Anomalous Cases of Conjugation and Spontaneous Autogamy in Stylonychia mytilus: A Note on the Asexuality of Early Conjugants

Four types of anomalous conjugation were documented in Stylonychia mytilus. Type I pairs were formed between mates of different sizes. These pairs exhibited an abnormal site of fusion in at least one of the mates, and the mates might face each other ventrally throughout conjugation instead of the normal side-by-side position. Type I pairs underwent sexual nuclear development and proceeded with the first cortical reorganization as in normal conjugants. Type II involved pairing at the anterior ends of mates with ventral surfaces facing the same direction. These pairs also underwent sexual nuclear development. Hence, aberrant orientation of the mates, and also ectopic sites of cytoplasmic fusion, if extensive, would permit sexual development. Type III pairs were united ventral-to-ventral with their anterior-left sides at the adoral zone of membranelles, and remained as such throughout conjugation. In these pairs, nuclear and cortical events were typical of the asexual development of physiological reorganization. In Type IV pairs, one mate of the pair possessed a fission furrow and developed two sets of ciliature typical of binary fission, while the other mate might undergo physiological reorganization or binary fission. Type III and Type IV pairs thus reveal the asexual state of early conjugants, which can pursue either one of the two modes of asexual cortical reorganization; these cases reinforce the notion of overlap of asexual and sexual cycles during conjugation of hypotrichs. Spontaneous autogamy was documented for the first time for this genus. The autogamonts proceeded with nuclear development and with the first cortical reorganization. Some probably underwent second and third reorganizations, as in conjugants, but accompanied by abnormalities, particularly in the stages beyond fertilization. Post-autogamous clones were nonviable except for one dubious case.     

Physiological Reorganization and Post-Traumatic Regeneration in Stylonychia mytilus: Reflections on Developmental Constraints and the Evolutionary Origin of Alternative Modes of Asexual Morphogenesis

We investigated development of cortical ciliature in Stylonychia mytilus during starvation-induced physiological reorganization, and during regeneration following amputation of the anterior part of the cell. Cortical reorganization in the two processes is generally similar. The posterior part of the adoral zone of membranelles is resorbed and replaced with newly assembled membranelles. The pre-existing set of ventral cirri and dorsal bristles is entirely resorbed and replaced with new ones. Regenerants exhibit posterior displacement of the frontal-ventral-transverse cirri primordium and the undulating membrane primordium, and recruit basal bodies from ectopic locations for the development of these ciliature. This illustrates flexibility in the initiation site of ciliary primordia, and opportunism in utilizing building blocks. Such morphogenetic versatility of hypotrichs provides the basis for the operation of a global control of pattern formation, which governs cortical reorganization in dividers, and additionally, in the absence of the prerequisites for binary fission, alternative modes of cortical development such as physiological reorganization or regeneration. These considerations suggest that the three processes are homologous and that physiological reorganization and regeneration have evolved from binary fission. In physiological reorganization and regeneration, the micro- and macronuclei reorganize to resemble that in binary fission; these nuclear events are considered evolutionary relics of the nuclear development of binary fission. Tetrahymena also exhibits such morphogenetic flexibility; stomatogenesis is under global control, so that asexual cells can replace its oral apparatus without undergoing binary fission. Paramecium , on the other hand, adopts a more rigid strategy in relying heavily on pre-existing structures for morphogenetic cues; this could have imposed constraints in the exploration of alternative modes of asexual development.     

A cdc2-Like Kinase Associated with Commitment to Division in Paramecium tetraurelia

ABSTRACT. Cell division in higher eukaryotes is mainly controlled by p34 ^cdc2 or related kinases and by other components of these kinase complexes. We present evidence that cdc2 -like kinases also occur in Paramecium. Two polypeptides reacted with an antibody directed against the perfectly conserved PSTAIR region found in cdc2 kinases in other eukaryotes. Only the less abundant peptide bound to p13 ^suc1 from Schizosaccharomyces pombe. Using centrifugal elutriation to select cells on the basis of size, we isolated highly synchronous Paramecium G1 cells. With this procedure, we demonstrated that the p13^suc1-associated cdc2 -like histone H1 kinase was activated before cell division at the point of commitment to division in Paramecium. Further, we show that Paramecium cdc2 -like proteins occurred principally as monomers and that these monomers were active as histone H1 kinases in vitro.     

An in silico investigation into the causes of telomere length heterogeneity and its implications for the Hayflick limit

In telomerase-negative cell populations the mean telomere length (TL) decreases with increasing population doubling number (PD). A critically small TL is believed to stop cell proliferation at a cell-, age- and species-specific PD thus defining the Hayflick limit. However, positively skewed TL distributions are broad compared to differences between initial and final mean TL and strongly overlap at middle and late PD, which is inconsistent with a limiting role of TL. We used computer-assisted modelling to define what set of premises may account for the above. Our model incorporates the following concepts. DNA end replication problem: telomeres loose 1 shortening unit (SU) upon each cell division. Free radical-caused TL decrease: telomeres experience random events resulting in the loss of a random SU number within a remaining TL. Stochasticity of gene expression and cell differentiation: cells experience random events inducing mitoses or committing cells to proliferation arrest, the latter option requiring a specified number of mitoses to be passed. Cells whose TL reaches 1SU cannot divide. The proliferation kinetics of such virtual cells conforms to the transition probability model of cell cycle. When no committing events occur and at realistic SU estimates of the initial TL, maximal PD values far exceed the Hayflick limit observed in normal cells and are consistent with the crisis stage entered by transformed cells that have surpassed the Hayflick limit. At intermediate PD, symmetrical TL distributions are yielded. Upon introduction of committing events making the ratio of the rates of proliferating and committing events (P/C) range from 1.10 to 1.25, TL distributions at intermediate PD become positively skewed, and virtual cell clones show bimodal size distributions. At P/C as high as 1.25 the majority of virtual cells at maximal PD contain telomeres with TL>1SU. A 10% increase in P/C within the 1.10-1.25 range produces a two-fold increase in the maximal PD, which can reach values of up to 25 observed in rodent and some human cells. Increasing the number of committed mitoses from 0 to 10 can increases PD to about 50 observed in human fibroblasts. Introduction of the random TL breakage makes the shapes of TL distributions quite dissimilar from those observed in real cells. CONCLUSIONS: Telomere length decrease is a correlate of cell proliferation that cannot alone account for the Hayflick limit, which primarily depends on parameters of cell population kinetics. Free radical damage influences the Hayflick limit not through TL but rather by affecting the ratio of the rates of events that commit cells to mitoses or to proliferation arrest.     

Insulin action mechanism for redox signaling in the cell cycle: Its alterations in diabetes

Several reports describe the existence of a redox cycle within the normal cell cycle that helps control the process of cell proliferation. According to some of these reports, this redox cycle comprises an intracellular redox potential E that oscillates above and below θ during the cell cycle process. θ is the threshold for dephosphorylation of protein regulators associated with serine residues such as the retinoblastoma protein. This article describes how insulin action may be the source of the redox cycle within the cell cycle. The relative lack of insulin action as a consequence of oxidative stress results in the hallmarks of type 2 diabetes.     

An autoradiographic study of cellular proliferaton,DNA synthesis and cell cycle variability in the rat liver caused by phenobarbital-induced oxidative stress: The protective role of melatonin

The protective effect of melatonin against phenobarbital-induced oxidative stress in the rat liver was measured based on lipid peroxidation levels (malondialedyde and 4-hydroxyalkenals). Cellular proliferation, DNA synthesis and cell cycle duration were quantitated by the incorporation of ³H-thymidine, detected by autoradiography, into newly synthesized DNA. Two experiments were carried out in this study, each on four equal-sized groups of male rats (control, melatonin [10 mg/kg], phenobabital [20 mg/kg] and phenobarbital plus melatonin). Experiment I was designed to study the proliferative activity and rate of DNA synthesis, and measure the levels of lipid peroxidation, while experiment II was for cell cycle time determination. Relative to the controls, the phenobarbital-treated rats showed a significant increase (P < 0.01) in the lipid peroxidation levels (30.7%), labelling index (69.4%) and rate of DNA synthesis (37.8%), and a decrease in the cell cycle time. Administering melatonin to the phenobarbital-treated rats significantly reduced (P < 0.01) the lipid peroxidation levels (23.5%), labelling index (38.2%) and rate of DNA synthesis (29.0%), and increased the cell cycle time. These results seem to indicate that the stimulatory effect of phenobarbital on the oxidized lipids, proliferative activity, kinetics of DNA synthesis and cell cycle time alteration in the liver may be one of the mechanisms by which the non-genotoxic mitogen induces its carcinogenic action. Furthermore, melatonin displayed powerful protection against the toxic effect of phenobarbital.     

When the caps fall off: Responses to telomere uncapping in yeast

Telomeres protect the ends of linear chromosomes from activities that cause sequence losses or challenge chromosome integrity. Furthermore, these ends must be hidden from detection by the DNA damage recognition and response pathways. In particular, they must not fuse with each other. These fundamental and very first functions attributed to telomeres are also summarized with the term ‘chromosome capping’. However, telomeres can become uncapped and the foremost cellular responses to such events aim to restore genome stability in the most conservative fashion possible. I will provide an outline of cellular responses to uncapping in budding yeast and briefly discuss the reverse, namely avoidance mechanisms that prevent telomere formation at inappropriate places.     

Reduction in p53 synthesis during differentiation of Friend-erythroleukemia cells. Correlation with the commitment to terminal cell division

The process of cell differentiation in Friend-erythroleukemia cells was accompanied by 80-90% inhibition of p53 synthesis. This decrease was found to be linked to changes in cell-cycle distribution characteristics of the growth arrest program during differentiation rather than to the induction of the globin genes. The shut-off in the expression of p53 always preceded the specific arrest of cells in the G0/G1 phase. Interferon did not modulate down the expression of p53 if added to transformed non-induced Friend-erythroleukemia cells; however, it slightly enhanced the extent of reduction in p53 synthesis if added during cell differentiation, thus suggesting a differential effect of interferon between cells at different stages of differentiation.     

Commitment to relationships and preferences for femininity and apparent health in faces are strongest on days of the menstrual cycle when progesterone level is high

Previous studies of changes in women's behavior during the menstrual cycle have offered insight into the motivations underpinning women's preferences for social cues associated with possible direct benefits (e.g., investment, low risk of infection) and indirect benefits (e.g., offspring viability). Here we sought to extend this work by testing for systematic variation in women's preferences for male and female faces and in their attitudes to their romantic relationship during the menstrual cycle. In Study 1, we found partnered women's reported commitment to their romantic relationship and preferences for femininity in male and female faces were strongest on days of the menstrual cycle when progesterone levels are increased (and fertility is low). Happiness in relationships did not change across the cycle. In Study 2, we found that the effect of cycle phase on women's preference for feminine faces was independent of increased attraction to apparent health in faces during the luteal phase. Collectively, these findings are further evidence that women's preferences for social cues associated with possible direct benefits and commitment to relationships are strongest during conditions characterized by raised progesterone level, while attraction to men displaying cues associated with possible indirect benefits is strongest when women are most fertile.     

An algorithm for a decomposition of weighted digraphs: with applications to life cycle analysis in ecology

In the analysis of organism life cycles in ecology, comparisons of life cycles between species or between different types of life cycles within species are frequently conducted. In matrix population models, partitioning of the elasticity matrix is used to quantify the separate contributions of different life cycles to the population growth rate. Such partition is equivalent to a decomposition of the life cycle graph of the population. A graph theoretic spanning tree method to carry out the decomposition was formalized by Wardle [Ecology 79(7), 2539–2549 (1998)]. However there are difficulties in realizing a suitable decomposition for complex life histories using the spanning-tree method. One of the problems is the occurrence of life cycles that contain contradictory directions that defy biological interpretation. We propose an algorithmic approach for decomposing a directed, weighted graph. The graph is to be decomposed into two parts. The first part is a set of simple cycles that contain no contradictory directions and that consist of edges of equal weight. The second part of the decomposition is a subgraph in which no such simple cycles are obtainable. When applied to life cycle analysis in ecology, the proposed method will guarantee a complete decomposition of the life cycle graph into individual life cycles containing no contradictory directions. Although the research described in this article has been funded in part by the United States Environmental Protection Agency through STAR cooperative agreement R-82940201-0 to the University of Chicago, it has not been subjected to the Agency’s required peer and policy review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred.     

Stochastic modeling of cellular colonies with quiescence: an application to drug resistance in cancer

Several cancers are thought to be driven by cells with stem cell like properties. An important characteristic of stem cells, which also applies to primitive tumor cells, is the ability to undergo quiescence, where cells can temporarily stop the cell cycle. Cellular quiescence can affect the kinetics of tumor growth, and the susceptibility of the cells to therapy. To study how quiescence affects treatment, we formulate a stochastic birth-death process with quiescence, on a combinatorial cellular mutation network, and consider the pre-treatment (growth) and treatment (decay) regimes. We find that, in the absence of mutations, treatment (if sufficiently strong) will proceed as a biphasic decline with the first (faster) phase driven by the elimination of the cycling cells and the second (slower) phase limited by the process of cell awakening. Other regimes are possible for weaker treatments. We also describe how the process of mutant generation is influenced by quiescence. Interestingly, for single-drug treatments, the probability to have resistance at start of treatment is independent of quiescence. For two or more drugs, the probability to have generated resistant mutants before treatment grows with quiescence. Finally, we study the influence of quiescence on the treatment phase. Starting from a given composition of mutants, the chances of treatment success are not influenced by the presence of quiescence.     

Deciphering the intricate regulatory mechanisms for the cellular choice between cell repair, apoptosis or senescence in response to damaging signals

In response to various types of stress, cells can undergo significant phenotypic changes, ranging from an increased DNA repair to senescence and apoptosis. The mechanisms by which p53 manages the choice between three possible cell fates in response to damaging stress remain poorly understood. p53 is not a simple switch that determines cell fate single-handedly; but rather as a component, albeit an important one, of an intricate signal network and molecular interactions. Thus, in addition to p53, fine-tuned interactions between growth- and division-activator molecules such as TGFβ, cMyc and FOXO are important determinants of the cellular fate. The aim of the paper is to resolve the complex interactions between these molecules and to elicit clear and reasonable working mechanisms for these diverse cellular processes.     

Techniques for Dual Staining of DNA and Intracellular Immunoglobulins in Murine Hybridoma Cells: Applications to Cell-Cycle Analysis of Hyperosmotic Cultures

Flow cytometry was used to evaluate the effects of hyperosmotic stress on cell-cycle distribution and cell-associated immunoglobulins for murine hybridoma cells grown in batch culture. Paraformaldehyde/methanol fixation substantially increased the fluorescence signal for intracellular immunoglobulins compared to ethanol fixation. For surface immunoglobulins, similar fluorescence signals were observed regardless of fixation method. Dual staining of immunoglobulins and cellular DNA was employed to determine immunoglobulin pool size as a function of cell-cycle phase. The intracellular immunoglobulin pool sizes increased as the cells progressed through the cell cycle for both control and hyperosmotic cultures. For control cultures, the immunoglobulin pool size increased during the exponential phase of culture, followed by a decrease as the cultures entered stationary phase. In contrast, hyperosmotic cultures showed an initial decrease in immunoglobulin pool size upon the application of osmotic shock, followed by an increase to a level above that of control cultures. This behavior was observed in all phases of the cell cycle. In addition, hyperosmotic cultures exhibited an increase in cell size when compared to control cultures. When normalized for cell size, the intracellular immunoglobulin concentration in hyperosmotic cultures was initially lower than in control cultures and subsequently increased to slightly above the level of control cells. Cells in all phases of the cell cycle behaved in a similar manner. There was no apparent relationship between the intracellular antibody concentration and the rate of antibody secretion.     

Age-dependent acute interference with stem and progenitor cell proliferation in the hippocampus after exposure to 1800 MHz electromagnetic radiation

To investigate the effects of exposure to an 1800 MHz electromagnetic field on cell death and cell proliferation in the developing brain, postnatal day 7 (P7) and P21 healthy Kunming mice were randomly assigned into the experimental and control groups. The experimental groups were exposed to an 1800 MHz electromagnetic field for 8 h daily for three consecutive days. The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally 1 h before each exposure session, and all animals were sacrificed 24 h after the last exposure. Cell death and proliferation markers were detected by immunohistochemistry in the dentate gyrus of the hippocampus. Electromagnetic exposure has no influence on cell death in the dentate gyrus of the hippocampus in P7 and P21 mice as indicated by active caspase-3 immunostaining and Fluoro-Jade labeling. The basal cell proliferation in the hippocampus was higher in P7 than in P21 mice as indicated by the number of cells labeled with BrdU and by immunohistochemical staining for phosphor-histone H3 (PHH3) and brain lipid-binding protein (BLBP). Electromagnetic exposure stimulated DNA synthesis in P7 neural stem and progenitor cells, but reduced cell division and the total number of stem cells in the hippocampus as indicated by increased BrdU labeling and reduced PHH3 and BLBP labeling compared to P7 control mice. There were no significant changes in cell proliferation in P21 mice after exposure to the electromagnetic field. These results indicate that interference with stem cell proliferation upon short-term exposure to an 1800 MHz electromagnetic field depends on the developmental stage of the brain.     

The role of the human DNA mismatch repair gene hMSH2 in DNA repair, cell cycle control and apoptosis: implications for pathogenesis, progression and therapy of cancer

The cellular DNA mismatch repair (MMR) pathway, involving the DNA mismatch repair genes MLH1 and MSH2, detects and repairs DNA replication errors. Defects in MSH2 and MLH1 account for most cases of hereditary non-polyposis colorectal cancer as well as for sporadic colorectal tumors. Additionally, increased expression of MSH2 RNA and/or protein has been reported in various malignancies. Loss of DNA MMR in mammalian cells has been linked to resistance to certain DNA damaging agents including clinically important cytotoxic chemotherapeutics. Due to other functions besides its role in DNA repair, that include regulation of cell proliferation and apoptosis, MSH2 has recently been shown to be of importance for pathogenesis and progression of cancer. This review summarizes our present understanding of the function of MSH2 for DNA repair, cell cycle control, and apoptosis and discusses its importance for pathogenesis, progression and therapy of cancer.     

A lentiviral vector with expression controlled by E2F-1: a potential tool for the study and treatment of proliferative diseases

We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for this factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis.     

First evidence for photoperiodic regulation of the life cycle in a millipede species, Polydesmus angustus (Diplopoda: Polydesmidae)

The hypothesis that the periods of dormancy previously described in the millipede Polydesmus angustus may be photoperiodically induced diapauses was tested experimentally. In this species, biennial individuals exhibit two successive periods of dormancy: aestivation in the penultimate stadium (stadium VII) and reproductive dormancy in the adults, which emerge in autumn. It was first established that the reproductive dormancy is not a thermally controlled state of quiescence. When adults emerging in autumn were kept at 16 °C under natural photoperiod, their reproduction was delayed for several months in comparison with adults emerging in spring at similar temperatures. This indicates that the reproductive dormancy begins with a period of diapause. Further experiments provided evidence of a photoperiodic induction of the adult diapause. When millipedes were reared under short day length (L:D 12:12 h) throughout their development, they required more time to reproduce than millipedes reared under long day length (L:D 16:8 h) at the same temperatures. Photoperiod influenced reproduction in females, but no significant effects were detected in adult males. On the other hand, stadium VII was markedly longer at L:D 16:8 h than at L:D 12:12 h in both sexes, which strongly suggests that aestivation is also induced by photoperiod. However, the effects on the duration of stadium VII varied among individuals, some of which showed no response to long days. This study is the first to document photoperiodic regulation of the life cycle in the class Diplopoda, a trait common in other classes of terrestrial arthropods.     

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.