Background

A two-stage, self-cycling process for the production of bacteriophages was developed. The first stage, containing only the uninfected host bacterium, was operated under self-cycling fermentation (SCF) conditions. This automated method, using the derivative of the carbon dioxide evolution rate (CER) as the control parameter, led to the synchronization of the host bacterium. The second stage, containing both the host and the phage, was operated using self-cycling infection (SCI) with CER and CER-derived data as the control parameters. When each infection cycle was terminated, phages were harvested and a new infection cycle was initiated by adding host cells from the SCF (first stage). This was augmented with fresh medium and the small amount of phages left from the previous cycle initiated the next infection cycle. Both stages were operated independently, except for this short period of time when the SCF harvest was added to the SCI to initiate the next cycle.

Results

It was demonstrated that this mode of operation resulted in stable infection cycles if the growth of the host cells in the SCF was synchronized. The final phage titers obtained were reproducible among cycles and were as good as those obtained in batch productions performed under the same conditions (medium, temperature, initial multiplicity of infection, etc.). Moreover, phages obtained in different cycles showed no important difference in infectivity. Finally, it was shown that cell synchronization of the host cells in the first stage (SCF) not only maintained the volumetric productivity (phages per volume) but also led to higher specific productivity (phage per cell per hour) in the second stage (SCI).

Conclusions

Production of bacteriophage T4 in the semi-continuous, automated SCF/SCI system was efficient and reproducible from cycle to cycle. Synchronization of the host in the first stage prior to infection led to improvements in the specific productivity of phages in the second stage while maintaining the volumetric productivity. These results demonstrate the significant potential of this approach for both upstream and downstream process optimization.     

Synchronization of cell cycle of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by using a cell chip platform

Cell synchrony is a critical requirement for the study of eukaryotic cells. Although several chemical and genetic methods of cell cycle synchronization are currently available, they have certain limitations, such as unnecessary perturbations to cells. We developed a novel cell cycle synchronization method that is based on a cell chip platform. The budding yeast, Saccharomyces cerevisiae, is a simple but useful model system to study cell biology and shares many similar features with higher eukaryotic cells. Single yeast cells were individually captured in the wells of a specially designed cell chip platform. When released from the cell chip, the yeast cells were synchronized, with all cells in the G1 phase. This method is non-invasive and causes minimal chemical and biological damage to cells. The capture and release of cells using cells chips with microwells of specific dimensions allows for the isolation of cells of a particular size and shape; this enables the isolation of cells of a given phase, because the size and shape of yeast cells vary with the phase of the cell cycle. To test the viability of synchronized cells, the yeast cells captured in the cell chip platform were assessed for response to mating pheromone (α-factor). The synchronized cells isolated using the cell chip were capable of mediating the mating signaling response and exhibited a dynamic and robust response behavior. By changing the dimensions of the well of the cell chip, cells of other cell cycle phases can also be isolated.     

Nucleoside phosphotransferase activity through the growth and cell cycle of Tetrahymena pyriformis GL-I

Tetrahymena pyriformis GL-I were synchronized by three different techniques and nucleoside phosphotransferase activity measured through the different cell cycles obtained. In cells that were starved and then refed, activity did not increase until 75 min after refeeding. This increase in activity occurred well before nuclear DNA synthesis and was not blocked by hydroxyurea. In cells synchronized by the induction technique of one heat shock per generation and the selection technique of differential density labelling, enzyme activity increased continuously over the cell cycle but did not double. However, during early logarithmic growth nucleoside phosphotransferase activity more than doubled over one cell cycle time while late in log growth phase less than a doubling was observed. Cycloheximide and mixed extract experiments suggest that the patterns of activity observed reflect the patterns of enzyme synthesis. These results are discussed with respect to the pattern of activity observed for thymidine kinase in other organisms.     

Synchronization of the cultures ofScenedesmus quadricauda by optimalizing the length of the light period

Experiments revealed that the synchronized cultures ofScenedesmus quadricauda, strainGreifswald/15, must be kept under continuous illumination throughout the whole growth phase (from the release of daughter coenobia up to the moment when all cells are capable of further reproduction). The length of the light period is among others the function of the suspension density. With regard to these facts a method of synchronization was worked out on the basis of a rhythmical repetition of the calculated light regime, corresponding to the physiological parameters of the synchronized strain under given cultivation conditions. This method enables to maintain synchronized cultures in adequate cycles for any required period, even in sufficiently dense, linearly growing suspensions. It may be applied to any chlorococcal algae; however, if coenobial types are studied, it is necessary to consider special peculiarities, which result from the regular arrangement of the cells in the coenobium.     

Spontaneous activity of olfactory bulb neurons in awake rabbits, with some observations on the effects of pentobarbital anaesthesia

A specially adapted microelectrode driver device has been used to record the spontaneous activity of neurons in the olfactory bulb of awake rabbits. Several parameters of this activity were studied in 78 neurons of conscious animals. A second experiment was performed to investigate anaesthetic-induced modifications of the spike discharge initially recorded in awake animals. 1. In unanaesthetized animals, the interspike interval distribution of all cells was found to be stable over short as well as long periods of time. 2. A periodical change in firing probability, correlated with respiratory activity, was observed in a high percentage of neurons. During inspiration, the discharge was markedly increased ("well synchronized" neurons, n = 2), slightly increased ("poorly synchronized" neurons, n = 15); or unchanged ("not synchronized" neurons, n = 8). 3. The passage from the awake to the anaesthetized state resulted in more regular cell activity with sudden changes from one steady firing level to another, without affecting the cell classification. As anaesthesia wore off, the cell units recovered the characteristic discharge pattern initially observed.     

A Diurnal Settling Rhythm in Platymonas subcordiformis Hazen*

SYNOPSIS. In cultures of Platymonas subcordiformis Hazen, grown in appropriate light-dark cycles, as many as 75% of the cells adhered to the surface of the glass culture vessel toward the end of the light period of each day. Cell division occurred primarily while the cells were attached. Subsequently, motile daughter cells were released into the growth medium by the rupture of the mother cell theca. The settling behavior appears to be an integral part of the life cycle being synchronized to the same extent as cell division.     

The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle

We describe a new method for synchronizing bacterial cells. Cells that have transiently expressed an inducible mutant 'sticky' flagellin are adhered to a volume of glass beads suspended in a chromatography column though which growth medium is pumped. Following repression of flagellin synthesis, newborn cells are eluted from the column in large quantities exceeding that of current baby machine techniques by approximately 10-fold. Eluted cultures of 'baby cells' are highly synchronous as determined by analysis of DNA replication, cell division and other events, over time after elution from the column. We also show that use of 'minutes after elution' as a time metric permits much greater temporal resolution among sequential chromosomal events than the commonly used metric of cell size (length). The former approach reveals the existence of transient intermediate stages that are missed by the latter approach. This finding has two important implications. First, at a practical level, the baby cell column is a particularly powerful method for temporal analysis. Second, at a conceptual level, replication-related events are more tightly linked to cell birth (i.e. cell division) than to absolute cell mass.     

Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells

Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time‐lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode‐locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev‐Erbα‐YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.     

Experiments with cultures of mammalian cells aboard the biosatellite "Cosmos-782"

A considerable contribution to the investigation on biological importance of weightlessness was made by the experiments with animals in the artificial Earth satelites (AES) of "Cosmos" type. Cell cultures can serve as an ideal model to get a direct cell response to the effect of external factors. For the experiment in the AES "Cosmos-782", two thoroughly examined cell strains (L and 237) were chosen, which differed in a number of parameters (for example, duration of their mitotic cycles). Density of cell seeding and temperature of their cultivation in the laboratory experiment were calculated in such a way that the whole cycle of the culture development should take place under the conditions of weightlessness: the beginning of lag-phase--before launching and the stationary phase--after landing. The weightlessness was not shown to result in any genetical shifts revealed at chromosomal level. When cultivated after the flight, the cells do not change their mitotic cycle parameters, mitotic course and structural organization. The data obtained in the experiments with AES "Cosmos-368" and "Cosmos-782" (increase of mitotic index, some forms of mitotic pathology during the first terms of cultivation after the flight and enlargement of cellular nuclei) demonstrate the changes in the cell population which have formed under the conditions of weightlessness. Similar changes are observed while the cells propagate in the laboratory conditions. Indirect data on an earlier cell culture aging during the flight do not exclued the possibility that under weightlessness the rate of cell propagation could differ from that under gravitation.