Cell Cycle Control in the Early Embryonic Development of Aquatic Animal Species

PubMed Central

Siefert, Joseph C.; Clowdus, Emily A.; Sansam, Christopher L.

2016-01-01

The cell cycle is integrated with many aspects of embryonic development. Not only is proper control over the pace of cell proliferation important, but also the timing of cell cycle progression is coordinated with transcription, cell migration, and cell differentiation. Due to the ease with which the embryos of aquatic organisms can be observed and manipulated, they have been a popular choice for embryologists throughout history. In the cell cycle field, aquatic organisms have been extremely important because they have played a major role in the discovery and analysis of key regulators of the cell cycle. In particular, the frog Xenopus laevis has been instrumental for understanding how the basic embryonic cell cycle is regulated. More recently, the zebrafish has been used to understand how the cell cycle is remodeled during vertebrate development and how it is regulated during morphogenesis. This review describes how some of the unique strengths of aquatic species have been leveraged for cell cycle research and suggests how species such as Xenopus and zebrafish will continue to reveal the roles of the cell cycle in human biology and disease. PMID:26475527

Cell cycle arrest in the jewel wasp Nasonia vitripennis in larval diapause.

PubMed

Shimizu, Yuta; Mukai, Ayumu; Goto, Shin G

2018-04-01

Insects enter diapause to synchronise their life cycle with biotic and abiotic environmental conditions favourable for their development, reproduction, and survival. One of the most noticeable characteristics of diapause is the blockage of ontogeny. Although this blockage should occur with the cessation of cellular proliferation, i.e. cell cycle arrest, it was confirmed only in a few insect species and information on the molecular pathways involved in cell cycle arrest is limited. In the present study, we investigated developmental and cell cycle arrest in diapause larvae of the jewel wasp Nasonia vitripennis. Developmental and cell cycle arrest occur in the early fourth instar larval stage of N. vitripennis under short days. By entering diapause, the S fraction of the cell cycle disappears and approximately 80% and 20% of cells arrest their cell cycle in the G0/G1 and G2 phases, respectively. We further investigated expression of cell cycle regulatory genes and some housekeeping genes to dissect molecular mechanisms underlying the cell cycle arrest. Copyright © 2016 Elsevier Ltd. All rights reserved.

The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex. III. Kinetics of cell proliferation in the external granular layer.

PubMed

Lauder, J M

1977-04-22

The effects of early hypo- and hyperthyroidism on the rates of cell acquisition and proliferation have been studied in the external granular layer (EGL) of the developing rat cerebellar cortex at 10 days of age using quantitative autoradiographic methods. Both altered thyroid states reduce the rate of cell acquisition in the EGL, but appear to do so for different reasons. Hyperthyroidism shortens the average length of the cell cycle by decreasing the duration of the pre-DNA synthetic phase (G1), indicating that excess thyroxine may exert a direct effect on the EGL. This action involves the early onset of neuronal differentiation (cessation of proliferation)46 which presumably leads to the observed decrease in the rate of cell acquisition (increased doubling time). Such differentiating cells do not, however, leave the proliferative zone or the EGL prematurely, resulting in a reduced labeling index, mitotic index, and growth fraction as non-dividing cells dilute the proliferating cell population. Hypothyroidism, on the other hand, leads to no significant change in the length of the cell cycle or in the mitotic index, but causes a decreased labeling index and growth fraction, as well as a reduced rate of cell acquisition (increased doubling time). No significant change in the amount of cell death in the EGL could be found to explain this apparent discrepancy between the rate of cell proliferation (cell cycle length) and cell acqusiition. The answer to this puzzle appears to lie in the mitotic index, which is not affected to the same extent as the labeling index, although it is also slightly reduced. If cells were to remain longer in mitosis, this could result in a decreased labeling index and growth fraction but nearly normal mitotic index and cell cycle length (as measured using the % labeled mitoses method), since those cells dropping out of the cycling population would be counted as mitoses...

Cell cycle proteins in brain in mild cognitive impairment: insights into progression to Alzheimer disease.

PubMed

Keeney, Jeriel T R; Swomley, Aaron M; Harris, Jessica L; Fiorini, Ada; Mitov, Mihail I; Perluigi, Marzia; Sultana, Rukhsana; Butterfield, D Allan

2012-10-01

Recent studies have demonstrated the re-emergence of cell cycle proteins in brain as patients progress from the early stages of mild cognitive impairment (MCI) into Alzheimer's disease (AD). Oxidative stress markers present in AD have also been shown to be present in MCI brain suggesting that these events occur in early stages of the disease. The levels of key cell cycle proteins, such as CDK2, CDK5, cyclin G1, and BRAC1 have all been found to be elevated in MCI brain compared to age-matched control. Further, peptidyl prolyl cis-trans isomerase (Pin1), a protein that plays an important role in regulating the activity of key proteins, such as CDK5, GSK3-β, and PP2A that are involved in both the phosphorylation state of Tau and in the cell cycle, has been found to be oxidatively modified and downregulated in both AD and MCI brain. Hyperphosphorylation of Tau then results in synapse loss and the characteristic Tau aggregation as neurofibrillary tangles, an AD hallmark. In this review, we summarized the role of cell cycle dysregulation in the progression of disease from MCI to AD. Based on the current literature, it is tempting to speculate that a combination of oxidative stress and cell cycle dysfunction conceivably leads to neurodegeneration.

Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans.

PubMed

Kocsisova, Zuzana; Kornfeld, Kerry; Schedl, Tim

2018-05-30

The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E. To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of-function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development. In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor cells is distinct from somatic cells, as it does not heavily rely on cyclic accumulation of classic cell cycle proteins.

A stochastic spatiotemporal model of a response-regulator network in the Caulobacter crescentus cell cycle

NASA Astrophysics Data System (ADS)

Li, Fei; Subramanian, Kartik; Chen, Minghan; Tyson, John J.; Cao, Yang

2016-06-01

The asymmetric cell division cycle in Caulobacter crescentus is controlled by an elaborate molecular mechanism governing the production, activation and spatial localization of a host of interacting proteins. In previous work, we proposed a deterministic mathematical model for the spatiotemporal dynamics of six major regulatory proteins. In this paper, we study a stochastic version of the model, which takes into account molecular fluctuations of these regulatory proteins in space and time during early stages of the cell cycle of wild-type Caulobacter cells. We test the stochastic model with regard to experimental observations of increased variability of cycle time in cells depleted of the divJ gene product. The deterministic model predicts that overexpression of the divK gene blocks cell cycle progression in the stalked stage; however, stochastic simulations suggest that a small fraction of the mutants cells do complete the cell cycle normally.

Temporal remodeling of the cell cycle accompanies differentiation in the Drosophila germline.

PubMed

Hinnant, Taylor D; Alvarez, Arturo A; Ables, Elizabeth T

2017-09-01

Development of multicellular organisms relies upon the coordinated regulation of cellular differentiation and proliferation. Growing evidence suggests that some molecular regulatory pathways associated with the cell cycle machinery also dictate cell fate; however, it remains largely unclear how the cell cycle is remodeled in concert with cell differentiation. During Drosophila oogenesis, mature oocytes are created through a series of precisely controlled division and differentiation steps, originating from a single tissue-specific stem cell. Further, germline stem cells (GSCs) and their differentiating progeny remain in a predominantly linear arrangement as oogenesis proceeds. The ability to visualize the stepwise events of differentiation within the context of a single tissue make the Drosophila ovary an exceptional model for study of cell cycle remodeling. To describe how the cell cycle is remodeled in germ cells as they differentiate in situ, we used the Drosophila Fluorescence Ubiquitin-based Cell Cycle Indicator (Fly-FUCCI) system, in which degradable versions of GFP::E2f1 and RFP::CycB fluorescently label cells in each phase of the cell cycle. We found that the lengths of the G1, S, and G2 phases of the cell cycle change dramatically over the course of differentiation, and identified the 4/8-cell cyst as a key developmental transition state in which cells prepare for specialized cell cycles. Our data suggest that the transcriptional activator E2f1, which controls the transition from G1 to S phase, is a key regulator of mitotic divisions in the early germline. Our data support the model that E2f1 is necessary for proper GSC proliferation, self-renewal, and daughter cell development. In contrast, while E2f1 degradation by the Cullin 4 (Cul4)-containing ubiquitin E3 ligase (CRL4) is essential for developmental transitions in the early germline, our data do not support a role for E2f1 degradation as a mechanism to limit GSC proliferation or self-renewal. Taken together, these findings provide further insight into the regulation of cell proliferation and the acquisition of differentiated cell fate, with broad implications across developing tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

Tumor suppressor Lzap regulates cell cycle progression, doming and zebrafish epiboly

PubMed Central

Liu, Dan; Wang, Wen-Der; Melville, David B.; Cha, Yong I.; Yin, Zhirong; Issaeva, Natalia; Knapik, Ela W.; Yarbrough, Wendell G.

2012-01-01

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly – the earliest morphogenetic movement in animal development – which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation. PMID:21523853

Cyclin B in mouse oocytes and embryos: importance for human reproduction and aneuploidy.

PubMed

Polański, Zbigniew; Homer, Hayden; Kubiak, Jacek Z

2012-01-01

Oocyte maturation and early embryo development require precise coordination between cell cycle progression and the developmental programme. Cyclin B plays a major role in this process: its accumulation and degradation is critical for driving the cell cycle through activation and inactivation of the major cell cycle kinase, CDK1. CDK1 activation is required for M-phase entry whereas its inactivation leads to exit from M-phase. The tempo of oocyte meiotic and embryonic mitotic divisions is set by the rate of cyclin B accumulation and the timing of its destruction. By controlling when cyclin B destruction is triggered and by co-ordinating this with the completion of chromosome alignment, the spindle assembly checkpoint (SAC) is a critical quality control system important for averting aneuploidy and for building in the flexibility required to better integrate cell cycle progression with development. In this review we focus on cyclin B metabolism in mouse oocytes and embryos and illustrate how the cell cycle-powered clock (in fact cyclin B-powered clock) controls oocyte maturation and early embryo development, thereby providing important insight into human reproduction and potential causes of Down syndrome.

Phospho-Rb mediating cell cycle reentry induces early apoptosis following oxygen-glucose deprivation in rat cortical neurons.

PubMed

Yu, Ying; Ren, Qing-Guo; Zhang, Zhao-Hui; Zhou, Ke; Yu, Zhi-Yuan; Luo, Xiang; Wang, Wei

2012-03-01

The aim of this study was to investigate the relationship between cell cycle reentry and apoptosis in cultured cortical neurons following oxygen-glucose deprivation (OGD). We found that the percentage of neurons with BrdU uptake, TUNEL staining, and colocalized BrdU uptake and TUNEL staining was increased relative to control 6, 12 and 24 h after 1 h of OGD. The number of neurons with colocalized BrdU and TUNEL staining was decreased relative to the number of TUNEL-positive neurons at 24 h. The expression of phosphorylated retinoblastoma protein (phospho-Rb) was significantly increased 6, 12 and 24 h after OGD, parallel with the changes in BrdU uptake. Phospho-Rb and TUNEL staining were colocalized in neurons 6 and 12 h after OGD. This colocalization was strikingly decreased 24 h after OGD. Treatment with the cyclin-dependent kinase inhibitor roscovitine (100 μM) decreased the expression of phospho-Rb and reduced neuronal apoptosis in vitro. These results demonstrated that attempted cell cycle reentry with phosphorylation of Rb induce early apoptosis in neurons after OGD and there must be other mechanisms involved in the later stages of neuronal apoptosis besides cell cycle reentry. Phosphoralated Rb may be an important factor which closely associates aberrant cell cycle reentry with the early stages of neuronal apoptosis following ischemia/hypoxia in vitro, and pharmacological interventions for neuroprotection may be useful directed at this keypoint.

A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

PubMed Central

Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

2012-01-01

In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

Expression of the proliferation marker Ki-67 during early mouse development.

PubMed

Winking, H; Gerdes, J; Traut, W

2004-01-01

In somatic tissues, the mouse Ki-67 protein (pKi-67) is expressed in proliferating cells only. Depending on the stage of the cell cycle, pKi-67 is associated with different nuclear domains: with euchromatin as part of the perichromosomal layer, with centromeric heterochromatin, and with the nucleolus. In gametes, sex-specific expression is evident. Mature MII oocytes contain pKi-67, whereas pKi-67 is not detectable in mature sperm. We investigated the re-establishment of the cell cycle-dependent distribution of pKi-67 during early mouse development. After fertilization, male and female pronuclei exhibited very little or no pKi-67, while polar bodies were pKi-67 positive. Towards the end of the first cell cycle, prophase chromosomes of male and female pronuclei simultaneously got decorated with pKi-67. In 2-cell embryos, the distribution pattern changed, presumably depending on the progress of development of the embryo, from a distribution all over the nucleus to a preferential location in the nucleolus precursor bodies (NPBs). From the 4-cell stage onwards, pKi-67 showed the regular nuclear relocations known from somatic tissues: during mitosis the protein was found covering the chromosome arms as a constituent of the perichromosomal layer, in early G1 it was distributed in the whole nucleus, and for the rest of the cell cycle it was associated with NPBs or with the nucleolus. Copyright 2004 S. Karger AG, Basel

Analysis of growth of tetraploid nuclei in roots of Vicia faba.

PubMed

Bansal, J; Davidson, D

1978-03-01

Growth of nuclei of a marked population of cells was determined from G1 to prophase in roots of Vicia faba. The cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G1 and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. Of the marked population of cells, about 65% had completed a cell cycle 14--15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.

Herpes simplex virus 1 regulatory protein ICP22 interacts with a new cell cycle-regulated factor and accumulates in a cell cycle-dependent fashion in infected cells.

PubMed

Bruni, R; Roizman, B

1998-11-01

The herpes simplex virus 1 infected cell protein 22 (ICP22), the product of the alpha22 gene, is a nucleotidylylated and phosphorylated nuclear protein with properties of a transcriptional factor required for the expression of a subset of viral genes. Here, we report the following. (i) ICP22 interacts with a previously unknown cellular factor designated p78 in the yeast two-hybrid system. The p78 cDNA encodes a polypeptide with a distribution of leucines reminiscent of a leucine zipper. (ii) In uninfected and infected cells, antibody to p78 reacts with two major bands with an apparent Mr of 78,000 and two minor bands with apparent Mrs of 62, 000 and 55,000. (ii) p78 also interacts with ICP22 in vitro. (iii) In uninfected cells, p78 was dispersed largely in the nucleoplasm in HeLa cells and in the nucleoplasm and cytoplasm in HEp-2 cells. After infection, p78 formed large dense bodies which did not colocalize with the viral regulatory protein ICP0. (iv) Accumulation of p78 was cell cycle dependent, being highest very early in S phase. (v) The accumulation of ICP22 in synchronized cells was highest in early S phase, in contrast to the accumulation of another protein, ICP27, which was relatively independent of the cell cycle. (vi) In the course of the cell cycle, ICP22 was transiently modified in an aberrant fashion, and this modification coincided with expression of p78. The results suggest that ICP22 interacts with and may be stabilized by cell cycle-dependent proteins.

Repeated cycles of 5-fluorouracil chemotherapy impaired anti-tumor functions of cytotoxic T cells in a CT26 tumor-bearing mouse model.

PubMed

Wu, Yanhong; Deng, Zhenling; Wang, Huiru; Ma, Wenbo; Zhou, Chunxia; Zhang, Shuren

2016-09-20

Recently, the immunostimulatory roles of chemotherapeutics have been increasingly revealed, although bone marrow suppression is still a common toxicity of chemotherapy. While the numbers and ratios of different immune subpopulations are analyzed after chemotherapy, changes to immune status after each cycle of treatment are less studied and remain unclear. To determine the tumor-specific immune status and functions after different cycles of chemotherapy, we treated CT26 tumor-bearing mice with one to four cycles of 5-fluorouracil (5-FU). Overall survival was not improved when more than one cycle of 5-FU was administered. Here we present data concerning the immune statuses after one and three cycles of chemotherapy. We analyzed the amount of spleen cells from mice treated with one and three cycles of 5-FU as well as assayed their proliferation and cytotoxicity against the CT26 tumor cell line. We found that the absolute numbers of CD8 T-cells and NK cells were not influenced significantly after either one or three cycles of chemotherapy. However, after three cycles of 5-FU, proliferated CD8 T-cells were decreased, and CT26-specific cytotoxicity and IFN-γ secretion of spleen cells were impaired in vitro. After one cycle of 5-FU, there was a greater percentage of tumor infiltrating CD8 T-cells. In addition, more proliferated CD8 T-cells, enhanced tumor-specific cytotoxicity as well as IFN-γ secretion of spleen cells against CT26 in vitro were observed. Given the increased expression of immunosuppressive factors, such as PD-L1 and TGF-β, we assessed the effect of early introduction of immunotherapy in combination with chemotherapy. We found that mice treated with cytokine induced killer cells and PD-L1 monoclonal antibodies after one cycle of 5-FU had a better anti-tumor performance than those treated with chemotherapy or immunotherapy alone. These data suggest that a single cycle of 5-FU treatment promoted an anti-tumor immune response, whereas repeated chemotherapy cycles impaired anti-tumor immune functions. Though the amount of immune cells could recover after chemotherapy suspension, their anti-tumor functions were damaged by multiple rounds of chemotherapy. These findings also point towards early implementation of immunotherapy to improve the anti-tumor effect.

xCT expression reduces the early cell cycle requirement for calcium signaling

PubMed Central

Lastro, Michele; Kourtidis, Antonis; Farley, Kate; Conklin, Douglas S.

2009-01-01

Calcium has long been recognized as an important regulator of cell cycle transitions although the mechanisms are largely unknown. A functional genomic screen has identified genes involved in the regulation of early cell cycle progression by calcium. These genes when overexpressed confer the ability to bypass the G1/S arrest induced by Ca2+- channel antagonists in mouse fibroblasts. Overexpression of the cystine-glutamate exchanger, xCT, had the greatest ability to evade calcium antagonist-induced cell cycle arrest. xCT carries out the rate limiting step of glutathione synthesis in many cell types and is responsible for the uptake of cystine in most human cancer cell lines. Functional analysis indicates that the cystine uptake activity of xCT overcomes the G1/S arrest induced by Ca2+- channel antagonists by bypassing the requirement for calcium signaling. Since cells overexpressing xCT were found to have increased levels and activity of the AP-1 transcription factor in G1, redox stimulation of AP-1 activity accounts for the observed growth of these cells in the presence of calcium channel antagonists. These results suggest that reduced calcium signaling impairs AP-1 activation and that xCT expression may directly affect cell proliferation. PMID:18054200

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

PubMed Central

Quinn, Laura L.; Williams, Luke R.; White, Claire; Forrest, Calum; Rowe, Martin

2015-01-01

ABSTRACT The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8+ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8+ cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8+ cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4+ cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8+ and CD4+ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8+ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8+ T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8+ T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4+ T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. PMID:26468525

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II.

PubMed

Quinn, Laura L; Williams, Luke R; White, Claire; Forrest, Calum; Zuo, Jianmin; Rowe, Martin

2016-01-01

The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

From egg to gastrula: How the cell cycle is remodeled during the Drosophila mid-blastula transition

PubMed Central

Farrell, Jeffrey A.; O’Farrell, Patrick H.

2015-01-01

Many, if not most, embryos begin development with extremely short cell cycles that exhibit unusually rapid DNA replication and no gap phases. The commitment to the cell cycle in the early embryo appears to preclude many other cellular processes which only emerge as the cell cycle slows, at a major embryonic transition known as the mid-blastula transition (MBT) just prior to gastrulation. As reviewed here, genetic and molecular studies in Drosophila have identified changes that extend S phase and introduce a post-replicative gap phase, G2, to slow the cell cycle. While many mysteries remain about the upstream regulators of these changes, we review the core mechanisms of the change in cell cycle regulation and discuss advances in our understanding of how these might be timed and triggered. Finally, we consider how the elements of this program may be conserved or changed in other organisms. PMID:25195504

Impact of cycling cells and cell cycle regulation on Hydra regeneration.

PubMed

Buzgariu, Wanda; Wenger, Yvan; Tcaciuc, Nina; Catunda-Lemos, Ana-Paula; Galliot, Brigitte

2018-01-15

Hydra tissues are made from three distinct populations of stem cells that continuously cycle and pause in G2 instead of G1. To characterize the role of cell proliferation after mid-gastric bisection, we have (i) used flow cytometry and classical markers to monitor cell cycle modulations, (ii) quantified the transcriptomic regulations of 202 genes associated with cell proliferation during head and foot regeneration, and (iii) compared the impact of anti-proliferative treatments on regeneration efficiency. We confirm two previously reported events: an early mitotic wave in head-regenerating tips, when few cell cycle genes are up-regulated, and an early-late wave of proliferation on the second day, preceded by the up-regulation of 17 cell cycle genes. These regulations appear more intense after mid-gastric bisection than after decapitation, suggesting a position-dependent regulation of cell proliferation during head regeneration. Hydroxyurea, which blocks S-phase progression, delays head regeneration when applied before but not after bisection. This result is consistent with the fact that the Hydra central region is enriched in G2-paused adult stem cells, poised to divide upon injury, thus forming a necessary constitutive pro-blastema. However a prolonged exposure to hydroxyurea does not block regeneration as cells can differentiate apical structures without traversing S-phase, and also escape in few days the hydroxyurea-induced S-phase blockade. Thus Hydra head regeneration, which is a fast event, is highly plastic, relying on large stocks of adult stem cells paused in G2 at amputation time, which immediately divide to proliferate and/or differentiate apical structures even when S-phase is blocked. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

KPU-300, a Novel Benzophenone–Diketopiperazine–Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase

PubMed Central

Okuyama, Kohei; Kaida, Atsushi; Hayashi, Yoshiki; Hayashi, Yoshio; Harada, Kiyoshi; Miura, Masahiko

2015-01-01

KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300. PMID:26716455

KPU-300, a Novel Benzophenone-Diketopiperazine-Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase.

PubMed

Okuyama, Kohei; Kaida, Atsushi; Hayashi, Yoshiki; Hayashi, Yoshio; Harada, Kiyoshi; Miura, Masahiko

2015-01-01

KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

Argonaute-1 functions as a mitotic regulator by controlling Cyclin B during Drosophila early embryogenesis.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Bag, Indira; Hunt, Clayton R; Ramaiah, M Janaki; Pandita, Tej K; Bhadra, Utpal; Pal-Bhadra, Manika

2014-02-01

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. In this study, we established evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. We identified involvement of 2 novel embryonic miRNAs--miR-981 and miR--317-for spatiotemporal regulation of cyclin B. In summary, our results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (mei-41 and grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, our results have established a novel role of Ago-1 as a regulator of the cell cycle.

Examination of a modified cell cycle synchronization method and bovine nuclear transfer using synchronized early G1 phase fibroblast cells.

PubMed

Urakawa, Manami; Ideta, Atsushi; Sawada, Tokihiko; Aoyagi, Yoshito

2004-08-01

Somatic cell nuclear transfer has a low success rate, due to a high incidence of fetal loss and increased perinatal morbidity/mortality. One factor that may affect the successful development of nuclear transfer embryos is the cell cycle stage of the donor cell. In order to establish a cell cycle synchronization method that can consistently produce cloned embryos and offspring, we examined the effects of different combinations of three cell treatments on the recovery rate of mitotic phase cells using bovine fetal fibroblasts. In the first experiment, we examined the recovery rate of mitotic phase cells by a combination of treatment with a metaphase arrestant (1 microM 2-methoxyestradiol), shaking the plate and selecting cells with a diameter of 20 microns. As a result, 99% of mitotic phase cells were recovered by repeating the combined treatment of metaphase arrestant and shaking, and collection of cells with a specific diameter. In the second experiment, nuclear transfer was carried out using early G1 phase cells by choosing pairs of bridged cells derived from mitotic phase cells recovered by the combined treatment of 1 microM 2-methoxyestradiol and shaking, and collection of cells with a diameter of 20 microns. The reconstructed embryos were transferred to recipient heifers to determine post-implantation development. Development of embryos reconstructed from early G1 phase cells from the >/=6 cells stage on Day 3 to the morula-blastocyst stage on Day 6 was 100%. Ten blastocysts constructed from two cell lines were transferred into 10 recipient heifers. Nine of the 10 recipients delivered single live calves. In conclusion, mitotic phase bovine fibroblast cells were easily recovered by the combined treatments of 1 microM 2-methoxyestradiol, shaking, and selecting cells of the appropriate diameter. Furthermore, nuclear transfer using cells in the early G1 phase as donor cells gave a high rate of offspring production.

Cell cycle inhibition limits development and maintenance of neuropathic pain following spinal cord injury

PubMed Central

Wu, Junfang; Zhao, Zaorui; Zhu, Xiya; Renn, Cynthia L.; Dorsey, Susan G.; Faden, Alan I.

2016-01-01

Chronic pain after spinal cord injury (SCI) may present as hyperalgesia, allodynia, and/or spontaneous pain and is often resistant to conventional pain medications. Identifying more effective interventions to manage SCI-Pain requires improved understanding of the pathophysiological mechanisms involved. Cell cycle activation (CCA) has been implicated as a key pathophysiological event following SCI. We have shown that early central or systemic administration of a cell cycle inhibitor reduces CCA, prevents glial changes, and limits SCI-induced hyperesthesia. Here we compared the effects of early versus late treatment with the pan-cyclin dependent kinase inhibitor flavopiridol on allodynia as well as spontaneous pain. Adult C57BL/6 male mice subjected to moderate SCI were treated with intraperitoneal injections of flavopiridol (1 mg/kg), daily for 7 days beginning either 3 h or 5 weeks after injury. Mechanical/thermal allodynia was evaluated, as well as spontaneous pain using the mouse grimace scale (MGS). We show that sensitivity to mechanical and thermal stimulation, and locomotor dysfunction were significantly reduced by early flavopiridol treatment compared to vehicle treated controls. SCI caused robust and extended increases of MGS up to 3 weeks after trauma. Early administration of flavopiridol significantly shortened duration of MGS changes. Late flavopiridol intervention significantly limited hyperesthesia at 7 days after treatment, associated with reduced glial changes, but without effect on locomotion. Thus, our data suggest that cell cycle modulation may provide an effective therapeutic strategy to reduce hyperesthesia after SCI, with a prolonged therapeutic window. PMID:26797506

Autophagic flux is highly active in early mitosis and differentially regulated throughout the cell cycle

PubMed Central

Li, Zhiyuan; Ji, Xinmiao; Wang, Dongmei; Liu, Juanjuan; Zhang, Xin

2016-01-01

Mitosis is a fast process that involves dramatic cellular remodeling and has a high energy demand. Whether autophagy is active or inactive during the early stages of mitosis in a naturally dividing cell is still debated. Here we aimed to use multiple assays to resolve this apparent discrepancy. Although the LC3 puncta number was reduced in mitosis, the four different cell lines we tested all have active autophagic flux in both interphase and mitosis. In addition, the autophagic flux was highly active in nocodazole-induced, double-thymidine synchronization released as well as naturally occurring mitosis in HeLa cells. Multiple autophagy proteins are upregulated in mitosis and the increased Beclin-1 level likely contributes to the active autophagic flux in early mitosis. It is interesting that although the autophagic flux is active throughout the cell cycle, early mitosis and S phase have relatively higher autophagic flux than G1 and late G2 phases, which might be helpful to degrade the damaged organelles and provide energy during S phase and mitosis. PMID:27213594

Autophagic flux is highly active in early mitosis and differentially regulated throughout the cell cycle.

PubMed

Li, Zhiyuan; Ji, Xinmiao; Wang, Dongmei; Liu, Juanjuan; Zhang, Xin

2016-06-28

Mitosis is a fast process that involves dramatic cellular remodeling and has a high energy demand. Whether autophagy is active or inactive during the early stages of mitosis in a naturally dividing cell is still debated. Here we aimed to use multiple assays to resolve this apparent discrepancy. Although the LC3 puncta number was reduced in mitosis, the four different cell lines we tested all have active autophagic flux in both interphase and mitosis. In addition, the autophagic flux was highly active in nocodazole-induced, double-thymidine synchronization released as well as naturally occurring mitosis in HeLa cells. Multiple autophagy proteins are upregulated in mitosis and the increased Beclin-1 level likely contributes to the active autophagic flux in early mitosis. It is interesting that although the autophagic flux is active throughout the cell cycle, early mitosis and S phase have relatively higher autophagic flux than G1 and late G2 phases, which might be helpful to degrade the damaged organelles and provide energy during S phase and mitosis.

Distinct chronology of neuronal cell cycle re-entry and tau pathology in the 3xTg-AD mouse model and Alzheimer's disease patients.

PubMed

Hradek, Alex C; Lee, Hyun-Pil; Siedlak, Sandra L; Torres, Sandy L; Jung, Wooyoung; Han, Ashley H; Lee, Hyoung-gon

2015-01-01

Cell cycle re-entry in Alzheimer's disease (AD) has emerged as an important pathological mechanism in the progression of the disease. This appearance of cell cycle related proteins has been linked to tau pathology in AD, but the causal and temporal relationship between the two is not completely clear. In this study, we found that hyperphosphorylated retinoblastoma protein (ppRb), a key regulator for G1/S transition, is correlated with a late marker for hyperphosphorylation of tau but not with other early markers for tau alteration in the 3xTg-AD mouse model. However, in AD brains, ppRb can colocalize with both early and later markers for tau alterations, and can often be found singly in many degenerating neurons, indicating the distinct development of pathology between the 3xTg-AD mouse model and human AD patients. The conclusions of this study are two-fold. First, our findings clearly demonstrate the pathological link between the aberrant cell cycle re-entry and tau pathology. Second, the chronological pattern of cell cycle re-entry with tau pathology in the 3xTg-AD mouse is different compared to AD patients suggesting the distinct pathogenic mechanism between the animal AD model and human AD patients.

Systems-level feedback regulation of cell cycle transitions in Ostreococcus tauri.

PubMed

Kapuy, Orsolya; Vinod, P K; Bánhegyi, Gábor; Novák, Béla

2018-05-01

Ostreococcus tauri is the smallest free-living unicellular organism with one copy of each core cell cycle genes in its genome. There is a growing interest in this green algae due to its evolutionary origin. Since O. tauri is diverged early in the green lineage, relatively close to the ancestral eukaryotic cell, it might hold a key phylogenetic position in the eukaryotic tree of life. In this study, we focus on the regulatory network of its cell division cycle. We propose a mathematical modelling framework to integrate the existing knowledge of cell cycle network of O. tauri. We observe that feedback loop regulation of both G1/S and G2/M transitions in O. tauri is conserved, which can make the transition bistable. This is essential to make the transition irreversible as shown in other eukaryotic organisms. By performing sequence analysis, we also predict the presence of the Greatwall/PP2A pathway in the cell cycle of O. tauri. Since O. tauri cell cycle machinery is conserved, the exploration of the dynamical characteristic of the cell division cycle will help in further understanding the regulation of cell cycle in higher eukaryotes. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Development of first generation aerospace NiMH cells

NASA Technical Reports Server (NTRS)

Tinker, Lawrence; Dell, Dan; Wu, Tony; Rampel, Guy

1993-01-01

Gates Aerospace Batteries in conjunction with Gates Energy Products (GEP) has been developing NiMH technology for aerospace use since 1990. GEP undertook the development of NiMH technology for commercial cell applications in 1987. This program focused on wound cell technology for replacement of current NiCd technology. As an off shoot of this program small, wound cells were used to evaluate initial design options for aerospace prismatic cell designs. Early in 1991, the first aerospace prismatic cell designs were built in a 6 Ah cell configuration. These cells were used to initially characterize performance in prismatic configurations and begin early life cycle testing. Soon after the 6 Ah cells were on test, several 22 Ah cells were built to test other options. The results of testing of these cells were used to identify potential problem areas for long lived cells and develop solutions to those problems. Following these two cell builds, a set of 7 Ah cells was built to evaluate improvements to the technology. To date results from these tests are very promising. Cycle lives in excess of 2,200 LEO cycles at 50 percent DoD were achieved with cells continuing on test. Results from these cell tests are discussed and data presented to demonstrate feasibility of this technology for aerospace programs.

The Expression Pattern of microRNAs in Granulosa Cells of Subordinate and Dominant Follicles during the Early Luteal Phase of the Bovine Estrous Cycle

PubMed Central

Gebremedhn, Samuel; Sahadevan, Sudeep; Hossain, MD Munir; Rings, Franca; Hoelker, Michael; Tholen, Ernst; Neuhoff, Christiane; Looft, Christian; Schellander, Karl; Tesfaye, Dawit

2014-01-01

This study aimed to investigate the miRNA expression patterns in granulosa cells of subordinate (SF) and dominant follicle (DF) during the early luteal phase of the bovine estrous cycle. For this, miRNA enriched total RNA isolated from granulosa cells of SF and DF obtained from heifers slaughtered at day 3 and day 7 of the estrous cycle was used for miRNAs deep sequencing. The results revealed that including 17 candidate novel miRNAs, several known miRNAs (n = 291–318) were detected in SF and DF at days 3 and 7 of the estrous cycle of which 244 miRNAs were common to all follicle groups. The let-7 families, bta-miR-10b, bta-miR-26a, bta-miR-99b and bta-miR-27b were among abundantly expressed miRNAs in both SF and DF at both days of the estrous cycle. Further analysis revealed that the expression patterns of 16 miRNAs including bta-miR-449a, bta-miR-449c and bta-miR-222 were differentially expressed between the granulosa cells of SF and DF at day 3 of the estrous cycle. However, at day 7 of the estrous cycle, 108 miRNAs including bta-miR-409a, bta-miR-383 and bta-miR-184 were differentially expressed between the two groups of granulosa cell revealing the presence of distinct miRNA expression profile changes between the two follicular stages at day 7 than day 3 of the estrous cycle. In addition, unlike the SF, marked temporal miRNA expression dynamics was observed in DF groups between day 3 and 7 of the estrous cycle. Target gene prediction and pathway analysis revealed that major signaling associated with follicular development including Wnt signaling, TGF-beta signaling, oocyte meiosis and GnRH signaling were affected by differentially expressed miRNAs. Thus, this study highlights the miRNA expression patterns of granulosa cells in subordinate and dominant follicles that could be associated with follicular recruitment, selection and dominance during the early luteal phase of the bovine estrous cycle. PMID:25192015

Cardiomyocyte cell cycle control and growth estimation in vivo--an analysis based on cardiomyocyte nuclei.

PubMed

Walsh, Stuart; Pontén, Annica; Fleischmann, Bernd K; Jovinge, Stefan

2010-06-01

Adult mammalian cardiomyocytes are traditionally viewed as being permanently withdrawn from the cell cycle. Whereas some groups have reported none, others have reported extensive mitosis in adult myocardium under steady-state conditions. Recently, a highly specific assay of 14C dating in humans has suggested a continuous generation of cardiomyocytes in the adult, albeit at a very low rate. Mice represent the most commonly used animal model for these studies, but their short lifespan makes them unsuitable for 14C studies. Herein, we investigate the cellular growth pattern for murine cardiomyocyte growth under steady-state conditions, addressed with new analytical and technical strategies, and we furthermore relate this to gene expression patterns. The observed levels of DNA synthesis in early life were associated with cardiomyocyte proliferation. Mitosis was prolonged into early life, longer than the most conservative previous estimates. DNA synthesis in neonatal life was attributable to bi-nucleation, therefore suggesting that cardiomyocytes withdraw from the cell cycle shortly after birth. No cell cycle activity was observed in adult cardiomyocytes and significant polyploidy was observed in cardiomyocyte nuclei. Gene analyses identified 32 genes whose expression was predicted to be particular to day 3-4 neonatal myocytes, compared with embryonic or adult cells. These cell cycle-associated genes are crucial to the understanding of the mechanisms of bi-nucleation and physiological cellular growth in the neonatal period.

Individual Pressure Vessel (PV) and Common Pressure Vessel (CPV) Nickel-Hydrogen Battery Performance Under LEO Cycling Conditions

NASA Technical Reports Server (NTRS)

Miller, Thomas B.; Lewis, Harlan L.

2004-01-01

LEO life cycle testing of Individual Pressure Vessel (PV) and Common Pressure Vessel (CPV) nickel-hydrogen cell packs have been sponsored by the NASA Aerospace Flight Battery Program. The cell packs have cycled under both 35% and 60% depth-of- discharge and temperature conditions of -5 C and +lO C. The packs have been on test since as early as 1992 and have generated a substantial database. This report will provide insight into performance trends as a function of the specific cell configuration and manufacturer for eight separate nickel-hydrogen battery cell packs.

Nucleosome architecture throughout the cell cycle

PubMed Central

Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto

2016-01-01

Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity. PMID:26818620

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

PubMed

Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A; Grant, Gavin D; Chao, Hui Yan; Cook, Jeanette G; Purvis, Jeremy E

2017-11-22

Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none. Copyright © 2017 Elsevier Inc. All rights reserved.

The role of a cell surface inhibitor in early signal transduction associated with the regulation of cell division and differentiation

NASA Technical Reports Server (NTRS)

Johnson, T. C.; Enebo, D. J.; Moos, P. J.; Fattaey, H. K.; Spooner, B. S. (Principal Investigator)

1992-01-01

Serum stimulation of quiescent human fibroblast cultures resulted in a hyperphosphorylation of the nuclear retinoblastoma gene susceptibility product (RB). However, serum stimulation in the presence of 9 x 10(-8) M of a purified bovine sialoglycopeptide (SGP) cell surface inhibitor abrogated the hyperphosphorylation of the RB protein and the subsequent progression of cells through the mitotic cycle. The experimental results suggest that the SGP mediated its cell cycle arrest at a site in the cell cycle that was at the time of RB phosphorylation or somewhat upstream of the modification of this regulatory protein of cell division. Both cells serum-deprived and serum stimulated in the presence of the SGP displayed only a hypophosphorylated RB protein, consistent with the SGP-mediated cell cycle arrest point being near the G1/S interface.

Multiprobe Study of the Solid Electrolyte Interphase on Silicon-Based Electrodes in Full-Cell Configuration

PubMed Central

Moreau, P.; De Vito, E.; Quazuguel, L.; Boniface, M.; Bordes, A.; Rudisch, C.; Bayle-Guillemaud, P.; Guyomard, D.

2016-01-01

The failure mechanism of silicon-based electrodes has been studied only in a half-cell configuration so far. Here, a combination of 7Li, 19F MAS NMR, XPS, TOF-SIMS, and STEM-EELS, provides an in-depth characterization of the solid electrolyte interphase (SEI) formation on the surface of silicon and its evolution upon aging and cycling with LiNi1/3Mn1/3Co1/3O2 as the positive electrode in a full Li-ion cell configuration. This multiprobe approach indicates that the electrolyte degradation process observed in the case of full Li-ion cells exhibits many similarities to what has been observed in the case of half-cells in previous works, in particular during the early stages of the cycling. Like in the case of Si/Li half-cells, the development of the inorganic part of the SEI mostly occurs during the early stage of cycling while an incessant degradation of the organic solvents of the electrolyte occurs upon cycling. However, for extended cycling, all the lithium available for cycling is consumed because of parasitic reactions and is either trapped in an intermediate part of the SEI or in the electrolyte. This nevertheless does not prevent the further degradation of the organic electrolyte solvents, leading to the formation of lithium-free organic degradation products at the extreme surface of the SEI. At this point, without any available lithium left, the cell cannot function properly anymore. Cycled positive and negative electrodes do not show any sign of particles disconnection or clogging of their porosity by electrolyte degradation products and can still function in half-cell configuration. The failure mechanism for full Li-ion cells appears then very different from that known for half-cells and is clearly due to a lack of cyclable lithium because of parasitic reactions occurring before the accumulation of electrolyte degradation products clogs the porosity of the composite electrode or disconnects the active material particles. PMID:27212791

Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

PubMed Central

Roy, Sujayita; He, Ran; Kapoor, Arun; Forman, Michael; Mazzone, Jennifer R.; Posner, Gary H.

2015-01-01

Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition. PMID:25870074

A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells.

PubMed

Schorpp, Kenji; Rothenaigner, Ina; Maier, Julia; Traenkle, Bjoern; Rothbauer, Ulrich; Hadian, Kamyar

2016-10-01

Many screening hits show relatively poor quality regarding later efficacy and safety. Therefore, small-molecule screening efforts shift toward high-content analysis providing more detailed information. Here, we describe a novel screening approach to identify cell cycle modulators with low toxicity by combining the Cell Cycle Chromobody (CCC) technology with the CytoTox-Glo (CTG) cytotoxicity assay. The CCC technology employs intracellularly functional single-domain antibodies coupled to a fluorescent protein (chromobodies) to visualize the cell cycle-dependent redistribution of the proliferating cell nuclear antigen (PCNA) in living cells. This image-based cell cycle analysis was combined with determination of dead-cell protease activity in cell culture supernatants by the CTG assay. We adopted this multiplex approach to high-throughput format and screened 960 Food and Drug Administration (FDA)-approved drugs. By this, we identified nontoxic compounds, which modulate different cell cycle stages, and validated selected hits in diverse cell lines stably expressing CCC. Additionally, we independently validated these hits by flow cytometry as the current state-of-the-art format for cell cycle analysis. This study demonstrates that CCC imaging is a versatile high-content screening approach to identify cell cycle modulators, which can be multiplexed with cytotoxicity assays for early elimination of toxic compounds during screening. © 2016 Society for Laboratory Automation and Screening.

Identification and Characterization of Three Differentially Expressed Genes, Encoding S-Adenosylhomocysteine Hydrolase, Methionine Aminopeptidase, and a Histone-Like Protein, in the Toxic Dinoflagellate Alexandrium fundyense†

PubMed Central

Taroncher-Oldenburg, Gaspar; Anderson, Donald M.

2000-01-01

Genes showing differential expression related to the early G1 phase of the cell cycle during synchronized circadian growth of the toxic dinoflagellate Alexandrium fundyense were identified and characterized by differential display (DD). The determination in our previous work that toxin production in Alexandrium is relegated to a narrow time frame in early G1 led to the hypothesis that transcriptionally up- or downregulated genes during this subphase of the cell cycle might be related to toxin biosynthesis. Three genes, encoding S-adenosylhomocysteine hydrolase (Sahh), methionine aminopeptidase (Map), and a histone-like protein (HAf), were isolated. Sahh was downregulated, while Map and HAf were upregulated, during the early G1 phase of the cell cycle. Sahh and Map encoded amino acid sequences with about 90 and 70% similarity to those encoded by several eukaryotic and prokaryotic Sahh and Map genes, respectively. The partial Map sequence also contained three cobalt binding motifs characteristic of all Map genes. HAf encoded an amino acid sequence with 60% similarity to those of two histone-like proteins from the dinoflagellate Crypthecodinium cohnii Biecheler. This study documents the potential of applying DD to the identification of genes that are related to physiological processes or cell cycle events in phytoplankton under conditions where small sample volumes represent an experimental constraint. The identification of an additional 21 genes with various cell cycle-related DD patterns also provides evidence for the importance of pretranslational or transcriptional regulation in dinoflagellates, contrary to previous reports suggesting the possibility that translational mechanisms are the primary means of circadian regulation in this group of organisms. PMID:10788388

40 CFR 86.1867-12 - Optional early CO2 credit programs.

Code of Federal Regulations, 2010 CFR

2010-07-01

... efficiency credits, early advanced technology credits, and early off-cycle technology credits. Manufacturers...) Carbon-related exhaust emission values for electric, fuel cell, and plug-in hybrid electric model types... extent that such vehicles are not being used to generate early advanced technology vehicle credits under...

40 CFR 86.1867-12 - Optional early CO2 credit programs.

Code of Federal Regulations, 2011 CFR

2011-07-01

... efficiency credits, early advanced technology credits, and early off-cycle technology credits. Manufacturers...) Carbon-related exhaust emission values for electric, fuel cell, and plug-in hybrid electric model types... extent that such vehicles are not being used to generate early advanced technology vehicle credits under...

Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant1[C][W

PubMed Central

Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

2014-01-01

The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants. PMID:25037213

Molecular mechanisms involved in the early steps of flavivirus cell entry.

PubMed

Kaufmann, Bärbel; Rossmann, Michael G

2011-01-01

Flaviviruses enter their host cells by receptor-mediated endocytosis, a well-orchestrated process of receptor recognition, penetration and uncoating. Recent findings on these early steps in the life cycle of flaviviruses are the focus of this review. Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

Cell cycle gene expression networks discovered using systems biology: Significance in carcinogenesis

PubMed Central

Scott, RE; Ghule, PN; Stein, JL; Stein, GS

2015-01-01

The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co-vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty-three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with p = e−13 to e−36. Cell cycle expression networks show species, sex and tissue variability and they are enriched in mRNA transcripts associated with mitosis many of which are associated with chromosomal instability. PMID:25808367

Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

PubMed Central

Schraufstätter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

1985-01-01

Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells. PMID:3840176

A dual-color marker system for in vivo visualization of cell cycle progression in Arabidopsis.

PubMed

Yin, Ke; Ueda, Minako; Takagi, Hitomi; Kajihara, Takehiro; Sugamata Aki, Shiori; Nobusawa, Takashi; Umeda-Hara, Chikage; Umeda, Masaaki

2014-11-01

Visualization of the spatiotemporal pattern of cell division is crucial to understand how multicellular organisms develop and how they modify their growth in response to varying environmental conditions. The mitotic cell cycle consists of four phases: S (DNA replication), M (mitosis and cytokinesis), and the intervening G1 and G2 phases; however, only G2/M-specific markers are currently available in plants, making it difficult to measure cell cycle duration and to analyze changes in cell cycle progression in living tissues. Here, we developed another cell cycle marker that labels S-phase cells by manipulating Arabidopsis CDT1a, which functions in DNA replication origin licensing. Truncations of the CDT1a coding sequence revealed that its carboxy-terminal region is responsible for proteasome-mediated degradation at late G2 or in early mitosis. We therefore expressed this region as a red fluorescent protein fusion protein under the S-specific promoter of a histone 3.1-type gene, HISTONE THREE RELATED2 (HTR2), to generate an S/G2 marker. Combining this marker with the G2/M-specific CYCB1-GFP marker enabled us to visualize both S to G2 and G2 to M cell cycle stages, and thus yielded an essential tool for time-lapse imaging of cell cycle progression. The resultant dual-color marker system, Cell Cycle Tracking in Plant Cells (Cytrap), also allowed us to identify root cells in the last mitotic cell cycle before they entered the endocycle. Our results demonstrate that Cytrap is a powerful tool for in vivo monitoring of the plant cell cycle, and thus for deepening our understanding of cell cycle regulation in particular cell types during organ development. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

The emerging role and targetability of the TCA cycle in cancer metabolism.

PubMed

Anderson, Nicole M; Mucka, Patrick; Kern, Joseph G; Feng, Hui

2018-02-01

The tricarboxylic acid (TCA) cycle is a central route for oxidative phosphorylation in cells, and fulfills their bioenergetic, biosynthetic, and redox balance requirements. Despite early dogma that cancer cells bypass the TCA cycle and primarily utilize aerobic glycolysis, emerging evidence demonstrates that certain cancer cells, especially those with deregulated oncogene and tumor suppressor expression, rely heavily on the TCA cycle for energy production and macromolecule synthesis. As the field progresses, the importance of aberrant TCA cycle function in tumorigenesis and the potentials of applying small molecule inhibitors to perturb the enhanced cycle function for cancer treatment start to evolve. In this review, we summarize current knowledge about the fuels feeding the cycle, effects of oncogenes and tumor suppressors on fuel and cycle usage, common genetic alterations and deregulation of cycle enzymes, and potential therapeutic opportunities for targeting the TCA cycle in cancer cells. With the application of advanced technology and in vivo model organism studies, it is our hope that studies of this previously overlooked biochemical hub will provide fresh insights into cancer metabolism and tumorigenesis, subsequently revealing vulnerabilities for therapeutic interventions in various cancer types.

The temporal organization of processes of cell reproduction and its connection with rhythms of radiosensitivity of the body

NASA Technical Reports Server (NTRS)

Druzhinin, Y. P.; Romanov, Y. A.; Vatsek, A.

1974-01-01

Radiosensitivity of individual phases of the mitotic cycle was studied in synchronous cell cultures and in several biological objects. It was found that radiosensitivity changed essentially according to phases of the mitotic cycle, depending on the kind of cells, evaluation criteria and the radiation dosage. Tests on partially synchronized HeLa cell populations, according to the criterion of survival, showed them most sensitive during mitosis, as well as in later G sub 1- or early DNA-synthesizing stages. With radiation in doses of 300 rad, the proportion of surviving cells showed a sensitivity directly before DNA synthesis of approximately 4 times higher than the later S-phase and during the major portion of G sub 1- and G sub 2-periods. Sensitivity of cells in mitosis was approximately 3 times higher than in late G sub 1- and early S-phases.

Regulation of the Embryonic Cell Cycle During Mammalian Preimplantation Development.

PubMed

Palmer, N; Kaldis, P

2016-01-01

The preimplantation development stage of mammalian embryogenesis consists of a series of highly conserved, regulated, and predictable cell divisions. This process is essential to allow the rapid expansion and differentiation of a single-cell zygote into a multicellular blastocyst containing cells of multiple developmental lineages. This period of development, also known as the germinal stage, encompasses several important developmental transitions, which are accompanied by dramatic changes in cell cycle profiles and dynamics. These changes are driven primarily by differences in the establishment and enforcement of cell cycle checkpoints, which must be bypassed to facilitate the completion of essential cell cycle events. Much of the current knowledge in this area has been amassed through the study of knockout models in mice. These mouse models are powerful experimental tools, which have allowed us to dissect the relative dependence of the early embryonic cell cycles on various aspects of the cell cycle machinery and highlight the extent of functional redundancy between members of the same gene family. This chapter will explore the ways in which the cell cycle machinery, their accessory proteins, and their stimuli operate during mammalian preimplantation using mouse models as a reference and how this allows for the usually well-defined stages of the cell cycle to be shaped and transformed during this unique and critical stage of development. © 2016 Elsevier Inc. All rights reserved.

Redox Changes During the Cell Cycle in the Embryonic Root Meristem of Arabidopsis thaliana.

PubMed

de Simone, Ambra; Hubbard, Rachel; de la Torre, Natanael Viñegra; Velappan, Yazhini; Wilson, Michael; Considine, Michael J; Soppe, Wim J J; Foyer, Christine H

2017-12-20

The aim of this study was to characterize redox changes in the nuclei and cytosol occurring during the mitotic cell cycle in the embryonic roots of germinating Arabidopsis seedlings, and to determine how redox cycling was modified in mutants with a decreased capacity for ascorbate synthesis. Using an in vivo reduction-oxidation (redox) reporter (roGFP2), we show that transient oxidation of the cytosol and the nuclei occurred at G1 in the synchronized dividing cells of the Arabidopsis root apical meristem, with reduction at G2 and mitosis. This redox cycle was absent from low ascorbate mutants in which nuclei were significantly more oxidized than controls. The cell cycle-dependent increase in nuclear size was impaired in the ascorbate-deficient mutants, which had fewer cells per unit area in the root proliferation zone. The transcript profile of the dry seeds and size of the imbibed seeds was strongly influenced by low ascorbate but germination, dormancy release and seed aging characteristics were unaffected. These data demonstrate the presence of a redox cycle within the plant cell cycle and that the redox state of the nuclei is an important factor in cell cycle progression. Controlled oxidation is a key feature of the early stages of the plant cell cycle. However, sustained mild oxidation restricts nuclear functions and impairs progression through the cell cycle leading to fewer cells in the root apical meristem. Antioxid. Redox Signal. 27, 1505-1519.

"Till Death Do Us Part": A Potential Irreversible Link Between Aberrant Cell Cycle Control and Neurodegeneration in the Adult Olfactory Bulb.

PubMed

Omais, Saad; Jaafar, Carine; Ghanem, Noël

2018-01-01

Adult neurogenesis (AN) is an ongoing developmental process that generates newborn neurons in the olfactory bulb (OB) and the hippocampus (Hi) throughout life and significantly contributes to brain plasticity. Adult neural stem and progenitor cells (aNSPCs) are relatively limited in number and fate and are spatially restricted to the subventricular zone (SVZ) and the subgranular zone (SGZ). During AN, the distinct roles played by cell cycle proteins extend beyond cell cycle control and constitute key regulatory mechanisms involved in neuronal maturation and survival. Importantly, aberrant cell cycle re-entry (CCE) in post-mitotic neurons has been strongly linked to the abnormal pathophysiology in rodent models of neurodegenerative diseases with potential implications on the etiology and progression of such diseases in humans. Here, we present an overview of AN in the SVZ-OB and olfactory epithelium (OE) in mice and humans followed by a comprehensive update of the distinct roles played by cell cycle proteins including major tumors suppressor genes in various steps during neurogenesis. We also discuss accumulating evidence underlining a strong link between abnormal cell cycle control, olfactory dysfunction and neurodegeneration in the adult and aging brain. We emphasize that: (1) CCE in post-mitotic neurons due to loss of cell cycle suppression and/or age-related insults as well as DNA damage can anticipate the development of neurodegenerative lesions and protein aggregates, (2) the age-related decline in SVZ and OE neurogenesis is associated with compensatory pro-survival mechanisms in the aging OB which are interestingly similar to those detected in Alzheimer's disease and Parkinson's disease in humans, and (3) the OB represents a well suitable model to study the early manifestation of age-related defects that may eventually progress into the formation of neurodegenerative lesions and, possibly, spread to the rest of the brain. Such findings may provide a novel approach to the modeling of neurodegenerative diseases in humans from early detection to progression and treatment as well.

Slow-cycling stem cells in hydra contribute to head regeneration

PubMed Central

Govindasamy, Niraimathi; Murthy, Supriya; Ghanekar, Yashoda

2014-01-01

ABSTRACT Adult stem cells face the challenge of maintaining tissue homeostasis by self-renewal while maintaining their proliferation potential over the lifetime of an organism. Continuous proliferation can cause genotoxic/metabolic stress that can compromise the genomic integrity of stem cells. To prevent stem cell exhaustion, highly proliferative adult tissues maintain a pool of quiescent stem cells that divide only in response to injury and thus remain protected from genotoxic stress. Hydra is a remarkable organism with highly proliferative stem cells and ability to regenerate at whole animal level. Intriguingly, hydra does not display consequences of high proliferation, such as senescence or tumour formation. In this study, we investigate if hydra harbours a pool of slow-cycling stem cells that could help prevent undesirable consequences of continuous proliferation. Hydra were pulsed with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) and then chased in the absence of EdU to monitor the presence of EdU-retaining cells. A significant number of undifferentiated cells of all three lineages in hydra retained EdU for about 8–10 cell cycles, indicating that these cells did not enter cell cycle. These label-retaining cells were resistant to hydroxyurea treatment and were predominantly in the G2 phase of cell cycle. Most significantly, similar to mammalian quiescent stem cells, these cells rapidly entered cell division during head regeneration. This study shows for the first time that, contrary to current beliefs, cells in hydra display heterogeneity in their cell cycle potential and the slow-cycling cells in this population enter cell cycle during head regeneration. These results suggest an early evolution of slow-cycling stem cells in multicellular animals. PMID:25432513

CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo

PubMed Central

Mende, Nicole; Kuchen, Erika E.; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D.; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico

2015-01-01

Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1–CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1–CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1–CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis. PMID:26150472

CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.

PubMed

Mende, Nicole; Kuchen, Erika E; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico; Waskow, Claudia

2015-07-27

Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1-CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1-CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1-CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis. © 2015 Mende et al.

The Deadbeat Paternal Effect of Uncapped Sperm Telomeres on Cell Cycle Progression and Chromosome Behavior in Drosophila melanogaster

PubMed Central

Yamaki, Takuo; Yasuda, Glenn K.; Wakimoto, Barbara T.

2016-01-01

Telomere-capping complexes (TCCs) protect the ends of linear chromosomes from illegitimate repair and end-to-end fusions and are required for genome stability. The identity and assembly of TCC components have been extensively studied, but whether TCCs require active maintenance in nondividing cells remains an open question. Here we show that Drosophila melanogaster requires Deadbeat (Ddbt), a sperm nuclear basic protein (SNBP) that is recruited to the telomere by the TCC and is required for TCC maintenance during genome-wide chromatin remodeling, which transforms spermatids to mature sperm. Ddbt-deficient males produce sperm lacking TCCs. Their offspring delay the initiation of anaphase as early as cycle 1 but progress through the first two cycles. Persistence of uncapped paternal chromosomes induces arrest at or around cycle 3. This early arrest can be rescued by selective elimination of paternal chromosomes and production of gynogenetic haploid or haploid mosaics. Progression past cycle 3 can also occur if embryos have reduced levels of the maternally provided checkpoint kinase Chk2. The findings provide insights into how telomere integrity affects the regulation of the earliest embryonic cell cycles. They also suggest that other SNBPs, including those in humans, may have analogous roles and manifest as paternal effects on embryo quality. PMID:27029731

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage

PubMed Central

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein–protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage. PMID:24675884

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage.

PubMed

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein-protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage.

Comprehensive single cell-resolution analysis of the role of chromatin regulators in early C. elegans embryogenesis.

PubMed

Krüger, Angela V; Jelier, Rob; Dzyubachyk, Oleh; Zimmerman, Timo; Meijering, Erik; Lehner, Ben

2015-02-15

Chromatin regulators are widely expressed proteins with diverse roles in gene expression, nuclear organization, cell cycle regulation, pluripotency, physiology and development, and are frequently mutated in human diseases such as cancer. Their inhibition often results in pleiotropic effects that are difficult to study using conventional approaches. We have developed a semi-automated nuclear tracking algorithm to quantify the divisions, movements and positions of all nuclei during the early development of Caenorhabditis elegans and have used it to systematically study the effects of inhibiting chromatin regulators. The resulting high dimensional datasets revealed that inhibition of multiple regulators, including F55A3.3 (encoding FACT subunit SUPT16H), lin-53 (RBBP4/7), rba-1 (RBBP4/7), set-16 (MLL2/3), hda-1 (HDAC1/2), swsn-7 (ARID2), and let-526 (ARID1A/1B) affected cell cycle progression and caused chromosome segregation defects. In contrast, inhibition of cir-1 (CIR1) accelerated cell division timing in specific cells of the AB lineage. The inhibition of RNA polymerase II also accelerated these division timings, suggesting that normal gene expression is required to delay cell cycle progression in multiple lineages in the early embryo. Quantitative analyses of the dataset suggested the existence of at least two functionally distinct SWI/SNF chromatin remodeling complex activities in the early embryo, and identified a redundant requirement for the egl-27 and lin-40 MTA orthologs in the development of endoderm and mesoderm lineages. Moreover, our dataset also revealed a characteristic rearrangement of chromatin to the nuclear periphery upon the inhibition of multiple general regulators of gene expression. Our systematic, comprehensive and quantitative datasets illustrate the power of single cell-resolution quantitative tracking and high dimensional phenotyping to investigate gene function. Furthermore, the results provide an overview of the functions of essential chromatin regulators during the early development of an animal. Copyright © 2014 Elsevier Inc. All rights reserved.

Biophysical modelling of early and delayed radiation damage at chromosome level

NASA Astrophysics Data System (ADS)

Andreev, S.; Eidelman, Y.

Exposure by ionising radiation increases cancer risk in human population Cancer is thought to originate from an altered expression of certain number of specific genes It is now widely recognised that chromosome aberrations CA are involved in stable change in expression of genes by gain or loss of their functions Thus CA can contribute to initiation or progression of cancer Therefore understanding mechanisms of CA formation in the course of cancer development might be valuable tool for quantification and prognosis of different stages of radiation carcinogenesis Early CA are defined as aberrations induced in first post-irradiation mitotic cycle The present work describes the original biophysical technique for early CA modelling It includes the following simulation steps the ionising particle track structure the structural organisation of all chromosomes in G 0 G 1 cell nucleus spatial distribution of radiation induced DNA double-strand breaks dsb within chromosomes dsb rejoining and misrejoining modelling cell cycle taking into account mitotic delay which results in complex time dependence of aberrant cells in first mitosis The results on prediction of dose-response curves for simple and complex CA measured in cells undergoing first division cycle are presented in comparison with recent experimental data There is increasing evidence that CA are also observed in descendents of irradiated cells many generations after direct DNA damage These delayed CA or chromosome instability CI are thought to be a manifestation of genome

Developmental Regulation of Nucleolus Size during Drosophila Eye Differentiation

PubMed Central

Baker, Nicholas E.

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals. PMID:23472166

Developmental regulation of nucleolus size during Drosophila eye differentiation.

PubMed

Baker, Nicholas E

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

The reproductive-cell cycle theory of aging: an update.

PubMed

Atwood, Craig S; Bowen, Richard L

2011-01-01

The Reproductive-Cell Cycle Theory posits that the hormones that regulate reproduction act in an antagonistic pleiotrophic manner to control aging via cell cycle signaling; promoting growth and development early in life in order to achieve reproduction, but later in life, in a futile attempt to maintain reproduction, become dysregulated and drive senescence. Since reproduction is the most important function of an organism from the perspective of the survival of the species, if reproductive-cell cycle signaling factors determine the rate of growth, determine the rate of development, determine the rate of reproduction, and determine the rate of senescence, then by definition they determine the rate of aging and thus lifespan. The theory is able to explain: 1) the simultaneous regulation of the rate of aging and reproduction as evidenced by the fact that environmental conditions and experimental interventions known to extend longevity are associated with decreased reproductive-cell cycle signaling factors, thereby slowing aging and preserving fertility in a hostile reproductive environment; 2) two phenomena that are closely related to species lifespan-the rate of growth and development and the ultimate size of the animal; 3). the apparent paradox that size is directly proportional to lifespan and inversely proportional to fertility between species but vice versa within a species; 4). how differing rates of reproduction between species is associated with differences in their lifespan; 5). why we develop aging-related diseases; and 6). an evolutionarily credible reason for why and how aging occurs-these hormones act in an antagonistic pleiotrophic manner via cell cycle signaling; promoting growth and development early in life in order to achieve reproduction, but later in life, in a futile attempt to maintain reproduction, become dysregulated and drive senescence (dyosis). In essence, the Reproductive-Cell Cycle Theory can explain aging in all sexually reproductive life forms. Published by Elsevier Inc.

40 CFR 86.1867-12 - Optional early CO2 credit programs.

Code of Federal Regulations, 2012 CFR

2012-07-01

... efficiency credits, early advanced technology credits, and early off-cycle technology credits. Manufacturers... an approved value. (F) Carbon-related exhaust emission values for electric, fuel cell, and plug-in hybrid electric model types shall be included in the fleet average determined under paragraph (a)(1) of...

40 CFR 86.1871-12 - Optional early CO2 credit programs.

Code of Federal Regulations, 2013 CFR

2013-07-01

... efficiency credits, early advanced technology credits, and early off-cycle technology credits. Manufacturers... manufacturer may use such an approved value. (F) Carbon-related exhaust emission values for electric, fuel cell, and plug-in hybrid electric model types shall be included in the fleet average determined under...

LPS-induced inflammatory response triggers cell cycle reactivation in murine neuronal cells through retinoblastoma proteins induction.

PubMed

D'Angelo, Barbara; Astarita, Carlo; Boffo, Silvia; Massaro-Giordano, Mina; Antonella Ianuzzi, Carmelina; Caporaso, Antonella; Macaluso, Marcella; Giordano, Antonio

2017-01-01

Cell cycle reactivation in adult neurons is an early hallmark of neurodegeneration. The lipopolysaccharide (LPS) is a well-known pro-inflammatory factor that provokes neuronal cell death via glial cells activation. The retinoblastoma (RB) family includes RB1/p105, retinoblastoma-like 1 (RBL1/p107), and retinoblastoma-like 2 (Rb2/p130). Several studies have indicated that RB proteins exhibit tumor suppressor activities, and play a central role in cell cycle regulation. In this study, we assessed LPS-mediated inflammatory effect on cell cycle reactivation and apoptosis of neuronally differentiated cells. Also, we investigated whether the LPS-mediated inflammatory response can influence the function and expression of RB proteins. Our results showed that LPS challenges triggered cell cycle reactivation of differentiated neuronal cells, indicated by an accumulation of cells in S and G2/M phase. Furthermore, we found that LPS treatment also induced apoptotic death of neurons. Interestingly, we observed that LPS-mediated inflammatory effect on cell cycle re-entry and apoptosis was concomitant with the aberrant expression of RBL1/p107 and RB1/p105. To the best of our knowledge, our study is the first to indicate a role of LPS in inducing cell cycle re-entry and/or apoptosis of differentiated neuronal cells, perhaps through mechanisms altering the expression of specific members of RB family proteins. This study provides novel information on the biology of post-mitotic neurons and could help in identifying novel therapeutic targets to prevent de novo cell cycle reactivation and/or apoptosis of neurons undergoing neurodegenerative processes.

A new cell culture model to genetically dissect the complete human papillomavirus life cycle.

PubMed

Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Myers, Julia E; Keiffer, Timothy R; DiGiuseppe, Stephen; Polk, Paula; Bodily, Jason M; Scott, Rona S; Sapp, Martin

2018-03-01

Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.

Diatom life cycles and ecology in the Cretaceous.

PubMed

Jewson, David H; Harwood, David M

2017-06-01

The earliest known diatom fossils with well-preserved siliceous frustules are from Lower Cretaceous neritic marine deposits in Antarctica. In this study, we analyzed the cell wall structure to establish whether their cell and life cycles were similar to modern forms. At least two filamentous species (Basilicostephanus ornatus and Archepyrgus melosiroides) had girdle band structures that functioned during cell division in a similar way to present day Aulacoseira species. Also, size analyses of cell diameter indicated that the cyclic process of size decline and size restoration used to time modern diatom life cycles was present in five species from the Lower Cretaceous (B. ornatus, A. melosiroides, Gladius antiquus, Ancylopyrgus reticulatus, Kreagra forfex) as well as two species from Upper Cretaceous deposits (Trinacria anissimowii and Eunotogramma fueloepi) from the Southwest Pacific. The results indicate that the "Diatom Sex Clock" was present from an early evolutionary stage. Other ecological adaptations included changes in mantle height and coiling. Overall, the results suggest that at least some of the species in these early assemblages are on a direct ancestral line to modern forms. © 2017 Phycological Society of America.

Two cell cycle blocks caused by iron chelation of neuroblastoma cells: separating cell cycle events associated with each block.

PubMed

Siriwardana, Gamini; Seligman, Paul A

2013-12-01

Studies have presented evidence that besides the well described S phase block, treatment of cancer cell lines with the iron chelator deferrioxamine (DFO) also results in an earlier block in G1 phase. In this article, measurements of cell cycle regulatory proteins define this block at a very specific point in G1. DFO treatment results in markedly decreased cyclin A protein levels. Cyclin E levels that accumulate in early to mid-G1 are increased in cells treated with DFO as compared to the resting cells. The DFO S phase block is shown after cells are arrested at G1/S by (aphidicolin) then released into DFO. The same S phase block occurs with DFO treatment of a neuroblastoma cell line relatively resistant to the G1 DFO block. These experiments clearly differentiate the S phase DFO block from the earlier block pinpointed to a point in mid-G1, before G1/S when cyclin E protein increases but before increased cyclin A synthesis. Apoptosis was observed in cells inhibited by DFO at both cell cycle arrest points.

Intermittent Stem Cell Cycling Balances Self-Renewal and Senescence of the C. elegans Germ Line.

PubMed

Cinquin, Amanda; Chiang, Michael; Paz, Adrian; Hallman, Sam; Yuan, Oliver; Vysniauskaite, Indre; Fowlkes, Charless C; Cinquin, Olivier

2016-04-01

Self-renewing organs often experience a decline in function in the course of aging. It is unclear whether chronological age or external factors control this decline, or whether it is driven by stem cell self-renewal-for example, because cycling cells exhaust their replicative capacity and become senescent. Here we assay the relationship between stem cell cycling and senescence in the Caenorhabditis elegans reproductive system, defining this senescence as the progressive decline in "reproductive capacity," i.e. in the number of progeny that can be produced until cessation of reproduction. We show that stem cell cycling diminishes remaining reproductive capacity, at least in part through the DNA damage response. Paradoxically, gonads kept under conditions that preclude reproduction keep cycling and producing cells that undergo apoptosis or are laid as unfertilized gametes, thus squandering reproductive capacity. We show that continued activity is in fact beneficial inasmuch as gonads that are active when reproduction is initiated have more sustained early progeny production. Intriguingly, continued cycling is intermittent-gonads switch between active and dormant states-and in all likelihood stochastic. Other organs face tradeoffs whereby stem cell cycling has the beneficial effect of providing freshly-differentiated cells and the detrimental effect of increasing the likelihood of cancer or senescence; stochastic stem cell cycling may allow for a subset of cells to preserve proliferative potential in old age, which may implement a strategy to deal with uncertainty as to the total amount of proliferation to be undergone over an organism's lifespan.

Ovarian Expression, Localization, and Function of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period of the Human Menstrual Cycle1

PubMed Central

Rosewell, Katherine L.; Li, Feixue; Puttabyatappa, Muraly; Akin, James W.; Brännström, Mats; Curry, Thomas E.

2013-01-01

ABSTRACT Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability. PMID:24048576

Ovarian expression, localization, and function of tissue inhibitor of metalloproteinase 3 (TIMP3) during the periovulatory period of the human menstrual cycle.

PubMed

Rosewell, Katherine L; Li, Feixue; Puttabyatappa, Muraly; Akin, James W; Brännström, Mats; Curry, Thomas E

2013-11-01

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Mitotic wavefronts mediated by mechanical signaling in early Drosophila embryos

NASA Astrophysics Data System (ADS)

Kang, Louis; Idema, Timon; Liu, Andrea; Lubensky, Tom

2013-03-01

Mitosis in the early Drosophila embryo demonstrates spatial and temporal correlations in the form of wavefronts that travel across the embryo in each cell cycle. This coordinated phenomenon requires a signaling mechanism, which we suggest is mechanical in origin. We have constructed a theoretical model that supports nonlinear wavefront propagation in a mechanically-excitable medium. Previously, we have shown that this model captures quantitatively the wavefront speed as it varies with cell cycle number, for reasonable values of the elastic moduli and damping coefficient of the medium. Now we show that our model also captures the displacements of cell nuclei in the embryo in response to the traveling wavefront. This new result further supports that mechanical signaling may play an important role in mediating mitotic wavefronts.

Single-cell analysis of transcription kinetics across the cell cycle

PubMed Central

Skinner, Samuel O; Xu, Heng; Nagarkar-Jaiswal, Sonal; Freire, Pablo R; Zwaka, Thomas P; Golding, Ido

2016-01-01

Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation. DOI: http://dx.doi.org/10.7554/eLife.12175.001 PMID:26824388

40 CFR 86.1871-12 - [Reserved

Code of Federal Regulations, 2014 CFR

2014-07-01

... credits, air conditioning leakage credits, air conditioning efficiency credits, early advanced technology credits, and early off-cycle technology credits. Manufacturers generating any credits under this section... value. (F) Carbon-related exhaust emission values for electric, fuel cell, and plug-in hybrid electric...

The Bone-specific Expression of Runx2 Oscillates during the Cell Cycle to Support a G1-related Antiproliferative Function in Osteoblasts*

PubMed Central

Galindo, Mario; Pratap, Jitesh; Young, Daniel W.; Hovhannisyan, Hayk; Im, Hee-Jeong; Choi, Je-Yong; Lian, Jane B.; Stein, Janet L.; Stein, Gary S.; van Wijnen, Andre J.

2010-01-01

The Runx2 (CBFA1/AML3/PEBP2αA) transcription factor promotes skeletal cell differentiation, but it also has a novel cell growth regulatory activity in osteoblasts. We addressed here whether Runx2 activity is functionally linked to cell cycle-related mechanisms that control normal osteoblast proliferation and differentiation. We found that the levels of Runx2 gene transcription, mRNA and protein, are each up-regulated with cessation of cell growth (i.e. G0/G1 transition) in preconfluent MC3T3 osteoblastic cells that do not yet express mature bone phenotypic gene expression. Cell growth regulation of Runx2 is also observed in primary calvarial osteoblasts and other osteoblastic cells with relatively normal cell growth characteristics, but not in osteosarcoma cells (e.g. SAOS-2 and ROS17/2.8). Runx2 levels are cell cycle-regulated in MC3T3 cells with respect to the G1/S and M/G1 transitions: expression oscillates from maximal levels during early G1 to minimal levels during early S phase and mitosis. However, in normal or immortalized (e.g. ATDC5) chondrocytic cells, Runx2 expression is suppressed during quiescence, and Runx2 levels are not regulated during G1 and S phase in ATDC5 cells. Antisense or small interfering RNA-mediated reduction of the low physiological levels of Runx2 in proliferating MC3T3 cells does not accelerate cell cycle progression. However, forced expression of Runx2 suppresses proliferation of MC3T3 preosteoblasts or C2C12 mesenchymal cells which have osteogenic potential. Forced elevation of Runx2 in synchronized MC3T3 cells causes a delay in G1. We propose that Runx2 levels and function are biologically linked to a cell growth-related G1 transition in osteoblastic cells. PMID:15781466

Intracellular and Extracellular pH and Ca Are Bound to Control Mitosis in the Early Sea Urchin Embryo via ERK and MPF Activities

PubMed Central

Ciapa, Brigitte; Philippe, Laetitia

2013-01-01

Studies aiming to predict the impact on marine life of ocean acidification and of altered salinity have shown altered development in various species including sea urchins. We have analyzed how external Na, Ca, pH and bicarbonate control the first mitotic divisions of sea urchin embryos. Intracellular free Ca (Cai) and pH (pHi) and the activities of the MAP kinase ERK and of MPF regulate mitosis in various types of cells including oocytes and early embryos. We found that intracellular acidification of fertilized eggs by Na-acetate induces a huge activation of ERK at time of mitosis. This also stops the cell cycle and leads to cell death, which can be bypassed by treatment with the MEK inhibitor U0126. Similar intracellular acidification induced in external medium containing low sodium or 5-(N-Methyl-N-isobutyl) amiloride, an inhibitor of the Na+/H+ exchanger, also stops the cell cycle and leads to cell death. In that case, an increase in Cai and in the phosphorylation of tyr-cdc2 occurs during mitosis, modifications that depend on external Ca. Our results indicate that the levels of pHi and Cai determine accurate levels of Ptyr-Cdc2 and P-ERK capable of ensuring progression through the first mitotic cycles. These intracellular parameters rely on external Ca, Na and bicarbonate, alterations of which during climate changes could act synergistically to perturb the early marine life. PMID:23785474

Intracellular and extracellular pH and Ca are bound to control mitosis in the early sea urchin embryo via ERK and MPF activities.

PubMed

Ciapa, Brigitte; Philippe, Laetitia

2013-01-01

Studies aiming to predict the impact on marine life of ocean acidification and of altered salinity have shown altered development in various species including sea urchins. We have analyzed how external Na, Ca, pH and bicarbonate control the first mitotic divisions of sea urchin embryos. Intracellular free Ca (Cai) and pH (pHi) and the activities of the MAP kinase ERK and of MPF regulate mitosis in various types of cells including oocytes and early embryos. We found that intracellular acidification of fertilized eggs by Na-acetate induces a huge activation of ERK at time of mitosis. This also stops the cell cycle and leads to cell death, which can be bypassed by treatment with the MEK inhibitor U0126. Similar intracellular acidification induced in external medium containing low sodium or 5-(N-Methyl-N-isobutyl) amiloride, an inhibitor of the Na(+)/H(+) exchanger, also stops the cell cycle and leads to cell death. In that case, an increase in Cai and in the phosphorylation of tyr-cdc2 occurs during mitosis, modifications that depend on external Ca. Our results indicate that the levels of pHi and Cai determine accurate levels of Ptyr-Cdc2 and P-ERK capable of ensuring progression through the first mitotic cycles. These intracellular parameters rely on external Ca, Na and bicarbonate, alterations of which during climate changes could act synergistically to perturb the early marine life.

A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom[C][W][OPEN

PubMed Central

Tulin, Frej; Cross, Frederick R.

2014-01-01

Research in yeast and animals has resulted in a well-supported consensus model for eukaryotic cell cycle control. The fit of this model to early diverging eukaryotes, such as the plant kingdom, remains unclear. Using the green alga Chlamydomonas reinhardtii, we developed an efficient pipeline, incorporating robotics, semiautomated image analysis, and deep sequencing, to molecularly identify >50 genes, mostly conserved in higher plants, specifically required for cell division but not cell growth. Mutated genes include the cyclin-dependent kinases CDKA (resembling yeast and animal Cdk1) and the plant-specific CDKB. The Chlamydomonas cell cycle consists of a long G1 during which cells can grow >10-fold, followed by multiple rapid cycles of DNA replication and segregation. CDKA and CDKB execute nonoverlapping functions: CDKA promotes transition between G1 and entry into the division cycle, while CDKB is essential specifically for spindle formation and nuclear division, but not for DNA replication, once CDKA-dependent initiation has occurred. The anaphase-promoting complex is required for similar steps in the Chlamydomonas cell cycle as in Opisthokonts; however, the spindle assembly checkpoint, which targets the APC in Opisthokonts, appears severely attenuated in Chlamydomonas, based on analysis of mutants affecting microtubule function. This approach allows unbiased integration of the consensus cell cycle control model with innovations specific to the plant lineage. PMID:25336509

“Till Death Do Us Part”: A Potential Irreversible Link Between Aberrant Cell Cycle Control and Neurodegeneration in the Adult Olfactory Bulb

PubMed Central

Omais, Saad; Jaafar, Carine; Ghanem, Noël

2018-01-01

Adult neurogenesis (AN) is an ongoing developmental process that generates newborn neurons in the olfactory bulb (OB) and the hippocampus (Hi) throughout life and significantly contributes to brain plasticity. Adult neural stem and progenitor cells (aNSPCs) are relatively limited in number and fate and are spatially restricted to the subventricular zone (SVZ) and the subgranular zone (SGZ). During AN, the distinct roles played by cell cycle proteins extend beyond cell cycle control and constitute key regulatory mechanisms involved in neuronal maturation and survival. Importantly, aberrant cell cycle re-entry (CCE) in post-mitotic neurons has been strongly linked to the abnormal pathophysiology in rodent models of neurodegenerative diseases with potential implications on the etiology and progression of such diseases in humans. Here, we present an overview of AN in the SVZ-OB and olfactory epithelium (OE) in mice and humans followed by a comprehensive update of the distinct roles played by cell cycle proteins including major tumors suppressor genes in various steps during neurogenesis. We also discuss accumulating evidence underlining a strong link between abnormal cell cycle control, olfactory dysfunction and neurodegeneration in the adult and aging brain. We emphasize that: (1) CCE in post-mitotic neurons due to loss of cell cycle suppression and/or age-related insults as well as DNA damage can anticipate the development of neurodegenerative lesions and protein aggregates, (2) the age-related decline in SVZ and OE neurogenesis is associated with compensatory pro-survival mechanisms in the aging OB which are interestingly similar to those detected in Alzheimer's disease and Parkinson's disease in humans, and (3) the OB represents a well suitable model to study the early manifestation of age-related defects that may eventually progress into the formation of neurodegenerative lesions and, possibly, spread to the rest of the brain. Such findings may provide a novel approach to the modeling of neurodegenerative diseases in humans from early detection to progression and treatment as well. PMID:29593485

3D chromatin conformation correlates with replication timing and is conserved in resting cells

PubMed Central

Moindrot, Benoit; Audit, Benjamin; Klous, Petra; Baker, Antoine; Thermes, Claude; de Laat, Wouter; Bouvet, Philippe; Mongelard, Fabien; Arneodo, Alain

2012-01-01

Although chromatin folding is known to be of functional importance to control the gene expression program, less is known regarding its interplay with DNA replication. Here, using Circular Chromatin Conformation Capture combined with high-throughput sequencing, we identified megabase-sized self-interacting domains in the nucleus of a human lymphoblastoid cell line, as well as in cycling and resting peripheral blood mononuclear cells (PBMC). Strikingly, the boundaries of those domains coincide with early-initiation zones in every cell types. Preferential interactions have been observed between the consecutive early-initiation zones, but also between those separated by several tens of megabases. Thus, the 3D conformation of chromatin is strongly correlated with the replication timing along the whole chromosome. We furthermore provide direct clues that, in addition to the timing value per se, the shape of the timing profile at a given locus defines its set of genomic contacts. As this timing-related scheme of chromatin organization exists in lymphoblastoid cells, resting and cycling PBMC, this indicates that it is maintained several weeks or months after the previous S-phase. Lastly, our work highlights that the major chromatin changes accompanying PBMC entry into cell cycle occur while keeping largely unchanged the long-range chromatin contacts. PMID:22879376

Identification of Novel Saccharomyces cerevisiae Proteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

PubMed Central

Jensen, Torben Heick; Neville, Megan; Rain, Jean Christophe; McCarthy, Terri; Legrain, Pierre; Rosbash, Michael

2000-01-01

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5′ RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G1-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G1 but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner. PMID:11027275

Sequence of neuron origin and neocortical laminar fate: relation to cell cycle of origin in the developing murine cerebral wall

NASA Technical Reports Server (NTRS)

Takahashi, T.; Goto, T.; Miyama, S.; Nowakowski, R. S.; Caviness, V. S. Jr

1999-01-01

Neurons destined for each region of the neocortex are known to arise approximately in an "inside-to-outside" sequence from a pseudostratified ventricular epithelium (PVE). This sequence is initiated rostrolaterally and propagates caudomedially. Moreover, independently of location in the PVE, the neuronogenetic sequence in mouse is divisible into 11 cell cycles that occur over a 6 d period. Here we use a novel "birth hour" method that identifies small cohorts of neurons born during a single 2 hr period, i.e., 10-20% of a single cell cycle, which corresponds to approximately 1.5% of the 6 d neuronogenetic period. This method shows that neurons arising with the same cycle of the 11 cycle sequence in mouse have common laminar fates even if they arise from widely separated positions on the PVE (neurons of fields 1 and 40) and therefore arise at different embryonic times. Even at this high level of temporal resolution, simultaneously arising cells occupy more than one cortical layer, and there is substantial overlap in the distributions of cells arising with successive cycles. We demonstrate additionally that the laminar representation of cells arising with a given cycle is little if at all modified over the early postnatal interval of histogenetic cell death. We infer from these findings that cell cycle is a neuronogenetic counting mechanism and that this counting mechanism is integral to subsequent processes that determine cortical laminar fate.

The Cdk4-E2f1 pathway regulates early pancreas development by targeting Pdx1+ progenitors and Ngn3+ endocrine precursors

PubMed Central

Kim, So Yoon; Rane, Sushil G.

2011-01-01

Cell division and cell differentiation are intricately regulated processes vital to organ development. Cyclin-dependent kinases (Cdks) are master regulators of the cell cycle that orchestrate the cell division and differentiation programs. Cdk1 is essential to drive cell division and is required for the first embryonic divisions, whereas Cdks 2, 4 and 6 are dispensable for organogenesis but vital for tissue-specific cell development. Here, we illustrate an important role for Cdk4 in regulating early pancreas development. Pancreatic development involves extensive morphogenesis, proliferation and differentiation of the epithelium to give rise to the distinct cell lineages of the adult pancreas. The cell cycle molecules that specify lineage commitment within the early pancreas are unknown. We show that Cdk4 and its downstream transcription factor E2f1 regulate mouse pancreas development prior to and during the secondary transition. Cdk4 deficiency reduces embryonic pancreas size owing to impaired mesenchyme development and fewer Pdx1+ pancreatic progenitor cells. Expression of activated Cdk4R24C kinase leads to increased Nkx2.2+ and Nkx6.1+ cells and a rise in the number and proliferation of Ngn3+ endocrine precursors, resulting in expansion of the β cell lineage. We show that E2f1 binds and activates the Ngn3 promoter to modulate Ngn3 expression levels in the embryonic pancreas in a Cdk4-dependent manner. These results suggest that Cdk4 promotes β cell development by directing E2f1-mediated activation of Ngn3 and increasing the pool of endocrine precursors, and identify Cdk4 as an important regulator of early pancreas development that modulates the proliferation potential of pancreatic progenitors and endocrine precursors. PMID:21490060

Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment.

PubMed

Yap, W H; Khoo, K S; Lim, S H; Yeo, C C; Lim, Y M

2012-01-15

Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells. Copyright © 2011 Elsevier GmbH. All rights reserved.

Effect of Different Carbon Sources on Relative Growth Rate, Internal Carbohydrates, and Mannitol 1-Oxidoreductase Activity in Celery Suspension Cultures.

PubMed Central

Stoop, JMH.; Pharr, D. M.

1993-01-01

Little information exists concerning the biochemical route of mannitol catabolism in higher plant cells. In this study, the role of a recently discovered mannitol 1-oxidoreductase (MDH) in mannitol catabolism was investigated. Suspension cultures of celery (Apium graveolens L. var dulce [Mill.] Pers.) were successfully grown on nutrient media with either mannitol, mannose, or sucrose as the sole carbon source. Cell cultures grown on any of the three carbon sources did not differ in relative growth rate, as measured by packed cell volume, but differed drastically in internal carbohydrate concentration. Mannitol-grown cells contained high concentrations of mannitol and extremely low concentrations of sucrose, fructose, glucose, and mannose. Sucrose-grown cells had high concentrations of sucrose early in the growth cycle and contained a substantial hexose pool. Mannose-grown cells had a high mannose concentration early in the cycle, which decreased during the growth cycle, whereas their internal sucrose concentrations remained relatively constant during the entire growth cycle. Celery suspension cultures on all three carbon substrates contained an NAD-dependent MDH. Throughout the growth cycle, MDH activity was 2- to 4-fold higher in mannitol-grown cells compared with sucrose- or mannose-grown cells, which did not contain detectable levels of mannitol, indicating that MDH functions pre-dominantly in an oxidative capacity in situ. The MDH activity observed in celery cells was 3-fold higher than the minimum amount required to account for the observed rate of mannitol utilization from the media. Cultures transferred from mannitol to mannose underwent a decrease in MDH activity over a period of days, and transfer from mannose to mannitol resulted in an increase in MDH activity. These data provide strong evidence that MDH plays an important role in mannitol utilization in celery suspension cultures. PMID:12231996

Assessment of cell death mechanisms triggered by 177Lu-anti-CD20 in lymphoma cells.

PubMed

Azorín-Vega, E; Rojas-Calderón, E; Martínez-Ventura, B; Ramos-Bernal, J; Serrano-Espinoza, L; Jiménez-Mancilla, N; Ordaz-Rosado, D; Ferro-Flores, G

2018-08-01

The aim of this research was to evaluate the cell cycle redistribution and activation of early and late apoptotic pathways in lymphoma cells after treatment with 177 Lu-anti-CD20. Experimental and computer models were used to calculate the radiation absorbed dose to cancer cell nuclei. The computer model (Monte Carlo, PENELOPE) consisted of twenty spheres representing cells with an inner sphere (cell nucleus) embedded in culture media. Radiation emissions of the radiopharmaceutical located in cell membranes and in culture media were considered for nuclei dose calculations. Flow cytometric analyses demonstrated that doses as low as 4.8Gy are enough to induce cell cycle arrest and activate late apoptotic pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multiparameter Cell Cycle Analysis.

PubMed

Jacobberger, James W; Sramkoski, R Michael; Stefan, Tammy; Woost, Philip G

2018-01-01

Cell cycle cytometry and analysis are essential tools for studying cells of model organisms and natural populations (e.g., bone marrow). Methods have not changed much for many years. The simplest and most common protocol is DNA content analysis, which is extensively published and reviewed. The next most common protocol, 5-bromo-2-deoxyuridine S phase labeling detected by specific antibodies, is also well published and reviewed. More recently, S phase labeling using 5'-ethynyl-2'-deoxyuridine incorporation and a chemical reaction to label substituted DNA has been established as a basic, reliable protocol. Multiple antibody labeling to detect epitopes on cell cycle regulated proteins, which is what this chapter is about, is the most complex of these cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. However, because this knowledge is relatively well presented methodologically in many papers and reviews, this chapter will present a minimal Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature. Most of the presented work involves how to segment the data to produce a complete, progressive, and compartmentalized cell cycle analysis from early G1 to late mitosis (telophase). A more recent development, using fluorescent proteins fused with proteins or peptides that are degraded by ubiquitination during specific periods of the cell cycle, termed "Fucci" (fluorescent, ubiquitination-based cell cycle indicators) provide an analysis similar in concept to multiple antibody labeling, except in this case cells can be analyzed while living and transgenic organisms can be created to perform cell cycle analysis ex or in vivo (Sakaue-Sawano et al., Cell 132:487-498, 2007). This technology will not be discussed.

Characterization of resistance to rhabdovirus and retrovirus infection in a human myeloid cell line.

PubMed

Boso, Guney; Somia, Nikunj V

2015-01-01

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.

Global increase in replication fork speed during a p57KIP2-regulated erythroid cell fate switch

PubMed Central

Hwang, Yung; Futran, Melinda; Hidalgo, Daniel; Pop, Ramona; Iyer, Divya Ramalingam; Scully, Ralph; Rhind, Nicholas; Socolovsky, Merav

2017-01-01

Cell cycle regulators are increasingly implicated in cell fate decisions, such as the acquisition or loss of pluripotency and self-renewal potential. The cell cycle mechanisms that regulate these cell fate decisions are largely unknown. We studied an S phase–dependent cell fate switch, in which murine early erythroid progenitors transition in vivo from a self-renewal state into a phase of active erythroid gene transcription and concurrent maturational cell divisions. We found that progenitors are dependent on p57KIP2-mediated slowing of replication forks for self-renewal, a novel function for cyclin-dependent kinase inhibitors. The switch to differentiation entails rapid down-regulation of p57KIP2 with a consequent global increase in replication fork speed and an abruptly shorter S phase. Our work suggests that cell cycles with specialized global DNA replication dynamics are integral to the maintenance of specific cell states and to cell fate decisions. PMID:28560351

Two cell cycle blocks caused by iron chelation of neuroblastoma cells: separating cell cycle events associated with each block

PubMed Central

Siriwardana, Gamini; Seligman, Paul A.

2013-01-01

Abstract Studies have presented evidence that besides the well described S phase block, treatment of cancer cell lines with the iron chelator deferrioxamine (DFO) also results in an earlier block in G1 phase. In this article, measurements of cell cycle regulatory proteins define this block at a very specific point in G1. DFO treatment results in markedly decreased cyclin A protein levels. Cyclin E levels that accumulate in early to mid‐G1 are increased in cells treated with DFO as compared to the resting cells. The DFO S phase block is shown after cells are arrested at G1/S by (aphidicolin) then released into DFO. The same S phase block occurs with DFO treatment of a neuroblastoma cell line relatively resistant to the G1 DFO block. These experiments clearly differentiate the S phase DFO block from the earlier block pinpointed to a point in mid‐G1, before G1/S when cyclin E protein increases but before increased cyclin A synthesis. Apoptosis was observed in cells inhibited by DFO at both cell cycle arrest points. PMID:24744856

Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubin, D.B.; Drab, E.A.; Ward, W.F.

1988-11-01

Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and anmore » increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous (3H)thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro.« less

The leaving or Q fraction of the murine cerebral proliferative epithelium: a general model of neocortical neuronogenesis

NASA Technical Reports Server (NTRS)

Takahashi, T.; Nowakowski, R. S.; Caviness, V. S. Jr

1996-01-01

Neurons of neocortical layers II-VI in the dorsomedial cortex of the mouse arise in the pseudostratified ventricular epithelium (PVE) through 11 cell cycles over the six embryonic days 11-17 (E11-E17). The present experiments measure the proportion of daughter cells that leave the cycle (quiescent or Q fraction or Q) during a single cell cycle and the complementary proportion that continues to proliferate (proliferative or P fraction or P; P = 1 - Q). Q and P for the PVE become 0.5 in the course of the eighth cycle, occurring on E14, and Q rises to approximately 0.8 (and P falls to approximately 0.2) in the course of the 10th cycle occurring on E16. This indicates that early in neuronogenesis, neurons are produced relatively slowly and the PVE expands rapidly but that the reverse happens in the final phase of neuronogenesis. The present analysis completes a cycle of analyses that have determined the four fundamental parameters of cell proliferation: growth fraction, lengths of cell cycle, and phases Q and P. These parameters are the basis of a coherent neuronogenetic model that characterizes patterns of growth of the PVE and mathematically relates the size of the initial proliferative population to the neuronal population of the adult neocortex.

Neisseria meningitidis causes cell cycle arrest of human brain microvascular endothelial cells at S phase via p21 and cyclin G2.

PubMed

Oosthuysen, Wilhelm F; Mueller, Tobias; Dittrich, Marcus T; Schubert-Unkmeir, Alexandra

2016-01-01

Microbial pathogens have developed several mechanisms to modulate and interfere with host cell cycle progression. In this study, we analysed the effect of the human pathogen Neisseria meningitidis on cell cycle in a brain endothelial cell line as well as in primary brain endothelial cells. We found that N.  Meningitidis causes an accumulation of cells in the S phase early at 3 and at 24 h post-infection that was paralleled by a decrease of cells in G2/M phase. Importantly, the outer membrane proteins of the colony opacity-associated (Opa) protein family as well as the Opc protein proved to trigger the accumulation of cells in the S phase. A focused cell cycle reverse transcription quantitative polymerase chain reaction-based array and integrated network analysis revealed changes in the abundance of several cell cycle regulatory mRNAs, including the cell cycle inhibitors p21(WAF1/CIP1) and cyclin G2. These alterations were reflected in changes in protein expression levels and/or relocalization in N. meningitidis-infected cells. Moreover, an increase in p21(WAF1/CIP1) expression was found to be p53 independent. Genetic ablation of p21(WAF1/CIP1) and cyclin G2 abrogated N. meningitidis-induced S phase accumulation. Finally, by measuring the levels of the biomarker 8-hydroxydeoxyguanosine and phosphorylation of the histone variant H2AX, we provide evidence that N. meningitidis induces oxidative DNA damage in infected cells. © 2015 John Wiley & Sons Ltd.

Running rescues defective adult neurogenesis by shortening the length of the cell cycle of neural stem and progenitor cells.

PubMed

Farioli-Vecchioli, Stefano; Mattera, Andrea; Micheli, Laura; Ceccarelli, Manuela; Leonardi, Luca; Saraulli, Daniele; Costanzi, Marco; Cestari, Vincenzo; Rouault, Jean-Pierre; Tirone, Felice

2014-07-01

Physical exercise increases the generation of new neurons in adult neurogenesis. However, only few studies have investigated the beneficial effects of physical exercise in paradigms of impaired neurogenesis. Here, we demonstrate that running fully reverses the deficient adult neurogenesis within the hippocampus and subventricular zone of the lateral ventricle, observed in mice lacking the antiproliferative gene Btg1. We also evaluated for the first time how running influences the cell cycle kinetics of stem and precursor subpopulations of wild-type and Btg1-null mice, using a new method to determine the cell cycle length. Our data show that in wild-type mice running leads to a cell cycle shortening only of NeuroD1-positive progenitor cells. In contrast, in Btg1-null mice, physical exercise fully reactivates the defective hippocampal neurogenesis, by shortening the S-phase length and the overall cell cycle duration of both neural stem (glial fibrillary acidic protein(+) and Sox2(+)) and progenitor (NeuroD1(+)) cells. These events are sufficient and necessary to reactivate the hyperproliferation observed in Btg1-null early-postnatal mice and to expand the pool of adult neural stem and progenitor cells. Such a sustained increase of cell proliferation in Btg1-null mice after running provides a long-lasting increment of proliferation, differentiation, and production of newborn neurons, which rescues the impaired pattern separation previously identified in Btg1-null mice. This study shows that running positively affects the cell cycle kinetics of specific subpopulations of newly generated neurons and suggests that the plasticity of neural stem cells without cell cycle inhibitory control is reactivated by running, with implications for the long-term modulation of neurogenesis. © 2014 AlphaMed Press.

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function

PubMed Central

Zhang, Lixia; Kendrick, Christina; Jülich, Dörthe; Holley, Scott A.

2010-01-01

Summary Cell division, differentiation and morphogenesis are coordinated during embryonic development and frequently in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and utilize the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is substantially driven by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1−/−. The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for normal segmental arrangement of epithelial borders and internal mesenchymal cells. PMID:18480162

Cell cycle control, checkpoint mechanisms, and genotoxic stress.

PubMed Central

Shackelford, R E; Kaufmann, W K; Paules, R S

1999-01-01

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling. Images Figure 3 PMID:10229703

Introducing Undergraduate Students to Real-Time PCR

ERIC Educational Resources Information Center

Hancock, Dale; Funnell, Alister; Jack, Briony; Johnston, Jill

2010-01-01

An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis,…

Global gene expression analysis of apple fruit development from the floral bud to ripe fruit

PubMed Central

Janssen, Bart J; Thodey, Kate; Schaffer, Robert J; Alba, Rob; Balakrishnan, Lena; Bishop, Rebecca; Bowen, Judith H; Crowhurst, Ross N; Gleave, Andrew P; Ledger, Susan; McArtney, Steve; Pichler, Franz B; Snowden, Kimberley C; Ward, Shayna

2008-01-01

Background Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45–55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple. Results Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes. Conclusion Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development. PMID:18279528

Global gene expression analysis of apple fruit development from the floral bud to ripe fruit.

PubMed

Janssen, Bart J; Thodey, Kate; Schaffer, Robert J; Alba, Rob; Balakrishnan, Lena; Bishop, Rebecca; Bowen, Judith H; Crowhurst, Ross N; Gleave, Andrew P; Ledger, Susan; McArtney, Steve; Pichler, Franz B; Snowden, Kimberley C; Ward, Shayna

2008-02-17

Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45-55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple. Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes. Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development.

Changes in cell-cycle kinetics responsible for limiting somatic growth in mice

PubMed Central

Chang, Maria; Parker, Elizabeth A.; Muller, Tessa J. M.; Haenen, Caroline; Mistry, Maanasi; Finkielstain, Gabriela P.; Murphy-Ryan, Maureen; Barnes, Kevin M.; Sundaram, Rajeshwari; Baron, Jeffrey

2009-01-01

In mammals, the rate of somatic growth is rapid in early postnatal life but then slows with age, approaching zero as the animal approaches adult body size. To investigate the underlying changes in cell-cycle kinetics, [methyl-3H]thymidine and 5’-bromo-2’deoxyuridine were used to double-label proliferating cells in 1-, 2-, and 3-week-old mice for four weeks. Proliferation of renal tubular epithelial cells and hepatocytes decreased with age. The average cell-cycle time did not increase in liver and increased only 1.7 fold in kidney. The fraction of cells in S-phase that will divide again declined approximately 10 fold with age. Concurrently, average cell area increased approximately 2 fold. The findings suggest that somatic growth deceleration primarily results not from an increase in cell-cycle time but from a decrease in growth fraction (fraction of cells that continue to proliferate). During the deceleration phase, cells appear to reach a proliferative limit and undergo their final cell divisions, staggered over time. Concomitantly, cells enlarge to a greater volume, perhaps because they are relieved of the size constraint imposed by cell division. In conclusion, a decline in growth fraction with age causes somatic growth deceleration and thus sets a fundamental limit on adult body size. PMID:18535488

CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells

PubMed Central

Valli, Emanuele; Trazzi, Stefania; Fuchs, Claudia; Erriquez, Daniela; Bartesaghi, Renata; Perini, Giovanni; Ciani, Elisabetta

2012-01-01

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G0/G1 phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5. PMID:22921766

The role of model organisms in the history of mitosis research.

PubMed

Yanagida, Mitsuhiro

2014-09-02

Mitosis is a cell-cycle stage during which condensed chromosomes migrate to the middle of the cell and segregate into two daughter nuclei before cytokinesis (cell division) with the aid of a dynamic mitotic spindle. The history of mitosis research is quite long, commencing well before the discovery of DNA as the repository of genetic information. However, great and rapid progress has been made since the introduction of recombinant DNA technology and discovery of universal cell-cycle control. A large number of conserved eukaryotic genes required for the progression from early to late mitotic stages have been discovered, confirming that DNA replication and mitosis are the two main events in the cell-division cycle. In this article, a historical overview of mitosis is given, emphasizing the importance of diverse model organisms that have been used to solve fundamental questions about mitosis. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

The Role of Model Organisms in the History of Mitosis Research

PubMed Central

Yanagida, Mitsuhiro

2014-01-01

Mitosis is a cell-cycle stage during which condensed chromosomes migrate to the middle of the cell and segregate into two daughter nuclei before cytokinesis (cell division) with the aid of a dynamic mitotic spindle. The history of mitosis research is quite long, commencing well before the discovery of DNA as the repository of genetic information. However, great and rapid progress has been made since the introduction of recombinant DNA technology and discovery of universal cell-cycle control. A large number of conserved eukaryotic genes required for the progression from early to late mitotic stages have been discovered, confirming that DNA replication and mitosis are the two main events in the cell-division cycle. In this article, a historical overview of mitosis is given, emphasizing the importance of diverse model organisms that have been used to solve fundamental questions about mitosis. PMID:25183827

Forty-five years of cell-cycle genetics

PubMed Central

Reid, Brian J.; Culotti, Joseph G.; Nash, Robert S.; Pringle, John R.

2015-01-01

In the early 1970s, studies in Leland Hartwell’s laboratory at the University of Washington launched the genetic analysis of the eukaryotic cell cycle and set the path that has led to our modern understanding of this centrally important process. This 45th-anniversary Retrospective reviews the steps by which the project took shape, the atmosphere in which this happened, and the possible morals for modern times. It also provides an up-to-date look at the 35 original CDC genes and their human homologues. PMID:26628751

Xenopus egg cytoplasm with intact actin.

PubMed

Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

2014-01-01

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

Midkine-A functions upstream of Id2a to regulate cell cycle kinetics in the developing vertebrate retina

PubMed Central

2012-01-01

Background Midkine is a small heparin binding growth factor expressed in numerous tissues during development. The unique midkine gene in mammals has two paralogs in zebrafish: midkine-a (mdka) and midkine-b (mdkb). In the zebrafish retina, during both larval development and adult photoreceptor regeneration, mdka is expressed in retinal stem and progenitor cells and functions as a molecular component of the retina’s stem cell niche. In this study, loss-of-function and conditional overexpression were used to investigate the function of Mdka in the retina of the embryonic zebrafish. Results The results show that during early retinal development Mdka functions to regulate cell cycle kinetics. Following targeted knockdown of Mdka synthesis, retinal progenitors cycle more slowly, and this results in microphthalmia, a diminished rate of cell cycle exit and a temporal delay of cell cycle exit and neuronal differentiation. In contrast, Mdka overexpression results in acceleration of the cell cycle and retinal overgrowth. Mdka gain-of-function, however, does not temporally advance cell cycle exit. Experiments to identify a potential Mdka signaling pathway show that Mdka functions upstream of the HLH regulatory protein, Id2a. Gene expression analysis shows Mdka regulates id2a expression, and co-injection of Mdka morpholinos and id2a mRNA rescues the Mdka loss-of-function phenotype. Conclusions These data show that in zebrafish, Mdka resides in a shared Id2a pathway to regulate cell cycle kinetics in retinal progenitors. This is the first study to demonstrate the function of Midkine during retinal development and adds Midkine to the list of growth factors that transcriptionally regulate Id proteins. PMID:23111152

Midkine-A functions upstream of Id2a to regulate cell cycle kinetics in the developing vertebrate retina.

PubMed

Luo, Jing; Uribe, Rosa A; Hayton, Sarah; Calinescu, Anda-Alexandra; Gross, Jeffrey M; Hitchcock, Peter F

2012-10-30

Midkine is a small heparin binding growth factor expressed in numerous tissues during development. The unique midkine gene in mammals has two paralogs in zebrafish: midkine-a (mdka) and midkine-b (mdkb). In the zebrafish retina, during both larval development and adult photoreceptor regeneration, mdka is expressed in retinal stem and progenitor cells and functions as a molecular component of the retina's stem cell niche. In this study, loss-of-function and conditional overexpression were used to investigate the function of Mdka in the retina of the embryonic zebrafish. The results show that during early retinal development Mdka functions to regulate cell cycle kinetics. Following targeted knockdown of Mdka synthesis, retinal progenitors cycle more slowly, and this results in microphthalmia, a diminished rate of cell cycle exit and a temporal delay of cell cycle exit and neuronal differentiation. In contrast, Mdka overexpression results in acceleration of the cell cycle and retinal overgrowth. Mdka gain-of-function, however, does not temporally advance cell cycle exit. Experiments to identify a potential Mdka signaling pathway show that Mdka functions upstream of the HLH regulatory protein, Id2a. Gene expression analysis shows Mdka regulates id2a expression, and co-injection of Mdka morpholinos and id2a mRNA rescues the Mdka loss-of-function phenotype. These data show that in zebrafish, Mdka resides in a shared Id2a pathway to regulate cell cycle kinetics in retinal progenitors. This is the first study to demonstrate the function of Midkine during retinal development and adds Midkine to the list of growth factors that transcriptionally regulate Id proteins.

Therapeutic Cell-Cycle-Decoy Efficacy of a Telomerase-Dependent Adenovirus in an Orthotopic Model of Chemotherapy-Resistant Human Stomach Carcinomatosis Peritonitis Visualized With FUCCI Imaging.

PubMed

Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M

2017-11-01

We have established an orthotopic nude-mouse model of gastric cancer carcinomatosis peritonitis, a recalcitrant disease in human patients. Human MKN45 poorly-differentiated human gastric cancer cells developed carcinomatosis peritonitis upon orthotopic transplantation in nude mice. The MKN45 cells expressed the fluorescent ubiquitination-based cell cycle indicator (FUCCI) that color codes the phases of the cell cycle. The intra-peritoneal tumors and ascites contained mostly quiescent G 1 /G o cancer cells visualized as red by FUCCI imaging. Cisplatinum (CDDP) treatment did not reduce bloody ascites, and larger tumors formed in the peritoneal cavity after CDDP treatment in an early-stage carcinomatosis peritonitis orthotopic mouse model. Paclitaxel-treated mice had reduced ascites, but also had large tumor masses in the peritonium after treatment with cancer cells mostly in G 0 /G 1 , visualized by FUCCI red. In contrast, OBP-301 telomerase-dependent adenovirus-treated mice had no ascites and only small tumor nodules consisting of cancer cells mostly in S/G 2 phases in the early-stage carcinomatosis peritonitis model, visualized by FUCCI green. Furthermore, OBP-301 significantly reduced the size of tumors (P < 0.01) and ascites even in a late-stage carcinomatosis peritonitis model. These results suggest that quiescent peritoneally-disseminated gastric cancer cells are resistant to conventional chemotherapy, but OBP-301 significantly reduced the weight of the tumors and increased survival, suggesting clinical potential. J. Cell. Biochem. 118: 3635-3642, 2017. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Synchrony of plant cellular circadian clocks with heterogeneous properties under light/dark cycles.

PubMed

Okada, Masaaki; Muranaka, Tomoaki; Ito, Shogo; Oyama, Tokitaka

2017-03-22

Individual cells in a plant can work independently as circadian clocks, and their properties are the basis of various circadian phenomena. The behaviour of individual cellular clocks in Lemna gibba was orderly under 24-h light/dark cycles despite their heterogeneous free-running periods (FRPs). Here, we reveal the entrainment habits of heterogeneous cellular clocks using non-24-h light/dark cycles (T-cycles). The cellular rhythms of AtCCA1::LUC under T = 16 h cycles showed heterogeneous entrainment that was associated with their heterogeneous FRPs. Under T = 12 h cycles, most cells showed rhythms having ~24-h periods. This suggested that the lower limit of entrainment to the light/dark cycles of heterogeneous cellular circadian clocks is set to a period longer than 12 h, which enables them to be synchronous under ~24-h daily cycles without being perturbed by short light/dark cycles. The entrainment habits of individual cellular clocks are likely to be the basis of the circadian behaviour of plant under the natural day-night cycle with noisy environmental fluctuations. We further suggest that modifications of EARLY FLOWERING3 (ELF3) in individual cells deviate the entrainability to shorter T-cycles possibly by altering both the FRPs and light responsiveness.

The invariant cleavage pattern displayed by ascidian embryos depends on spindle positioning along the cell's longest axis in the apical plane and relies on asynchronous cell divisions

PubMed Central

Dumollard, Rémi; Minc, Nicolas; Salez, Gregory; Aicha, Sameh Ben; Bekkouche, Faisal; Hebras, Céline; Besnardeau, Lydia; McDougall, Alex

2017-01-01

The ascidian embryo is an ideal system to investigate how cell position is determined during embryogenesis. Using 3D timelapse imaging and computational methods we analyzed the planar cell divisions in ascidian early embryos and found that spindles in every cell tend to align at metaphase in the long length of the apical surface except in cells undergoing unequal cleavage. Furthermore, the invariant and conserved cleavage pattern of ascidian embryos was found to consist in alternate planar cell divisions between ectoderm and endomesoderm. In order to test the importance of alternate cell divisions we manipulated zygotic transcription induced by β-catenin or downregulated wee1 activity, both of which abolish this cell cycle asynchrony. Crucially, abolishing cell cycle asynchrony consistently disrupted the spindle orienting mechanism underpinning the invariant cleavage pattern. Our results demonstrate how an evolutionary conserved cell cycle asynchrony maintains the invariant cleavage pattern driving morphogenesis of the ascidian blastula. DOI: http://dx.doi.org/10.7554/eLife.19290.001 PMID:28121291

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

PubMed Central

Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

2015-01-01

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues. PMID:26067441

Phosphorylation of mitogen-activated protein kinase (MAPK) is required for cytokinesis and progression of cell cycle in tobacco BY-2 cells.

PubMed

Ma, Zhaowu; Yu, Guanghui

2010-02-15

The role of mitogen-activated protein kinase (MAPK) in plant cytokinesis remains largely uncharacterized. To elucidate its role, tobacco Bright Yellow-2 (BY-2) cells have been synchronized using a two-step procedure, and the different phases of the cell cycle identified by Histone 4 gene expression and the mitotic index. MAPK expression was analyzed by semi-quantitative (SQ) RT-PCR and protein gel blot analysis for phosphorylated MAPK during cell cycle progression. The SQ RT-PCR analysis indicated that MAPK expression is lower in mitosis than in interphase (G1, G2 and S). However, the amount of phosphorylated MAPK remained stable throughout the cell cycle, indicating that MAPK activity is predominantly regulated at the post-translational level and that phosphorylation of MAPK plays an important role in mitosis. Application of the specific MAPK phosphorylation inhibitor U0126 revealed that while U0126 treatment decreases the phosphorylation of MAPK and the progression from telophase to early cytokinesis is significantly inhibited. The formation of the phragmoplast is also negatively affected at this stage. These results demonstrate that MAPK phosphorylation is involved in the formation of the cell plate within the phragmoplast during cytokinesis and that MAPK predominantly functions during the cytokinesis stage of the cell cycle in tobacco BY-2 cells. Copyright 2009 Elsevier GmbH. All rights reserved.

Phosphatidylcholine catabolism in the MCF-7 cell cycle.

PubMed

Lin, Weiyang; Arthur, Gilbert

2006-10-01

The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.

Chk1 and Wee1 kinases coordinate DNA replication, chromosome condensation, and anaphase entry

PubMed Central

Fasulo, Barbara; Koyama, Carol; Yu, Kristina R.; Homola, Ellen M.; Hsieh, Tao S.; Campbell, Shelagh D.; Sullivan, William

2012-01-01

Defects in DNA replication and chromosome condensation are common phenotypes in cancer cells. A link between replication and condensation has been established, but little is known about the role of checkpoints in monitoring chromosome condensation. We investigate this function by live analysis, using the rapid division cycles in the early Drosophila embryo. We find that S-phase and topoisomerase inhibitors delay both the initiation and the rate of chromosome condensation. These cell cycle delays are mediated by the cell cycle kinases chk1 and wee1. Inhibitors that cause severe defects in chromosome condensation and congression on the metaphase plate result in delayed anaphase entry. These delays are mediated by wee1 and are not the result of spindle assembly checkpoint activation. In addition, we provide the first detailed live analysis of the direct effect of widely used anticancer agents (aclarubicin, ICRF-193, VM26, doxorubicin, camptothecin, aphidicolin, hydroxyurea, cisplatin, mechlorethamine and x-rays) on key nuclear and cytoplasmic cell cycle events. PMID:22262459

Differential response of cell-cycle and cell-expansion regulators to heat stress in apple (Malus domestica) fruitlets.

PubMed

Flaishman, Moshe A; Peles, Yuval; Dahan, Yardena; Milo-Cochavi, Shira; Frieman, Aviad; Naor, Amos

2015-04-01

Temperature is one of the most significant factors affecting physiological and biochemical aspects of fruit development. Current and progressing global warming is expected to change climate in the traditional deciduous fruit tree cultivation regions. In this study, 'Golden Delicious' trees, grown in a controlled environment or commercial orchard, were exposed to different periods of heat treatment. Early fruitlet development was documented by evaluating cell number, cell size and fruit diameter for 5-70 days after full bloom. Normal activities of molecular developmental and growth processes in apple fruitlets were disrupted under daytime air temperatures of 29°C and higher as a result of significant temporary declines in cell-production and cell-expansion rates, respectively. Expression screening of selected cell cycle and cell expansion genes revealed the influence of high temperature on genetic regulation of apple fruitlet development. Several core cell-cycle and cell-expansion genes were differentially expressed under high temperatures. While expression levels of B-type cyclin-dependent kinases and A- and B-type cyclins declined moderately in response to elevated temperatures, expression of several cell-cycle inhibitors, such as Mdwee1, Mdrbr and Mdkrps was sharply enhanced as the temperature rose, blocking the cell-cycle cascade at the G1/S and G2/M transition points. Moreover, expression of several expansin genes was associated with high temperatures, making them potentially useful as molecular platforms to enhance cell-expansion processes under high-temperature regimes. Understanding the molecular mechanisms of heat tolerance associated with genes controlling cell cycle and cell expansion may lead to the development of novel strategies for improving apple fruit productivity under global warming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Blockade of lipid accumulation by silibinin in adipocytes and zebrafish.

PubMed

Suh, Hyung Joo; Cho, So Young; Kim, Eun Young; Choi, Hyeon-Son

2015-02-05

Silibinin is a compound present mainly in milk thistle. In this study, we investigated the mechanism by which silibinin suppresses adipogenesis of 3T3-L1 cells, and evaluated the anti-adipogenic effect of silibinin in zebrafish. Silibinin reduced lipid accumulation by downregulating adipogenic factors, such as, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer binding protein α (C/EBPα), and fatty acid-binding protein 4 (FABP4). The reduction of these adipogenic protein levels was associated with the regulation of early adipogenic factors, such as, C/EBPβ and Krüppel-like factor 2 (KLF2), and was reflected in downregulation of lipid synthetic enzymes. Silibinin arrested cells in the G0/G1 phase of the cell cycle, accompanied by downregulation of cyclins and upregulation of p27, a cell cycle inhibitor. These results correlated with the finding of deactivation of extracellular signal-regulated kinase (ERK) and AKT, a serine/threonine-specific kinase. In addition, silibinin activated AMP-activated protein kinase α (AMPKα) to inhibit fatty acid synthesis. As observed in 3T3-L1 cells, silibinin inhibited lipid accumulation in zebrafish with the reduction of adipogenic factors and triglyceride levels. Our data revealed that silibinin inhibited lipid accumulation in 3T3-L1 cells and zebrafish, and this inhibitory effect was associated with abrogation of early adipogenesis via regulation of cell cycle and AMPKα signaling. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Characterization of the replication cycle of the Lymantria dispar nuclear polyhedrosis virus

Treesearch

Christopher I. Riegel; James M. Slavicek

1997-01-01

The life cycle of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) was characterized through analysis of budded virus (BV) release, the temporal formation of polyhedra, the temporal transcription pattern of representative early, late, and hyper-expressed late genes, and the onset of DNA replication in the Ld652Y cell line. Transcripts from...

Protein farnesyltransferase in plants: molecular characterization and involvement in cell cycle control.

PubMed Central

Qian, D; Zhou, D; Ju, R; Cramer, C L; Yang, Z

1996-01-01

Farnesylation is required for membrane targeting, protein-protein interactions, and the biological activity of key regulatory proteins, such as Ras small GTPases and protein kinases in a wide range of eukaryotes. In this report, we describe the molecular identification of a plant protein farnesyltransferase (FTase) and evidence for its role in the control of the cell cycle in plants. A pea gene encoding a homolog of the FTase beta subunit was previously cloned using a polymerase chain reaction-based strategy. A similar approach was used to clone a pea gene encoding a homolog of the FTase alpha subunit. The biochemical function of the pea FTase homologs was demonstrated by the reconstitution of FTase enzyme activity using FTase fusion proteins coexpressed in Escherichia coll. RNA gel blot analyses showed that levels of FTase mRNAs are generally higher in tissues, such as those of nodules, that are active in cell division. The relationship of FTase to cell division was further analyzed during the growth of suspension-cultured tobacco BY-2 cells. A biphasic fluctuation of FTase enzyme activity preceded corresponding changes in mitotic activity at the early log phase of cell growth. Moreover, manumycin, a specific inhibitor of FTase, was effective in inhibiting mitosis and growth in these cells. Using synchronized BY-2 cells, manumycin completely blocked mitosis when added at the early S phase but not when added at the G2 phase. These data suggest that FTase is required for the plant cell cycle, perhaps by modulating the progression through the S phase and the transition from G1 to the S phase. PMID:8989889

Prolonged early G(1) arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle-coupled loss of IRF4.

PubMed

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C; Staudt, Louis M; Niesvizky, Ruben; Moore, Malcolm A S; Chen-Kiang, Selina

2012-08-02

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.

Computation Molecular Kinetics Model of HZE Induced Cell Cycle Arrest

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Ren, Lei

2004-01-01

Cell culture models play an important role in understanding the biological effectiveness of space radiation. High energy and charge (HZE) ions produce prolonged cell cycle arrests at the G1/S and G2/M transition points in the cell cycle. A detailed description of these phenomena is needed to integrate knowledge of the expression of DNA damage in surviving cells, including the determination of relative effectiveness factors between different types of radiation that produce differential types of DNA damage and arrest durations. We have developed a hierarchical kinetics model that tracks the distribution of cells in various cell phase compartments (early G1, late G1, S, G2, and M), however with transition rates that are controlled by rate-limiting steps in the kinetics of cyclin-cdk's interactions with their families of transcription factors and inhibitor molecules. The coupling of damaged DNA molecules to the downstream cyclin-cdk inhibitors is achieved through a description of the DNA-PK and ATM signaling pathways. For HZE irradiations we describe preliminary results, which introduce simulation of the stochastic nature of the number of direct particle traversals per cell in the modulation of cyclin-cdk and cell cycle population kinetics. Comparison of the model to data for fibroblast cells irradiated photons or HZE ions are described.

Cellular distribution of cell cycle-related molecules in the renal tubules of rats treated with renal carcinogens for 28 days: relationship between cell cycle aberration and carcinogenesis.

PubMed

Taniai, Eriko; Hayashi, Hitomi; Yafune, Atsunori; Watanabe, Maiko; Akane, Hirotoshi; Suzuki, Kazuhiko; Mitsumori, Kunitoshi; Shibutani, Makoto

2012-09-01

Some renal carcinogens can induce karyomegaly, which reflects aberrant cell division in the renal tubules, from the early stages of exposure. To clarify the cell cycle-related changes during the early stages of renal carcinogenesis, we performed immunohistochemical analysis of tubular cells in male F344 rats treated with carcinogenic doses of representative renal carcinogens for 28 days. For this purpose, the karyomegaly-inducing carcinogens ochratoxin A (OTA), ferric nitrilotriacetic acid, and monuron, and the non-karyomegaly-inducing carcinogens tris(2-chloroethyl) phosphate and potassium bromate were examined. For comparison, a karyomegaly-inducing non-carcinogen, p-nitrobenzoic acid, and a non-carcinogenic non-karyomegaly-inducing renal toxicant, acetaminophen, were also examined. The outer stripe of the outer medulla (OSOM) and the cortex + OSOM were subjected to morphometric analysis of immunoreactive proximal tubular cells. Renal carcinogens, irrespective of their karyomegaly-inducing potential, increased proximal tubular cell proliferation accompanied by an increase in topoisomerase IIα-immunoreactive cells, suggesting a reflection of cell proliferation. Karyomegaly-inducing carcinogens increased nuclear Cdc2-, γH2AX-, and phosphorylated Chk2-immunoreactive cells in both areas, the former two acting in response to DNA damage and the latter one suggestive of sustained G₂. OTA, an OSOM-targeting carcinogen, could easily be distinguished from untreated controls and non-carcinogens by evaluation of molecules responding to DNA damage and G₂/M transition in the OSOM. Thus, all renal carcinogens examined facilitated proximal tubular proliferation by repeated short-term treatment. Among these, karyomegaly-inducing carcinogens may cause DNA damage and G₂ arrest in the target tubular cells.

Sulforaphane inhibits mitotic clonal expansion during adipogenesis through cell cycle arrest.

PubMed

Choi, Kyeong-Mi; Lee, Youn-Sun; Sin, Dong-Mi; Lee, Seunghyun; Lee, Mi Kyeong; Lee, Yong-Moon; Hong, Jin-Tae; Yun, Yeo-Pyo; Yoo, Hwan-Soo

2012-07-01

Obesity is a risk factor for numerous metabolic disorders such as type 2 diabetes, hypertension, and coronary heart disease. Adipocyte differentiation is triggered by adipocyte hyperplasia, which leads to obesity. In this study, the inhibitory effect of sulforaphane, an isothiocyanate, on adipogenesis in 3T3-L1 cells was investigated. Sulforaphane decreased the accumulation of lipid droplets stained with Oil Red O and inhibited the elevation of triglycerides in the adipocytes (half-maximal inhibitory concentration = 7.3 µmol/l). The expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), major transcription factors for adipocyte differentiation, was significantly reduced by sulforaphane. The major effects of sulforaphane on the inhibition of adipocyte differentiation occurred during the early stage of adipogenesis. Thus, the expression of C/EBPβ, an early-stage biomarker of adipogenesis, decreased in a concentration-dependent manner when the adipocytes were exposed to sulforaphane (0, 5, 10, and 20 µmol/l). The proliferation of adipocytes treated with 20 µmol/l sulforaphane for 24 and 48 h was also suppressed. These results indicate that sulforaphane may specifically affect mitotic clonal expansion to inhibit adipocyte differentiation. Sulforaphane arrested the cell cycle at the G(0)/G(1) phase, increased p27 expression, and decreased retinoblastoma (Rb) phosphorylation. Additionally, sulforaphane modestly decreased the phosphorylation of ERK1/2 and Akt. Our results indicate that the inhibition of early-stage adipocyte differentiation by sulforaphane may be associated with cell cycle arrest at the G(0)/G(1) phase through upregulation of p27 expression.

Ultrastructural changes of goat corpus luteum during the estrous cycle.

PubMed

Jiang, Yi-Fan; Hsu, Meng-Chieh; Cheng, Chiung-Hsiang; Tsui, Kuan-Hao; Chiu, Chih-Hsien

2016-07-01

The present study was designed to study the ultrastructure of goat corpora lutea (CL, n=10) and structural changes as related to steroidogenic functions during the estrous cycle. The reproduction status of goats was estimated by analyzing serum progesterone concentrations. The CL at various stages was surgically collected. To characterize ultrastructural features associated with steroidogenesis, tissue and cellular structures were studied. Blood supplies were examined based on features of the endothelial cells and capillary structures in the CL. Activated endothelial cells and developing vessels were observed in the early stage, whereas mature endothelial cells, accumulating extracellular matrix fibers, and stabilized vessels were observed in the middle and late stages of assessment. In the late stage of assessment, shrunken goat luteal cells scattered around the capillaries were detected and formed circular regression areas. Features of autophagy and luteal cell apoptosis were noted. In large luteal cells, steroidogenic organelles were present, including microvillar channels, endoplasmic reticulum, and mitochondria. Conformational changes in the endoplasmic reticulum and increased mitochondria with tubular cristae were observed in the early-middle CL transitions. In contrast, mitochondria swelled and the cristae transformed to the lamellar type in the late stage, suggesting that organelle plasticity could contribute to steroidogenesis in goat CL. In conclusion, results suggest angiogenesis occurs in early developing CL and programmed cell death occurred in the late stage of CL assessment in the present study. Structures and quantiles of steroidogenic organelles are correlated with the steroidogenic functions in goats. Copyright © 2016 Elsevier B.V. All rights reserved.

Neurogenic transdifferentiation of human adipose-derived stem cells? A critical protocol reevaluation with special emphasis on cell proliferation and cell cycle alterations.

PubMed

Kompisch, Kai Michael; Lange, Claudia; Steinemann, Doris; Skawran, Britta; Schlegelberger, Brigitte; Müller, Reinhard; Schumacher, Udo

2010-11-01

Adipose-derived stem cells (ASCs) are reported to display multilineage differentiation potential, including neuroectodermal pathways. The aim of the present study was to critically re-evaluate the potential neurogenic (trans-)differentiation capacity of ASCs using a neurogenic induction protocol based on the combination of isobutylmethylxanthine (IBMX), indomethacin and insulin. ASCs isolated from lipo-aspirate samples of five healthy female donors were characterized and potential neurogenic (trans-)differentiation was assessed by means of immunohistochemistry and gene expression analyses. Cell proliferation and cell cycle alterations were studied, and the expression of CREB/ATF transcription factors was analyzed. ASCs expressed CD59, CD90 and CD105, and were tested negative for CD34 and CD45. Under neurogenic induction, ASCs adopted a characteristic morphology comparable to neur(on)al progenitors and expressed musashi1, β-III-tubulin and nestin. Gene expression analyses revealed an increased expression of β-III-tubulin, GFAP, vimentin and BDNF, as well as SOX4 in induced ASCs. Cell proliferation was significantly reduced under neurogenic induction; cell cycle analyses showed a G2-cell cycle arrest accompanied by differential expression of key regulators of cell cycle progression. Differential expression of CREB/ATF transcription factors could be observed on neurogenic induction, pointing to a decisive role of the cAMP-CREB/ATF system. Our findings may point to a potential neurogenic (trans-)differentiation of ASCs into early neur(on)al progenitors, but do not present definite evidence for it. Especially, the adoption of a neural progenitor cell-like morphology must not automatically be misinterpreted as a specific characteristic of a respective (trans-)differentiation process, as this may as well be caused by alterations of cell cycle progression.

Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanginakudru, Sriramana, E-mail: skangina@iu.edu; DeSmet, Marsha, E-mail: mdesmet@iupui.edu; Thomas, Yanique, E-mail: ysthomas@umail.iu.edu

2015-04-15

The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reducedmore » viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.« less

Up-regulation of tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 in human colon cancer Caco-2 cells following repetitive exposure to dietary levels of a polyphenol-rich chokeberry juice.

PubMed

Bermúdez-Soto, María J; Larrosa, Mar; Garcia-Cantalejo, Jesús M; Espín, Juan C; Tomás-Barberan, Francisco A; García-Conesa, María T

2007-04-01

Consumption of berries and red fruits rich in polyphenols may contribute to the reduction of colon cancer through mechanisms not yet understood. In this study, we investigated the response of subconfluent Caco-2 cells (a human colon carcinoma model) to repetitive exposure (2 h a day for a 4-day period) of a subtoxic dose of a chokeberry (Aronia melanocarpa) juice containing mixed polyphenols. To mimic physiological conditions, we subjected the chokeberry juice to in vitro gastric and pancreatic digestion. The effects on viability, proliferation and cell cycle were determined, and changes in the expression of genes in response to the chokeberry treatment were screened using Affymetrix oligonucleotide microarrays. Exposure to the chokeberry juice inhibited Caco-2 cell proliferation by causing G(2)/M cell cycle arrest. We detected changes in the expression of a group of genes involved in cell growth and proliferation and cell cycle regulation, as well as those associated to colorectal cancer. A selection of these genes was further confirmed by quantitative RT-PCR. Among these, the tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), whose expression is known to be reduced in the majority of early adenomas and carcinomas, was up-regulated by the treatment both at the mRNA and protein levels (as shown by flow cytometry analysis). CEACAM1, with a significant regulatory role on cell proliferation of particular interest at early stages of cancer development, may be a potential target for chemoprevention by food components such as those present in polyphenol-rich fruits.

In situ engineering of the electrode-electrolyte interface for stabilized overlithiated cathodes

DOE PAGES

Evans, Tyler; Piper, Daniela Molina; Sun, Huaxing; ...

2017-01-05

Here, the first-ever demonstration of stabilized Si/lithium-manganese-rich full cells, capable of retaining >90% energy over early cycling and >90% capacity over more than 750 cycles at the 1C rate (100% depth-of-discharge), is made through the utilization of a modified ionic liquid electrolyte capable of forming a favorable cathode-electrolyte interface.

CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells.

PubMed

Valli, Emanuele; Trazzi, Stefania; Fuchs, Claudia; Erriquez, Daniela; Bartesaghi, Renata; Perini, Giovanni; Ciani, Elisabetta

2012-01-01

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G(0)/G(1) phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5. Copyright © 2012 Elsevier B.V. All rights reserved.

Increased leaf mesophyll porosity following transient retinoblastoma-related protein silencing is revealed by microcomputed tomography imaging and leads to a system-level physiological response to the altered cell division pattern

PubMed Central

Dorca-Fornell, Carmen; Pajor, Radoslaw; Lehmeier, Christoph; Pérez-Bueno, Marísa; Bauch, Marion; Sloan, Jen; Osborne, Colin; Rolfe, Stephen; Sturrock, Craig; Mooney, Sacha; Fleming, Andrew

2013-01-01

The causal relationship between cell division and growth in plants is complex. Although altered expression of cell-cycle genes frequently leads to altered organ growth, there are many examples where manipulation of the division machinery leads to a limited outcome at the level of organ form, despite changes in constituent cell size. One possibility, which has been under-explored, is that altered division patterns resulting from manipulation of cell-cycle gene expression alter the physiology of the organ, and that this has an effect on growth. We performed a series of experiments on retinoblastoma-related protein (RBR), a well characterized regulator of the cell cycle, to investigate the outcome of altered cell division on leaf physiology. Our approach involved combination of high-resolution microCT imaging and physiological analysis with a transient gene induction system, providing a powerful approach for the study of developmental physiology. Our investigation identifies a new role for RBR in mesophyll differentiation that affects tissue porosity and the distribution of air space within the leaf. The data demonstrate the importance of RBR in early leaf development and the extent to which physiology adapts to modified cellular architecture resulting from altered cell-cycle gene expression. PMID:24118480

A theory that may explain the Hayflick limit--a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts.

PubMed

Naveilhan, P; Baudet, C; Jabbour, W; Wion, D

1994-09-01

A model that may explain the limited division potential of certain cells such as human fibroblasts in culture is presented. The central postulate of this theory is that there exists, prior to certain key exons that code for materials needed for cell division, a unique sequence of specific repeating segments of DNA. One copy of such repeating segments is deleted during each cell cycle in cells that are not protected from such deletion through methylation of their cytosine residues. According to this theory, the means through which such repeated sequences are removed, one per cycle, is through the sequential action of enzymes that act much as bacterial restriction enzymes do--namely to produce scissions in both strands of DNA in areas that correspond to the DNA base sequence recognition specificities of such enzymes. After the first scission early in a replicative cycle, that enzyme becomes inhibited, but the cleavage of the first site exposes the closest site in the repetitive element to the action of a second restriction enzyme after which that enzyme also becomes inhibited. Then repair occurs, regenerating the original first site. Through this sequential activation and inhibition of two different restriction enzymes, only one copy of the repeating sequence is deleted during each cell cycle. In effect, the repeating sequence operates as a precise counter of the numbers of cell doubling that have occurred since the cells involved differentiated during development.

N-acetylcysteine normalizes the urea cycle and DNA repair in cells from patients with Batten disease.

PubMed

Kim, June-Bum; Lim, Nary; Kim, Sung-Jo; Heo, Tae-Hwe

2012-12-01

Batten disease is an inherited disorder characterized by early onset neurodegeneration due to the mutation of the CLN3 gene. The function of the CLN3 protein is not clear, but an association with oxidative stress has been proposed. Oxidative stress and DNA damage play critical roles in the pathogenesis of neurodegenerative diseases. Antioxidants are of interest because of their therapeutic potential for treating neurodegenerative diseases. We tested whether N-acetylcysteine (NAC), a well-known antioxidant, improves the pathology of cells from patients with Batten disease. At first, the expression levels of urea cycle components and DNA repair enzymes were compared between Batten disease cells and normal cells. We used both mRNA expression levels and Western blot analysis. We found that carbamoyl phosphate synthetase 1, an enzyme involved in the urea cycle, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta, enzymes involved in DNA repair, were expressed at higher levels in Batten disease cells than in normal cells. The treatment of Batten disease cells with NAC for 48 h attenuated activities of the urea cycle and of DNA repair, as indicated by the substantially decreased expression levels of carbamoyl phosphate synthetase 1, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta proteins compared with untreated Batten cells. NAC may serve in alleviating the burden of urea cycle and DNA repair processes in Batten disease cells. We propose that NAC may have beneficial effects in patients with Batten disease. Copyright © 2012 John Wiley & Sons, Ltd.

Telomere lengthening early in development.

PubMed

Liu, Lin; Bailey, Susan M; Okuka, Maja; Muñoz, Purificación; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L

2007-12-01

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

[Technologies for hair reconstruction and their applicability for pharmaceutical research].

PubMed

Matsuzaki, Takashi

2008-01-01

Hair follicles are the organs that produce hair shafts. They periodically regenerate throughout the life of the organisms, which is called the hair cycle. To develop new drugs to treat hair disorders and diseases, reproducible and high throughput assays or screening methods have been required to estimate the efficacy of various factors on hair follicle function. Although organ culture of hair follicles is one of the useful ways to carry out such research, it is not suitable for manipulating the genes or cells present in hair follicles. Patch assay is a method used to reconstruct hair follicles from enzymatically dissociated skin cells and has many advantages compared to the conventional Chamber method. Using the Patch method, transferring genes into follicular cells becomes easier than ever before. Chimeric follicles could be produced with dissociated cells by modifying the combination of cells or by simply merging cells of different origins. These applications certainly help the progress of hair research. However, we recently found that some functions of dermal papillae and follicular epithelia change during the growing phase (anagen) of the hair cycle. Dermal papillae produce different factors in early anagen and mid anagen. The signals from dermal papillae in early anagen could produce hair bulbs with clonogenic epithelial precursors but not with dormant epithelial precursors. On the other hand, the signals from dermal papillae in mid anagen strongly promote hair formation with dormant epithelial precursors. Therefore, more attention should be given to the hair cycle stages when using organ culture of hair follicles and conducting reconstruction experiments with follicularly derived cells.

The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

PubMed Central

Fimia, Gian Maria; Gottifredi, Vanesa; Bellei, Barbara; Ricciardi, Maria Rosaria; Tafuri, Agostino; Amati, Paolo; Maione, Rossella

1998-01-01

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis. PMID:9614186

40 CFR 600.514-12 - Reports to the Environmental Protection Agency.

Code of Federal Regulations, 2012 CFR

2012-07-01

...-cycle technology, and various early credit programs; (vi) A description of the method which will be used to calculate the carbon-related exhaust emissions for any electric vehicles, fuel cell vehicles and... number of electric vehicles, fuel cell vehicles and plug-in hybrid vehicles using (or projected to use...

Experimental evolution of an alternating uni- and multicellular life cycle in Chlamydomonas reinhardtii

PubMed Central

Ratcliff, William C.; Herron, Matthew D.; Howell, Kathryn; Pentz, Jennifer T.; Rosenzweig, Frank; Travisano, Michael

2013-01-01

The transition to multicellularity enabled the evolution of large, complex organisms, but early steps in this transition remain poorly understood. Here we show that multicellular complexity, including development from a single cell, can evolve rapidly in a unicellular organism that has never had a multicellular ancestor. We subject the alga Chlamydomonas reinhardtii to conditions that favour multicellularity, resulting in the evolution of a multicellular life cycle in which clusters reproduce via motile unicellular propagules. While a single-cell genetic bottleneck during ontogeny is widely regarded as an adaptation to limit among-cell conflict, its appearance very early in this transition suggests that it did not evolve for this purpose. Instead, we find that unicellular propagules are adaptive even in the absence of intercellular conflict, maximizing cluster-level fecundity. These results demonstrate that the unicellular bottleneck, a trait essential for evolving multicellular complexity, can arise rapidly via co-option of the ancestral unicellular form. PMID:24193369

The intermediate filament network protein, vimentin, is required for parvoviral infection.

PubMed

Fay, Nikta; Panté, Nelly

2013-09-01

Intermediate filaments (IFs) have recently been shown to serve novel roles during infection by many viruses. Here we have begun to study the role of IFs during the early steps of infection by the parvovirus minute virus of mice (MVM). We found that during early infection with MVM, after endosomal escape, the vimentin IF network was considerably altered, yielding collapsed immunofluorescence staining near the nuclear periphery. Furthermore, we found that vimentin plays an important role in the life cycle of MVM. The number of cells, which successfully replicated MVM, was reduced in infected cells in which the vimentin network was genetically or pharmacologically modified; viral endocytosis, however, remained unaltered. Perinuclear accumulation of MVM-containing vesicles was reduced in cells lacking vimentin. Our data suggests that vimentin is required for the MVM life cycle, presenting possibly a dual role: (1) following MVM escape from endosomes and (2) during endosomal trafficking of MVM. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

The drosophila fragile X protein dFMR1 is required during early embryogenesis for pole cell formation and rapid nuclear division cycles.

PubMed

Deshpande, Girish; Calhoun, Gretchen; Schedl, Paul

2006-11-01

The FMR family of KH domain RNA-binding proteins is conserved from invertebrates to humans. In humans, inactivation of the X-linked FMR gene fragile X is the most common cause of mental retardation and leads to defects in neuronal architecture. While there are three FMR family members in humans, there is only a single gene, dfmr1, in flies. As in humans, inactivation of dfmr1 causes defects in neuronal architecture and in behavior. dfmr1 has other functions in the fly in addition to neurogenesis. Here we have analyzed its role during early embryonic development. We found that dfmr1 embryos display defects in the rapid nuclear division cycles that precede gastrulation in nuclear migration and in pole cell formation. While the aberrations in nuclear division are correlated with a defect in the assembly of centromeric/centric heterochromatin, the defects in pole cell formation are associated with alterations in the actin-myosin cytoskeleton.

Time-Lapse Analysis of Human Embryonic Stem Cells Reveals Multiple Bottlenecks Restricting Colony Formation and Their Relief upon Culture Adaptation

PubMed Central

Barbaric, Ivana; Biga, Veronica; Gokhale, Paul J.; Jones, Mark; Stavish, Dylan; Glen, Adam; Coca, Daniel; Andrews, Peter W.

2014-01-01

Summary Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns. PMID:25068128

Outcome with lenalidomide plus dexamethasone followed by early autologous stem cell transplantation in patients with newly diagnosed multiple myeloma on the ECOG-ACRIN E4A03 randomized clinical trial: long-term follow-up.

PubMed

Biran, N; Jacobus, S; Vesole, D H; Callander, N S; Fonseca, R; Williams, M E; Abonour, R; Katz, M S; Rajkumar, S V; Greipp, P R; Siegel, D S

2016-09-02

In Eastern Cooperative Oncology Group-ACRIN E4A03, on completion of four cycles of therapy, newly diagnosed multiple myeloma patients had the option of proceeding to autologous peripheral blood stem cell transplant (ASCT) or continuing on their assigned therapy lenalidomide plus low-dose dexamethasone (Ld) or lenalidomide plus high-dose dexamethasone (LD). This landmark analysis compared the outcome of 431 patients surviving their first four cycles of therapy pursuing early ASCT to those continuing on their assigned therapy. Survival distributions were estimated using the Kaplan-Meier method and compared with log-rank test. Ninety patients (21%) opted for early ASCT. The 1-, 2-, 3-, 4- and 5-year survival probability estimates were higher for early ASCT versus no early ASCT at 99, 93, 91, 85 and 80% versus 94, 84, 75, 65 and 57%, respectively. The median overall survival (OS) in the early versus no early ASCT group was not reached (NR) versus 5.78 years. In patients <65 years of age, median OS in the early versus no early ASCT groups was NR in both, hazard ratio 0.79, 95% confidence interval: (0.50, 0.25). In patients ⩾65 years of age, median OS in the early versus no early ASCT was NR versus 5.11 years. ASCT dropped out of statistical significance (P=0.080). Patients opting for ASCT after induction Ld/LD had a higher survival probability and improvement in OS regardless of dexamethasone dose density.

Utility of the PET-CT in the evaluation of early response to treatment in the diffuse large B-cell lymphoma. Preliminary results.

PubMed

Cortés Romera, M; Gámez Cenzano, C; Caresia Aróztegui, A P; Martín-Comín, J; González-Barca, E; Ricart Brulles, Y; Palacios Abufón, A; Robles Barba, J; Rodríguez-Bel, L; Rossi Seoane, S; Fernández de Sevilla, A

2012-01-01

To assess the role of FDG-PET/CT performed after the first cycles of chemotherapy in the prediction of response to treatment in patients with diffuse large B-cell lymphoma. Twenty patients (mean age: 48 years) were included, 16 initial staging and 4 relapse. All patients underwent PET/CT at 3 times: 1) Baseline, 2) After 1-3 cycles of chemotherapy (early response assessment), and 3) End of treatment (evaluation of final response). Early PET/CT findings were correlated to the end-treatment PET/CT and follow-up. The evaluation of the response was established according to the decrease in uptake of the lesions (SUVmax). In the early assessment, a good response indicator (GRI) was obtained when the lesion disappeared or had more than 50% reduction in SUVmax. At the end of the treatment, a complete metabolic response (CMR) was determined in negative PET scans. Follow-up was superior to 19 months and final outcome was established as progression/relapse or no evidence of disease (NED). At the early treatment evaluation, 16/16 patients of initial staging (100%) and 2/4 of relapse (50%) achieved GRI. At the end of treatment evaluation, 14/16 patients of initial staging with GRI achieved CMR and 1/16 PMR: 14 were alive with NED in the follow-up while 1 relapsed. In the second group, 2/2 patients with GRI achieved CMR (100%): 1 continued with NED in the follow-up and another relapsed. FDG-PET/CT after the first cycles of chemotherapy is useful to monitor treatment due to its high negative predictive value (87.5%), using it to modify treatment early in the non-responders. Copyright © 2011 Elsevier España, S.L. y SEMNIM. All rights reserved.

The MYC Road to Hearing Restoration

PubMed Central

Kopecky, Benjamin; Fritzsch, Bernd

2012-01-01

Current treatments for hearing loss, the most common neurosensory disorder, do not restore perfect hearing. Regeneration of lost organ of Corti hair cells through forced cell cycle re-entry of supporting cells or through manipulation of stem cells, both avenues towards a permanent cure, require a more complete understanding of normal inner ear development, specifically the balance of proliferation and differentiation required to form and to maintain hair cells. Direct successful alterations to the cell cycle result in cell death whereas regulation of upstream genes is insufficient to permanently alter cell cycle dynamics. The Myc gene family is uniquely situated to synergize upstream pathways into downstream cell cycle control. There are three Mycs that are embedded within the Myc/Max/Mad network to regulate proliferation. The function of the two ear expressed Mycs, N-Myc and L-Myc were unknown less than two years ago and their therapeutic potentials remain speculative. In this review, we discuss the roles the Mycs play in the body and what led us to choose them to be our candidate gene for inner ear therapies. We will summarize the recently published work describing the early and late effects of N-Myc and L-Myc on hair cell formation and maintenance. Lastly, we detail the translational significance of our findings and what future work must be performed to make the ultimate hearing aid: the regeneration of the organ of Corti. PMID:24710525

Functions of the Type 1 BMP Receptor Acvr1 (Alk2) in Lens Development: Cell Proliferation, Terminal Differentiation, and Survival

PubMed Central

Rajagopal, Ramya; Dattilo, Lisa K.; Kaartinen, Vesa; Deng, Chu-Xia; Umans, Lieve; Zwijsen, An; Roberts, Anita B.; Bottinger, Erwin P.; Beebe, David C.

2009-01-01

Purpose Bone morphogenetic protein (BMP) signaling is essential for the induction and subsequent development of the lens. The purpose of this study was to analyze the function(s) of the type 1 BMP receptor, Acvr1, in lens development. Methods Acvr1 was deleted from the surface ectoderm of mouse embryos on embryonic day 9 using the Cre-loxP method. Cell proliferation, cell cycle exit, and apoptosis were measured in tissue sections by immunohistochemistry, immunofluorescence, and TUNEL staining. Results Lenses formed in the absence of Acvr1. However, Acvr1CKO (conditional knockout) lenses were small. Acvr1 signaling promoted proliferation at early stages of lens formation but inhibited proliferation at later stages. Inhibition of cell proliferation by Acvr1 was necessary for the proper regionalization of the lens epithelium and promoted the withdrawal of lens fiber cells from the cell cycle. In spite of the failure of all Acvr1CKO fiber cells to withdraw from the cell cycle, they expressed proteins characteristic of differentiated fiber cells. Although the stimulation of proliferation was Smad independent, the ability of Acvr1 to promote cell cycle exit later in development depended on classical R-Smad-Smad4 signaling. Loss of Acvr1 led to an increase in apoptosis of lens epithelial and fiber cells. Increased cell death, together with the initial decrease in proliferation, appeared to account for the smaller sizes of the Acvr1CKO lenses. Conclusions This study revealed a novel switch in the functions of Acvr1 in regulating lens cell proliferation. Previously unknown functions mediated by this receptor included regionalization of the lens epithelium and cell cycle exit during fiber cell differentiation. PMID:18566469

Drug-Free Approach To Study the Unusual Cell Cycle of Giardia intestinalis

PubMed Central

Horlock-Roberts, Kathleen; Reaume, Chase; Dayer, Guillem; Ouellet, Christine; Cook, Nicholas

2017-01-01

ABSTRACT Giardia intestinalis is a protozoan parasite that causes giardiasis, a form of severe and infectious diarrhea. Despite the importance of the cell cycle in the control of proliferation and differentiation during a giardia infection, it has been difficult to study this process due to the absence of a synchronization procedure that would not induce cellular damage resulting in artifacts. We utilized counterflow centrifugal elutriation (CCE), a size-based separation technique, to successfully obtain fractions of giardia cultures enriched in G1, S, and G2. Unlike drug-induced synchronization of giardia cultures, CCE did not induce double-stranded DNA damage or endoreplication. We observed increases in the appearance and size of the median body in the cells from elutriation fractions corresponding to the progression of the cell cycle from early G1 to late G2. Consequently, CCE could be used to examine the dynamics of the median body and other structures and organelles in the giardia cell cycle. For the cell cycle gene expression studies, the actin-related gene was identified by the program geNorm as the most suitable normalizer for reverse transcription-quantitative PCR (RT-qPCR) analysis of the CCE samples. Ten of 11 suspected cell cycle-regulated genes in the CCE fractions have expression profiles in giardia that resemble those of higher eukaryotes. However, the RNA levels of these genes during the cell cycle differ less than 4-fold to 5-fold, which might indicate that large changes in gene expression are not required by giardia to regulate the cell cycle. IMPORTANCE Giardias are among the most commonly reported intestinal protozoa in the world, with infections seen in humans and over 40 species of animals. The life cycle of giardia alternates between the motile trophozoite and the infectious cyst. The regulation of the cell cycle controls the proliferation of giardia trophozoites during an active infection and contains the restriction point for the differentiation of trophozoite to cyst. Here, we developed counterflow centrifugal elutriation as a drug-free method to obtain fractions of giardia cultures enriched in cells from the G1, S, and G2 stages of the cell cycle. Analysis of these fractions showed that the cells do not show side effects associated with the drugs used for synchronization of giardia cultures. Therefore, counterflow centrifugal elutriation would advance studies on key regulatory events during the giardia cell cycle and identify potential drug targets to block giardia proliferation and transmission. PMID:28959734

Genetic variants in the cell cycle control pathways contribute to early onset colorectal cancer in Lynch syndrome.

PubMed

Chen, Jinyun; Etzel, Carol J; Amos, Christopher I; Zhang, Qing; Viscofsky, Nancy; Lindor, Noralane M; Lynch, Patrick M; Frazier, Marsha L

2009-11-01

Lynch syndrome is an autosomal dominant syndrome of familial malignancies resulting from germ line mutations in DNA mismatch repair (MMR) genes. Our goal was to take a pathway-based approach to investigate the influence of polymorphisms in cell cycle-related genes on age of onset for Lynch syndrome using a tree model. We evaluated polymorphisms in a panel of cell cycle-related genes (AURKA, CDKN2A, TP53, E2F2, CCND1, TP73, MDM2, IGF1, and CDKN2B) in 220 MMR gene mutation carriers from 129 families. We applied a novel statistical approach, tree modeling (Classification and Regression Tree), to the analysis of data on patients with Lynch syndrome to identify individuals with a higher probability of developing colorectal cancer at an early age and explore the gene-gene interactions between polymorphisms in cell cycle genes. We found that the subgroup with CDKN2A C580T wild-type genotype, IGF1 CA-repeats >or=19, E2F2 variant genotype, AURKA wild-type genotype, and CCND1 variant genotype had the youngest age of onset, with a 45-year median onset age, while the subgroup with CDKN2A C580T wild-type genotype, IGF1 CA-repeats >or=19, E2F2 wild-type genotype, and AURKA variant genotype had the latest median age of onset, which was 70 years. Furthermore, we found evidence of a possible gene-gene interaction between E2F2 and AURKA genes related to CRC age of onset. Polymorphisms in these cell cycle-related genes work together to modify the age at the onset of CRC in patients with Lynch syndrome. These studies provide an important part of the foundation for development of a model for stratifying age of onset risk among those with Lynch syndrome.

The Epstein-Barr virus (EBV) nuclear antigen 1 BamHI F promoter is activated on entry of EBV-transformed B cells into the lytic cycle.

PubMed Central

Lear, A L; Rowe, M; Kurilla, M G; Lee, S; Henderson, S; Kieff, E; Rickinson, A B

1992-01-01

In Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cell lines exhibiting the latency I form of infection (i.e., EBV nuclear antigen 1 [EBNA1] positive in the absence of other latent proteins), the EBNA1 mRNA has a unique BamHI Q/U/K splice structure and is expressed from a novel promoter, Fp, located near the BamHI FQ boundary. This contrasts with the situation in EBV-transformed lymphoblastoid cell lines (LCLs) exhibiting the latency III form of infection (i.e., positive for all latent proteins), in which transcription from the upstream Cp or Wp promoters is the principal source of EBNA mRNAs. We carried out cDNA amplifications with oligonucleotide primer-probe combinations to determine whether Fp is ever active in an LCL environment. The results clearly showed that some LCLs express a Q/U/K-spliced EBNA1 mRNA in addition to the expected Cp/Wp-initiated transcripts; this seemed inconsistent with the concept of Cp/Wp and Fp as mutually exclusive promoters. Here we show that Fp is indeed silent in latency III cells but is activated at an early stage following the switch from latency III into the virus lytic cycle. Four pieces of evidence support this conclusion: (i) examples of coincident Cp/Wp and Fp usage in LCLs are restricted to those lines in which a small subpopulation of cells have spontaneously entered the lytic cycle; (ii) transcripts initiating from Fp can readily be demonstrated in spontaneously productive lines by S1 nuclease protection; (iii) the presence of Fp-initiated transcripts is not affected by acyclovir blockade of the late lytic cycle; and (iv) infection of latently infected LCLs with a recombinant vaccinia virus encoding the EBV immediate-early protein BZLF1, a transcriptional transactivator which normally initiates the lytic cycle, results in the appearance of the diagnostic Q/U/K-spliced transcripts. Images PMID:1331531

Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells

PubMed Central

Pauwels, Laurens; Morreel, Kris; De Witte, Emilie; Lammertyn, Freya; Van Montagu, Marc; Boerjan, Wout; Inzé, Dirk; Goossens, Alain

2008-01-01

Jasmonates (JAs) are plant-specific signaling molecules that steer a diverse set of physiological and developmental processes. Pathogen attack and wounding inflicted by herbivores induce the biosynthesis of these hormones, triggering defense responses both locally and systemically. We report on alterations in the transcriptome of a fast-dividing cell culture of the model plant Arabidopsis thaliana after exogenous application of methyl JA (MeJA). Early MeJA response genes encoded the JA biosynthesis pathway proteins and key regulators of MeJA responses, including most JA ZIM domain proteins and MYC2, together with transcriptional regulators with potential, but yet unknown, functions in MeJA signaling. In a second transcriptional wave, MeJA reprogrammed cellular metabolism and cell cycle progression. Up-regulation of the monolignol biosynthesis gene set resulted in an increased production of monolignols and oligolignols, the building blocks of lignin. Simultaneously, MeJA repressed activation of M-phase genes, arresting the cell cycle in G2. MeJA-responsive transcription factors were screened for their involvement in early signaling events, in particular the regulation of JA biosynthesis. Parallel screens based on yeast one-hybrid and transient transactivation assays identified both positive (MYC2 and the AP2/ERF factor ORA47) and negative (the C2H2 Zn finger proteins STZ/ZAT10 and AZF2) regulators, revealing a complex control of the JA autoregulatory loop and possibly other MeJA-mediated downstream processes. PMID:18216250

Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro.

PubMed

Arcangeletti, Maria-Cristina; Rodighiero, Isabella; Mirandola, Prisco; De Conto, Flora; Covan, Silvia; Germini, Diego; Razin, Sergey; Dettori, Giuseppe; Chezzi, Carlo

2011-01-01

The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.

The intermediate filament network protein, vimentin, is required for parvoviral infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fay, Nikta; Panté, Nelly, E-mail: pante@zoology.ubc.ca

Intermediate filaments (IFs) have recently been shown to serve novel roles during infection by many viruses. Here we have begun to study the role of IFs during the early steps of infection by the parvovirus minute virus of mice (MVM). We found that during early infection with MVM, after endosomal escape, the vimentin IF network was considerably altered, yielding collapsed immunofluorescence staining near the nuclear periphery. Furthermore, we found that vimentin plays an important role in the life cycle of MVM. The number of cells, which successfully replicated MVM, was reduced in infected cells in which the vimentin network wasmore » genetically or pharmacologically modified; viral endocytosis, however, remained unaltered. Perinuclear accumulation of MVM-containing vesicles was reduced in cells lacking vimentin. Our data suggests that vimentin is required for the MVM life cycle, presenting possibly a dual role: (1) following MVM escape from endosomes and (2) during endosomal trafficking of MVM. - Highlights: • MVM infection changes the distribution of the vimentin network to perinuclear regions. • Disrupting the vimentin network with acrylamide decreases MVM replication. • MVM replication is significantly reduced in vimentin-null cells. • Distribution of MVM-containing vesicles is affected in MVM infected vimentin-null cells.« less

The sow endometrium at different stages of the oestrous cycle: studies on morphological changes and infiltration by cells of the immune system.

PubMed

Kaeoket, K; Persson, E; Dalin, A M

2001-01-31

The aim of this study was to investigate the distribution of leukocytes and the morphological changes of the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire), with an average parity number of 3.4 +/- 0.7 (mean +/- S.D.) were used. Blood samples were collected from the jugular vein 1h before slaughter for analyses of oestradiol-17beta and progesterone levels. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus, prooestrus, oestrus, early dioestrus and dioestrus, were fixed, embedded in plastic resin and stained with toluidine blue. The surface and glandular epithelium as well as subepithelial and glandular connective tissue layers were examined by light microscopy. The significantly highest surface and the glandular epithelium were observed at oestrus and dioestrus, respectively. The largest number of capillaries (underneath the surface epithelium) was found at oestrus. In the surface epithelium, the largest number of intraepithelial lymphocytes (IELs, round nucleus) was found at early dioestrus. The largest number of lymphocytes and macrophages within the glandular epithelium were found at early dioestrus and oestrus, respectively. In the subepithelial connective tissue layer, the most common type of leukocytes during all stages was the lymphocyte. The largest numbers of lymphocytes and neutrophils were found at oestrus while the largest number of eosinophils was found at dioestrus. The dominating cells of the immune system in the connective tissue of the glandular layer were lymphocytes and macrophages. The significantly largest numbers of lymphocytes and plasma cells were found at early dioestrus and dioestrus, respectively. The number of lymphocytes in the connective tissue of the glandular layer and the number of plasma cells in the subepithelial layer were positively correlated with the plasma level of progesterone (P < or = 0.05). The numbers of capillaries and neutrophils in the subepithelial layer underneath the surface epithelium as well as the number of macrophages in both surface and glandular epithelium were positively correlated with the plasma level of oestradiol-17beta (P < or = 0.05). In conclusion, the present study showed a variation in the infiltration and distribution of lymphocytes, neutrophils, eosinophils, macrophages, mast cells and plasma cells in the sow endometrium during different stages of the oestrous cycle. Also morphological parameters (e.g. height of surface and glandular epithelium, capillaries density and degree of oedema) varied throughout the oestrous cycle.

Corrigendum to "The sow endometrium at different stages of the oestrus cycle: studies on morphological changes and infiltration by cells of the immune system." [Anim. Reprod. Sci. 65 (2001) 95-114].

PubMed

Kaeoket, K; Persson, E; Dalin, A-M

2002-09-16

The aim of this study was to investigate the distribution of leukocytes and the morphological changes of the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire), with an average parity number of 3.4+/-0.7 (mean+/-S.D.) were used. Blood samples were collected from the jugular vein 1 h before slaughter for analyses of oestradiol-17beta and progesterone levels. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus, prooestrus, oestrus, early dioestrus and dioestrus, were fixed, embedded in plastic resin and stained with toluidine blue. The surface and glandular epithelium as well as subepithelial and glandular connective tissue layers were examined by light microscopy (LM). The significantly highest surface and the glandular epithelium were observed at oestrus and dioestrus, respectively. The largest number of capillaries (underneath the surface epithelium) was found at oestrus. In the surface epithelium, the largest number of intraepithelial lymphocytes (IELs, round nucleus) was found at early dioestrus. The largest number of lymphocytes and macrophages within the glandular epithelium were found at early dioestrus and oestrus, respectively. In the subepithelial connective tissue layer, the most common type of leukocytes during all stages was the lymphocyte. The largest numbers of lymphocytes and neutrophils were found at oestrus while the largest number of eosinophils was found at dioestrus. The dominating cells of the immune system in the connective tissue of the glandular layer were lymphocytes and macrophages. The significantly largest numbers of lymphocytes and plasma cells were found at early dioestrus and dioestrus, respectively. The number of lymphocytes in the connective tissue of the glandular layer and the number of plasma cells in the subepithelial layer were positively correlated with the plasma level of progesterone (P < or = 0.05). The numbers of capillaries and neutrophils in the subepithelial layer underneath the surface epithelium as well as the number of macrophages in both surface and glandular epithelium were positively correlated with the plasma level of oestradiol-17beta (P < or = 0.05). In conclusion, the present study showed a variation om the infiltration and distrobution of lymphocytes, neutrophils, eosinophils, macrophages, mast cells, and plasma cells in the sow endometrium during different stages of the oestrous cycle. Also morphological parameters (e.g. height of surface and glandular epithelium, capillaries density and degree of oedema) varied throughout the oestrous cycle. Copyright 2002 Elsevier Science B.V.

Rotaviruses induce an early membrane permeabilization of MA104 cells and do not require a low intracellular Ca2+ concentration to initiate their replication cycle.

PubMed Central

Cuadras, M A; Arias, C F; López, S

1997-01-01

In this work, we found that rotavirus infection induces an early membrane permeabilization of MA104 cells and promotes the coentry of toxins, such as alpha-sarcin, into the cell. This cell permeability was shown to depend on infectious virus and was also shown to be virus dose dependent, with 10 infectious particles per cell being sufficient to achieve maximum permeability; transient, lasting no more than 15 min after virus entry and probably occurring concomitantly with virus penetration; and specific, since cells that are poorly permissive for rotavirus were not permeabilized. The rotavirus-mediated coentry of toxins was not blocked by the endocytosis inhibitors dansylcadaverine and cytochalasin D or by the vacuolar proton-ATPase inhibitor bafilomycin A1, suggesting that neither endocytocis nor an intraendosomal acidic pH or a proton gradient is required for permeabilization of the cells. Compounds that raise the intracellular concentration of calcium ([Ca2+]i) by different mechanisms, such as the calcium ionophores A23187 and ionomycin and the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, did not block the coentry of alpha-sarcin or affect the onset of viral protein synthesis, suggesting that a low [Ca2+]i is not essential for the initial steps of the virus life cycle. Since the entry of alpha-sarcin correlates with virus penetration in all parameters tested, the assay for permeabilization to toxins might be a useful tool for studying and characterizing the route of entry and the mechanism used by rotaviruses to traverse the cell membrane and initiate a productive replication cycle. PMID:9371563

PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.

PubMed

Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E

2001-10-11

Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

PubMed

Wang, Tao; Xie, Zi-ping; Huang, Zhan-sen; Li, Hao; Wei, An-yang; Di, Jin-ming; Xiao, Heng-jun; Zhang, Zhi-gang; Cai, Liu-hong; Tao, Xin; Qi, Tao; Chen, Di-ling; Chen, Jun

2015-10-01

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Mechanisms underlying regulation of cell cycle and apoptosis by hnRNP B1 in human lung adenocarcinoma A549 cells.

PubMed

Han, Juan; Tang, Feng-ming; Pu, Dan; Xu, Dan; Wang, Tao; Li, Weimin

2014-01-01

Overexpression of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a nuclear RNA binding protein, has been reported to occur in early-stage lung cancer and in premalignant lesions. DNA-dependent protein kinase (DNA-PK) is known to be involved in the repair of double-strand DNA breaks. Reduced capacity to repair DNA has been associated with the risk of lung cancer. We investigated a link between hnRNP B1 and DNA-PK and their effects on proliferation, cell cycle, and apoptosis in the human lung adenocarcinoma cell line A549. We found that hnRNP B1 and DNA-PK interact with each other in a complex fashion. Reducing hnRNP B1 expression in A549 cells with the use of RNAi led to upregulation of p53 activity through upregulation of DNA-PK activity but without inducing p53 expression. Further, suppression of hnRNP B1 in A549 cells slowed cell proliferation, promoted apoptosis, and induced cell cycle arrest at the G1 stage. The presence of NU7026 reduced the arrest of cells at the G1 stage and reduced the apoptosis rate while promoting cell growth. Taken together, our results demonstrate that by regulating DNA-PK activity, hnRNP B1 can affect p53-mediated cell cycle progression and apoptosis, resulting in greater cell survival and subsequent proliferation.

The use of cell cycle arrest biomarkers in the early detection of acute kidney injury. Is this the new renal troponin?

PubMed

Ortega, Luis M; Heung, Michael

2018-04-05

Acute kidney injury (AKI) has a high prevalence in critical care patients. Early detection might prevent patients from developing chronic kidney disease and requirement for renal replacement therapy. If we compare AKI with acute coronary syndrome, in which an increase in cardiac troponin may trigger early diagnosis and therapeutic intervention, we could extrapolate a similar technique in patients with early AKI without changes in urinary frequency or serum creatinine. The objective is to identify biomarker-positive, creatinine-negative patients that would allow therapeutic interventions to be initiated before finding changes in serum creatinine, preventing kidney damage. Tissue inhibitor of metalloproteinase 2 and insulin-like growth factor binding protein 7 are cell cycle arrest biomarkers that have demonstrated, in recent clinical trials, to have good sensitivity and specificity for early detection of AKI. Other recent studies have shown that the joint use of these biomarkers with serum creatinine and urine production could improve the prognosis of AKI in critical patients. The application of these biomarkers in clinical practice would enable the early identification of patients at risk of AKI, establishing interventions that would improve the survival of renal function. Copyright © 2017 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

Cyclooxygenase activity is important for efficient replication of mouse hepatitis virus at an early stage of infection

PubMed Central

Raaben, Matthijs; Einerhand, Alexandra WC; Taminiau, Lucas JA; van Houdt, Michel; Bouma, Janneke; Raatgeep, Rolien H; Büller, Hans A; de Haan, Cornelis AM; Rossen, John WA

2007-01-01

Cyclooxygenases (COXs) play a significant role in many different viral infections with respect to replication and pathogenesis. Here we investigated the role of COXs in the mouse hepatitis coronavirus (MHV) infection cycle. Blocking COX activity by different inhibitors or by RNA interference affected MHV infection in different cells. The COX inhibitors reduced MHV infection at a post-binding step, but early in the replication cycle. Both viral RNA and viral protein synthesis were affected with subsequent loss of progeny virus production. Thus, COX activity appears to be required for efficient MHV replication, providing a potential target for anti-coronaviral therapy. PMID:17555580

Role of Cyclin E as an Early Event in Ovarian Carcinogenesis

DTIC Science & Technology

2012-04-01

degradation . P27 is a powerful negative regulator of the cell cycle, preventing activation of cyclin E- cdk2 or cyclin D-cdk4 complexes and cell cycle...Ahmed M, Bavi P, et al. Bortezomib (Velcade) induces p27Kip1 expression through S-phase kinase protein 2 degradation in colorectal cancer. Cancer Res...CA1251CA72·4 CA 125 CA72-4 M-CSF CA 125/CA 72-4/M-CSFICA 15-3 CA125 CA 125/mesothelin CA 125/IL·6JIL·8NEGF;EGF CA 125/IL-6,G-CSFNEGF/EGF Leptin

Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID

PubMed Central

Le, Quy; Maizels, Nancy

2015-01-01

AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome. PMID:26355458

Symbiotic Origin of Aging.

PubMed

Greenberg, Edward F; Vatolin, Sergei

2018-06-01

Normally aging cells are characterized by an unbalanced mitochondrial dynamic skewed toward punctate mitochondria. Genetic and pharmacological manipulation of mitochondrial fission/fusion cycles can contribute to both accelerated and decelerated cellular or organismal aging. In this work, we connect these experimental data with the symbiotic theory of mitochondrial origin to generate new insight into the evolutionary origin of aging. Mitochondria originated from autotrophic α-proteobacteria during an ancient endosymbiotic event early in eukaryote evolution. To expand beyond individual host cells, dividing α-proteobacteria initiated host cell lysis; apoptosis is a product of this original symbiont cell lytic exit program. Over the course of evolution, the host eukaryotic cell attenuated the harmful effect of symbiotic proto-mitochondria, and modern mitochondria are now functionally interdependent with eukaryotic cells; they retain their own circular genomes and independent replication timing. In nondividing differentiated or multipotent eukaryotic cells, intracellular mitochondria undergo repeated fission/fusion cycles, favoring fission as organisms age. The discordance between cellular quiescence and mitochondrial proliferation generates intracellular stress, eventually leading to a gradual decline in host cell performance and age-related pathology. Hence, aging evolved from a conflict between maintenance of a quiescent, nonproliferative state and the evolutionarily conserved propagation program driving the life cycle of former symbiotic organisms: mitochondria.

[The mechanisms of p21WAF1/Cip-1 expression in MOLT-4 cell line induced by TSA].

PubMed

Song, Yi; Liu, Mei-Ju; Zhao, Guo-Wei; Qian, Jun-Jie; Dong, Yan; Liu, Hua; Sun, Guo-Jing; Mei, Zhu-Zhong; Liu, Bin; Tian, Bao-Lei; Sun, Zhi-Xian

2005-04-01

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.

DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from kashmiri saffron in a human pancreatic cancer cell line.

PubMed

Bakshi, Hamid; Sam, Smitha; Rozati, Roya; Sultan, Phalisteen; Islam, Tajamul; Rathore, Babita; Lone, Zahoor; Sharma, Manik; Triphati, Jagrati; Saxena, Ramesh Chand

2010-01-01

Apoptosis, a widely important mechanism that contributes to cell growth reduction, is reported to be induced by Crocus sativus in different cancer types. The present study was designed to elucidate apoptosis induction by crocin, a main component of Crocus sativus in a human pancreatic cancer cell line (BxPC-3). Cell viability was measured by MTT assay, Hoechest33258 staining was used to detect the chromatin condensation characteristic of apoptosis, and DNA fragmentation was assessed by gel electrophoresis and cell cycle analysis by flow cytometry. Crocin induced apoptosis and G1-phase cell cycle arrest of BxPC-3 cells, while decreasing cell viability in a dose dependent and time dependent manner. Cells treated with 10μg/L crocin exhibited apoptotic morphology (brightly blue-fluorescent condensed nuclei on Hoechst 33258 staining) and reduction of volume. DNA analysis revealed typical ladders as early as 12 hours after treatment indicative of apoptosis. Our preclinical study demonstrated a pancreatic cancer cell line to be highly sensitive to crocin-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of crocin action are not yet clearly understood, it appears to have potential as a therapeutic agent.

Centrosome-Based Mechanisms, Prognostics and Therapeutics in Prostate Cancer

DTIC Science & Technology

2006-12-01

progression of prostate carcinomas. The specific aims of the original proposal were designed to test several features of this model . 1. Are centrosome...features of this model . 1. Are centrosome defects present in early prostate cancer and can they predict aggressive disease? 2. Do pericentrin’s...cells, supports this model . The ability to block the cell cycle in prostate cells by depletion of any of 14 centrosome proteins identifies several

Genetic interactions between the hedgehog co-receptors Gas1 and Boc regulate cell proliferation during murine palatogenesis

PubMed Central

Xavier, Guilherme M.; Seppala, Maisa; Papageorgiou, Spyridon N.; Fan, Chen-Ming; Cobourne, Martyn T.

2016-01-01

Abnormal regulation of Sonic hedgehog (Shh) signaling has been described in a variety of human cancers and developmental anomalies, which highlights the essential role of this signaling molecule in cell cycle regulation and embryonic development. Gas1 and Boc are membrane co-receptors for Shh, which demonstrate overlapping domains of expression in the early face. This study aims to investigate potential interactions between these co-receptors during formation of the secondary palate. Mice with targeted mutation in Gas1 and Boc were used to generate Gas1; Boc compound mutants. The expression of key Hedgehog signaling family members was examined in detail during palatogenesis via radioactive in situ hybridization. Morphometric analysis involved computational quantification of BrdU-labeling and cell packing; whilst TUNEL staining was used to assay cell death. Ablation of Boc in a Gas1 mutant background leads to reduced Shh activity in the palatal shelves and an increase in the penetrance and severity of cleft palate, associated with failed elevation, increased proliferation and reduced cell death. Our findings suggest a dual requirement for Boc and Gas1 during early development of the palate, mediating cell cycle regulation during growth and subsequent fusion of the palatal shelves. PMID:27811357

Biological Characteristics and Genetic Heterogeneity between Carcinoma-Associated Fibroblasts and Their Paired Normal Fibroblasts in Human Breast Cancer

PubMed Central

Hou, Yixuan; Sun, Yan; Wang, Liyang; Luo, Haojun; Peng, Huimin; Liu, Manran

2013-01-01

Background The extensional signals in cross-talk between stromal cells and tumor cells generated from extracellular matrix molecules, soluble factor, and cell-cell adhesion complexes cooperate at the extra- and intracellular level in the tumor microenvironment. CAFs are the primary type of stromal cells in the tumor microenvironment and play a pivotal role in tumorigenesis and development. Hitherto, there is hardly any systematic analysis of the intrinsic relationship between CAFs function and its abnormal signaling pathway. The extreme complexity of CAFs’ features and their role in tumor development are needed to be further investigated. Methodology/Principal Findings We primary cultured CAFs and NFs from early stages of breast cancer tissue and identified them using their biomarker by immunohistochemistry for Fibronectin, α-SMA and FAP. Microarray was applied to analyze gene expression profiles of human breast CAFs and the paired NFs. The Up-regulated genes classified by Gene Ontology, signal pathways enriched by DAVID pathway analysis. Abnormal signaling pathways in breast cancer CAFs are involved in cell cycle, cell adhesion, signal transduction and protein transport being reported in CAFs derived from other tumors. Significantly, the altered ATM signaling pathway, a set of cell cycle regulated signaling, and immune associated signaling are identified to be changed in CAFs. Conclusions/Significance CAFs have the vigorous ability of proliferation and potential of invasion and migration comparing with NFs. CAFs could promote breast cancer cell invasion under co-culture conditions through up-regulated CCL18 and CXCL12. Consistently with its biologic behavior, the gene expression profiling analyzed by microarray shows that some of key signaling pathways, such as cell cycle, cell adhesion, and secreting factors play an important role in CAFs. The altered ATM signaling pathway is abnormally active in the early stage of breast cancer. The set of immune associated signaling may be involved in tumor cell immune evasion. PMID:23577100

Skin transcriptome reveals the intrinsic molecular mechanisms underlying hair follicle cycling in Cashmere goats under natural and shortened photoperiod conditions.

PubMed

Yang, Min; Song, Shen; Dong, Kunzhe; Chen, XiaoFei; Liu, Xuexue; Rouzi, Marhaba; Zhao, Qianjun; He, Xiaohong; Pu, Yabin; Guan, Weijun; Ma, Yuehui; Jiang, Lin

2017-10-18

The growth of cashmere exhibits a seasonal pattern arising from photoperiod change. However, the underlying molecular mechanism remains unclear. We profiled the skin transcriptome of six goats at seven time points during hair follicle cycling via RNA-seq. The six goats comprised three goats exposed to a natural photoperiod and three exposed to a shortened photoperiod. During hair cycle transition, 1713 genes showed differential expression, and 332 genes showed a pattern of periodic expression. Moreover, a short photoperiod induced the hair follicle to enter anagen early, and 246 genes overlapped with the periodic genes. Among these key genes, cold-shock domain containing C2 (CSDC2) was highly expressed in the epidermis and dermis of Cashmere goat skin, although its function in hair-follicle development remains unknown. CSDC2 silencing in mouse fibroblasts resulted in the decreased mRNA expression of two key hair-follicle factors, leading to reduced cell numbers and a lower cell density. Cashmere growth or molting might be controlled by a set of periodic regulatory genes. The appropriate management of short light exposure can induce hair follicles to enter full anagen early through the activation of these regulators. The CSDC2 gene is a potentially important transcription factor in the hair growth cycle.

Simulated Carbon Cycling in a Model Microbial Mat.

NASA Astrophysics Data System (ADS)

Decker, K. L.; Potter, C. S.

2006-12-01

We present here the novel addition of detailed organic carbon cycling to our model of a hypersaline microbial mat ecosystem. This ecosystem model, MBGC (Microbial BioGeoChemistry), simulates carbon fixation through oxygenic and anoxygenic photosynthesis, and the release of C and electrons for microbial heterotrophs via cyanobacterial exudates and also via a pool of dead cells. Previously in MBGC, the organic portion of the carbon cycle was simplified into a black-box rate of accumulation of simple and complex organic compounds based on photosynthesis and mortality rates. We will discuss the novel inclusion of fermentation as a source of carbon and electrons for use in methanogenesis and sulfate reduction, and the influence of photorespiration on labile carbon exudation rates in cyanobacteria. We will also discuss the modeling of decomposition of dead cells and the ultimate release of inorganic carbon. The detailed modeling of organic carbon cycling is important to the accurate representation of inorganic carbon flux through the mat, as well as to accurate representation of growth models of the heterotrophs under different environmental conditions. Because the model ecosystem is an analog of ancient microbial mats that had huge impacts on the atmosphere of early earth, this MBGC can be useful as a biological component to either early earth models or models of other planets that potentially harbor life.

Transition in Survival From Low-Dose Hyper-Radiosensitivity to Increased Radioresistance Is Independent of Activation of ATM SER1981 Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krueger, Sarah A.; Collis, Spencer J.; Joiner, Michael C.

2007-11-15

Purpose: The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G{sub 2}-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance. Methods and Materials: Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 {mu}g/mL chloroquine, 15 {mu}M genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eightmore » cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G{sub 2}-phase cells entering mitosis, using histone H3 phosphorylation analysis. Results: The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G{sub 2}-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range. Conclusion: Overcoming HRS is reliant on the function of the early G{sub 2}-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.« less

Synchrony of clonal cell proliferation and contiguity of clonally related cells: production of mosaicism in the ventricular zone of developing mouse neocortex

NASA Technical Reports Server (NTRS)

Cai, L.; Hayes, N. L.; Nowakowski, R. S.

1997-01-01

We have analyzed clonal cell proliferation in the ventricular zone (VZ) of the early developing mouse neocortex with a replication-incompetent retrovirus encoding human placental alkaline phosphatase (AP). The retrovirus was injected into the lateral ventricles on embryonic day 11 (E11), i.e., at the onset of neuronogenesis. Three days postinjection, on E14, a total of 259 AP-labeled clones of various sizes were found in 7 fetal brains. There are approximately 7 cell cycles between E11 and E14 (), and there is a 1-2 cell cycle delay between retroviral injection and the production of a retrovirally labeled "founder" cell; thus, we estimate that the "age" of the clones was about 5-6 cell cycles. Almost one-half of the clones (48.3%) identified were pure proliferating clones containing cells only in the VZ. Another 18.5% contained both proliferating and postproliferative cells, and 33.2% contained only postproliferative cells. It was striking that over 90% of the clonally related proliferating cells occurred in clusters of two or more apparently contiguous cells, and about 73% of the proliferating cells occurred in clusters of three or more cells. Regardless of the number of cells in the clone, these clusters were tightly packed and confined to a single level of the VZ. This clustering of proliferating cells indicates that clonally related cells maintain neighbor-neighbor relationships as they undergo interkinetic nuclear migration and progress through several cell cycles, and, as a result, the ventricular zone is a mosaic of small clusters of clonally related and synchronously cycling cells. In addition, cells in the intermediate zone and the cortical plate were also frequently clustered, indicating that they became postproliferative at a similar time and that the output of the VZ is influenced by its mosaic structure.

Analysis of cardiomyocyte movement in the developing murine heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Hisayuki; Yuasa, Shinsuke, E-mail: yuasa@a8.keio.jp; Tabata, Hidenori

The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cellmore » cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.« less

Donor cell differentiation, reprogramming, and cloning efficiency: elusive or illusive correlation?

PubMed

Oback, B; Wells, D N

2007-05-01

Compared to other assisted reproductive technologies, mammalian nuclear transfer (NT) cloning is inefficient in generating viable offspring. It has been postulated that nuclear reprogramming and cloning efficiency can be increased by choosing less differentiated cell types as nuclear donors. This hypothesis is mainly supported by comparative mouse cloning experiments using early blastomeres, embryonic stem (ES) cells, and terminally differentiated somatic donor cells. We have re-evaluated these comparisons, taking into account different NT procedures, the use of donor cells from different genetic backgrounds, sex, cell cycle stages, and the lack of robust statistical significance when post-blastocyst development is compared. We argue that while the reprogrammability of early blastomeres appears to be much higher than that of somatic cells, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic donor cell lineages. Copyright (c) 2006 Wiley-Liss, Inc.

pRb phosphorylation regulates the proliferation of supporting cells in gentamicin-damaged neonatal avian utricle.

PubMed

Wu, Jingfang; Sun, Shan; Li, Wenyan; Chen, Yan; Li, Huawei

2014-10-01

The ability of nonmammalian vertebrates to regenerate hair cells (HCs) after damage-induced HC loss has stimulated and inspired research in the field of HC regeneration. The protein pRb encoded by retinoblastoma gene Rb1 forces sensory progenitor cells to exit cell cycle and maintain differentiated HCs and supporting cells (SCs) in a quiescent state. pRb function is regulated by phosphorylation through the MEK/ERK or the pRb/Raf-1 signaling pathway. In our previous study, we have shown that pRb phosphorylation is crucial for progenitor cell proliferation and survival during the early embryonic stage of avian otocyst sensory epithelium development. However, in damaged avian utricle, the role of pRb in regulating the cell cycling of SCs or HCs regeneration still remains unclear. To further elucidate the function of pRb phosphorylation on SCs re-entering the cell cycle triggered by gentamycin-induced HCs damage, we isolated neonatal chicken utricles and treated them with the MEK inhibitor U0126 or the pRb/Raf-1 inhibitor RRD-251, respectively in vitro. We found that after gentamycin-induced HCs damage, pRb phosphorylation is important for the quiescent SCs re-entering the cell cycle in the neonatal chicken utricle. In addition, the proliferation of SCs decreased in a dose-dependent manner in response to both U0126 and RRD-251, which indicates that both the MEK/ERK and the pRb/Raf-1 signaling pathway play important roles in pRb phosphorylation in damaged neonatal chicken utricle. Together, these findings on the function of pRb in damaged neonatal chicken utricle improve our understanding of the regulation of the cell cycle of SCs after HCs loss and may shed light on the mammalian HC regeneration from SCs in damaged organs.

Peptide promotes overcoming of the division limit in human somatic cell.

PubMed

Khavinson, V Kh; Bondarev, I E; Butyugov, A A; Smirnova, T D

2004-05-01

We previously showed that treatment of normal human diploid cells with Epithalon (Ala-Glu-Asp-Gly) induced expression of telomerase catalytic subunit, its enzymatic activity, and elongation of telomeres. Here we studied the effect of this peptide on proliferative potential of human fetal fibroblasts. Primary pulmonary fibroblasts derived from a 24-week fetus lost the proliferative potential at the 34th passage. The mean size of telomeres in these cells was appreciably lower than during early passages (passage 10). Addition of Epithalon to aging cells in culture induced elongation of telomeres to the size comparable to their length during early passages. Peptide-treated cells with elongated telomeres made 10 extra divisions (44 passages) in comparison with the control and continued dividing. Hence, Epithalon prolonged the vital cycle of normal human cells due to overcoming the Heyflick limit.

Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development

PubMed Central

Fox, Catherine A.; Dowdle, Megan E.; Blaser, Susanne Imboden; Chung, Andy; Park, Sookhee

2017-01-01

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation. PMID:27975270

Cell cycle regulator E2F4 is essential for the development of the ventral telencephalon.

PubMed

Ruzhynsky, Vladimir A; McClellan, Kelly A; Vanderluit, Jacqueline L; Jeong, Yongsu; Furimsky, Marosh; Park, David S; Epstein, Douglas J; Wallace, Valerie A; Slack, Ruth S

2007-05-30

Early forebrain development is characterized by extensive proliferation of neural precursors coupled with complex structural transformations; however, little is known regarding the mechanisms by which these processes are integrated. Here, we show that deficiency of the cell cycle regulatory protein, E2F4, results in the loss of ventral telencephalic structures and impaired self-renewal of neural precursor cells. The mechanism underlying aberrant ventral patterning lies in a dramatic loss of Sonic hedgehog (Shh) expression specifically in this region. The E2F4-deficient phenotype can be recapitulated by interbreeding mice heterozygous for E2F4 with those lacking one allele of Shh, suggesting a genetic interaction between these pathways. Treatment of E2F4-deficient cells with a Hh agonist rescues stem cell self-renewal and cells expressing the homeodomain proteins that specify the ventral telencephalic structures. Finally, we show that E2F4 deficiency results in impaired activity of Shh forebrain-specific enhancers. In conclusion, these studies establish a novel requirement for the cell cycle regulatory protein, E2F4, in the development of the ventral telencephalon.

How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase I inhibitor.

PubMed

Zuma, Aline Araujo; Mendes, Isabela Cecília; Reignault, Lissa Catherine; Elias, Maria Carolina; de Souza, Wanderley; Machado, Carlos Renato; Motta, Maria Cristina M

2014-02-01

The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, which affects approximately 8 million people in Latin America. This parasite contains a single nucleus and a kinetoplast, which harbors the mitochondrial DNA (kDNA). DNA topoisomerases act during replication, transcription and repair and modulate DNA topology by reverting supercoiling in the DNA double-strand. In this work, we evaluated the effects promoted by camptothecin, a topoisomerase I inhibitor that promotes protozoan proliferation impairment, cell cycle arrest, ultrastructure alterations and DNA lesions in epimastigotes of T. cruzi. The results showed that inhibition of cell proliferation was reversible only at the lowest drug concentration (1μM) used. The unpacking of nuclear heterochromatin and mitochondrion swelling were the main ultrastructural modifications observed. Inhibition of parasite proliferation also led to cell cycle arrest, which was most likely caused by nuclear DNA lesions. Following camptothecin treatment, some of the cells restored their DNA, whereas others entered early apoptosis but did not progress to late apoptosis, indicating that the protozoa stay alive in a "senescence-like" state. This programmed cell death may be associated with a decrease in mitochondrial membrane potential and an increase in the production of reactive oxygen species. Taken together, these results indicate that the inhibition of T. cruzi proliferation is related to events capable of affecting cell cycle, DNA organization and mitochondrial activity. Copyright © 2014. Published by Elsevier B.V.

The sow endometrium at different stages of the oestrous cycle: immunohistochemical study on the distribution of SWC3-expressing cells (granulocytes, monocytes and macrophages).

PubMed

Kaeoket, K; Dalin, A M; Magnusson, U; Persson, E

2001-10-01

Uterine samples from sows taken immediately after slaughter at late di-oestrus, pro-oestrus, oestrus, early di-oestrus and di-ocstrus, were analysed by immunohistochemistry with an avidinbiotin-peroxidase method using a monoclonal antibody (anti-SWC3) to granulocyte, monocyte and macrophage populations. The endometrium was then examined by light microscopy. In the surface and glandular epithelium, the largest numbers of SWC3-expressing cells (P < or = 0.01 and P < or = 0.05) were found at oestrus, and at pro-oestrus and oestrus, respectively. The numbers of SWC3-expressing cells in the epithelium were positively correlated with the plasma levels of oestradiol-17beta. In the connective tissue of the subepithelial and glandular layers, no significant effect of the oestrous Cycle stage was found on the number of SWC3-expressing cells. The present study showed a variation in the distribution of SWC3-expressing cells in the sow endometrium, especially in the surface and glandular epithelium, during different stages of the oestrous cycle.

Cell cycle-related fluctuations in transcellular ionic currents and plasma membrane Ca2+/Mg2+ ATPase activity during early cleavages of Lymnaea stagnalis embryos.

PubMed

Zivkovic, Danica; Créton, Robbert; Dohmen, René

1991-08-01

During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca 2+ -stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 μA/cm 2 have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca 2+ /Mg 2+ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca 2+ -stimulated ATPase. The ionic currents and Ca 2+ -stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca 2+ -stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca 2+ -stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase.

The effect of Bcr-Abl protein tyrosine kinase on maturation and proliferation of primitive haematopoietic cells.

PubMed Central

Buckle, A. M.; Mottram, R.; Pierce, A.; Lucas, G. S.; Russell, N.; Miyan, J. A.; Whetton, A. D.

2000-01-01

BACKGROUND: Chronic Myeloid Leukaemia (CML) is characterised by the chromosomal translocation resulting in expression of the Bcr-Abl protein tyrosine kinase (PTK) in early stem cells and their progeny. However the precise nature of Bcr-Abl effects in primitive CML stem cells remains a matter of active debate. MATERIALS AND METHODS: Extremely primitive Bcr-Abl fusion positive cells were purified from patients with CML using multiparameter flow cytometric analysis of CD34, Thy, and lineage marker (Lin) expression, plus rhodamine-123 (Rh-123) brightness. Progenitor cells of increasing maturity were examined for cycling status by flow cytometry and their proliferative status directly correlated with cell phenotype. The activation status of a key transcription factor, signal transducers and activators of transcription (STAT-5), was also analyzed by immunocytochemistry. RESULTS: The most primitive stem cells currently defined (CD34+Lin-Thy+ Rh-1231o) were present as a lower proportion of the stem cell compartment (CD34+Lin-) of CML patients at presentation than of normal individuals (2.3% +/- 0.4 compared with 5.1% +/- 0.6 respectively). Conversely there was a significantly higher proportion of the more mature cells (CD34+Lin-Thy-Rh-123 hi) in CML patients than in normal individuals (79.3 +/- 1.8 compared with 70.9 +/- 3.3). No primitive subpopulation of CML CD34+Lin- cells was cycling to a significantly greater degree than cells from normal donors, in fact, late progenitor cells (CD34+Lin+) were cycling significantly less in CML samples than normal samples. STAT5, however, was observed to be activated in CML cells. CONCLUSIONS: We conclude that no subpopulation of CML stem cells displays significantly increased cell cycling. Thus, increased cycling cannot be a direct consequence of Bcr-Abl PTK acquisition in highly enriched stem cells from patients with CML. In vivo CML need not be considered a disease of unbridled stem cell proliferation, but a subtle defect in the balance between self renewal and maturation. PMID:11126203

Cytokinetics of adult rat SVZ after EAE.

PubMed

Sajad, Mir; Chawla, Raman; Zargan, Jamil; Umar, Sadiq; Sadaqat, Mir; Khan, Haider A

2011-01-31

Cytokinetics regulating cell cycle division can be modulated by several endogenous factors. EAE (experimental autoimmune encephalomyelitis) increases proliferation of progenitor cells in the subventricular zone (SVZ). Using cumulative and single S phase labeling with 5-bromo-2-deoxyuridine, we examined cell cycle kinetics of neural progenitor cells in the SVZ after EAE. 20% of the SVZ cell population was proliferating in adjuvant control rats. However, EAE significantly increased them up to 27% and these cells had a cell cycle length (TC) of 15.6h, significantly (P<0.05) shorter than the 19 h TC in non EAE SVZ cells. Few TUNEL (+) cells were detected in the SVZ cells of adjuvant controls. EAE increased (P<0.05) TUNEL (+) nuclei in SVZ suggesting early stage progenitor cell death. Cell cycle phase analysis revealed that EAE substantially shortened the length of the G1 phase (9.6h) compared with the G1 phase of 12.25 h in adjuvant control SVZ cells (P<0.05). This reduction in G1 contributes to EAE-induced reduction of TC because no significant changes were detected on the length of S, G2 and M phases between the two groups. Our results show a surge in proliferating progenitor cells in the SVZ with concomitant increase in apoptotic cell death after EAE. Furthermore, increase in the SVZ proliferation contributes to EAE-induced neurogenesis and this increase is regulated by shortening the G1 phase. Our investigation suggests the activation of quiescent cells in SVZ to generate actively proliferating progenitors. Moreover, the increase in the cell death in proliferating population may contribute towards negative regulation of proliferative cell number and hence diminished regenerative capacity of CNS following EAE. Copyright © 2010 Elsevier B.V. All rights reserved.

Adaptor proteins NUMB and NUMBL promote cell cycle withdrawal by targeting ERBB2 for degradation

PubMed Central

Hirai, Maretoshi; Arita, Yoh; McGlade, C. Jane; Lee, Kuo-Fen; Chen, Ju; Evans, Sylvia M.

2017-01-01

Failure of trabecular myocytes to undergo appropriate cell cycle withdrawal leads to ventricular noncompaction and heart failure. Signaling of growth factor receptor ERBB2 is critical for myocyte proliferation and trabeculation. However, the mechanisms underlying appropriate downregulation of trabecular ERBB2 signaling are little understood. Here, we have found that the endocytic adaptor proteins NUMB and NUMBL were required for downregulation of ERBB2 signaling in maturing trabeculae. Loss of NUMB and NUMBL resulted in a partial block of late endosome formation, resulting in sustained ERBB2 signaling and STAT5 activation. Unexpectedly, activated STAT5 overrode Hippo-mediated inhibition and drove YAP1 to the nucleus. Consequent aberrant cardiomyocyte proliferation resulted in ventricular noncompaction that was markedly rescued by heterozygous loss of function of either ERBB2 or YAP1. Further investigations revealed that NUMB and NUMBL interacted with small GTPase Rab7 to transition ERBB2 from early to late endosome for degradation. Our studies provide insight into mechanisms by which NUMB and NUMBL promote cardiomyocyte cell cycle withdrawal and highlight previously unsuspected connections between pathways that are important for cardiomyocyte cell cycle reentry, with relevance to ventricular noncompaction cardiomyopathy and regenerative medicine. PMID:28067668

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

PubMed Central

Arzumanyan, Alla; Kulathinal, Rob J.; Blain, Stacy W.; Holcombe, Randall F.; Mahajna, Jamal; Marino, Maria; Martinez-Chantar, Maria L.; Nawroth, Roman; Sanchez-Garcia, Isidro; Sharma, Dipali; Saxena, Neeraj K.; Singh, Neetu; Vlachostergios, Panagiotis J.; Guo, Shanchun; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G.; Amedei, Amedeo; Niccolai, Elena; Amin, Amr; Ashraf, S. Salman; Boosani, Chandra S.; Guha, Gunjan; Ciriolo, Maria Rosa; Aquilano, Katia; Chen, Sophie; Mohammed, Sulma I.; Azmi, Asfar S.; Bhakta, Dipita; Halicka, Dorota; Nowsheen, Somaira

2016-01-01

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). This data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression. PMID:25892662

Meioc maintains an extended meiotic prophase I in mice

PubMed Central

Soh, Y. Q. Shirleen; Godfrey, Alexander K.; de Rooij, Dirk G.; Page, David C.

2017-01-01

The meiosis-specific chromosomal events of homolog pairing, synapsis, and recombination occur over an extended meiotic prophase I that is many times longer than prophase of mitosis. Here we show that, in mice, maintenance of an extended meiotic prophase I requires the gene Meioc, a germ-cell specific factor conserved in most metazoans. In mice, Meioc is expressed in male and female germ cells upon initiation of and throughout meiotic prophase I. Mouse germ cells lacking Meioc initiate meiosis: they undergo pre-meiotic DNA replication, they express proteins involved in synapsis and recombination, and a subset of cells progress as far as the zygotene stage of prophase I. However, cells in early meiotic prophase—as early as the preleptotene stage—proceed to condense their chromosomes and assemble a spindle, as if having progressed to metaphase. Meioc-deficient spermatocytes that have initiated synapsis mis-express CYCLIN A2, which is normally expressed in mitotic spermatogonia, suggesting a failure to properly transition to a meiotic cell cycle program. MEIOC interacts with YTHDC2, and the two proteins pull-down an overlapping set of mitosis-associated transcripts. We conclude that when the meiotic chromosomal program is initiated, Meioc is simultaneously induced so as to extend meiotic prophase. Specifically, MEIOC, together with YTHDC2, promotes a meiotic (as opposed to mitotic) cell cycle program via post-transcriptional control of their target transcripts. PMID:28380054

Changes of ploidy during the Azotobacter vinelandii growth cycle.

PubMed Central

Maldonado, R; Jiménez, J; Casadesús, J

1994-01-01

The size of the Azotobacter vinelandii chromosome is approximately 4,700 kb, as calculated by pulsed-field electrophoretic separation of fragments digested with the rarely cutting endonucleases SpeI and SwaI. Surveys of DNA content per cell by flow cytometry indicated the existence of ploidy changes during the A. vinelandii growth cycle in rich medium. Early-exponential-phase cells have a ploidy level similar to that of Escherichia coli or Salmonella typhimurium (probably ca. four chromosomes per cell), but a continuous increase of DNA content per cell is observed during growth. Late-exponential-phase cells may contain > 40 chromosomes per cell, while cells in the early stationary stage may contain > 80 chromosomes per cell. In late-stationary-phase cultures, the DNA content per cell is even higher, probably over 100 chromosome equivalents per cell. A dramatic change is observed in old stationary-phase cultures, when the population of highly polyploid bacteria segregates cells with low ploidy. The DNA content of the latter cells resembles that of cysts, suggesting that the process may reflect the onset of cyst differentiation. Cells with low ploidy are also formed when old stationary-phase cultures are diluted into fresh medium. Addition of rifampin to exponential-phase cultures causes a rapid increase in DNA content, indicating that A. vinelandii initiates multiple rounds of chromosome replication per cell division. Growth in minimal medium does not result in the spectacular changes of ploidy observed during rapid growth; this observation suggests that the polyploidy of A. vinelandii may not exist outside the laboratory. Images PMID:8021173

Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression.

PubMed

Nordin, Noraziah; Majid, Nazia Abdul; Hashim, Najihah Mohd; Rahman, Mashitoh Abd; Hassan, Zalila; Ali, Hapipah Mohd

2015-01-01

Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent.

Isolation, characterization, and genetic complementation of a cellular mutant resistant to retroviral infection

PubMed Central

Agarwal, Sumit; Harada, Josephine; Schreifels, Jeffrey; Lech, Patrycja; Nikolai, Bryan; Yamaguchi, Tomoyuki; Chanda, Sumit K.; Somia, Nikunj V.

2006-01-01

By using a genetic screen, we have isolated a mammalian cell line that is resistant to infection by retroviruses that are derived from the murine leukemia virus, human immunodeficiency virus type 1, and feline immunodeficiency virus. We demonstrate that the cell line is genetically recessive for the resistance, and hence it is lacking a factor enabling infection by retroviruses. The block to infection is early in the life cycle, at the poorly understood uncoating stage. We implicate the proteasome at uncoating by completely rescuing the resistant phenotype with the proteasomal inhibitor MG-132. We further report on the complementation cloning of a gene (MRI, modulator of retrovirus infection) that can also act to reverse the inhibition of infection in the mutant cell line. These data implicate a role for the proteasome during uncoating, and they suggest that MRI is a regulator of this activity. Finally, we reconcile our findings and other published data to suggest a model for the involvement of the proteasome in the early phase of the retroviral life cycle. PMID:17043244

Qualification testing of general electric 50 Ah nickel-cadmium cells with new separator and new positive plate processing

NASA Astrophysics Data System (ADS)

Morrow, G. W.

1986-09-01

Forty-two 50 Ah aerospace nickel-cadmium cells were delivered to Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985, for the purpose of evaluating and qualifying a new nylon separator material Pellon 2536, and the new GE Positive Plate Nickel Attack Control Passivation process. Testing began in May, 1985, at the Naval Weapons Support Center (NWSC) in Crane, Indiana with standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985, with approximately 1200 LEO cycles complete at this writting. Early test results show that cells with positive plate passivation exhibit higher than normal charge voltage characteristics. Other aspects of performance were nominal.

Qualification Testing of General Electric 50 Ah Nickel-Cadmium Cells with New Separator and New Positive Plate Processing

NASA Technical Reports Server (NTRS)

Morrow, G. W.

1986-01-01

Forty-two 50 Ah aerospace nickel-cadmium cells were delivered to Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985, for the purpose of evaluating and qualifying a new nylon separator material Pellon 2536, and the new GE Positive Plate Nickel Attack Control Passivation process. Testing began in May, 1985, at the Naval Weapons Support Center (NWSC) in Crane, Indiana with standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985, with approximately 1200 LEO cycles complete at this writting. Early test results show that cells with positive plate passivation exhibit higher than normal charge voltage characteristics. Other aspects of performance were nominal.

HPV-16 virions can remain infectious for 2 weeks on senescent cells but require cell cycle re-activation to allow virus entry.

PubMed

Broniarczyk, Justyna; Ring, Nadja; Massimi, Paola; Giacca, Mauro; Banks, Lawrence

2018-01-16

Successful infection with Human Papillomaviruses requires mitosis, when incoming viral genomes gain access to nuclear components. However, very little is known about how long HPV particles can remain infectious in non-dividing cells or in which cellular compartments these viruses may reside. To investigate these questions we have used BJ cells as a reversible model of senescence and show that HPV-16 can only infect early-passage proliferating cells. Late-passage senescent cells are resistant to HPV infection, but this can be reversed by inducing cell cycle re-entry with a p53 siRNA. In senescent cells we find that efficient virus entry can be attained upon cell cycle re-entry 16 days after infection, demonstrating that HPV can persist for 2 weeks prior to induction of mitosis. However, exposing cells to anti-HPV-16 L1 neutralising antibody blocks infection at these late time points, suggesting that the virions reside near the cell surface. Indeed, immunofluorescence analysis shows that virions accumulate on the cell surface of senescent cells and only enter endocytic vesicles upon stimulation with p53 siRNA. These results demonstrate that HPV-16 virions can remain viable on a non-dividing cell for extended periods of time, but are nonetheless vulnerable to antibody-induced neutralisation throughout.

Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism

PubMed Central

Foe, Ian T.; Foster, Scott A.; Cheung, Stephanie K.; DeLuca, Steven Z.; Morgan, David O.; Toczyski, David P.

2012-01-01

SUMMARY Background Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the Anaphase Promoting Complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood. Results Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule, but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20’s C-box. Cdc20 turnover by this intramolecular mechanism is cell cycle-regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination. Conclusion We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle. PMID:22079111

Polyoma small T antigen triggers cell death via mitotic catastrophe

PubMed Central

Fernando, Arun T Pores; Andrabi, Shaida; Cizmecioglu, Onur; Zhu, Cailei; Livingston, David M.; Higgins, Jonathan M.G; Schaffhausen, Brian S; Roberts, Thomas M

2014-01-01

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST-expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, resulting in the activation of the Spindle Assembly Checkpoint (SAC). Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed that, PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors. PMID:24998850

Temporally distinct transcriptional regulation of myocyte dedifferentiation and Myofiber growth during muscle regeneration.

PubMed

Louie, Ke'ale W; Saera-Vila, Alfonso; Kish, Phillip E; Colacino, Justin A; Kahana, Alon

2017-11-09

Tissue regeneration requires a series of steps, beginning with generation of the necessary cell mass, followed by cell migration into damaged area, and ending with differentiation and integration with surrounding tissues. Temporal regulation of these steps lies at the heart of the regenerative process, yet its basis is not well understood. The ability of zebrafish to dedifferentiate mature "post-mitotic" myocytes into proliferating myoblasts that in turn regenerate lost muscle tissue provides an opportunity to probe the molecular mechanisms of regeneration. Following subtotal excision of adult zebrafish lateral rectus muscle, dedifferentiating residual myocytes were collected at two time points prior to cell cycle reentry and compared to uninjured muscles using RNA-seq. Functional annotation (GAGE or K-means clustering followed by GO enrichment) revealed a coordinated response encompassing epigenetic regulation of transcription, RNA processing, and DNA replication and repair, along with protein degradation and translation that would rewire the cellular proteome and metabolome. Selected candidate genes were phenotypically validated in vivo by morpholino knockdown. Rapidly induced gene products, such as the Polycomb group factors Ezh2 and Suz12a, were necessary for both efficient dedifferentiation (i.e. cell reprogramming leading to cell cycle reentry) and complete anatomic regeneration. In contrast, the late activated gene fibronectin was important for efficient anatomic muscle regeneration but not for the early step of myocyte cell cycle reentry. Reprogramming of a "post-mitotic" myocyte into a dedifferentiated myoblast requires a complex coordinated effort that reshapes the cellular proteome and rewires metabolic pathways mediated by heritable yet nuanced epigenetic alterations and molecular switches, including transcription factors and non-coding RNAs. Our studies show that temporal regulation of gene expression is programmatically linked to distinct steps in the regeneration process, with immediate early expression driving dedifferentiation and reprogramming, and later expression facilitating anatomical regeneration.

Testicular histology and germ cell cytology during spermatogenesis in the Mississippi map turtle, Graptemys pseudogeographica kohnii, from Northeast Arkansas.

PubMed

Lancaster, Kelsey; Trauth, Stanley E; Gribbins, Kevin M

2014-01-01

The testicular histology and cytology of spermatogenesis in Graptemys pseudogeographica kohnii were examined using specimens collected between July 1996 and May 2004 from counties in northeastern Arkansas. A histological examination of the testes and germ cell cytology indicates a postnuptial testicular cycle of spermatogenesis and a major fall spermiation event. The majority of the germ cell populations in May and June specimens are represented by resting spermatogonia, type A spermatogonia, type B spermatogonia, pre-leptotene spermatocytes, and numerous Sertoli cell nuclei near the basement membrane. The start of proliferation is evident as spermatogonia in metaphase are present near the basal lamina and many of these germ cells have entered meiosis in June seminiferous tubules. Major spermatogenic events occur in the June and July specimens and result in an increased height of the seminiferous epithelium and increased diameter of the seminiferous tubules. The germ cell population during this time is represented by spermatogonia (type A, B, and resting), hypertrophic cells, large populations of early primary spermatocytes, and early round spermatids. By September, the major germ cell population has progressed past meiosis with abundant round and early elongating spermatids dominating the seminiferous epithelium. October seminiferous epithelia are marked by a decreas in height and mature spermatozoa fill the luminal space. Round and elongating spermatids constitute the largest portion of the germ cell population. Following the spermiation event, the testes enter a period of quiescence that lasts till the next spermatogenic cycle, which begins in the subsequent spring. Based on the cytological development of the seminiferous tubules revealed by our study, Graptemys pseudogeographica kohnii demonstrates a temporal germ cell development strategy similar to other temperate reptiles. A single major generation of germ cells progresses through spermatogenesis each year resulting in a single spermiation event with sperm stored within the epididymis until the next spring mating season.

Testicular histology and germ cell cytology during spermatogenesis in the Mississippi map turtle, Graptemys pseudogeographica kohnii, from Northeast Arkansas

PubMed Central

Lancaster, Kelsey; Trauth, Stanley E; Gribbins, Kevin M

2014-01-01

The testicular histology and cytology of spermatogenesis in Graptemys pseudogeographica kohnii were examined using specimens collected between July 1996 and May 2004 from counties in northeastern Arkansas. A histological examination of the testes and germ cell cytology indicates a postnuptial testicular cycle of spermatogenesis and a major fall spermiation event. The majority of the germ cell populations in May and June specimens are represented by resting spermatogonia, type A spermatogonia, type B spermatogonia, pre-leptotene spermatocytes, and numerous Sertoli cell nuclei near the basement membrane. The start of proliferation is evident as spermatogonia in metaphase are present near the basal lamina and many of these germ cells have entered meiosis in June seminiferous tubules. Major spermatogenic events occur in the June and July specimens and result in an increased height of the seminiferous epithelium and increased diameter of the seminiferous tubules. The germ cell population during this time is represented by spermatogonia (type A, B, and resting), hypertrophic cells, large populations of early primary spermatocytes, and early round spermatids. By September, the major germ cell population has progressed past meiosis with abundant round and early elongating spermatids dominating the seminiferous epithelium. October seminiferous epithelia are marked by a decreas in height and mature spermatozoa fill the luminal space. Round and elongating spermatids constitute the largest portion of the germ cell population. Following the spermiation event, the testes enter a period of quiescence that lasts till the next spermatogenic cycle, which begins in the subsequent spring. Based on the cytological development of the seminiferous tubules revealed by our study, Graptemys pseudogeographica kohnii demonstrates a temporal germ cell development strategy similar to other temperate reptiles. A single major generation of germ cells progresses through spermatogenesis each year resulting in a single spermiation event with sperm stored within the epididymis until the next spring mating season. PMID:26413408

Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation

PubMed Central

Sellers, Katherine; Fox, Matthew P.; Bousamra, Michael; Slone, Stephen P.; Higashi, Richard M.; Miller, Donald M.; Wang, Yali; Yan, Jun; Yuneva, Mariia O.; Deshpande, Rahul; Lane, Andrew N.; Fan, Teresa W.-M.

2015-01-01

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non–small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation. PMID:25607840

Inhibition of cell proliferation by nobiletin, a dietary phytochemical, associated with apoptosis and characteristic gene expression, but lack of effect on early rat hepatocarcinogenesis in vivo.

PubMed

Ohnishi, Hiroyuki; Asamoto, Makoto; Tujimura, Kazunari; Hokaiwado, Naomi; Takahashi, Satoru; Ogawa, Kumiko; Kuribayashi, Masanori; Ogiso, Tadashi; Okuyama, Harumi; Shirai, Tomoyuki

2004-12-01

Dietary phytochemicals can inhibit the development of certain types of tumors. We here investigated the effects of nobiletin (Nob), garcinol (Gar), auraptene (Aur), beta-cryptoxanthin- and hesperidine-rich pulp (CHRP) and 1,1'-acetoxychavicol acetate (ACA) on hepatocarcinogenesis in a rat medium-term liver bioassay, and also examined their influence on cell proliferation, cell cycle kinetics, apoptosis and cell invasion of rat and human hepatocellular carcinoma (HCC) cells, MH1C1 and HepG2, respectively. While there were no obvious suppressive effects on the development of putative preneoplastic liver lesions, inhibition of hepatocarcinoma cell proliferation was evident in the Nob group. Nob also caused G2/M cell cycle arrest and apoptosis. Microarray analysis identified a set of genes specifically regulated by Nob, and these are likely to be involved in the observed growth suppression of HCC cells. These results suggest that phytochemicals might have chemopreventive potential in late stages of hepatocarcinogenesis.

BM88 is an early marker of proliferating precursor cells that will differentiate into the neuronal lineage.

PubMed

Koutmani, Yassemi; Hurel, Catherine; Patsavoudi, Evangelia; Hack, Michael; Gotz, Magdalena; Thomaidou, Dimitra; Matsas, Rebecca

2004-11-01

Progression of progenitor cells towards neuronal differentiation is tightly linked with cell cycle control and the switch from proliferative to neuron-generating divisions. We have previously shown that the neuronal protein BM88 drives neuroblastoma cells towards exit from the cell cycle and differentiation into a neuronal phenotype in vitro. Here, we explored the role of BM88 during neuronal birth, cell cycle exit and the initiation of differentiation in vivo. By double- and triple-labelling with the S-phase marker BrdU or the late G2 and M-phase marker cyclin B1, antibodies to BM88 and markers of the neuronal or glial cell lineages, we demonstrate that in the rodent forebrain, BM88 is expressed in multipotential progenitor cells before terminal mitosis and in their neuronal progeny during the neurogenic interval, as well as in the adult. Further, we defined at E16 a cohort of proliferative progenitors that exit S phase in synchrony, and by following their fate for 24 h we show that BM88 is associated with the dynamics of neuron-generating divisions. Expression of BM88 was also evident in cycling cortical radial glial cells, which constitute the main neurogenic population in the cerebral cortex. In agreement, BM88 expression was markedly reduced and restricted to a smaller percentage of cells in the cerebral cortex of the Small eye mutant mice, which lack functional Pax6 and exhibit severe neurogenesis defects. Our data show an interesting correlation between BM88 expression and the progression of progenitor cells towards neuronal differentiation during the neurogenic interval.

Inhibition of p38 MAP kinase pathway induces apoptosis and prevents Epstein Barr virus reactivation in Raji cells exposed to lytic cycle inducing compounds

PubMed Central

Matusali, Giulia; Arena, Giuseppe; De Leo, Alessandra; Di Renzo, Livia; Mattia, Elena

2009-01-01

Background EBV lytic cycle activators, such as phorbol esters, anti-immunoglobulin, transforming growth factor β (TGFβ), sodium butyrate, induce apoptosis in EBV-negative but not in EBV-positive Burkitt's lymphoma (BL) cells. To investigate the molecular mechanisms allowing EBV-infected cells to be protected, we examined the expression of viral and cellular antiapoptotic proteins as well as the activation of signal transduction pathways in BL-derived Raji cells exposed to lytic cycle inducing agents. Results Our data show that, following EBV activation, the latent membrane protein 1 (LMP1) and the cellular anti-apoptotic proteins MCL-1 and BCL-2 were quickly up-regulated and that Raji cells remained viable even when exposed simultaneously to P(BU)2, sodium butyrate and TGFβ. We report here that inhibition of p38 pathway, during EBV activation, led to a three fold increment of apoptosis and largely prevented lytic gene expression. Conclusion These findings indicate that, during the switch from the latent to the lytic phase of EBV infection, p38 MAPK phosphorylation plays a key role both for protecting the host cells from apoptosis as well as for inducing viral reactivation. Because Raji cells are defective for late antigens expression, we hypothesize that the increment of LMP1 gene expression in the early phases of EBV lytic cycle might contribute to the survival of the EBV-positive cells. PMID:19272151

MRI surveillance of cancer cell fate in a brain metastasis model after early radiotherapy.

PubMed

Murrell, Donna H; Zarghami, Niloufar; Jensen, Michael D; Dickson, Fiona; Chambers, Ann F; Wong, Eugene; Foster, Paula J

2017-10-01

Incidence of brain metastasis attributed to breast cancer is increasing and prognosis is poor. It is thought that disseminated dormant cancer cells persist in metastatic organs and may evade treatments, thereby facilitating a mechanism for recurrence. Radiotherapy is used to treat brain metastases clinically, but assessment has been limited to macroscopic tumor volumes detectable by clinical imaging. Here, we use cellular MRI to understand the concurrent responses of metastases and nonproliferative or slowly cycling cancer cells to radiotherapy. MRI cell tracking was used to investigate the impact of early cranial irradiation on the fate of individual iron-labeled cancer cells and outgrowth of breast cancer brain metastases in the human MDA-MB-231-BR-HER2 cell model. Early whole-brain radiotherapy significantly reduced the outgrowth of metastases from individual disseminated cancer cells in treated animals compared to controls. However, the numbers of nonproliferative iron-retaining cancer cells in the brain were not significantly different. Radiotherapy, when given early in cancer progression, is effective in preventing the outgrowth of solitary cancer cells to brain metastases. Future studies of the nonproliferative cancer cells' clonogenic potentials are warranted, given that their persistent presence suggests that they may have evaded treatment. Magn Reson Med 78:1506-1512, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

PubMed

Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

2011-02-15

At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

Prevalence of Circulating Tumor Cells After Adjuvant Chemotherapy With or Without Anthracyclines in Patients With HER2-negative, Hormone Receptor-positive Early Breast Cancer.

PubMed

Schramm, Amelie; Schochter, Fabienne; Friedl, Thomas W P; de Gregorio, Nikolaus; Andergassen, Ulrich; Alunni-Fabbroni, Marianna; Trapp, Elisabeth; Jaeger, Bernadette; Heinrich, Georg; Camara, Oumar; Decker, Thomas; Ober, Angelika; Mahner, Sven; Fehm, Tanja N; Pantel, Klaus; Fasching, Peter A; Schneeweiss, Andreas; Janni, Wolfgang; Rack, Brigitte K

2017-07-01

Use of anthracycline-based chemotherapy in patients with early breast cancer (EBC) has been well-established but is often associated with cardiotoxicity. Based on data suggesting a limited benefit of anthracyclines in human epidermal growth factor receptor 2 (HER2)-negative patients, the Simultaneous Study of Docetaxel Based Anthracycline Free Adjuvant Treatment Evaluation, as well as Life Style Intervention Strategies (SUCCESS) C study randomized patients to either anthracycline-containing or anthracycline-free chemotherapy. Given the proven prognostic value of circulating tumor cells (CTCs) in EBC, we compared the prevalence of CTCs after chemotherapy between both treatment arms for a preliminary efficacy assessment. The SUCCESS C trial (NCT00847444) is an open-label, phase III study randomizing 3547 patients with HER2-negative EBC to either 3 cycles of epirubicin, 5-fluorouracil, and cyclophosphamide followed by 3 cycles of docetaxel (FEC-DOC) or 6 cycles of docetaxel and cyclophosphamide (DOC-C). CTC status was prospectively evaluated in hormone receptor-positive patients at the time of last chemotherapy cycle using the US Food and Drug Administration-approved CellSearch System (Janssen Diagnostics). Data on CTC status were available for 1766 patients. Overall, CTCs were found in 221 (12.5%) patients. Univariate analyses revealed that presence of CTCs at time of last chemotherapy cycle was not significantly associated with tumor or patient characteristics (all P > .1). There was no significant difference with respect to presence of CTCs between patients randomized to FEC-DOC or DOC-C (11.5% vs. 13.6%; P = .18). The comparable prevalence of CTCs at the time of last chemotherapy cycle may indicate that anthracycline-free chemotherapy is equally effective to anthracycline-containing chemotherapy in HER2-negative, hormone receptor-positive EBC. However, efficacy data from the final survival analysis of SUCCESS C have to be awaited to confirm these preliminary findings. Copyright © 2017 Elsevier Inc. All rights reserved.

Circadian expression of clock genes in human oral mucosa and skin: association with specific cell-cycle phases.

PubMed

Bjarnason, G A; Jordan, R C; Wood, P A; Li, Q; Lincoln, D W; Sothern, R B; Hrushesky, W J; Ben-David, Y

2001-05-01

We studied the relative RNA expression of clock genes throughout one 24-hour period in biopsies obtained from the oral mucosa and skin from eight healthy diurnally active male study participants. We found that the human clock genes hClock, hTim, hPer1, hCry1, and hBmal1 are expressed in oral mucosa and skin, with a circadian profile consistent with that found in the suprachiasmatic nuclei and the peripheral tissues of rodents. hPer1, hCry1, and hBmal1 have a rhythmic expression, peaking early in the morning, in late afternoon, and at night, respectively, whereas hClock and hTim are not rhythmic. This is the first human study to show a circadian profile of expression for all five clock genes as documented in rodents, suggesting their functional importance in man. In concurrent oral mucosa biopsies, thymidylate synthase enzyme activity, a marker for DNA synthesis, had a circadian variation with peak activity in early afternoon, coinciding with the timing of S phase in our previous study on cell-cycle timing in human oral mucosa. The major peak in hPer1 expression occurs at the same time of day as the peak in G(1) phase in oral mucosa, suggesting a possible link between the circadian clock and the mammalian cell cycle.

Prognostic significance of cell cycle proteins and genomic instability in borderline, early and advanced stage ovarian carcinomas.

PubMed

Blegen, H.; Einhorn, N.; Sjövall, K.; Roschke, A.; Ghadimi, B. M.; McShane, L. M.; Nilsson, B.; Shah, K.; Ried, T.; Auer, G.

2000-11-01

Disturbed cell cycle-regulating checkpoints and impairment of genomic stability are key events during the genesis and progression of malignant tumors. We analyzed 80 epithelial ovarian tumors of benign (n = 10) and borderline type (n = 18) in addition to carcinomas of early (n = 26) and advanced (n = 26) stages for the expression of Ki67, cyclin A and cyclin E, p21WAF-1, p27KIP-1 and p53 and correlated the results with the clinical course. Genomic instability was assessed by DNA ploidy measurements and, in 35 cases, by comparative genomic hybridization. Overexpression of cyclin A and cyclin E was observed in the majority of invasive carcinomas, only rarely in borderline tumors and in none of the benign tumors. Similarly, high expression of p53 together with undetectable p21 or loss of chromosome arm 17p were frequent events only in adenocarcinomas. Both borderline tumors and adenocarcinomas revealed a high number of chromosomal gains and losses. However, regional chromosomal amplifications were found to occur 13 times more frequently in the adenocarcinomas than in the borderline tumors. The expression pattern of low p27 together with high Ki67 was found to be an independent predictor of poor outcome in invasive carcinomas. The results provide a link between disturbed cell cycle regulatory proteins, chromosomal aberrations and survival in ovarian carcinomas.

Serum bioactive and immunoreactive luteinizing hormone and follicle-stimulating hormone levels in women with cycle abnormalities, with or without polycystic ovarian disease.

PubMed

Fauser, B C; Pache, T D; Lamberts, S W; Hop, W C; de Jong, F H; Dahl, K D

1991-10-01

Serum steroid, gonadotropin, and alpha-subunit levels were assessed in 35 women with cycle abnormalities [11 with and 24 without polycystic ovarian disease (PCOD) according to strict clinical and biochemical criteria] and 8 regularly cycling women in the early (cycle day 3 or 4) and mid (cycle day 7 or 8) follicular phase. LH and FSH levels were estimated using two immunological techniques [RIA and immunoradiometric assay (IRMA)] and in vitro bioassays (BIO), using mouse Leydig cells and rat granulosa cells, respectively. In PCOD patients mean alpha-subunit, free androgen index [FAI; testosterone x 100/sex hormone-binding globulin (SHBG)], androstenedione, estrone, and estradiol (E2) were significantly elevated compared to levels in the early follicular phase of control cycles and non-PCOD patients. In addition, in PCOD patients mean IRMA-LH and RIA-LH levels were distinctly increased (2.8- to 3.6 fold, respectively; both comparisons, P less than 0.001) compared to control values, but in the same order of magnitude (1.3- to 1.4-fold increments) as that in non-PCOD patients. However, the median BIO-LH level in PCOD patients was 5.9-fold higher than that in non-PCOD patients and 4.0-fold higher than the BIO-LH in the early follicular phase of control women. Consequently, the median BIO/IRMA-LH ratio was 4.8-fold higher in PCOD patients compared to non-PCOD patients. In women with cycle abnormalities, individual BIO/IRMA-LH ratios correlated with BIO-LH (rs = 0.48), FAI (rs = 0.39), free estrogens (E2/SHBG ratios; rs = 0 0.47), and dehydroepiandrosterone sulfate (rs = 0.60) concentrations. Mean IRMA-, RIA-, and BIO-FSH levels and BIO/IRMA-FSH ratios were not significantly different when various groups were compared. Although RIA- and IRMA-LH levels showed good correlation (rs = 0.88), RIA-LH levels were consistently higher, resulting in distinctly higher RIA-LH/FSH ratios (mean, 4.5) compared to IRMA-LH/FSH ratios (median, 1.8) in PCOD patients.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

PubMed

Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

2015-01-01

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

PubMed Central

Dorfman, Mauricio D.; Kerr, Bredford; Garcia-Rudaz, Cecilia; Paredes, Alfonso H.; Dissen, Gregory A.

2011-01-01

Tropomyosin-related kinase (TRK) receptor B (TRKB) mediates the supportive actions of neurotrophin 4/5 and brain-derived neurotrophic factor on early ovarian follicle development. Absence of TRKB receptors reduces granulosa cell (GC) proliferation and delays follicle growth. In the present study, we offer mechanistic insights into this phenomenon. DNA array and quantitative PCR analysis of ovaries from TrkB-null mice revealed that by the end of the first week of postnatal life, Jagged1, Hes1, and Hey2 mRNA abundance is reduced in the absence of TRKB receptors. Although Jagged1 encodes a NOTCH receptor ligand, Hes1 and Hey2 are downstream targets of the JAGGED1-NOTCH2 signaling system. Jagged1 is predominantly expressed in oocytes, and the abundance of JAGGED1 is decreased in TrkB−/− oocytes. Lack of TRKB receptors also resulted in reduced expression of c-Myc, a NOTCH target gene that promotes entry into the cell cycle, but did not alter the expression of genes encoding core regulators of cell-cycle progression. Selective restoration of JAGGED1 synthesis in oocytes of TrkB−/− ovaries via lentiviral-mediated transfer of the Jagged1 gene under the control of the growth differentiation factor 9 (Gdf9) promoter rescued c-Myc expression, GC proliferation, and follicle growth. These results suggest that neurotrophins acting via TRKB receptors facilitate early follicle growth by supporting a JAGGED1-NOTCH2 oocyte-to-GC communication pathway, which promotes GC proliferation via a c-MYC-dependent mechanism. PMID:22028443

Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

PubMed Central

Datta, Antara; Silverman, Lee; Phipps, Andrew J; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D

2007-01-01

Background Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. Results Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. Conclusion Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival. PMID:17634129

Evolution and the origin of the visual retinoid cycle in vertebrates.

PubMed

Kusakabe, Takehiro G; Takimoto, Noriko; Jin, Minghao; Tsuda, Motoyuki

2009-10-12

Absorption of a photon by visual pigments induces isomerization of 11-cis-retinaldehyde (RAL) chromophore to all-trans-RAL. Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called 'visual cycle'. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Here we compare visual retinoid cycles between different photoreceptors of vertebrates, including rods, cones and non-visual photoreceptors, as well as between vertebrates and invertebrates. The visual cycle systems in ascidians, the closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.

Histone modifications in the male germ line of Drosophila.

PubMed

Hennig, Wolfgang; Weyrich, Alexandra

2013-02-22

In the male germ line of Drosophila chromatin remains decondensed and highly transcribed during meiotic prophase until it is rapidly compacted. A large proportion of the cell cycle-regulated histone H3.1 is replaced by H3.3, a histone variant encoded outside the histone repeat cluster and not subject to cell cycle controlled expression. We investigated histone modification patterns in testes of D. melanogaster and D. hydei. In somatic cells of the testis envelope and in germ cells these modification patterns differ from those typically seen in eu- and heterochromatin of other somatic cells. During the meiotic prophase some modifications expected in active chromatin are not found or are found at low level. The absence of H4K16ac suggests that dosage compensation does not take place. Certain histone modifications correspond to either the cell cycle-regulated histone H3.1 or to the testis-specific variant H3.3. In spermatogonia we found H3K9 methylation in cytoplasmic histones, most likely corresponding to the H3.3 histone variant. Most histone modifications persist throughout the meiotic divisions. The majority of modifications persist until the early spermatid nuclei, and only a minority further persist until the final chromatin compaction stages before individualization of the spermatozoa. Histone modification patterns in the male germ line differ from expected patterns. They are consistent with an absence of dosage compensation of the X chromosome during the male meiotic prophase. The cell cycle-regulated histone variant H3.1 and H3.3, expressed throughout the cell cycle, also vary in their modification patterns. Postmeiotically, we observed a highly complex pattern of the histone modifications until late spermatid nuclear elongation stages. This may be in part due to postmeiotic transcription and in part to differential histone replacement during chromatin condensation.

Parvovirus-Induced Depletion of Cyclin B1 Prevents Mitotic Entry of Infected Cells

PubMed Central

Adeyemi, Richard O.; Pintel, David J.

2014-01-01

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication. PMID:24415942

Parvovirus-induced depletion of cyclin B1 prevents mitotic entry of infected cells.

PubMed

Adeyemi, Richard O; Pintel, David J

2014-01-01

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication.

The DNA damage response during mitosis.

PubMed

Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

2013-10-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Foxi3 deficiency compromises hair follicle stem cell specification and activation

PubMed Central

Shirokova, Vera; Biggs, Leah C.; Jussila, Maria; Ohyama, Takahiro; Groves, Andrew K.; Mikkola, Marja L.

2017-01-01

The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ. Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary hair germ marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary hair germ activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. PMID:26992132

Embryonic Cleavage Cycles: How Is a Mouse Like a Fly?

PubMed Central

O’Farrell, Patrick H.; Stumpff, Jason; Su, Tin Tin

2009-01-01

The evolutionary advent of uterine support of embryonic growth in mammals is relatively recent. Nonetheless, striking differences in the earliest steps of embryogenesis make it difficult to draw parallels even with other chordates. We suggest that use of fertilization as a reference point misaligns the earliest stages and masks parallels that are evident when development is aligned at conserved stages surrounding gastrulation. In externally deposited eggs from representatives of all the major phyla, gastrulation is preceded by specialized extremely rapid cleavage cell cycles. Mammals also exhibit remarkably fast cell cycles in close association with gastrulation, but instead of beginning development with these rapid cycles, the mammalian egg first devotes itself to the production of extraembryonic structures. Previous attempts to identify common features of cleavage cycles focused on post-fertilization divisions of the mammalian egg. We propose that comparison to the rapid peri-gastrulation cycles is more appropriate and suggest that these cycles are related by evolutionary descent to the early cleavage stages of embryos such as those of frog and fly. The deferral of events in mammalian embryogenesis might be due to an evolutionary shift in the timing of fertilization. PMID:14711435

Efficacy of plasma vascular endothelial growth factor in monitoring first-line chemotherapy in patients with advanced non-small cell lung cancer

PubMed Central

2009-01-01

Background Along with the development of new cancer therapeutics, more effective tools for the estimation of response to therapy and prediction of disease progression are required for the better management of inoperable cancer patients. Methods We studied 134 newly diagnosed and primarily untreated advanced non-small cell lung cancer patients and 100 controls. Forty two patients received platinum-based chemotherapy. Plasma VEGF levels were quantified in all samples at baseline and also before second and third chemotherapy cycle in 42 patients and correlated with response to therapy as assessed by computed tomography after the third chemotherapy cycle. Results We observed that, patients who went into remission had significantly lower baseline VEGF levels before second and third cycles of chemotherapy when compared with patients with no change and progression. Plasma VEGF levels showed a greater decrease from cycle 1 to 2 and from cycle 1 to 3 in patients who showed remission in comparison to those with no change or progression. Plasma VEGF levels before the second cycle detected poor response to therapy with a sensitivity and specificity of 76.9% and 75.0%, respectively (area under the ROC curve = 0.724). Early prediction of disease progression was achieved with a sensitivity and specificity of 71.4% for plasma VEGF before cycle 2 (area under the ROC curve = 0.805). The kinetics of VEGF form cycle 1 to 2 and cycle 1 to 3 also gave significant information for predicting disease progression as well as insufficient therapy response. Conclusion Monitoring of plasma VEGF levels during the course of first-line chemotherapy could identify patients who are likely to have insufficient response to therapy and disease progression at an early stage. This may help in individualizing treatment and could lead to better management of the advanced stage lung cancer. PMID:19958548

Apigetrin inhibits adipogenesis in 3T3-L1 cells by downregulating PPARγ and CEBP-α.

PubMed

Hadrich, Fatma; Sayadi, Sami

2018-04-25

Apigetrin, a flavonoid found in many plant leaves and seeds, has been known to possess antimutagenic, anti-cancer, antioxidant and anti-inflammatory properties. Here, we are investigating the effect of the apigetrin on adipocytes differentiation in 3T3-L1 adipocytes, and elucidating the mechanism of its action. Lipids accumulation was measured by Oil Red O staining and cell cycle was analyzed by flow cytometry. The antioxidant effect of apigetrin was evaluated against hydrogen peroxide. The expression of various genes, involved in adipogenesis and inflammation, was studied by real-time PCR. Our results showed that apigterin treatment inhibited significantly lipid accumulation without effect on cell viability at 100 μM, and it exerted the anti-adipogenic effect during the early stages of differentiation. Flow cytometry analysis showed that apigenin-7-O-glucoside (Ap7G) inhibited cell proliferation during mitotic clonal expansion and caused cell cycle delay. Quantitative PCR analysis revealed that the mRNA levels of C/EBP-α, PPAR-γ, SREBP-1c and FAS were suppressed after apigetrin treatment at 100 μM. Moreover, the mRNA level of pro-inflammatory genes (TNF-α and IL-6) were suppressed after apigterin treatment, at high concentration preadipocyte cells. Taken together, these results indicated that apigenin-7-O-glucoside inhibits adipogenesis of 3T3-L1 preadipocytes at early stage of adipogenesis.

Neuronal Cell Death Induced by Mechanical Percussion Trauma in Cultured Neurons is not Preceded by Alterations in Glucose, Lactate and Glutamine Metabolism.

PubMed

Jayakumar, A R; Bak, L K; Rama Rao, K V; Waagepetersen, H S; Schousboe, A; Norenberg, M D

2016-02-01

Traumatic brain injury (TBI) is a devastating neurological disorder that usually presents in acute and chronic forms. Brain edema and associated increased intracranial pressure in the early phase following TBI are major consequences of acute trauma. On the other hand, neuronal injury, leading to neurobehavioral and cognitive impairments, that usually develop months to years after single or repetitive episodes of head trauma, are major consequences of chronic TBI. The molecular mechanisms responsible for TBI-induced injury, however, are unclear. Recent studies have suggested that early mitochondrial dysfunction and subsequent energy failure play a role in the pathogenesis of TBI. We therefore examined whether oxidative metabolism of (13)C-labeled glucose, lactate or glutamine is altered early following in vitro mechanical percussion-induced trauma (5 atm) to neurons (4-24 h), and whether such events contribute to the development of neuronal injury. Cell viability was assayed using the release of the cytoplasmic enzyme lactate dehydrogenase (LDH), together with fluorescence-based cell staining (calcein and ethidium homodimer-1 for live and dead cells, respectively). Trauma had no effect on the LDH release in neurons from 1 to 18 h. However, a significant increase in LDH release was detected at 24 h after trauma. Similar findings were identified when traumatized neurons were stained with fluorescent markers. Additionally (13)C-labeling of glutamate showed a small, but statistically significant decrease at 14 h after trauma. However, trauma had no effect on the cycling ratio of the TCA cycle at any time-period examined. These findings indicate that trauma does not cause a disturbance in oxidative metabolism of any of the substrates used for neurons. Accordingly, such metabolic disturbance does not appear to contribute to the neuronal death in the early stages following trauma.

Anaphase-Promoting Complex/Cyclosome-Cdh1-Mediated Proteolysis of the Forkhead Box M1 Transcription Factor Is Critical for Regulated Entry into S Phase▿

PubMed Central

Park, Hyun Jung; Costa, Robert H.; Lau, Lester F.; Tyner, Angela L.; Raychaudhuri, Pradip

2008-01-01

The forkhead box M1 (FoxM1) transcription factor is overexpressed in many cancers, and in mouse models it is required for tumor progression. FoxM1 activates expression of the cell cycle genes required for both S and M phase progression. Here we demonstrate that FoxM1 is degraded in late mitosis and early G1 phase by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. FoxM1 interacts with the APC/C complex and its adaptor, Cdh1. Expression of Cdh1 stimulated degradation of the FoxM1 protein, and depletion of Cdh1 resulted in stabilization of the FoxM1 protein in late mitosis and in early G1 phase of the cell cycle. Cdh1 has been implicated in regulating S phase entry. We show that codepletion of FoxM1 inhibits early S phase entry observed in Cdh1-depleted cells. The N-terminal region of FoxM1 contains both destruction box (D box) and KEN box sequences that are required for targeting by Cdh1. Mutation of either the D box sequence or the KEN box sequence stabilized FoxM1 and blocked Cdh1-induced proteolysis. Cells expressing a nondegradable form of FoxM1 entered S phase rapidly following release from M phase arrest. Together, our observations show that FoxM1 is one of the targets of Cdh1 in late M or early G1 phase and that its proteolysis is important for regulated entry into S phase. PMID:18573889

Anaphase-promoting complex/cyclosome-CDH1-mediated proteolysis of the forkhead box M1 transcription factor is critical for regulated entry into S phase.

PubMed

Park, Hyun Jung; Costa, Robert H; Lau, Lester F; Tyner, Angela L; Raychaudhuri, Pradip

2008-09-01

The forkhead box M1 (FoxM1) transcription factor is overexpressed in many cancers, and in mouse models it is required for tumor progression. FoxM1 activates expression of the cell cycle genes required for both S and M phase progression. Here we demonstrate that FoxM1 is degraded in late mitosis and early G(1) phase by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. FoxM1 interacts with the APC/C complex and its adaptor, Cdh1. Expression of Cdh1 stimulated degradation of the FoxM1 protein, and depletion of Cdh1 resulted in stabilization of the FoxM1 protein in late mitosis and in early G(1) phase of the cell cycle. Cdh1 has been implicated in regulating S phase entry. We show that codepletion of FoxM1 inhibits early S phase entry observed in Cdh1-depleted cells. The N-terminal region of FoxM1 contains both destruction box (D box) and KEN box sequences that are required for targeting by Cdh1. Mutation of either the D box sequence or the KEN box sequence stabilized FoxM1 and blocked Cdh1-induced proteolysis. Cells expressing a nondegradable form of FoxM1 entered S phase rapidly following release from M phase arrest. Together, our observations show that FoxM1 is one of the targets of Cdh1 in late M or early G(1) phase and that its proteolysis is important for regulated entry into S phase.

The long-term label retaining population of the renal papilla arises through divergent regional growth of the kidney.

PubMed

Adams, Derek C; Oxburgh, Leif

2009-09-01

Long-term pulse chase experiments previously identified a sizable population of BrdU-retaining cells within the renal papilla. The origin of these cells has been unclear, and in this work we test the hypothesis that they become quiescent early during the course of kidney development and organ growth. Indeed, we find that BrdU-retaining cells of the papilla can be labeled only by pulsing with BrdU from embryonic (E) day 11.25 to postnatal (P) day 7, the approximate period of kidney development in the mouse. BrdU signal in the cortex and outer medulla is rapidly diluted by cellular proliferation during embryonic development and juvenile growth, whereas cells within the papilla differentiate and exit the cell cycle during organogenesis. Indeed, by E17.5, little or no active proliferation can be seen in the distal papilla, indicating maturation of this structure in a distal-to-proximal manner during organogenesis. We conclude that BrdU-retaining cells of the papilla represent a population of cells that quiesce during embryonic development and localize within a region of the kidney that matures early. We therefore propose that selective papillary retention of BrdU arises through a combination of regionalized slowing of, and exit from, the cell cycle within the papilla during the period of ongoing kidney development, and extensive proliferative growth of the juvenile kidney resulting in dilution of BrdU below the detection level in extra-papillary regions.

Expression of progesterone receptor protein in the ovine uterus during the estrous cycle: Effects of nutrition, arginine and FSH.

PubMed

Grazul-Bilska, Anna T; Thammasiri, Jiratti; Kraisoon, Aree; Reyaz, Arshi; Bass, Casie S; Kaminski, Samantha L; Navanukraw, Chainarong; Redmer, Dale A

2018-03-01

To evaluate expression of progesterone receptor (PGR) AB in follicle stimulating hormone (FSH)-treated or non-treated sheep administered with arginine (Arg) or saline (Sal) fed a control (C), excess (O) or restricted (U) diet, uterine tissues were collected at the early, mid and/or late luteal phases. In exp. 1, ewes from each diet were randomly assigned to one of two treatments, Arg or Sal administration three times daily from day 0 of the first estrous cycle until uterine tissue collection. In exp. 2, ewes were injected twice daily with FSH on days 13-15 of the first estrous cycle. Uterine tissues were immunostained to detect PGR followed by image analysis. PGR were detected in luminal epithelium (LE), endometrial glands (EG), endometrial stroma (ES), myometrium (Myo), and endometrial and myometrial blood vessels. The percentage of PR-positive cells and/or intensity of staining were affected by phase of the estrous cycle, plane of nutrition, and/or FSH but not by Arg. In exp. 1, percentage of PGR-positive cells in LE and EG but not in ES and Myo was greater at the early and mid than late luteal phase, was not affected by plane of nutrition, and was similar in LE and EG. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in LE, EG and Myo, and was the greatest in LE, less in EG, and least in ES and Myo. In exp. 2, percentage of PGR-positive cells in LE, EG, ES and Myo was affected by phase of the estrous cycle, but not by plane of nutrition; was greater at the early than mid luteal phase; and was greatest in LE and EG, less in luminal (superficial) ES and Myo and least in deep ES. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in all compartments but ES, and was the greatest in LE and luminal EG, less in deep EG, and least in ES and Myo. Comparison of data for FSH (superovulated) and Sal-treated (non-superovulated) ewes demonstrated that FSH affected PR expression in all evaluated uterine compartments depending on plane of nutrition and phase of the estrous cycle. Thus, PGR are differentially distributed in uterine compartments, and PGR expression is affected by nutritional plane and FSH, but not Arg depending on phase of the estrous cycle. Such changes in dynamics of PGR expression indicate that diet plays a regulatory role and that FSH-treatment may alter uterine functions. Copyright © 2017 Elsevier Inc. All rights reserved.

Abnormal neural precursor cell regulation in the early postnatal Fragile X mouse hippocampus.

PubMed

Sourial, Mary; Doering, Laurie C

2017-07-01

The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST + CD15 + CD133 + ) and an increased proportion of neuroblasts (PSA-NCAM + CD15 + ) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G 2 /M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

ISG15 Functions as an Interferon-Mediated Antiviral Effector Early in the Murine Norovirus Life Cycle

PubMed Central

Rodriguez, Marisela R.; Monte, Kristen; Thackray, Larissa B.

2014-01-01

ABSTRACT Human noroviruses (HuNoV) are the leading cause of nonbacterial gastroenteritis worldwide. Similar to HuNoV, murine noroviruses (MNV) are enteric pathogens spread via the fecal-oral route and have been isolated from numerous mouse facilities worldwide. Type I and type II interferons (IFN) restrict MNV-1 replication; however, the antiviral effectors impacting MNV-1 downstream of IFN signaling are largely unknown. Studies using dendritic cells, macrophages, and mice deficient in free and conjugated forms of interferon-stimulated gene 15 (ISG15) revealed that ISG15 conjugation contributes to protection against MNV-1 both in vitro and in vivo. ISG15 inhibited a step early in the viral life cycle upstream of viral genome transcription. Directly transfecting MNV-1 RNA into IFN-stimulated mouse embryonic fibroblasts (MEFs) and bone marrow-derived dendritic cells (BMDC) lacking ISG15 conjugates bypassed the antiviral activity of ISG15, further suggesting that ISG15 conjugates restrict the MNV-1 life cycle at the viral entry/uncoating step. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of early stages of MNV-1 replication. IMPORTANCE Type I IFNs are important in controlling murine norovirus 1 (MNV-1) infections; however, the proteins induced by IFNs that restrict viral growth are largely unknown. This report reveals that interferon-stimulated gene 15 (ISG15) mitigates MNV-1 replication both in vitro and in vivo. In addition, it shows that ISG15 inhibits MNV-1 replication by targeting an early step in the viral life cycle, MNV-1 entry and/or uncoating. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of viral entry/uncoating. PMID:24899198

DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

PubMed

Dutertre, Martin; Vagner, Stéphan

2017-10-27

Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differences between T cell-type and natural killer cell-type chronic active Epstein-Barr virus infection.

PubMed

Kimura, Hiroshi; Hoshino, Yo; Hara, Shinya; Sugaya, Naomi; Kawada, Jun-Ichi; Shibata, Yukiko; Kojima, Seiji; Nagasaka, Tetsuro; Kuzushima, Kiyotaka; Morishima, Tsuneo

2005-02-15

Infections of T cells and natural killer (NK) cells play a central role in the pathogenesis of chronic active Epstein-Barr virus (CAEBV) infection. To characterize the virologic and cytokine profiles of T cell-type and NK cell-type infection, 39 patients with CAEBV infection were analyzed. Patients with T cell-type infection had higher titers of immunoglobulin G against early and late EBV antigens, suggesting lytic cycle infection. However, the pattern of EBV gene expression was latency type II; BZLF1, which is a hallmark of lytic cycle infection, could not be detected in any patients, regardless of infection type. Patients with CAEBV infection had high concentrations of proinflammatory, T helper cell type 1, and anti-inflammatory cytokines. The cytokine profile in patients with NK cell-type infection was similar to that in patients with T cell-type infection, but the concentration of IL-13 was high in patients with NK cell-type infection. These findings should help to clarify the pathogenesis of CAEBV infection and facilitate the development of more-effective treatments.

FOXO1 opposition of CD8+ T cell effector programming confers early memory properties and phenotypic diversity.

PubMed

Delpoux, Arnaud; Lai, Chen-Yen; Hedrick, Stephen M; Doedens, Andrew L

2017-10-17

The factors and steps controlling postinfection CD8 + T cell terminal effector versus memory differentiation are incompletely understood. Whereas we found that naive TCF7 (alias "Tcf-1") expression is FOXO1 independent, early postinfection we report bimodal, FOXO1-dependent expression of the memory-essential transcription factor TCF7 in pathogen-specific CD8 + T cells. We determined the early postinfection TCF7 high population is marked by low TIM3 expression and bears memory signature hallmarks before the appearance of established memory precursor marker CD127 (IL-7R). These cells exhibit diminished TBET, GZMB, mTOR signaling, and cell cycle progression. Day 5 postinfection, TCF7 high cells express higher memory-associated BCL2 and EOMES, as well as increased accumulation potential and capacity to differentiate into memory phenotype cells. TCF7 retroviral transduction opposes GZMB expression and the formation of KLRG1 pos phenotype cells, demonstrating an active role for TCF7 in extinguishing the effector program and forestalling terminal differentiation. Past the peak of the cellular immune response, we report a gradient of FOXO1 and TCF7 expression, which functions to oppose TBET and orchestrate a continuum of effector-to-memory phenotypes.

Productive infection of Epstein-Barr virus (EBV) in EBV-genome-positive epithelial cell lines (GT38 and GT39) derived from gastric tissues.

PubMed

Takasaka, N; Tajima, M; Okinaga, K; Satoh, Y; Hoshikawa, Y; Katsumoto, T; Kurata, T; Sairenji, T

1998-08-01

We characterized the expression of Epstein-Barr virus (EBV) on two epithelial cell lines, GT38 and GT39, derived from human gastric tissues. The EBV nuclear antigen (EBNA) was detected in all cells of both cell lines. The EBV immediate-early BZLF 1 protein (ZEBRA), the early antigen diffuse component (EA-D), and one of the EBV envelope proteins (gp350/220) were expressed spontaneously in small proportions in the cells. EBNA 1, EBNA2, latent membrane protein 1, ZEBRA, and EA-D molecules were then observed by Western blotting in the cells. The lytic cycle was enhanced with treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or n-butyrate. The virus particles were observed in the TPA treated GT38 cells by electron microscopy. Infectious EBV was detected with the transformation of cord blood lymphocytes and also with the induction of early antigen to Raji cells by the supernatants of both cells lines. A major single and minor multiple fused terminal fragments and a ladder of smaller fragments of the EBV genome were detected with a Xhol probe in both cell lines. These epithelial cells lines and viruses will be useful in studying their association with EBV in gastric epithelial cells.

Ultrastructural features of vagina at different phases of the oestrous cycle in the female African giant rat (Cricetomys gambianus Waterhouse).

PubMed

Akinloye, Adebayo K; Oke, Bankole O

2014-06-01

The ultrastructures of the vagina at various stages of the oestrous cycle in female African giant rats (Cricetomys gambianus Waterhouse) were described in the present study. At mid-proestrus, late proestrus (LP)/early estrus (EE) and mid-estrus (ME) as well as late metestrus (LM)/early diestrus (ED) and mid-diestrus (MD), complex interface of epithelium and lamina propria were observed. Cells of the stratum basale formed finger-like extensions into the underlying lamina propria and tips of the extensions displayed hemidesmosome while basal lamina followed the contour of the extensions. At mid-metestrus (MM) and late diestrus/early proestrus, well developed, relatively straight basal lamina interfaced between the stratum basale and the lamina propria without finger-like projections. Polygonal cells with indented nuclei and, cytoplasm containing ribosomes, polysomes, intermediate filaments, and mitochondria were observed in stratum spinosum at all the phases of the oestrus cycle. At MM, LM/ED, and MD, the stratum spinosum had numerous desmosomes with tonofilaments, large microvilli that intermingled at the intercellular spaces and evidence of trapped/migrating neutrophils and lymphocytes. The superficial layer displayed short microvilli at mid-proestrus, cornification at LP/EE and desquamation at ME while it showed condensation of intermediate filaments; projections of large microvilli into the luminal surface at MM, and embeddement of neutrophils at LM/ED as well as MD. This study looked into the reproductive biology of female African giant rats to produce baseline information on its reproductive organs and represented the first comprehensive description of the vagina at the ultrastructural level during oestrous cycle. © 2014 Wiley Periodicals, Inc.

Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

PubMed Central

Amodeo, Amanda A.; Jukam, David; Straight, Aaron F.; Skotheim, Jan M.

2015-01-01

During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development. PMID:25713373

Kaempferol suppresses lipid accumulation by inhibiting early adipogenesis in 3T3-L1 cells and zebrafish.

PubMed

Lee, Yeon-Joo; Choi, Hyeon-Son; Seo, Min-Jung; Jeon, Hui-Jeon; Kim, Kui-Jin; Lee, Boo-Yong

2015-08-01

Kaempferol is a flavonoid present in Kaempferia galanga and Opuntia ficus indica var. saboten. Recent studies have suggested that it has anti-oxidant, anti-inflammatory, anti-cancer, and anti-obesity effects. In this study, we focused on the anti-adipogenic effects of kaempferol during adipocyte differentiation. The results showed that kaempferol inhibits lipid accumulation in adipocytes and zebrafish. Oil Red O and Nile Red staining showed that the number of intracellular lipid droplets decreased in adipocytes and zebrafish treated with kaempferol. LPAATθ (lysophosphatidic acid acyltransferase), lipin1, and DGAT1 (triglyceride synthetic enzymes) and FASN and SREBP-1C (fatty acid synthetic proteins) showed decreased expression levels in the presence of kaempferol. In addition, treatment of kaempferol showed an inhibitory activity on cell cycle progression. Kaempferol delayed cell cycle progression from the S to G2/M phase through the regulation of cyclins in a dose-dependent manner. Kaempferol blocked the phosphorylation of AKT (protein kinase B) and mammalian target of rapamycin (mTOR) signaling pathway during the early stages of adipogenesis. In addition, kaempferol down-regulated pro-early adipogenic factors such as CCAAT-enhancer binding proteins β (C/EBPβ), and Krüppel-like factors (KLFs) 4 and 5, while anti-early adipogenic factors, such as KLF2 and pref-1(preadipocyte factor-1), were upregulated. These kaempferol-mediated regulations of early adipogenic factors resulted in the attenuation of late adipogenic factors such as C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ). These results were supported in zebrafish based on the decrease in lipid accumulation and expression of adipogenic factors. Our results indicated that kaempferol might have an anti-obesity effect by regulating lipid metabolism.

Singlet oxygen generation as a major cause for parasitic reactions during cycling of aprotic lithium-oxygen batteries

NASA Astrophysics Data System (ADS)

Mahne, Nika; Schafzahl, Bettina; Leypold, Christian; Leypold, Mario; Grumm, Sandra; Leitgeb, Anita; Strohmeier, Gernot A.; Wilkening, Martin; Fontaine, Olivier; Kramer, Denis; Slugovc, Christian; Borisov, Sergey M.; Freunberger, Stefan A.

2017-03-01

Non-aqueous metal-oxygen batteries depend critically on the reversible formation/decomposition of metal oxides on cycling. Irreversible parasitic reactions cause poor rechargeability, efficiency, and cycle life, and have predominantly been ascribed to the reactivity of reduced oxygen species with cell components. These species, however, cannot fully explain the side reactions. Here we show that singlet oxygen forms at the cathode of a lithium-oxygen cell during discharge and from the onset of charge, and accounts for the majority of parasitic reaction products. The amount increases during discharge, early stages of charge, and charging at higher voltages, and is enhanced by the presence of trace water. Superoxide and peroxide appear to be involved in singlet oxygen generation. Singlet oxygen traps and quenchers can reduce parasitic reactions effectively. Awareness of the highly reactive singlet oxygen in non-aqueous metal-oxygen batteries gives a rationale for future research towards achieving highly reversible cell operation.

FLASH is essential during early embryogenesis and cooperates with p73 to regulate histone gene transcription.

PubMed

De Cola, A; Bongiorno-Borbone, L; Bianchi, E; Barcaroli, D; Carletti, E; Knight, R A; Di Ilio, C; Melino, G; Sette, C; De Laurenzi, V

2012-02-02

Replication-dependent histone gene expression is a fundamental process occurring in S-phase under the control of the cyclin-E/CDK2 complex. This process is regulated by a number of proteins, including Flice-Associated Huge Protein (FLASH) (CASP8AP2), concentrated in specific nuclear organelles known as HLBs. FLASH regulates both histone gene transcription and mRNA maturation, and its downregulation in vitro results in the depletion of the histone pull and cell-cycle arrest in S-phase. Here we show that the transcription factor p73 binds to FLASH and is part of the complex that regulates histone gene transcription. Moreover, we created a novel gene trap to disrupt FLASH in mice, and we show that homozygous deletion of FLASH results in early embryonic lethality, owing to arrest of FLASH(-/-) embryos at the morula stage. These results indicate that FLASH is an essential, non-redundant regulator of histone transcription and cell cycle during embryogenesis.

JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNα.

PubMed

Hasan, Salma; Lacout, Catherine; Marty, Caroline; Cuingnet, Marie; Solary, Eric; Vainchenker, William; Villeval, Jean-Luc

2013-08-22

The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2(V617F)) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2(V617F) on early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2(V617F) clone in humans by using knock-in mice with conditional expression of JAK2(V617F) in hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48-/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2(V617F) SLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2(V617F) cells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2(V617F) in mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.

Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification

PubMed Central

Wilson, Korey A.; Elefanty, Andrew G.; Stanley, Edouard G.; Gilbert, David M.

2016-01-01

ABSTRACT Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification. PMID:27433885

SB225002 Induces Cell Death and Cell Cycle Arrest in Acute Lymphoblastic Leukemia Cells through the Activation of GLIPR1

PubMed Central

Leal, Paulo C.; Bhasin, Manoj K.; Zenatti, Priscila Pini; Nunes, Ricardo J.; Yunes, Rosendo A.; Nowill, Alexandre E.; Libermann, Towia A.; Zerbini, Luiz Fernando; Yunes, José Andrés

2015-01-01

Acute Lymphoblastic Leukemia (ALL) is the most frequent childhood malignancy. In the effort to find new anti-leukemic agents, we evaluated the small drug SB225002 (N-(2-hydroxy-4-nitrophenyl)-N’-(2-bromophenyl)urea). Although initially described as a selective antagonist of CXCR2, later studies have identified other cellular targets for SB225002, with potential medicinal use in cancer. We found that SB225002 has a significant pro-apoptotic effect against both B- and T-ALL cell lines. Cell cycle analysis demonstrated that treatment with SB225002 induces G2-M cell cycle arrest. Transcriptional profiling revealed that SB225002-mediated apoptosis triggered a transcriptional program typical of tubulin binding agents. Network analysis revealed the activation of genes linked to the JUN and p53 pathways and inhibition of genes linked to the TNF pathway. Early cellular effects activated by SB225002 included the up-regulation of GLIPR1, a p53-target gene shown to have pro-apoptotic activities in prostate and bladder cancer. Silencing of GLIPR1 in B- and T-ALL cell lines resulted in increased resistance to SB225002. Although SB225002 promoted ROS increase in ALL cells, antioxidant N-Acetyl Cysteine pre-treatment only modestly attenuated cell death, implying that the pro-apoptotic effects of SB225002 are not exclusively mediated by ROS. Moreover, GLIPR1 silencing resulted in increased ROS levels both in untreated and SB225002-treated cells. In conclusion, SB225002 induces cell cycle arrest and apoptosis in different B- and T-ALL cell lines. Inhibition of tubulin function with concurrent activation of the p53 pathway, in particular, its downstream target GLIPR1, seems to underlie the anti-leukemic effect of SB225002. PMID:26302043

MAP kinase dependent cyclinE/cdk2 activity promotes DNA replication in early sea urchin embryos

PubMed Central

Kisielewska, J.; Philipova, R.; Huang, J.-Y.; Whitaker, M.

2009-01-01

Sea urchins provide an excellent model for studying cell cycle control mechanisms governing DNA replication in vivo. Fertilization and cell cycle progression are tightly coordinated by Ca2+ signals, but the mechanisms underlying the onset of DNA replication after fertilization remain less clear. In this study we demonstrate that calcium-dependent activation of ERK1 promotes accumulation of cyclinE/cdk2 into the male and female pronucleus and entry into first S-phase. We show that cdk2 activity rises quickly after fertilization to a maximum at 4 min, corresponding in timing to the early ERK1 activity peak. Abolishing MAP kinase activity after fertilization with MEK inhibitor, U0126, substantially reduces the early peak of cdk2 activity and prevents cyclinE and cdk2 accumulation in both sperm pronucleus and zygote nucleus in vivo. Both p27kip1 and roscovitine, cdk2 inhibitors, prevented DNA replication suggesting cdk2 involvement in this process in sea urchin. Inhibition of cdk2 activity using p27kip1 had no effect on the phosphorylation of MBP by ERK, but completely abolished phosphorylation of retinoblastoma protein, a cdk2 substrate, indicating that cdk2 activity is downstream of ERK1 activation. This pattern of regulation of DNA synthesis conforms to the pattern observed in mammalian somatic cells. PMID:19665013

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development.

PubMed

Xu, Peng; Johnson, Tamara L; Stoller-Conrad, Jessica R; Schulz, Robert A

2012-01-01

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.

Area/latency optimized early output asynchronous full adders and relative-timed ripple carry adders.

PubMed

Balasubramanian, P; Yamashita, S

2016-01-01

This article presents two area/latency optimized gate level asynchronous full adder designs which correspond to early output logic. The proposed full adders are constructed using the delay-insensitive dual-rail code and adhere to the four-phase return-to-zero handshaking. For an asynchronous ripple carry adder (RCA) constructed using the proposed early output full adders, the relative-timing assumption becomes necessary and the inherent advantages of the relative-timed RCA are: (1) computation with valid inputs, i.e., forward latency is data-dependent, and (2) computation with spacer inputs involves a bare minimum constant reverse latency of just one full adder delay, thus resulting in the optimal cycle time. With respect to different 32-bit RCA implementations, and in comparison with the optimized strong-indication, weak-indication, and early output full adder designs, one of the proposed early output full adders achieves respective reductions in latency by 67.8, 12.3 and 6.1 %, while the other proposed early output full adder achieves corresponding reductions in area by 32.6, 24.6 and 6.9 %, with practically no power penalty. Further, the proposed early output full adders based asynchronous RCAs enable minimum reductions in cycle time by 83.4, 15, and 8.8 % when considering carry-propagation over the entire RCA width of 32-bits, and maximum reductions in cycle time by 97.5, 27.4, and 22.4 % for the consideration of a typical carry chain length of 4 full adder stages, when compared to the least of the cycle time estimates of various strong-indication, weak-indication, and early output asynchronous RCAs of similar size. All the asynchronous full adders and RCAs were realized using standard cells in a semi-custom design fashion based on a 32/28 nm CMOS process technology.

Gene expression profiling reveals underlying molecular mechanisms of the early stages of tamoxifen-induced rat hepatocarcinogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pogribny, Igor P.; Bagnyukova, Tetyana V.; Tryndyak, Volodymyr P.

2007-11-15

Tamoxifen is a widely used anti-estrogenic drug for chemotherapy and, more recently, for the chemoprevention of breast cancer. Despite the indisputable benefits of tamoxifen in preventing the occurrence and re-occurrence of breast cancer, the use of tamoxifen has been shown to induce non-alcoholic steatohepatitis, which is a life-threatening fatty liver disease with a risk of progression to cirrhosis and hepatocellular carcinoma. In recent years, the high-throughput microarray technology for large-scale analysis of gene expression has become a powerful tool for increasing the understanding of the molecular mechanisms of carcinogenesis and for identifying new biomarkers with diagnostic and predictive values. Inmore » the present study, we used the high-throughput microarray technology to determine the gene expression profiles in the liver during early stages of tamoxifen-induced rat hepatocarcinogenesis. Female Fisher 344 rats were fed a 420 ppm tamoxifen containing diet for 12 or 24 weeks, and gene expression profiles were determined in liver of control and tamoxifen-exposed rats. The results indicate that early stages of tamoxifen-induced liver carcinogenesis are characterized by alterations in several major cellular pathways, specifically those involved in the tamoxifen metabolism, lipid metabolism, cell cycle signaling, and apoptosis/cell proliferation control. One of the most prominent changes during early stages of tamoxifen-induced hepatocarcinogenesis is dysregulation of signaling pathways in cell cycle progression from the G{sub 1} to S phase, evidenced by the progressive and sustained increase in expression of the Pdgfc, Calb3, Ets1, and Ccnd1 genes accompanied by the elevated level of the PI3K, p-PI3K, Akt1/2, Akt3, and cyclin B, D1, and D3 proteins. The early appearance of these alterations suggests their importance in the mechanism of neoplastic cell transformation induced by tamoxifen.« less

Human Enterovirus 68 Interferes with the Host Cell Cycle to Facilitate Viral Production

PubMed Central

Wang, Zeng-yan; Zhong, Ting; Wang, Yue; Song, Feng-mei; Yu, Xiao-feng; Xing, Li-ping; Zhang, Wen-yan; Yu, Jing-hua; Hua, Shu-cheng; Yu, Xiao-fang

2017-01-01

Enterovirus D68 (EV-D68) is an emerging pathogen that recently caused a large outbreak of severe respiratory disease in the United States and other countries. Little is known about the relationship between EV-D68 virus and host cells. In this study, we assessed the effect of the host cell cycle on EV-D68 viral production, as well as the ability of EV-D68 to manipulate host cell cycle progression. The results suggest that synchronization in G0/G1 phase, but not S phase, promotes viral production, while synchronization in G2/M inhibits viral production. Both an early EV-D68 isolate and currently circulating strains of EV-D68 can manipulate the host cell cycle to arrest cells in the G0/G1 phase, thus providing favorable conditions for virus production. Cell cycle regulation by EV-D68 was associated with corresponding effects on the expression of cyclins and CDKs, which were observed at the level of the protein and/or mRNA. Furthermore, the viral non-structural protein 3D of EV-D68 prevents progression from G0/G1 to S. Interestingly, another member of the Picornaviridae family, EV-A71, differs from EV-D68 in that G0/G1 synchronization inhibits, rather than promotes, EV-A71 viral replication. However, these viruses are similar in that G2/M synchronization inhibits the production and activity of both viruses, which is suggestive of a common therapeutic target for both types of enterovirus. These results further clarify the pathogenic mechanisms of enteroviruses and provide a potential strategy for the treatment and prevention of EV-D68-related disease. PMID:28229049

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDK-dependent sensor of the host cell cycle and differentiation state.

PubMed

Bogdanow, Boris; Weisbach, Henry; von Einem, Jens; Straschewski, Sarah; Voigt, Sebastian; Winkler, Michael; Hagemeier, Christian; Wiebusch, Lüder

2013-10-22

Upon cell entry, herpesviruses deliver a multitude of premade virion proteins to their hosts. The interplay between these incoming proteins and cell-specific regulatory factors dictates the outcome of infections at the cellular level. Here, we report a unique type of virion-host cell interaction that is essential for the cell cycle and differentiation state-dependent onset of human cytomegalovirus (HCMV) lytic gene expression. The major tegument 150-kDa phosphoprotein (pp150) of HCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in its cyclin A2-dependent phosphorylation. Alanine substitution of the RXL/Cy motif prevents this interaction and allows the virus to fully escape the cyclin-dependent kinase (CDK)-mediated block of immediate early (IE) gene expression in S/G2 phase that normally restricts the onset of the HCMV replication cycle to G0/G1. Furthermore, the cyclin A2-CDK-pp150 axis is also involved in the establishment of HCMV quiescence in NTera2 cells, showing the importance of this molecular switch for differentiation state-dependent regulation of IE gene expression. Consistent with the known nucleocapsid-binding function of pp150, its RXL/Cy-dependent phosphorylation affects gene expression of the parental virion only, suggesting a cis-acting, virus particle-associated mechanism of control. The pp150 homologs of other primate and mammalian CMVs lack an RXL/Cy motif and accordingly even the nearest relative of HCMV, chimpanzee CMV, starts its lytic cycle in a cell cycle-independent manner. Thus, HCMV has evolved a molecular sensor for cyclin A2-CDK activity to restrict its IE gene expression program as a unique level of self-limitation and adaptation to its human host.

The retinoid X receptor agonist, 9-cis UAB30, inhibits cutaneous T-cell lymphoma proliferation through the SKP2-p27kip1 axis.

PubMed

Chou, Chu-Fang; Hsieh, Yu-Hua; Grubbs, Clinton J; Atigadda, Venkatram R; Mobley, James A; Dummer, Reinhard; Muccio, Donald D; Eto, Isao; Elmets, Craig A; Garvey, W Timothy; Chang, Pi-Ling

2018-06-01

Bexarotene (Targretin ® ) is currently the only FDA approved retinoid X receptor (RXR) -selective agonist for the treatment of cutaneous T-cell lymphomas (CTCLs). The main side effects of bexarotene are hypothyroidism and elevation of serum triglycerides (TGs). The novel RXR ligand, 9-cis UAB30 (UAB30) does not elevate serum TGs or induce hypothyroidism in normal subjects. To assess preclinical efficacy and mechanism of action of UAB30 in the treatment of CTCLs and compare its action with bexarotene. With patient-derived CTCL cell lines, we evaluated UAB30 function in regulating growth, apoptosis, cell cycle check points, and cell cycle-related markers. Compared to bexarotene, UAB30 had lower half maximal inhibitory concentration (IC 50 ) values and was more effective in inhibiting the G1 cell cycle checkpoint. Both rexinoids increased the stability of the cell cycle inhibitor, p27kip1 protein, in part, through targeting components involved in the ubiquitination-proteasome system: 1) decreasing SKP2, a F-box protein that binds and targets p27kip1 for degradation by 26S proteasome and 2) suppressing 20S proteasome activity (cell line-dependent) through downregulation of PSMA7, a component of the 20S proteolytic complex in 26S proteasome. UAB30 and bexarotene induce both early cell apoptosis and suppress cell proliferation. Inhibition of the G1 to S cell cycle transition by rexinoids is mediated, in part, through downregulation of SKP2 and/or 20S proteasome activity, leading to increased p27kip1 protein stability. Because UAB30 has minimal effect in elevating serum TGs and inducing hypothyroidism, it is potentially a better alternative to bexarotene for the treatment of CTCLs. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Minichromosome maintenance (Mcm) proteins, cyclin B1 and D1, phosphohistone H3 and in situ DNA replication for functional analysis of vulval intraepithelial neoplasia.

PubMed

Davidson, E J; Morris, L S; Scott, I S; Rushbrook, S M; Bird, K; Laskey, R A; Wilson, G E; Kitchener, H C; Coleman, N; Stern, P L

2003-01-27

Vulval intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. To investigate the functional properties of VIN, the expression of several proteins involved in the regulation of the cell cycle as well as in situ DNA replication competence was analysed by immunohistochemistry. Snap-frozen vulval biopsies were graded as normal squamous epithelium (n=6), undifferentiated HPV positive VIN 1 (n=3), VIN 2 (n=8) and VIN 3 (n=20). Immunohistochemistry was performed using the following markers: cyclin D1 (expressed in middle/late G1), cyclin B1 (expressed in G2/early M), phosphorylated histone H3 (expressed during mitosis) and minichromosome maintenance (Mcm) proteins 2 and 5 (expressed during the cell cycle, but not in differentiated or quiescent cells). In situ DNA replication competence was used to identify S-phase cells. The percentage of positively stained nuclei in three representative microscopic fields was calculated per biopsy. In normal vulva, the expression of all markers was restricted to the proliferative compartment of the basal layer of the epithelium. In contrast in high-grade VIN, the majority of epithelial cells expressed the Mcm proteins from basal to superficial layer. The detection of cyclins B1 and D1, phospho-histone H3 and in situ DNA replication was also found through the full thickness of these lesions but by a lower proportion of the cells. This is consistent with these markers providing a series of 'snapshots' of the cell cycle status of individual cells. The low-grade VIN showed reduced expression of the cell cycle markers in relation to the level of dysplasia. The combination of these analyses establishes that the majority of VIN cells remain in a functional replicative or prereplicative state of the cell cycle. Clinical application of these analyses may provide a basis for improved diagnosis of VIN.

Ultrastructural and cellular basis for the development of abnormal myocardial mechanics during the transition from hypertension to heart failure.

PubMed

Shah, Sanjiv J; Aistrup, Gary L; Gupta, Deepak K; O'Toole, Matthew J; Nahhas, Amanda F; Schuster, Daniel; Chirayil, Nimi; Bassi, Nikhil; Ramakrishna, Satvik; Beussink, Lauren; Misener, Sol; Kane, Bonnie; Wang, David; Randolph, Blake; Ito, Aiko; Wu, Megan; Akintilo, Lisa; Mongkolrattanothai, Thitipong; Reddy, Mahendra; Kumar, Manvinder; Arora, Rishi; Ng, Jason; Wasserstrom, J Andrew

2014-01-01

Although the development of abnormal myocardial mechanics represents a key step during the transition from hypertension to overt heart failure (HF), the underlying ultrastructural and cellular basis of abnormal myocardial mechanics remains unclear. We therefore investigated how changes in transverse (T)-tubule organization and the resulting altered intracellular Ca(2+) cycling in large cell populations underlie the development of abnormal myocardial mechanics in a model of chronic hypertension. Hearts from spontaneously hypertensive rats (SHRs; n = 72) were studied at different ages and stages of hypertensive heart disease and early HF and were compared with age-matched control (Wistar-Kyoto) rats (n = 34). Echocardiography, including tissue Doppler and speckle-tracking analysis, was performed just before euthanization, after which T-tubule organization and Ca(2+) transients were studied using confocal microscopy. In SHRs, abnormalities in myocardial mechanics occurred early in response to hypertension, before the development of overt systolic dysfunction and HF. Reduced longitudinal, circumferential, and radial strain as well as reduced tissue Doppler early diastolic tissue velocities occurred in concert with T-tubule disorganization and impaired Ca(2+) cycling, all of which preceded the development of cardiac fibrosis. The time to peak of intracellular Ca(2+) transients was slowed due to T-tubule disruption, providing a link between declining cell ultrastructure and abnormal myocardial mechanics. In conclusion, subclinical abnormalities in myocardial mechanics occur early in response to hypertension and coincide with the development of T-tubule disorganization and impaired intracellular Ca(2+) cycling. These changes occur before the development of significant cardiac fibrosis and precede the development of overt cardiac dysfunction and HF.

Molecular Analysis of the Inheritance of Transcriptional Silencing

DTIC Science & Technology

2007-04-01

arrest, to synchronize the cells for hydroxyurea (HU) addition, and then released into an alpha factor/0.2M HU arrest (Fig. 1A). 2 hours after release...restrictive conditions to degrade both Sir1td and Asf1td proteins. Cells were then release for 4 hours into 0.2M hydroxyurea (HU), an early S phase arrest...on three experiments. The “Block” row describes the cell cycle inhibitor used in each time point (alpha factor, hydroxyurea and nocodozole). 24

Mir Cooperative Solar Array Project Accelerated Life Thermal Cycling Test

NASA Technical Reports Server (NTRS)

Hoffman, David J.; Scheiman, David A.

1996-01-01

The Mir Cooperative Solar Array (MCSA) project was a joint U.S./Russian effort to build a photovoltaic (PV) solar array and deliver it to the Russian space station Mir. The MCSA will be used to increase the electrical power on Mir and provide PV array performance data in support of Phase 1 of the International Space Station. The MCSA was brought to Mir by space shuttle Atlantis in November 1995. This report describes an accelerated thermal life cycle test which was performed on two samples of the MCSA. In eight months time, two MCSA solar array 'mini' panel test articles were simultaneously put through 24,000 thermal cycles. There was no significant degradation in the structural integrity of the test articles and no electrical degradation, not including one cell damaged early and removed from consideration. The nature of the performance degradation caused by this one cell is briefly discussed. As a result of this test, changes were made to improve some aspects of the solar cell coupon-to-support frame interface on the flight unit. It was concluded from the results that the integration of the U.S. solar cell modules with the Russian support structure would be able to withstand at least 24,000 thermal cycles (4 years on-orbit). This was considered a successful development test.

Neuroblastoma cells depend on HDAC11 for mitotic cell cycle progression and survival

PubMed Central

Thole, Theresa M; Lodrini, Marco; Fabian, Johannes; Wuenschel, Jasmin; Pfeil, Sebastian; Hielscher, Thomas; Kopp-Schneider, Annette; Heinicke, Ulrike; Fulda, Simone; Witt, Olaf; Eggert, Angelika; Fischer, Matthias; Deubzer, Hedwig E

2017-01-01

The number of long-term survivors of high-risk neuroblastoma remains discouraging, with 10-year survival as low as 20%, despite decades of considerable international efforts to improve outcome. Major obstacles remain and include managing resistance to induction therapy, which causes tumor progression and early death in high-risk patients, and managing chemotherapy-resistant relapses, which can occur years after the initial diagnosis. Identifying and validating novel therapeutic targets is essential to improve treatment. Delineating and deciphering specific functions of single histone deacetylases in neuroblastoma may support development of targeted acetylome-modifying therapeutics for patients with molecularly defined high-risk neuroblastoma profiles. We show here that HDAC11 depletion in MYCN-driven neuroblastoma cell lines strongly induces cell death, mostly mediated by apoptotic programs. Genes necessary for mitotic cell cycle progression and cell division were most prominently enriched in at least two of three time points in whole-genome expression data combined from two cell systems, and all nine genes in these functional categories were strongly repressed, including CENPA, KIF14, KIF23 and RACGAP1. Enforced expression of one selected candidate, RACGAP1, partially rescued the induction of apoptosis caused by HDAC11 depletion. High-level expression of all nine genes in primary neuroblastomas significantly correlated with unfavorable overall and event-free survival in patients, suggesting a role in mediating the more aggressive biological and clinical phenotype of these tumors. Our study identified a group of cell cycle-promoting genes regulated by HDAC11, being both predictors of unfavorable patient outcome and essential for tumor cell viability. The data indicate a significant role of HDAC11 for mitotic cell cycle progression and survival of MYCN-amplified neuroblastoma cells, and suggests that HDAC11 could be a valuable drug target. PMID:28252645

Pause, play, repeat

PubMed Central

Sansó, Miriam; Fisher, Robert P

2013-01-01

Cyclin-dependent kinases (CDKs) play a central role in governing eukaryotic cell division. It is becoming clear that the transcription cycle of RNA polymerase II (RNAP II) is also regulated by CDKs; in metazoans, the cell cycle and transcriptional CDK networks even share an upstream activating kinase, which is itself a CDK. From recent chemical-genetic analyses we know that CDKs and their substrates control events both early in transcription (the transition from initiation to elongation) and late (3′ end formation and transcription termination). Moreover, mutual dependence on CDK activity might couple the “beginning” and “end” of the cycle, to ensure the fidelity of mRNA maturation and the efficient recycling of RNAP II from sites of termination to the transcription start site (TSS). As is the case for CDKs involved in cell cycle regulation, different transcriptional CDKs act in defined sequence on multiple substrates. These phosphorylations are likely to influence gene expression by several mechanisms, including direct, allosteric effects on the transcription machinery, co-transcriptional recruitment of proteins needed for mRNA-capping, splicing and 3′ end maturation, dependent on multisite phosphorylation of the RNAP II C-terminal domain (CTD) and, perhaps, direct regulation of RNA-processing or histone-modifying machinery. Here we review these recent advances, and preview the emerging challenges for transcription-cycle research. PMID:23756342

Micro Calorimeter for Batteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santhanagopalan, Shriram

2017-08-01

As battery technology forges ahead and consumer demand for safer, more affordable, high-performance batteries grows, the National Renewable Energy Laboratory (NREL) has added a patented Micro Calorimeter to its existing family of R&D 100 Award-winning Isothermal Battery Calorimeters (IBCs). The Micro Calorimeter examines the thermal signature of battery chemistries early on in the design cycle using popular coin cell and small pouch cell designs, which are simple to fabricate and study.

Operando lithium plating quantification and early detection of a commercial LiFePO4 cell cycled under dynamic driving schedule

NASA Astrophysics Data System (ADS)

Anseán, D.; Dubarry, M.; Devie, A.; Liaw, B. Y.; García, V. M.; Viera, J. C.; González, M.

2017-07-01

Lithium plating is considered one of the most detrimental phenomenon in lithium ion batteries (LIBs), as it increases cell degradation and might lead to safety issues. Plating induced LIB failure presents a major concern for emerging applications in transportation and electrical energy storage. Hence, the necessity to operando monitor, detect and analyze lithium plating becomes critical for safe and reliable usage of LIB systems. Here, we report in situ lithium plating analyses for a commercial graphite||LiFePO4 cell cycled under dynamic stress test (DST) driving schedule. We designed a framework based on incremental capacity (IC) analysis and mechanistic model simulations to quantify degradation modes, relate their effects to lithium plating occurrence and assess cell degradation. The results show that lithium plating was induced by large loss of active material on the negative electrode that eventually led the electrode to over-lithiate. Moreover, when lithium plating emerged, we quantified that the loss of lithium inventory pace was increased by a factor of four. This study illustrates the benefits of the proposed framework to improve lithium plating analysis. It also discloses the symptoms of lithium plating formation, which prove valuable for novel, online strategies on early lithium plating detection.

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds.

PubMed

Schuck, Desirée Cigaran; Ribeiro, Ramira Yuri; Nery, Arthur A; Ulrich, Henning; Garcia, Célia R S

2011-11-01

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds. Copyright © 2011 International Society for Advancement of Cytometry.

Report of the NASA Mammalian Developmental Biology Working Group

NASA Technical Reports Server (NTRS)

Keefe, J. R.

1985-01-01

Development is considered to encompass all aspects of the mammalian life span from initial initial germ cell production through the complete life cycle to death of the organism. Thus, gamete production, fertilization, embryogenesis, implantation, fetogenesis, birth, peri- and postnatal maturation, and aging were all considered as stages of a development continuum relevant to problems of Space Biology. Deliberations thus far have been limited to stages of the development cycle from fertilization to early postnatal life. The deliberations are detailed.

Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

PubMed Central

Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

2016-01-01

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis. PMID:26493868

Aberrant activation of M phase proteins by cell proliferation-evoking carcinogens after 28-day administration in rats.

PubMed

Yafune, Atsunori; Taniai, Eriko; Morita, Reiko; Hayashi, Hitomi; Suzuki, Kazuhiko; Mitsumori, Kunitoshi; Shibutani, Makoto

2013-06-07

We have previously reported that hepatocarcinogens increase liver cells expressing p21(Cip1), a G1 checkpoint protein and M phase proteins after 28-day treatment in rats. This study aimed to identify early prediction markers of carcinogens available in many target organs after 28-day treatment in rats. Immunohistochemical analysis was performed on Ki-67, p21(Cip1) and M phase proteins [nuclear Cdc2, phospho-Histone H3 (p-Histone H3), Aurora B and heterochromatin protein 1α (HP1α)] with carcinogens targeting different organs. Carcinogens targeting thyroid (sulfadimethoxine; SDM), urinary bladder (phenylethyl isothiocyanate), forestomach (butylated hydroxyanisole; BHA), glandular stomach (catechol; CC), and colon (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and chenodeoxycholic acid) were examined using a non-carcinogenic toxicant (caprolactam) and carcinogens targeting other organs as negative controls. All carcinogens increased Ki-67(+), nuclear Cdc2(+), p-Histone H3(+) or Aurora B(+) carcinogenic target cells, except for both colon carcinogens, which did not increase cell proliferation. On the other hand, p21(Cip1+) cells increased with SDM and CC. HP1α responded only to BHA. Results revealed carcinogens evoking cell proliferation concurrently induced cell cycle arrest at M phase or showing chromosomal instability reflecting aberration in cell cycle regulation, irrespective of target organs, after 28-day treatment. Therefore, M phase proteins may be early prediction markers of carcinogens evoking cell proliferation in many target organs. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The temporal and spatial distribution of the proliferation associated Ki-67 protein during female and male meiosis.

PubMed

Traut, Walther; Endl, Elmar; Scholzen, Thomas; Gerdes, Johannes; Winking, Heinz

2002-09-01

We used immunolocalization in tissue sections and cytogenetic preparations of female and male gonads to study the distribution of the proliferation marker pKi-67 during meiotic cell cycles of the house mouse, Mus musculus. During male meiosis, pKi-67 was continuously present in nuclei of all stages from the spermatogonium through spermatocytes I and II up to the earliest spermatid stage (early round spermatids) when it appeared to fade out. It was not detected in later spermatid stages or sperm. During female meiosis, pKi-67 was present in prophase I oocytes of fetal ovaries. It was absent in oocytes from newborn mice and most oocytes of primordial follicles from adults. The Ki-67 protein reappeared in oocytes of growing follicles and was continuously present up to metaphase II. Thus, pKi-67 was present in all stages of cell growth and cell division while it was absent from resting oocytes and during the main stages of spermiocytogenesis. Progression through the meiotic cell cycle was associated with extensive intranuclear relocation of pKi-67. In the zygotene and pachytene stages, most of the pKi-67 colocalized with centromeric (centric and pericentric) heterochromatin and adjacent nucleoli; the heterochromatic XY body in male pachytene, however, was free of pKi-67. At early diplotene, pKi-67 was mainly associated with nucleoli. At late diplotene, diakinesis, metaphase I and metaphase II of meiosis, pKi-67 preferentially bound to the perichromosomal layer and was almost absent from the heterochromatic centromeric regions of the chromosomes. After the second division of male meiosis, the protein reappeared at the centromeric heterochromatin and an adjacent region in the earliest spermatid stage and then faded out. The general patterns of pKi-67 distribution were comparable to those in mitotic cell cycles. With respect to the timing, it is interesting to note that relocation from the nucleolus to the perichromosomal layer takes place at the G2/M-phase transition in the mitotic cell cycle but at late diplotene of prophase I in meiosis, suggesting physiological similarity of these stages.

Mitotic effects of monochromatic ultraviolet radiation at 225, 265, and 280 nm on eleven stages of the cell cycle of the grasshopper neuroblast in culture. I. Overall retardation from the stage irradiated to nuclear membrane breakdown

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlson, J.G.

1976-10-01

Neuroblasts of Chortophaga viridifasciata (DeGeer) in culture were exposed to different doses of 225, 265, or 280 nm ultraviolet radiations at 11 different stages and substages of the mitotic cycle and individually selected cells were timed to breakdown of the nuclear membrane. Comparisons of the effectiveness of different wavelengths on the different stages were based on the dose that reduced the cell progression rate to 67 percent of normal (D/sub 67/) and the slope of the regression line, i.e., the control to treated time (C/T) ratio change/erg/mm/sup 2/ at the D/sub 67/ level. Cells of the prereplication period (metaphase +more » anaphase + early telophase) and the S phase (middle and late telophase + interphase + very early prophase) are equally sensitive to uv and contrast sharply with the much lower sensitivity of those in the postreplication period (early and middle prophase). This can best be interpreted if chromosomal DNA is the chromophore for uv-induced mitotic retardation. Cells in the prereplication period at exposure show no wavelength effect. In the S phase all stages except middle telophase and all stages combined are significantly more sensitive to 265 and 280 nm than to 225 nm. Of the postreplication stages, early prophase is retarded significantly more by 280 than by 225 or 265 nm. The C/T ratio/erg/mm/sup 2/ is greater after exposure to 265 nm at all prereplication and replication stages, but exhibits no consistent wavelength pattern during the postreplication period. Evidence based on the orientation of the neuroblast with respect to the uv-source suggests that the chromophore for mitotic retardation does not reside within the centrosome and related structures, but may be present, at least partly, in the nucleolus.« less

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo.

PubMed

Wang, Guang; Chen, En-Ni; Liang, Chang; Liang, Jianxin; Gao, Lin-Rui; Chuai, Manli; Münsterberg, Andrea; Bao, Yongping; Cao, Liu; Yang, Xuesong

2018-04-01

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7 + neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7 + and HNK-1 + cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1 + neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU + neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Ren-Jie

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G{sub 2}/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosismore » in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G{sub 2}/M cell cycle regulators, ABT-751 induced G{sub 2}/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. - Highlights: • An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells. • ABT-751 induces early autophagy and delays apoptosis. • ABT-751 induces autophagy via modulations of nuclear TP53 and AKT/MTOR pathways. • ABT-751-induced apoptosis is ROS-, mitochondria- and caspase-dependent. • Inhibition of autophagy enhances ABT-751-induced apoptosis.« less

BRCA1 and FancJ cooperatively promote interstrand crosslinker induced centrosome amplification through the activation of polo-like kinase 1

PubMed Central

Zou, Jianqiu; Zhang, Deli; Qin, Guang; Chen, Xiangming; Wang, Hongmin; Zhang, Dong

2014-01-01

DNA damage response (DDR) and the centrosome cycle are 2 of the most critical cellular processes affecting the genome stability in animal cells. Yet the cross-talks between DDR and the centrosome are poorly understood. Here we showed that deficiency of the breast cancer 1, early onset gene (BRCA1) induces centrosome amplification in non-stressed cells as previously reported while attenuating DNA damage-induced centrosome amplification (DDICA) in cells experiencing prolonged genotoxic stress. Mechanistically, the function of BRCA1 in promoting DDICA is through binding and recruiting polo-like kinase 1 (PLK1) to the centrosome. In a recent study, we showed that FancJ also suppresses centrosome amplification in non-stressed cells while promoting DDICA in both hydroxyurea and mitomycin C treated cells. FancJ is a key component of the BRCA1 B-complex. Here, we further demonstrated that, in coordination with BRCA1, FancJ promotes DDICA by recruiting both BRCA1 and PLK1 to the centrosome in the DNA damaged cells. Thus, we have uncovered a novel role of BRCA1 and FancJ in the regulation of DDICA. Dysregulation of DDR or centrosome cycle leads to aneuploidy, which is frequently seen in both solid and hematological cancers. BRCA1 and FancJ are known tumor suppressors and have well-recognized functions in DNA damage checkpoint and DNA repair. Together with our recent findings, we demonstrated here that BRCA1 and FancJ also play an important role in centrosome cycle especially in DDICA. DDICA is thought to be an alternative fail-safe mechanism to prevent cells experiencing severe DNA damage from becoming carcinogenic. Therefore, BRCA1 and FancJ are potential liaisons linking early DDR with the DDICA. We propose that together with their functions in DDR, the role of BRCA1 and FancJ in the activation of DDICA is also crucial for their tumor suppression functions in vivo. PMID:25483079

Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle

PubMed Central

Khurana, Simran; Warburton, Alix

2017-01-01

We have shown previously that Sp100 (a component of the ND10 nuclear body) represses transcription, replication and establishment of incoming human papillomavirus (HPV) DNA in the early stages of infection. In this follow up study, we show that Sp100 does not substantially regulate viral infection in the maintenance phase, however at late stages of infection Sp100 interacts with amplifying viral genomes to repress viral processes. We find that Sp100 localizes to HPV16 replication foci generated in primary keratinocytes, to HPV31 replication foci that form in differentiated cells, and to HPV16 replication foci in CIN 1 cervical biopsies. To analyze this further, Sp100 was down regulated by siRNA treatment of differentiating HPV31 containing cells and levels of viral transcription and replication were assessed. This revealed that Sp100 represses viral transcription and replication in differentiated cells. Analysis of Sp100 binding to viral chromatin showed that Sp100 bound across the viral genome, and that binding increased at late stages of infection. Therefore, Sp100 represses the HPV life cycle at both early and late stages of infection. PMID:28968443

Increased Postnatal Cardiac Hyperplasia Precedes Cardiomyocyte Hypertrophy in a Model of Hypertrophic Cardiomyopathy

PubMed Central

Farrell, Emily T.; Grimes, Adrian C.; de Lange, Willem J.; Armstrong, Annie E.; Ralphe, J. Carter

2017-01-01

Rationale: Hypertrophic cardiomyopathy (HCM) occurs in ~0.5% of the population and is a leading cause of sudden cardiac death (SCD) in young adults. Cardiomyocyte hypertrophy has been the accepted mechanism for cardiac enlargement in HCM, but the early signaling responsible for initiating hypertrophy is poorly understood. Mutations in cardiac myosin binding protein C (MYBPC3) are among the most common HCM-causing mutations. Ablation of Mybpc3 in an HCM mouse model (cMyBP-C−/−) rapidly leads to cardiomegaly by postnatal day (PND) 9, though hearts are indistinguishable from wild-type (WT) at birth. This model provides a unique opportunity to explore early processes involved in the dramatic postnatal transition to hypertrophy. Methods and Results: We performed microarray analysis, echocardiography, qPCR, immunohistochemistry (IHC), and isolated cardiomyocyte measurements to characterize the perinatal cMyBP-C−/− phenotype before and after overt hypertrophy. cMyBP-C−/− hearts showed elevated cell cycling at PND1 that transitioned to hypertrophy by PND9. An expanded time course revealed that increased cardiomyocyte cycling was associated with elevated heart weight to body weight ratios prior to cellular hypertrophy, suggesting that cell cycling resulted in cardiomyocyte proliferation. Animals heterozygous for the cMyBP-C deletion trended in the direction of the homozygous null, yet did not show increased heart size by PND9. Conclusions: Results indicate that altered regulation of the cell cycling pathway and elevated proliferation precedes hypertrophy in the cMyBP-C−/− HCM model, and suggests that increased cardiomyocyte number contributes to increased heart size in cMyBP-C−/− mice. This pre-hypertrophic period may reflect a unique time during which the commitment to HCM is determined and disease severity is influenced. PMID:28659827

Capacity Decay Mitigation by Asymmetric Positive/Negative Electrolyte Volumes in Vanadium Redox Flow Batteries.

PubMed

Park, Jong Ho; Park, Jung Jin; Park, O Ok; Yang, Jung Hoon

2016-11-23

Capacity decay in vanadium redox flow batteries during charge-discharge cycling has become an important issue because it lowers the practical energy density of the battery. The battery capacity tends to drop rapidly within the first tens of cycles and then drops more gradually over subsequent cycles during long-term operation. This paper analyzes and discusses the reasons for this early capacity decay. The imbalanced crossover rate of vanadium species was found to remain high until the total difference in vanadium concentration between the positive and negative electrolytes reached almost 1 mol dm -3 . To minimize the initial crossover imbalance, we introduced an asymmetric volume ratio between the positive and negative electrolytes during cell operation. Changing this ratio significantly reduced the capacity fading rate of the battery during the early cycles and improved its capacity retention at steady state. As an example, the practical energy density of the battery increased from 15.5 to 25.2 Wh L -1 simply after reduction of the positive volume by 25 %. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrostimulation of rat callus cells and human lymphocytes in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aro, H.; Eerola, E.; Aho, A.J.

1984-01-01

Asymmetrical pulsing low voltage current was supplied via electrodes to cultured rat fracture callus cells and human peripheral blood lymphocytes. The (/sup 3/H)thymidine incorporation of the callus cells and 5-(/sup 125/I)iodo-2'-deoxyuridine incorporation of the lymphocytes were determined. The growth pattern of callus cells (estimated by cellular density) did not respond to electrical stimulation. However, the uptake of (/sup 3/H)thymidine was increased at the early phase of cell proliferation and inhibited at later phases of proliferation. The (/sup 3/H)thymidine uptake of confluent callus cell cultures did not respond to electrical stimulation. Lymphocytes reacted in a similar way; stimulated cells took upmore » more DNA precursor than control cells at the early phase of stimulation. During cell division, induced by the mitogens phytohemagglutinin and Concanavalin-A, the uptake of DNA precursor by stimulated cells was constantly inhibited. The results suggest that electrical stimuli affect the uptake mechanisms of cell membranes. The duality of the effect seems to be dependent on the cell cycle.« less

Analysis of Gene Expression Changes in PHA-M Stimulated Lymphocytes - Unraveling PHA Activity as Prerequisite for Dicentric Chromosome Analysis.

PubMed

Beinke, C; Port, M; Ullmann, R; Gilbertz, K; Majewski, M; Abend, M

2018-06-01

Dicentric chromosome analysis (DCA) is the gold standard for individual radiation dose assessment. However, DCA is limited by the time-consuming phytohemagglutinin (PHA)-mediated lymphocyte activation. In this study using human peripheral blood lymphocytes, we investigated PHA-associated whole genome gene expression changes to elucidate this process and sought to identify suitable gene targets as a means of meeting our long-term objective of accelerating cell cycle kinetics to reduce DCA culture time. Human peripheral whole blood from three healthy donors was separately cultured in RPMI/FCS/antibiotics with BrdU and PHA-M. Diluted whole blood samples were transferred into PAXgene tubes at 0, 12, 24 and 36 h culture time. RNA was isolated and aliquots were used for whole genome gene expression screening. Microarray results were validated using qRT-PCR and differentially expressed genes [significantly (FDR corrected) twofold different from the 0 h value reference] were analyzed using several bioinformatic tools. The cell cycle positions and DNA-synthetic activities of lymphocytes were determined by analyzing the correlated total DNA content and incorporated BrdU level with flow cytometry after continued BrdU incubation. From 42,545 transcripts of the whole genome microarray 47.6%, on average, appeared expressed. The number of differentially expressed genes increased linearly from 855 to 2,858 and 4,607 at 12, 24 and 36 h after PHA addition, respectively. Approximately 2-3 times more up- than downregulated genes were observed with several hundred genes differentially expressed at each time point. Earliest enrichment was observed for gene sets related to the nucleus (12 h) followed by genes assigned to intracellular structures such as organelles (24 h) and finally genes related to the membrane and the extracellular matrix were enriched (36 h). Early gene expression changes at 12 h, in particular, were associated with protein classes such as chemokines/cytokines (e.g., CXCL1, CXCL2) and chaperones. Genes coding for biological processes involved in cell cycle control (e.g., MYBL2, RBL1, CCNA, CCNE) and DNA replication (e.g., POLA, POLE, MCM) appeared enriched at 24 h and later, but many more biological processes (42 altogether) showed enrichment as well. Flow cytometry data fit together with gene expression and bioinformatic analyses as cell cycle transition into S phase was observed with interindividual differences from 12 h onward, whereas progression into G 2 as well as into the second G 1 occurred from 36 h onward after activation. Gene set enrichment analysis over time identifies, in particular, two molecular categories of PHA-responsive gene targets (cytokine and cell cycle control genes). Based on that analysis target genes for cell cycle acceleration in lymphocytes have been identified ( CDKN1A/B/C, RBL-1/RBL-2, E2F2, Deaf-1), and it remains undetermined whether the time expenditure for DCA can be reduced by influencing gene expression involved in the regulatory circuits controlling PHA-associated cell cycle entry and/or progression at a specific early cell cycle phase.

Specific requirement of the chromatin modifier mSin3B in cell cycle exit and cellular differentiation

PubMed Central

David, Gregory; Grandinetti, Kathryn B.; Finnerty, Patricia M.; Simpson, Natalie; Chu, Gerald C.; DePinho, Ronald A.

2008-01-01

The Sin3-histone deacetylase (HDAC) corepressor complex is conserved from yeast to humans. Mammals possess two highly related Sin3 proteins, mSin3A and mSin3B, which serve as scaffolds tethering HDAC enzymatic activity, and numerous sequence-specific transcription factors to enable local chromatin regulation at specific gene targets. Despite broad overlapping expression of mSin3A and mSin3B, mSin3A is cell-essential and vital for early embryonic development. Here, genetic disruption of mSin3B reveals a very different phenotype characterized by the survival of cultured cells and lethality at late stages of embryonic development with defective differentiation of multiple lineages—phenotypes that are strikingly reminiscent of those associated with loss of retinoblastoma family members or E2F transcriptional repressors. Additionally, we observe that, whereas mSin3B−/− cells cycle normally under standard growth conditions, they show an impaired ability to exit the cell cycle with limiting growth factors. Correspondingly, mSin3B interacts physically with the promoters of known E2F target genes, and its deficiency is associated with derepression of these gene targets in vivo. Together, these results reveal a critical role for mSin3B in the control of cell cycle exit and terminal differentiation in mammals and establish contrasting roles for the mSin3 proteins in the growth and development of specific lineages. PMID:18332431

Early-life antibiotic treatment enhances the pathogenicity of CD4+ T cells during intestinal inflammation.

PubMed

Scheer, Sebastian; Medina, Tiago S; Murison, Alex; Taves, Matthew D; Antignano, Frann; Chenery, Alistair; Soma, Kiran K; Perona-Wright, Georgia; Lupien, Mathieu; Arrowsmith, Cheryl H; De Carvalho, Daniel D; Zaph, Colby

2017-04-01

The incidence of inflammatory bowel diseases (IBDs) has steadily increased in recent decades-a phenomenon that cannot be explained by genetic mutations alone. Other factors, including the composition of the intestinal microbiome, are potentially important contributors to the increased occurrence of this group of diseases. Previous reports have shown a correlation between early-life antibiotic (Abx) treatment and an increased incidence of IBD. In this report, we investigated the effects of early-life Abx treatments on the pathogenicity of CD4 + T cells using an experimental T cell transfer model of IBD. Our results show that CD4 + T cells isolated from adult mice that had been treated with Abx during gestation and in early life induced a faster onset of IBD in Rag1 -deficient mice compared with CD4 + T cells of untreated mice. Ex vivo functional analyses of IBD-inducing CD4 + T cells did not show significant differences in their immunologic potential ex vivo, despite their in vivo phenotype. However, genome-wide gene-expression analysis revealed that these cells displayed dysregulated expression of genes associated with cell-cycle regulation, metabolism, and cellular stress. Analysis of Abx-treated CD4 + T cell donors showed systemically elevated levels of the stress hormone corticosterone throughout life compared with untreated donors. The cohousing of Abx-treated mice with untreated mice decreased serum corticosterone, and a consequent transfer of the cells from cohoused mice into Rag1 -deficient mice restored the onset and severity of disease to that of untreated animals. Thus, our results suggest that early-life Abx treatment results in a stress response with high levels of corticosterone that influences CD4 + T cell function. © Society for Leukocyte Biology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Suna, E-mail: wangs3@mail.nih.gov; Zhou, Yifu; Andreyev, Oleg

Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, andmore » the differential expression of FABP3 was tested by quantitative {sup RT}PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions. - Highlights: • FABP3 expression pattern was studied in 12 human hypoxic-MSCs. • FABP3 mRNA and proteins are upregulated in the MSCs under hypoxic conditions. • Overexpression of FABP3 inhibits cell growth but advanced the MSC survival under hypoxia. • Overexpression of FABP3 down-regulate the cell cycle and stem cell signaling pathways.« less

Hydra myc2, a unique pre-bilaterian member of the myc gene family, is activated in cell proliferation and gametogenesis

PubMed Central

Hartl, Markus; Glasauer, Stella; Valovka, Taras; Breuker, Kathrin; Hobmayer, Bert; Bister, Klaus

2014-01-01

ABSTRACT The myc protooncogene encodes the Myc transcription factor which is the essential part of the Myc–Max network controlling fundamental cellular processes. Deregulation of myc leads to tumorigenesis and is a hallmark of many human cancers. We have recently identified homologs of myc (myc1, myc2) and max in the early diploblastic cnidarian Hydra and have characterized myc1 in detail. Here we show that myc2 is transcriptionally activated in the interstitial stem cell system. Furthermore, in contrast to myc1, myc2 expression is also detectable in proliferating epithelial stem cells throughout the gastric region. myc2 but not myc1 is activated in cycling precursor cells during early oogenesis and spermatogenesis, suggesting that the Hydra Myc2 protein has a possible non-redundant function in cell cycle progression. The Myc2 protein displays the principal design and properties of vertebrate Myc proteins. In complex with Max, Myc2 binds to DNA with similar affinity as Myc1–Max heterodimers. Immunoprecipitation of Hydra chromatin revealed that both Myc1 and Myc2 bind to the enhancer region of CAD, a classical Myc target gene in mammals. Luciferase reporter gene assays showed that Myc1 but not Myc2 transcriptionally activates the CAD promoter. Myc2 has oncogenic potential when tested in primary avian fibroblasts but to a lower degree as compared to Myc1. The identification of an additional myc gene in Cnidaria, a phylum that diverged prior to bilaterians, with characteristic expression patterns in tissue homeostasis and developmental processes suggests that principle functions of myc genes have arisen very early in metazoan evolution. PMID:24771621

Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

PubMed Central

Davies, Erin L; Lei, Kai; Seidel, Christopher W; Kroesen, Amanda E; McKinney, Sean A; Guo, Longhua; Robb, Sofia MC; Ross, Eric J; Gotting, Kirsten; Alvarado, Alejandro Sánchez

2017-01-01

Planarian neoblasts are pluripotent, adult somatic stem cells and lineage-primed progenitors that are required for the production and maintenance of all differentiated cell types, including the germline. Neoblasts, originally defined as undifferentiated cells residing in the adult parenchyma, are frequently compared to embryonic stem cells yet their developmental origin remains obscure. We investigated the provenance of neoblasts during Schmidtea mediterranea embryogenesis, and report that neoblasts arise from an anarchic, cycling piwi-1+ population wholly responsible for production of all temporary and definitive organs during embryogenesis. Early embryonic piwi-1+ cells are molecularly and functionally distinct from neoblasts: they express unique cohorts of early embryo enriched transcripts and behave differently than neoblasts in cell transplantation assays. Neoblast lineages arise as organogenesis begins and are required for construction of all major organ systems during embryogenesis. These subpopulations are continuously generated during adulthood, where they act as agents of tissue homeostasis and regeneration. DOI: http://dx.doi.org/10.7554/eLife.21052.001 PMID:28072387

Distinct but Concerted Roles of ATR, DNA-PK, and Chk1 in Countering Replication Stress during S Phase

PubMed Central

Buisson, Rémi; Boisvert, Jessica L.; Benes, Cyril H.; Zou, Lee

2015-01-01

The ATR-Chk1 pathway is critical for DNA damage responses and cell cycle progression. Chk1 inhibition is more deleterious to cycling cells than ATR inhibition, raising questions about ATR and Chk1 functions in the absence of extrinsic replication stress. Here, we show that a key role of ATR in S phase is to coordinate RRM2 accumulation and origin firing. ATR inhibitor (ATRi) induces massive ssDNA accumulation and replication catastrophe in a fraction of early S-phase cells. In other S-phase cells, however, ATRi induces moderate ssDNA and triggers a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. The backup pathway creates a threshold such that ATRi selectively kills cells under high replication stress, whereas Chk1 inhibitor induces cell death at a lower threshold. The levels of ATRi-induced ssDNA correlate with ATRi sensitivity in a panel of cell lines, suggesting that ATRi-induced ssDNA could be predictive of ATRi sensitivity in cancer cells. PMID:26365377

A new concept for high-cycle-life LEO: Rechargeable MnO2-hydrogen

NASA Technical Reports Server (NTRS)

Appleby, A. J.; Dhar, H. P.; Kim, Y. J.; Murphy, O. J.

1989-01-01

The nickel-hydrogen secondary battery system, developed in the early 1970s, has become the system of choice for geostationary earth orbit (GEO) applications. However, for low earth orbit (LEO) satellites with long expected lifetimes the nickel positive limits performance. This requires derating of the cell to achieve very long cycle life. A new system, rechargeable MnO2-Hydrogen, which does not require derating, is described here. For LEO applications, it promises to have longer cycle life, high rate capability, a higher effective energy density, and much lower self-discharge behavior than those of the nickel-hydrogen system.

Luna, a Drosophila KLF6/KLF7, Is Maternally Required for Synchronized Nuclear and Centrosome Cycles in the Preblastoderm Embryo

PubMed Central

Weber, Ursula; Rodriguez, Estefania; Martignetti, John; Mlodzik, Marek

2014-01-01

Krüppel like factors (KLFs) are conserved transcription factors that have been implicated in many developmental processes including differentiation, organ patterning, or regulation of stem cell pluripotency. We report the generation and analysis of loss-of-function mutants of Drosophila Klf6/7, the luna gene. We demonstrate that luna mutants are associated with very early embryonic defects prior to cellularization at the syncytial stage and cause DNA separation defects during the rapid mitotic cycles resulting in un-coupled DNA and centrosome cycles. These defects manifest themselves, both in animals that are maternally homozygous and heterozygous mutant. Surprisingly, luna is only required during the syncytial stages and not later in development, suggesting that the DNA segregation defect is linked to centrosomes, since centrosomes are dispensable for later cell divisions. PMID:24915236

Colon Cancer-Upregulated Long Non-Coding RNA lincDUSP Regulates Cell Cycle Genes and Potentiates Resistance to Apoptosis.

PubMed

Forrest, Megan E; Saiakhova, Alina; Beard, Lydia; Buchner, David A; Scacheri, Peter C; LaFramboise, Thomas; Markowitz, Sanford; Khalil, Ahmad M

2018-05-09

Long non-coding RNAs (lncRNAs) are frequently dysregulated in many human cancers. We sought to identify candidate oncogenic lncRNAs in human colon tumors by utilizing RNA sequencing data from 22 colon tumors and 22 adjacent normal colon samples from The Cancer Genome Atlas (TCGA). The analysis led to the identification of ~200 differentially expressed lncRNAs. Validation in an independent cohort of normal colon and patient-derived colon cancer cell lines identified a novel lncRNA, lincDUSP, as a potential candidate oncogene. Knockdown of lincDUSP in patient-derived colon tumor cell lines resulted in significantly decreased cell proliferation and clonogenic potential, and increased susceptibility to apoptosis. The knockdown of lincDUSP affects the expression of ~800 genes, and NCI pathway analysis showed enrichment of DNA damage response and cell cycle control pathways. Further, identification of lincDUSP chromatin occupancy sites by ChIRP-Seq demonstrated association with genes involved in the replication-associated DNA damage response and cell cycle control. Consistent with these findings, lincDUSP knockdown in colon tumor cell lines increased both the accumulation of cells in early S-phase and γH2AX foci formation, indicating increased DNA damage response induction. Taken together, these results demonstrate a key role of lincDUSP in the regulation of important pathways in colon cancer.

Involvement of apoptotic cell death and cell cycle perturbation in retinoic acid-induced cleft palate in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okano, Junko; Suzuki, Shigehiko; Shiota, Kohei

2007-05-15

Retinoic acid (RA), a metabolite of vitamin A, plays a key role in a variety of biological processes and is essential for normal embryonic development. On the other hand, exogenous RA could cause cleft palate in offspring when it is given to pregnant animals at either the early or late phases of palatogenesis, but the pathogenetic mechanism of cleft palate caused by excess RA remains not fully elucidated. The aim of the present study was to investigate the effects of excess of RA on early palatogenesis in mouse fetuses and analyze the teratogenic mechanism, especially at the stage prior tomore » palatal shelf elevation. We gave all-trans RA (100 mg/kg) orally to E11.5 ICR pregnant mice and observed the changes occurring in the palatal shelves of their fetuses. It was found that apoptotic cell death increased not only in the epithelium of the palatal shelves but also in the tongue primordium, which might affect tongue withdrawal movement during palatogenesis and impair the horizontal elevation of palatal shelves. In addition, RA was found to prevent the G{sub 1}/S progression of palatal mesenchymal cells through upregulation of p21 {sup Cip1}, leading to Rb hypophospholylation. Thus, RA appears to cause G{sub 1} arrest in palatal mesenchymal cells in a similar manner as in various cancer and embryonic cells. It is likely that apoptotic cell death and cell cycle disruption are involved in cleft palate formation induced by RA.« less

Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration

PubMed Central

Pajaud, J; Ribault, C; Ben Mosbah, I; Rauch, C; Henderson, C; Bellaud, P; Aninat, C; Loyer, P; Morel, F; Corlu, A

2015-01-01

Glutathione transferases (GST) are phase II enzymes catalyzing the detoxification of endogenous noxious compounds and xenobiotics. They also regulate phosphorylation activities of MAPKinases in a catalytic-independent manner. Previous studies have demonstrated the regulation of JNK-dependent pathway by GSTP1/2. Considering the crucial role of JNK in the early steps of the hepatocyte cell cycle, we sought to determine whether GSTP1/2 were essential for hepatocyte proliferation following partial hepatectomy (PH). Using a conventional double knockout mouse model for the Gstp1 and Gstp2 genes, we found that the lack of GSTP1/P2 reduced the rate of DNA replication and mitotic index during the first wave of hepatocyte proliferation. The lowered proliferation was associated with the decrease in TNFalpha and IL-6 plasma concentrations, reduced hepatic HGF expression and delayed and/or altered activation of STAT3, JNK and ERK1/2 signaling pathways. In addition, the expression and/or activation of cell cycle regulators such as Cyclin D1, CDK4, E2F1 and MCM7 was postponed demonstrating that the absence of GSTP1/2 delayed the entry into and progression through the G1 phase of the cell cycle and impaired the synchrony of proliferation in hepatocytes following PH. Furthermore, while JNK and its downstream targets c-Jun and ATF2 were activated during the early steps of the liver regeneration in wild-type animals, the constitutively active JNK found in the quiescent liver of Gstp1/2 knockout mice underwent a decrease in its activity after PH. Transient induction of antioxidant enzymes and nitric oxide synthase were also delayed or repressed during the regenerative response. Altogether our results demonstrate that GSTP1/2 are a critical regulators of hepatocyte proliferation in the initial phases of liver regeneration. PMID:25590808

Different rates of DNA replication at early versus late S-phase sections: multiscale modeling of stochastic events related to DNA content/EdU (5-ethynyl-2'deoxyuridine) incorporation distributions.

PubMed

Li, Biao; Zhao, Hong; Rybak, Paulina; Dobrucki, Jurek W; Darzynkiewicz, Zbigniew; Kimmel, Marek

2014-09-01

Mathematical modeling allows relating molecular events to single-cell characteristics assessed by multiparameter cytometry. In the present study we labeled newly synthesized DNA in A549 human lung carcinoma cells with 15-120 min pulses of EdU. All DNA was stained with DAPI and cellular fluorescence was measured by laser scanning cytometry. The frequency of cells in the ascending (left) side of the "horseshoe"-shaped EdU/DAPI bivariate distributions reports the rate of DNA replication at the time of entrance to S phase while their frequency in the descending (right) side is a marker of DNA replication rate at the time of transition from S to G2 phase. To understand the connection between molecular-scale events and scatterplot asymmetry, we developed a multiscale stochastic model, which simulates DNA replication and cell cycle progression of individual cells and produces in silico EdU/DAPI scatterplots. For each S-phase cell the time points at which replication origins are fired are modeled by a non-homogeneous Poisson Process (NHPP). Shifted gamma distributions are assumed for durations of cell cycle phases (G1, S and G2 M), Depending on the rate of DNA synthesis being an increasing or decreasing function, simulated EdU/DAPI bivariate graphs show predominance of cells in left (early-S) or right (late-S) side of the horseshoe distribution. Assuming NHPP rate estimated from independent experiments, simulated EdU/DAPI graphs are nearly indistinguishable from those experimentally observed. This finding proves consistency between the S-phase DNA-replication rate based on molecular-scale analyses, and cell population kinetics ascertained from EdU/DAPI scatterplots and demonstrates that DNA replication rate at entrance to S is relatively slow compared with its rather abrupt termination during S to G2 transition. Our approach opens a possibility of similar modeling to study the effect of anticancer drugs on DNA replication/cell cycle progression and also to quantify other kinetic events that can be measured during S-phase. © 2014 International Society for Advancement of Cytometry.

Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression

PubMed Central

Nordin, Noraziah; Majid, Nazia Abdul; Hashim, Najihah Mohd; Rahman, Mashitoh Abd; Hassan, Zalila; Ali, Hapipah Mohd

2015-01-01

Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent. PMID:25792804

In Situ Stress Evolution in Li 1+x Mn 2 O 4 Thin Films during Electrochemical Cycling in Li-Ion Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheth, Jay; Karan, Naba K.; Abraham, Daniel P.

2016-01-01

Real time monitoring of stress evolution in electrodes during electrochemical cycling can help quantify the driving forces that dictate their mechanical degradation. In the present work, in-situ stress evolution in thin films of spinel Li 1+x Mn 2 O 4 (LMO) was measured by monitoring the change in the elastic substrate curvature during electrochemical cycling in a specially designed beaker cell in the 3.5–4.3 V (vs. Li/Li+) voltage range. The LMO thin films were prepared using a solution deposition technique and their structures and morphologies were characterized by X-ray diffraction (XRD), Raman spectroscopy and scanning electron microscopy (SEM). The stressmore » evolution in the early part of the first delithiation cycle (<4.05 V) was consistent with the XRD data. However, stress evolution during later stages of the first delithiation cycle (>4.05 V) was not consistent with the XRD results, and showed irreversible behavior, suggesting irreversible changes in the electrode. Beyond the first delithiation cycle, the stress evolution was reversible, with a steady buildup of compressive and tensile stress during lithium insertion and extraction, respectively. Measurements on LMO films of varying thicknesses suggest that the first cycle irreversibility in stress response arises primarily from the electrode bulk.« less

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo.

PubMed

Speksnijder, J E; Dohmen, M R; Tertoolen, L G; de Laat, S W

1985-07-01

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1'-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine iodide (C14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lobe area as compared to the animal plasma membrane area (on average 30%), demonstrating the existence of an animal-vegetal polarity in plasma membrane properties. At third cleavage, the differences between animal and vegetal plasma membrane region become even more pronounced; in the four animal micromeres the diffusion coefficient (D) and mobile fraction (MF) are 2.9 +/- 0.2 X 10(-9) cm2/sec and 51 +/- 2%, respectively, while in the four vegetal macromeres D = 5.0 +/- 0.3 X 10(-9) cm2/sec and MF = 78 +/- 2%. Superimposed upon the observed animal-vegetal polarity, the lateral diffusion in the polar lobe membrane area shows a cell-cycle-dependent modulation. The highest mean values for D are reached during the S phase (ranging from 7.0 to 7.8 X 10(-9) cm2/sec in the three cycles measured), while at the end of G2 phase and during early mitosis mean values for D have decreased significantly (ranging from 5.0 to 5.9 X 10(-9) cm2/sec). Diffusion rates in the animal membranes of the embryo are constant during the three successive cell cycles (D = 4.3-5.0 X 10(-9) cm2/sec), except for a peak at the S phase of the first cell cycle (D = 6.0 X 10(-9) cm2/sec). These results are discussed in relation with previously observed ultrastructural heterogeneities in the Nassarius egg plasma membrane. It is speculated that the observed animal-vegetal polarity in the organization of the egg membrane might play an important role in the process of cell diversification during early development.

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen.

PubMed

Wang, Gangduo; Wang, Jianling; Khan, M Firoze

2017-09-01

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1 mmol/kg/day by gavage) for 7 days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.

The pathobiology and mechanisms of infection of HPV.

PubMed

Wood, N H; Khammissa, R A G; Chikte, U M E; Meyerov, R; Lemmer, J; Feller, L

2010-04-01

There are more than 120 types of low-risk and high-risk human papillomaviruses, all of which are epitheliotropic. HPV infection may be latent, or active in a subclinical form or a symptomatic form, the latter manifesting as benign or malignant neoplasms. In basal cells with non-productive HPV infection some early HPV proteins are expressed independently of cell maturation: the productive cycle of HPV replication depends upon specific cellular factors of the maturation of the infected keratinocytes. In HPV-mediated oncogenesis, the combined pathobiological effects of E6 and E7 oncoproteins of high-risk HPV culminate in cellular genomic instability and transformation of persistently infected cells, that progress to the development of a malignant phenotype. In this article we provide insights into the stages of HPV infection, and into the viral genomic organization and replicative cycle.

Propolis from the Stingless Bee Trigona incisa from East Kalimantan, Indonesia, Induces In Vitro Cytotoxicity and Apoptosis in Cancer Cell lines.

PubMed

Kustiawan, Paula M; Phuwapraisirisan, Preecha; Puthong, Songchan; Palaga, Tanapat; Arung, Enos T; Chanchao, Chanpen

2015-01-01

Previously, stingless bee (Trigona spp.) products from East Kalimantan, Indonesia, were successfully screened for in vitro antiproliferative activity against human cancer derived cell lines. It was established that propolis from T. incisa presented the highest in vitro cytotoxicity against the SW620 colon cancer cell line (6% cell survival in 20 μg/mL). Propolis from T. incisa was extracted with methanol and further partitioned with n-hexane, ethyl acetate and methanol. The in vitro cytotoxicity of the extracts was assessed by the MTT assay against human colon (SW620), liver (Hep-G2), gastric (KATO-III), lung (Chago) and breast (BT474) cancer derived cell lines. The active fractions were further enriched by silica gel quick column, absorption and size exclusion chromatography. The purity of each fraction was checked by thin layer chromatography. Cytotoxicity in BT-474 cells induced by cardanol compared to doxorubicin were evaluated by MTT assay, induction of cell cycle arrest and cell death by flow cytometric analysis of propidium iodide and annexin-V stained cells. A cardol isomer was found to be the major compound in one active fraction (F45) of T. incisa propolis, with a cytotoxicity against the SW620 (IC50 of 4.51±0.76 μg/mL), KATO-III (IC50 of 6.06±0.39 μg/mL), Hep-G2 (IC50 of 0.71±0.22 μg/mL), Chago I (IC50 of 0.81±0.18 μg/mL) and BT474 (IC50 of 4.28±0.14 μg/mL) cell lines. Early apoptosis (programmed cell death) of SW620 cells was induced by the cardol containing F45 fraction at the IC50 and IC80 concentrations, respectively, within 2-6 h of incubation. In addition, the F45 fraction induced cell cycle arrest at the G1 subphase. Indonesian stingless bee (T. incisa) propolis had moderately potent in vitro anticancer activity on human cancer derived cell lines. Cardol or 5-pentadecyl resorcinol was identified as a major active compound and induced apoptosis in SW620 cells in an early period (≤6 h) and cell cycle arrest at the G1 subphase. Thus, cardol is a potential candidate for cancer chemotherapy.

Cell Type-Specific Gene Expression Analyses by RNA Sequencing Reveal Local High Nitrate-Triggered Lateral Root Initiation in Shoot-Borne Roots of Maize by Modulating Auxin-Related Cell Cycle Regulation1[OPEN

PubMed Central

Yu, Peng; Eggert, Kai; von Wirén, Nicolaus; Li, Chunjian; Hochholdinger, Frank

2015-01-01

Plants have evolved a unique plasticity of their root system architecture to flexibly exploit heterogeneously distributed mineral elements from soil. Local high concentrations of nitrate trigger lateral root initiation in adult shoot-borne roots of maize (Zea mays) by increasing the frequency of early divisions of phloem pole pericycle cells. Gene expression profiling revealed that, within 12 h of local high nitrate induction, cell cycle activators (cyclin-dependent kinases and cyclin B) were up-regulated, whereas repressors (Kip-related proteins) were down-regulated in the pericycle of shoot-borne roots. In parallel, a ubiquitin protein ligase S-Phase Kinase-Associated Protein1-cullin-F-box proteinS-Phase Kinase-Associated Protein 2B-related proteasome pathway participated in cell cycle control. The division of pericycle cells was preceded by increased levels of free indole-3-acetic acid in the stele, resulting in DR5-red fluorescent protein-marked auxin response maxima at the phloem poles. Moreover, laser-capture microdissection-based gene expression analyses indicated that, at the same time, a significant local high nitrate induction of the monocot-specific PIN-FORMED9 gene in phloem pole cells modulated auxin efflux to pericycle cells. Time-dependent gene expression analysis further indicated that local high nitrate availability resulted in PIN-FORMED9-mediated auxin efflux and subsequent cell cycle activation, which culminated in the initiation of lateral root primordia. This study provides unique insights into how adult maize roots translate information on heterogeneous nutrient availability into targeted root developmental responses. PMID:26198256

Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F.

PubMed Central

Bandara, L R; Buck, V M; Zamanian, M; Johnston, L H; La Thangue, N B

1993-01-01

It is widely believed that the cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus because during cell cycle progression in mammalian cells it interacts with molecules that are important regulators of cellular proliferation, such as the retinoblastoma tumour suppressor gene product (pRb), p107, cyclins and cyclin-dependent kinases. Thus, pRb, which negatively regulates early cell cycle progression and is frequently mutated in tumour cells, and the Rb-related protein p107, bind to and repress the transcriptional activity of DRTF1/E2F. Viral oncoproteins, such as adenovirus E1a and SV40 large T antigen, overcome such repression by sequestering pRb and p107 and in so doing are likely to activate genes regulated by DRTF1/E2F, such as cdc2, c-myc and DHFR. Two sequence-specific DNA binding proteins, E2F-1 and DP-1, which bind to the E2F site, contain a small region of similarity. The functional relationship between them has, however, been unclear. We report here that DP-1 and E2F-1 exist in a DNA binding complex in vivo and that they bind efficiently and preferentially as a heterodimer to the E2F site. Moreover, studies in yeast and Drosophila cells indicate that DP-1 and E2F-1 interact synergistically in E2F site-dependent transcriptional activation. Images PMID:8223441

Bragg-cell receiver study

NASA Technical Reports Server (NTRS)

Wilson, Lonnie A.

1987-01-01

Bragg-cell receivers are employed in specialized Electronic Warfare (EW) applications for the measurement of frequency. Bragg-cell receiver characteristics are fully characterized for simple RF emitter signals. This receiver is early in its development cycle when compared to the IFM receiver. Functional mathematical models are derived and presented in this report for the Bragg-cell receiver. Theoretical analysis is presented and digital computer signal processing results are presented for the Bragg-cell receiver. Probability density function analysis are performed for output frequency. Probability density function distributions are observed to depart from assumed distributions for wideband and complex RF signals. This analysis is significant for high resolution and fine grain EW Bragg-cell receiver systems.

Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells*

PubMed Central

Lin, Shu; Yuan, Zuo-Fei; Han, Yumiao; Marchione, Dylan M.; Garcia, Benjamin A.

2016-01-01

How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2. PMID:27226594

Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells.

PubMed

Lin, Shu; Yuan, Zuo-Fei; Han, Yumiao; Marchione, Dylan M; Garcia, Benjamin A

2016-07-15

How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Energetics and genetics across the prokaryote-eukaryote divide

PubMed Central

2011-01-01

Background All complex life on Earth is eukaryotic. All eukaryotic cells share a common ancestor that arose just once in four billion years of evolution. Prokaryotes show no tendency to evolve greater morphological complexity, despite their metabolic virtuosity. Here I argue that the eukaryotic cell originated in a unique prokaryotic endosymbiosis, a singular event that transformed the selection pressures acting on both host and endosymbiont. Results The reductive evolution and specialisation of endosymbionts to mitochondria resulted in an extreme genomic asymmetry, in which the residual mitochondrial genomes enabled the expansion of bioenergetic membranes over several orders of magnitude, overcoming the energetic constraints on prokaryotic genome size, and permitting the host cell genome to expand (in principle) over 200,000-fold. This energetic transformation was permissive, not prescriptive; I suggest that the actual increase in early eukaryotic genome size was driven by a heavy early bombardment of genes and introns from the endosymbiont to the host cell, producing a high mutation rate. Unlike prokaryotes, with lower mutation rates and heavy selection pressure to lose genes, early eukaryotes without genome-size limitations could mask mutations by cell fusion and genome duplication, as in allopolyploidy, giving rise to a proto-sexual cell cycle. The side effect was that a large number of shared eukaryotic basal traits accumulated in the same population, a sexual eukaryotic common ancestor, radically different to any known prokaryote. Conclusions The combination of massive bioenergetic expansion, release from genome-size constraints, and high mutation rate favoured a protosexual cell cycle and the accumulation of eukaryotic traits. These factors explain the unique origin of eukaryotes, the absence of true evolutionary intermediates, and the evolution of sex in eukaryotes but not prokaryotes. Reviewers This article was reviewed by: Eugene Koonin, William Martin, Ford Doolittle and Mark van der Giezen. For complete reports see the Reviewers' Comments section. PMID:21714941

Dynamic Epstein-Barr Virus Gene Expression on the Path to B-Cell Transformation

PubMed Central

Price, Alexander M.; Luftig, Micah A.

2016-01-01

Epstein-Barr Virus is an oncogenic human herpesvirus in the γ-herpesvirinae sub-family that contains a 170–180 kb double stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B cell compartment of the peripheral blood. EBV can be reactivated from its latent state leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome as well as structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady state viral gene expression within EBV immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection EBV only expressed the well-characterized latency associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation as well as delayed responses in the known latency genes. This review summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, inhibition of apoptosis, and control of innate and adaptive immune responses. PMID:24373315

Perturbation of host-cell membrane is a primary mechanism of HIV cytopathology.

PubMed

Cloyd, M W; Lynn, W S

1991-04-01

Cytopathic viruses injure cells by a number of different mechanisms. The mechanism by which HIV-1 injures T cells was studied by temporally examining host-cell macromolecular syntheses, stages of the cell cycle, and membrane permeability following acute infection. T cells cytopathically infected at an m.o.i. of 1-5 grew normally for 24-72 hr, depending on the cell line, followed by the first manifestation of cell injury, slowing of cell division. At that time significant amounts of unintegrated HIV DNA and p24 core protein became detectable, and acridine orange flow cytometric cell cycle studies demonstrated the presence of fewer cells in the G2/M stage of the cell cycle. There was no change in the frequency of cells in the S-stage, and metabolic pulsing with radioactive precursors demonstrated that host-cell DNA, RNA, and protein syntheses were normal at that time and normal up to the time cells started to die (approximately 24 hr later), when all three decreased. Cellular lipid synthesis, however, was perturbed when cell multiplication slowed, with phospholipid synthesis reduced and neutral lipid synthesis enhanced. Permeability of the host-cell membrane to small molecules, such as Ca2+ and sucrose, was slightly enhanced early postinfection, and by the time of slowing of cell division, host membrane permeability was greatly increased to both Ca2+ and sucrose (Stokes radius 5.2 A) but not to inulin (Stokes radium 20 A). These changes in host-cell membrane permeability and phospholipid synthesis were not observed in acutely infected H9 cells, which are not susceptible to HIV cytopathology. Thus, HIV-1 appeared to predominantly injure T cells by perturbing host-cell membrane permeability and lipid synthesis, which is similar to the cytopathic mechanisms of paramyxoviruses.

Aglycone Ebselen and β-d-Xyloside Primed Glycosaminoglycans Co-contribute to Ebselen β-d-Xyloside-Induced Cytotoxicity.

PubMed

Tang, Yang; Zhang, Siqi; Chang, Yajing; Fan, Dacheng; Agostini, Ariane De; Zhang, Lijuan; Jiang, Tao

2018-04-12

Most β-d-xylosides with hydrophobic aglycones are nontoxic primers for glycosaminoglycan assembly in animal cells. However, when Ebselen was conjugated to d-xylose, d-glucose, d-galactose, and d-lactose (8A-D), only Ebselen β-d-xyloside (8A) showed significant cytotoxicity in human cancer cells. The following facts indicated that the aglycone Ebselen and β-d-xyloside primed glycosaminoglycans co-contributed to the observed cytotoxicity: 1. Ebselen induced S phase cell cycle arrest, whereas 8A induced G2/M cell cycle arrest; 2. 8A augmented early and late phase cancer cell apoptosis significantly compared to that of Ebselen and 8B-D; 3. Both 8A and phenyl-β-d-xyloside primed glycosaminoglycans with similar disaccharide compositions in CHO-pgsA745 cells; 4. Glycosaminoglycans could be detected inside of cells only when treated with 8A, indicating Ebselen contributed to the unique property of intracellular localization of the primed glycosaminoglycans. Thus, 8A represents a lead compound for the development of novel antitumor strategy by targeting glycosaminoglycans.

Identification of conserved genes triggering puberty in European sea bass males (Dicentrarchus labrax) by microarray expression profiling.

PubMed

Blázquez, Mercedes; Medina, Paula; Crespo, Berta; Gómez, Ana; Zanuy, Silvia

2017-06-05

Spermatogenesis is a complex process characterized by the activation and/or repression of a number of genes in a spatio-temporal manner. Pubertal development in males starts with the onset of the first spermatogenesis and implies the division of primary spermatogonia and their subsequent entry into meiosis. This study is aimed at the characterization of genes involved in the onset of puberty in European sea bass, and constitutes the first transcriptomic approach focused on meiosis in this species. European sea bass testes collected at the onset of puberty (first successful reproduction) were grouped in stage I (resting stage), and stage II (proliferative stage). Transition from stage I to stage II was marked by an increase of 11ketotestosterone (11KT), the main fish androgen, whereas the transcriptomic study resulted in 315 genes differentially expressed between the two stages. The onset of puberty induced 1) an up-regulation of genes involved in cell proliferation, cell cycle and meiosis progression, 2) changes in genes related with reproduction and growth, and 3) a down-regulation of genes included in the retinoic acid (RA) signalling pathway. The analysis of GO-terms and biological pathways showed that cell cycle, cell division, cellular metabolic processes, and reproduction were affected, consistent with the early events that occur during the onset of puberty. Furthermore, changes in the expression of three RA nuclear receptors point at the importance of the RA-signalling pathway during this period, in agreement with its role in meiosis. The results contribute to boost our knowledge of the early molecular and endocrine events that trigger pubertal development and the onset of spermatogenesis in fish. These include an increase in 11KT plasma levels and changes in the expression of several genes involved in cell proliferation, cell cycle progression, meiosis or RA-signalling pathway. Moreover, the results can be applied to study meiosis in this economically important fish species for Mediterranean countries, and may help to develop tools for its sustainable aquaculture.

Targeted deletion of p97 (VCP/CDC48) in mouse results in early embryonic lethality.

PubMed

Müller, J M M; Deinhardt, K; Rosewell, I; Warren, G; Shima, D T

2007-03-09

The highly conserved AAA ATPase p97 (VCP/CDC48) has well-established roles in cell cycle progression, proteasome degradation and membrane dynamics. Gene disruption in Saccromyces cerevisiae, Drosophila melanogaster and Trypanosoma brucei demonstrated that p97 is essential in unicellular and multicellular organisms. To explore the requirement for p97 in mammalian cell function and embryogenesis, we disrupted the p97 locus by gene targeting. Heterozygous p97+/- mice were indistinguishable from their wild-type littermates, whereas homozygous mutants did not survive to birth and died at a peri-implantation stage. These results show that p97 is an essential gene for early mouse development.

Characterization of the 20-Ah nickel-cadmium cell used for energy storage on the Orbiting Astronomical Observatory

NASA Technical Reports Server (NTRS)

Ford, F. E.

1972-01-01

Tests were conducted on 20-Ah sealed nickel cadmium cells to evaluate initial and long-term performance at various charge rates, temperatures and voltage-control levels. An average ampere-hour recharge of 103 percent per orbit at 13 C was able to maintain cell capacity; required watt-hour recharge on an orbital basis was 8 to 10 percent greater than required ampere-hour recharge. Cells exhibited an early life burn-in characteristic. A discharge after periods of repetitive cycling yielded two voltage plateaus which were temporarily eliminated by the discharge.

Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

PubMed

Frémin, Christophe; Bessard, Anne; Ezan, Frédéric; Gailhouste, Luc; Régeard, Morgane; Le Seyec, Jacques; Gilot, David; Pagès, Gilles; Pouysségur, Jacques; Langouët, Sophie; Baffet, Georges

2009-03-01

We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes.

Expression patterns of wnt8 orthologs in two sand dollar species with different developmental modes.

PubMed

Nakata, Hidewo; Minokawa, Takuya

2009-03-01

Two wnt8 orthologs, Smwnt8 and Pjwnt8, were isolated from an indirect developing sand dollar, Scaphechinus mirabilis, and a direct developing sand dollar, Peronella japonica, respectively. The expression patterns of two genes during early development were examined by whole mount in situ hybridization. The expression of Smwnt8 was initiated in the micromeres at the late 16-cell stage and expanded at the 64-cell stage to the whole vegetal hemisphere, including the presumptive endomesodermal regions. The timing of the initiation of Pjwnt8 transcription in the presumptive endomesoderm region was delayed by 2-3 cell cycles compared to that of Smwnt8. The delay, or molecular heterochrony, of Pjwnt8 transcription strongly suggests the existence of a substantial evolutionary change in the early endomesodermal specification of P. japonica. In addition to the endomesodermal expression during early embryogenesis, bilateral expressions were observed commonly in the ectoderm of two sand dollar species during larval stages.

Early BrdU-responsive genes constitute a novel class of senescence-associated genes in human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minagawa, Sachi; Nakabayashi, Kazuhiko; Fujii, Michihiko

2005-04-01

We identified genes that immediately respond to 5-bromodeoxyuridine (BrdU) in SUSM-1, an immortal fibroblastic line, with DNA microarray and Northern blot analysis. At least 29 genes were found to alter gene expression greater than twice more or less than controls within 36 h after addition of BrdU. They took several different expression patterns upon addition of BrdU, and the majority showed a significant alteration within 12 h. When compared among SUSM-1, HeLa, and TIG-7 normal human fibroblasts, 19 genes behaved similarly upon addition of BrdU. In addition, 14 genes, 9 of which are novel as regards senescence, behaved similarly inmore » senescent TIG-7 cells. The genes do not seem to have a role in proliferation or cell cycle progression. These results suggest that the early BrdU-responsive genes represent early signs of cellular senescence and can be its new biomarkers.« less

PM2.5-induced alterations of cell cycle associated gene expression in lung cancer cells and rat lung tissues.

PubMed

Zhao, Hui; Yang, Biao; Xu, Jia; Chen, Dong-Mei; Xiao, Chun-Ling

2017-06-01

The aim of the current study was to investigate the expression of cell cycle-associated genes induced by fine particulate matter (PM 2.5 ) in lung cancer cell line and tissues. The pulmonary lymph node metastasis cells (H292) were treated with PM 2.5 in vitro. Wistar rats were used to perform an in vivo study. Rats were randomly assigned to experiment and control groups and those in the experiment group were exposed to PM 2.5 once every 15 d, while those in the control group were exposed to normal saline. The cell cycle-associated genes expression was analyzed by real-time PCR. Trachea and lung tissues of rats were processed for scanning electron microscopic (SEM) examinations. Exposure of H292 cells to PM 2.5 dramatically increased the expressions of p53 and cyclin-dependent kinase 2 (CDK2) after 24h of exposure (p<0.01) and markedly increased the expressions of the cell division cycle 2 (Cdc2) and cyclin B after 48h of exposure (p<0.01), while those genes expressions were significantly reduced after 72h of exposure, at which time the expression of p21 was predominant (p<0.01). In vivo studies further demonstrated these results. The results of SEM suggested that both of the trachea and lung tissues were damaged and the degree of damage was time-dependent. In conclusion, PM 2.5 can induce significantly alterations of p53 and CDK2 in the early phase, Cdc2 and cyclin B in mid-term and p21 in long-term exposure. The degree of PM 2.5 -induced damage to the trachea and lung tissue was time-dependent. Copyright © 2017. Published by Elsevier B.V.

Alteration of the carbohydrate for deoxyguanosine analogs markedly changes DNA replication fidelity, cell cycle progression and cytotoxicity

PubMed Central

O’Konek, Jessica J.; Ladd, Brendon; Flanagan, Sheryl A.; Im, Mike M.; Boucher, Paul D.; Thepsourinthone, Tico S.; Secrist, John A.; Shewach, Donna S.

2011-01-01

Nucleoside analogs are efficacious cancer chemotherapeutics due to their incorporation into tumor cell DNA. However, they exhibit vastly different antitumor efficacies, suggesting that incorporation produces divergent effects on DNA replication. Here we have evaluated the consequences of incorporation on DNA replication and its fidelity for three structurally related deoxyguanosine analogs: ganciclovir (GCV), currently in clinical trials in a suicide gene therapy approach for cancer, D-carbocyclic 2′-deoxyguanosine (CdG) and penciclovir (PCV). GCV and CdG elicited similar cytotoxicity at low concentrations, whereas PCV was 10–100-fold less cytotoxic in human tumor cells. DNA replication fidelity was evaluated using a supF plasmid-based mutation assay. Only GCV induced a dose-dependent increase in mutation frequency, predominantly GC→TA transversions, which contributed to cytotoxicity and implicated the ether oxygen in mutagenicity. Activation of mismatch repair with hydroxyurea decreased mutations but failed to repair the GC→TA transversions. GCV slowed S-phase progression and CdG also induced a G2/M block, but both drugs allowed completion of one cell cycle after drug treatment followed by cell death in the second cell cycle. In contrast, PCV induced a lengthy early S-phase block due to profound suppression of DNA synthesis, with cell death in the first cell cycle after drug treatment. These data suggest that GCV and CdG elicit superior cytotoxicity due to their effects in template DNA, whereas strong inhibition of nascent strand synthesis by PCV may protect against cytotoxicity. Nucleoside analogs based on the carbohydrate structures of GCV and CdG is a promising area for antitumor drug development. PMID:20004674

PRM1 and KAR5 function in cell-cell fusion and karyogamy to drive distinct bisexual and unisexual cycles in the Cryptococcus pathogenic species complex

PubMed Central

Fu, Ci; Heitman, Joseph

2017-01-01

Sexual reproduction is critical for successful evolution of eukaryotic organisms in adaptation to changing environments. In the opportunistic human fungal pathogens, the Cryptococcus pathogenic species complex, C. neoformans primarily undergoes bisexual reproduction, while C. deneoformans undergoes both unisexual and bisexual reproduction. During both unisexual and bisexual cycles, a common set of genetic circuits regulates a yeast-to-hyphal morphological transition, that produces either monokaryotic or dikaryotic hyphae. As such, both the unisexual and bisexual cycles can generate genotypic and phenotypic diversity de novo. Despite the similarities between these two cycles, genetic and morphological differences exist, such as the absence of an opposite mating-type partner and monokaryotic instead of dikaryotic hyphae during C. deneoformans unisexual cycle. To better understand the similarities and differences between these modes of sexual reproduction, we focused on two cellular processes involved in sexual reproduction: cell-cell fusion and karyogamy. We identified orthologs of the plasma membrane fusion protein Prm1 and the nuclear membrane fusion protein Kar5 in both Cryptococcus species, and demonstrated their conserved roles in cell fusion and karyogamy during C. deneoformans α-α unisexual reproduction and C. deneoformans and C. neoformans a-α bisexual reproduction. Notably, karyogamy occurs inside the basidum during bisexual reproduction in C. neoformans, but often occurs earlier following cell fusion during bisexual reproduction in C. deneoformans. Characterization of these two genes also showed that cell fusion is dispensable for solo unisexual reproduction in C. deneoformans. The blastospores produced along hyphae during C. deneoformans unisexual reproduction are diploid, suggesting that diploidization occurs early during hyphal development, possibly through either an endoreplication pathway or cell fusion-independent karyogamy events. Taken together, our findings suggest distinct mating mechanisms for unisexual and bisexual reproduction in Cryptococcus, exemplifying distinct evolutionary trajectories within this pathogenic species complex. PMID:29176784

Pax6 Exerts Regional Control of Cortical Progenitor Proliferation via Direct Repression of Cdk6 and Hypophosphorylation of pRb

PubMed Central

Mi, Da; Carr, Catherine B.; Georgala, Petrina A.; Huang, Yu-Ting; Manuel, Martine N.; Jeanes, Emily; Niisato, Emi; Sansom, Stephen N.; Livesey, Frederick J.; Theil, Thomas; Hasenpusch-Theil, Kerstin; Simpson, T. Ian; Mason, John O.; Price, David J.

2013-01-01

Summary The mechanisms by which early spatiotemporal expression patterns of transcription factors such as Pax6 regulate cortical progenitors in a region-specific manner are poorly understood. Pax6 is expressed in a gradient across the developing cortex and is essential for normal corticogenesis. We found that constitutive or conditional loss of Pax6 increases cortical progenitor proliferation by amounts that vary regionally with normal Pax6 levels. We compared the gene expression profiles of equivalent Pax6-expressing progenitors isolated from Pax6+/+ and Pax6−/− cortices and identified many negatively regulated cell-cycle genes, including Cyclins and Cdks. Biochemical assays indicated that Pax6 directly represses Cdk6 expression. Cyclin/Cdk repression inhibits retinoblastoma protein (pRb) phosphorylation, thereby limiting the transcription of genes that directly promote the mechanics of the cell cycle, and we found that Pax6 inhibits pRb phosphorylation and represses genes involved in DNA replication. Our results indicate that Pax6’s modulation of cortical progenitor cell cycles is regional and direct. PMID:23622063

Seed Germination and Seedling Growth under Simulated Microgravity Causes Alterations in Plant Cell Proliferation and Ribosome Biogenesis

NASA Astrophysics Data System (ADS)

Matía, Isabel; van Loon, Jack W. A.; Carnero-Díaz, Eugénie; Marco, Roberto; Medina, Francisco Javier

2009-01-01

The study of the modifications induced by altered gravity in functions of plant cells is a valuable tool for the objective of the survival of terrestrial organisms in conditions different from those of the Earth. We have used the system "cell proliferation-ribosome biogenesis", two inter-related essential cellular processes, with the purpose of studying these modifications. Arabidopsis seedlings belonging to a transformed line containing the reporter gene GUS under the control of the promoter of the cyclin gene CYCB1, a cell cycle regulator, were grown in a Random Positioning Machine, a device known to accurately simulate microgravity. Samples were taken at 2, 4 and 8 days after germination and subjected to biometrical analysis and cellular morphometrical, ultrastructural and immunocytochemical studies in order to know the rates of cell proliferation and ribosome biogenesis, plus the estimation of the expression of the cyclin gene, as an indication of the state of cell cycle regulation. Our results show that cells divide more in simulated microgravity in a Random Positioning Machine than in control gravity, but the cell cycle appears significantly altered as early as 2 days after germination. Furthermore, higher proliferation is not accompanied by an increase in ribosome synthesis, as is the rule on Earth, but the functional markers of this process appear depleted in simulated microgravity-grown samples. Therefore, the alteration of the gravitational environmental conditions results in a considerable stress for plant cells, including those not specialized in gravity perception.

DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchetti, Francesco; Wyrobek, Andrew J.

Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization.more » During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.« less

Complexes of D-type cyclins with CDKs during maize germination

PubMed Central

Vázquez-Ramos, Jorge M.

2013-01-01

The importance of cell proliferation in plant growth and development has been well documented. The majority of studies on basic cell cycle mechanisms in plants have been at the level of gene expression and much less knowledge has accumulated in terms of protein interactions and activation. Two key proteins, cyclins and cyclin-dependent kinases (CDKs) are fundamental for cell cycle regulation and advancement. Our aim has been to understand the role of D-type cyclins and type A and B CDKs in the cell cycle taking place during a developmental process such as maize seed germination. Results indicate that three maize D-type cyclins—D2;2, D4;2, and D5;3—(G1-S cyclins by definition) bind and activate two different types of CDK—A and B1;1—in a differential way during germination. Whereas CDKA–D-type cyclin complexes are more active at early germination times than at later times, it was surprising to observe that CDKB1;1, a supposedly G2-M kinase, bound in a differential way to all D-type cyclins tested during germination. Binding to cyclin D2;2 was detectable at all germination times, forming a complex with kinase activity, whereas binding to D4;2 and D5;3 was more variable; in particular, D5;3 was only detected at late germination times. Results are discussed in terms of cell cycle advancement and its importance for seed germination. PMID:24127516

Influence of homologous recombinational repair on cell survival and chromosomal aberration induction during the cell cycle in γ-irradiated CHO cells

PubMed Central

Wilson, Paul F.; Hinz, John M.; Urbin, Salustra S.; Nham, Peter B.; Thompson, Larry H.

2010-01-01

The repair of DNA double-strand breaks (DSB) by homologous recombinational repair (HRR) underlies the high radioresistance and low mutability observed in S-phase mammalian cells. To evaluate the contributions of HRR and nonhomologous end-joining (NHEJ) to overall DSB repair capacity throughout the cell cycle after γ-irradiation, we compared HRR-deficient RAD51D-knockout 51D1 to CgRAD51D-complemented 51D1 (51D1.3) CHO cells for survival and chromosomal aberrations (CAs). Asynchronous cultures were irradiated with 150 or 300 cGy and separated by cell size using centrifugal elutriation. Cell survival of each synchronous fraction (~20 fractions total from early G1 to late G2/M) was measured by colony formation. 51D1.3 cells were most resistant in S, while 51D1 cells were most resistant in early G1 (with survival and chromosome-type CA levels similar to 51D1.3) and became progressively more sensitive throughout S and G2. Both cell lines experienced significantly reduced survival from late S into G2. Metaphases were collected from every third elutriation fraction at the first post-irradiation mitosis and scored for CAs. 51D1 cells irradiated in S and G2 had ~2-fold higher chromatid-type CAs and a remarkable ~25-fold higher level of complex chromatid-type exchanges compared to 51D1.3 cells. Complex exchanges in 51D1.3 cells were only observed in G2. These results show an essential role for HRR in preventing gross chromosomal rearrangements in proliferating cells and, with our previous report of reduced survival of G2-phase NHEJ-deficient prkdc CHO cells [Hinz et al. DNA Repair 4, 782–792, 2005], imply reduced activity/efficiency of both HRR and NHEJ as cells transition from S to G2. PMID:20434408

CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis.

PubMed

Zitouni, Sihem; Francia, Maria E; Leal, Filipe; Montenegro Gouveia, Susana; Nabais, Catarina; Duarte, Paulo; Gilberto, Samuel; Brito, Daniela; Moyer, Tyler; Kandels-Lewis, Steffi; Ohta, Midori; Kitagawa, Daiju; Holland, Andrew J; Karsenti, Eric; Lorca, Thierry; Lince-Faria, Mariana; Bettencourt-Dias, Mónica

2016-05-09

Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nucleoporins redistribute inside the nucleus after cell cycle arrest induced by histone deacetylases inhibition.

PubMed

Pérez-Garrastachu, Miguel; Arluzea, Jon; Andrade, Ricardo; Díez-Torre, Alejandro; Urtizberea, Marta; Silió, Margarita; Aréchaga, Juan

2017-09-03

Nucleoporins are the main components of the nuclear-pore complex (NPC) and were initially considered as mere structural elements embedded in the nuclear envelope, being responsible for nucleocytoplasmic transport. Nevertheless, several recent scientific reports have revealed that some nucleoporins participate in nuclear processes such as transcription, replication, DNA repair and chromosome segregation. Thus, the interaction of NPCs with chromatin could modulate the distribution of chromosome territories relying on the epigenetic state of DNA. In particular, the nuclear basket proteins Tpr and Nup153, and the FG-nucleoporin Nup98 seem to play key roles in all these novel functions. In this work, histone deacetylase inhibitors (HDACi) were used to induce a hyperacetylated state of chromatin and the behavior of the mentioned nucleoporins was studied. Our results show that, after HDACi treatment, Tpr, Nup153 and Nup98 are translocated from the nuclear pore toward the interior of the cell nucleus, accumulating as intranuclear nucleoporin clusters. These transitory structures are highly dynamic, and are mainly present in the population of cells arrested at the G0/G1 phase of the cell cycle. Our results indicate that the redistribution of these nucleoporins from the nuclear envelope to the nuclear interior may be implicated in the early events of cell cycle initialization, particularly during the G1 phase transition.

Detection of Serum Lysophosphatidic Acids Using Affinity Binding and Surface Enhanced Laser Deorption/Ionization (SELDI) Time of Flight Mass Spectrometry

DTIC Science & Technology

2006-04-01

Schmidt, S. A., Clark, K. J. & Murray, A. W. Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin - 43 in...hours later adherent and floating cells were collected and analyzed for cell cycle and apoptosis (hypodiploid peak) using flow cytometry of propidium...pathophysiology of ovarian cancer, provides a major opportunity to identify markers that could contribute to early diagnosis. We have demonstrated that the

Comparison of Accelerated Testing with Modeling to Predict Lifetime of CPV Solder Layers (Presentation)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silverman, T. J.; Bosco, N.; Kurtz, S.

2012-03-01

Concentrating photovoltaic (CPV) cell assemblies can fail due to thermomechanical fatigue in the die-attach layer. In this presentation, we show the latest results from our computational model of thermomechanical fatigue. The model is used to estimate the relative lifetime of cell assemblies exposed to various temperature histories consistent with service and with accelerated testing. We also present early results from thermal cycling experiments designed to help validate the computational model.

Temporal Control of Plant Organ Growth by TCP Transcription Factors.

PubMed

Huang, Tengbo; Irish, Vivian F

2015-06-29

The Arabidopsis petal is a simple laminar organ whose development is largely impervious to environmental effects, making it an excellent model for dissecting the regulation of cell-cycle progression and post-mitotic cell expansion that together sculpt organ form. Arabidopsis petals grow via basipetal waves of cell division, followed by a phase of cell expansion. RABBIT EARS (RBE) encodes a C2H2 zinc finger transcriptional repressor and is required for petal development. During the early phase of petal initiation, RBE regulates a microRNA164-dependent pathway that controls cell proliferation at the petal primordium boundaries. The effects of rbe mutations on petal lamina growth suggest that RBE is also required to regulate later developmental events during petal organogenesis. Here, we demonstrate that, early in petal development, RBE represses the transcription of a suite of CIN-TCP genes that in turn act to inhibit the number and duration of cell divisions; the temporal alleviation of that repression results in the transition from cell division to post-mitotic cell expansion and concomitant petal maturation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

PubMed

Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

2013-10-17

Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

Radiation induces premature chromatid separation via the miR-142-3p/Bod1 pathway in carcinoma cells.

PubMed

Pan, Dong; Du, Yarong; Ren, Zhenxin; Chen, Yaxiong; Li, Xiaoman; Wang, Jufang; Hu, Burong

2016-09-13

Radiation-induced genomic instability plays a vital role in carcinogenesis. Bod1 is required for proper chromosome biorientation, and Bod1 depletion increases premature chromatid separation. MiR-142-3p influences cell cycle progression and inhibits proliferation and invasion in cervical carcinoma cells. We found that radiation induced premature chromatid separation and altered miR-142-3p and Bod1 expression in 786-O and A549 cells. Overexpression of miR-142-3p increased premature chromatid separation and G2/M cell cycle arrest in 786-O cells by suppressing Bod1 expression. We also found that either overexpression of miR-142-3p or knockdown of Bod1 sensitized 786-O and A549 cells to X-ray radiation. Overexpression of Bod1 inhibited radiation- and miR-142-3p-induced premature chromatid separation and increased resistance to radiation in 786-O and A549 cells. Taken together, these results suggest that radiation alters miR-142-3p and Bod1 expression in carcinoma cells, and thus contributes to early stages of radiation-induced genomic instability. Combining ionizing radiation with epigenetic regulation may help improve cancer therapies.

The Osteogenic Niche Promotes Early-Stage Bone Colonization of Disseminated Breast Cancer Cells

PubMed Central

Wang, Hai; Yu, Cuijuan; Gao, Xia; Welte, Thomas; Muscarella, Aaron M.; Tian, Lin; Zhao, Hong; Zhao, Zhen; Du, Shiyu; Tao, Jianning; Lee, Brendan; Westbrook, Thomas F.; Wong, Stephen T. C.; Jin, Xin; Rosen, Jeffrey M.; Osborne, C. Kent; Zhang, Xiang H.-F.

2014-01-01

Summary Breast cancer bone micrometastases can remain asymptomatic for years before progressing into overt lesions. The biology of this process, including the microenvironment niche and supporting pathways, is unclear. We find that bone micrometastases predominantly reside in a niche that exhibits features of osteogenesis. Niche interactions are mediated by heterotypic adherens junctions (hAJs) involving cancer-derived E-cadherin and osteogenic N-cadherin, the disruption of which abolishes niche-conferred advantages. We further elucidate that hAJ activates the mTOR pathway in cancer cells, which drives the progression from single cells to micrometastases. Human datasets analyses support the roles of AJ and the mTOR pathway in bone colonization. Our study illuminates the initiation of bone colonization, and provides potential therapeutic targets to block progression toward osteolytic metastases. Significance In advanced stages, breast cancer bone metastases are driven by paracrine crosstalk among cancer cells, osteoblasts, and osteoclasts, which constitute a vicious osteolytic cycle. Current therapies targeting this process limit tumor progression, but do not improve patient survival. On the other hand, bone micrometastases may remain indolent for years before activating the vicious cycle, providing a therapeutic opportunity to prevent macrometastases. Here, we show that bone colonization is initiated in a microenvironment niche exhibiting active osteogenesis. Cancer and osteogenic cells form heterotypic adherens junctions, which enhance mTOR activity and drive early-stage bone colonization prior to osteolysis. These results reveal a strong connection between osteogenesis and micrometastasis and suggest potential therapeutic targets to prevent bone macrometastases. PMID:25600338

Regenerative hair waves in aging mice and extra-follicular modulators follistatin, dkk1, and sfrp4.

PubMed

Chen, Chih-Chiang; Murray, Philip J; Jiang, Ting Xin; Plikus, Maksim V; Chang, Yun-Ting; Lee, Oscar K; Widelitz, Randall B; Chuong, Cheng-Ming

2014-08-01

Hair cycling is modulated by factors both intrinsic and extrinsic to hair follicles. Cycling defects lead to conditions such as aging-associated alopecia. Recently, we demonstrated that mouse skin exhibits regenerative hair waves, reflecting a coordinated regenerative behavior in follicle populations. Here, we use this model to explore the regenerative behavior of aging mouse skin. Old mice (>18 months) tracked over several months show that with progressing age, hair waves slow down, wave propagation becomes restricted, and hair cycle domains fragment into smaller domains. Transplanting aged donor mouse skin to a young host can restore donor cycling within a 3 mm range of the interface, suggesting that changes are due to extracellular factors. Therefore, hair stem cells in aged skin can be reactivated. Molecular studies show that extra-follicular modulators Bmp2, Dkk1, and Sfrp4 increase in early anagen. Further, we identify follistatin as an extra-follicular modulator, which is highly expressed in late telogen and early anagen. Indeed, follistatin induces hair wave propagation and its level decreases in aging mice. We present an excitable medium model to simulate the cycling behavior in aging mice and illustrate how the interorgan macroenvironment can regulate the aging process by integrating both "activator" and "inhibitor" signals.

Hypoxia-inducible factor-1α plays roles in Epstein-Barr virus’s natural life cycle and tumorigenesis by inducing lytic infection through direct binding to the immediate-early BZLF1 gene promoter

PubMed Central

Kraus, Richard J.; Cordes, Blue-leaf A.; Nawandar, Dhananjay M.; Ma, Shidong; McChesney, Kyle G.; Lin, Zhen; Makielski, Kathleen R.; Lee, Denis L.; Lambert, Paul F.; Johannsen, Eric C.; Kenney, Shannon C.

2017-01-01

When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs). We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV). Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE) located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK) and gingival epithelial (hGET) cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV’s natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for development of a lytic-induction therapy for treating some EBV-associated malignancies. PMID:28617871

Regulation of early gene expression from the bovine papillomavirus genome in transiently transfected C127 cells.

PubMed Central

Szymanski, P; Stenlund, A

1991-01-01

Expression of bovine papillomavirus (BPV) early gene products is required for viral DNA replication and establishment of the transformed phenotype. By the use of a highly efficient electroporation system, we have examined for the first time the transcriptional activity of BPV promoters in their natural genomic context in a replication-permissive cell line. We have determined that a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. This suggests that the transcriptional activity of the BPV genome in stably transformed cells represents the early stage of BPV gene expression. Quantitative differences in promoter activity between transiently transfected and stably transformed cells suggest that subtle changes in gene expression may control progression of the viral life cycle. Deletion analysis demonstrated that the E2 transactivator protein stimulates all of the early promoters through sequences located in the upstream regulatory region. This E2-dependent enhancer was found to be highly redundant, and particular E2 binding sites did not display a preference for particular promoters. Despite this dependence on a common cis-acting sequence, the various promoters displayed different sensitivities to the E2 transactivator. The findings that E2 regulates all promoters and, with the exception of the E2 repressors, that no other known viral gene product appears to affect transcription indicate that the E2 system functions as the master regulator of BPV early gene expression. Images PMID:1656065

Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids.

PubMed

L'Espérance, Sylvain; Bachvarova, Magdalena; Tetu, Bernard; Mes-Masson, Anne-Marie; Bachvarov, Dimcho

2008-02-26

Chemotherapy (CT) resistance in ovarian cancer (OC) is broad and encompasses diverse unrelated drugs, suggesting more than one mechanism of resistance. To better understand the molecular mechanisms controlling the immediate response of OC cells to CT exposure, we have performed gene expression profiling in spheroid cultures derived from six OC cell lines (OVCAR3, SKOV3, TOV-112, TOV-21, OV-90 and TOV-155), following treatment with 10,0 microM cisplatin, 2,5 microM paclitaxel or 5,0 microM topotecan for 72 hours. Exposure of OC spheroids to these CT drugs resulted in differential expression of genes associated with cell growth and proliferation, cellular assembly and organization, cell death, cell cycle control and cell signaling. Genes, functionally involved in DNA repair, DNA replication and cell cycle arrest were mostly overexpressed, while genes implicated in metabolism (especially lipid metabolism), signal transduction, immune and inflammatory response, transport, transcription regulation and protein biosynthesis, were commonly suppressed following all treatments. Cisplatin and topotecan treatments triggered similar alterations in gene and pathway expression patterns, while paclitaxel action was mainly associated with induction of genes and pathways linked to cellular assembly and organization (including numerous tubulin genes), cell death and protein synthesis. The microarray data were further confirmed by pathway and network analyses. Most alterations in gene expression were directly related to mechanisms of the cytotoxics actions in OC spheroids. However, the induction of genes linked to mechanisms of DNA replication and repair in cisplatin- and topotecan-treated OC spheroids could be associated with immediate adaptive response to treatment. Similarly, overexpression of different tubulin genes upon exposure to paclitaxel could represent an early compensatory effect to this drug action. Finally, multicellular growth conditions that are known to alter gene expression (including cell adhesion and cytoskeleton organization), could substantially contribute in reducing the initial effectiveness of CT drugs in OC spheroids. Results described in this study underscore the potential of the microarray technology for unraveling the complex mechanisms of CT drugs actions in OC spheroids and early cellular response to treatment.

Tectonic-1 contributes to the growth and migration of prostate cancer cells in vitro

PubMed Central

WANG, ZHIJUN; GAO, YI; LIU, YUSHAN; CHEN, JIE; WANG, JUNKAI; GAN, SISHUN; XU, DANFENG; CUI, XINGANG

2015-01-01

Tectonic-1 (TCTN1) is an upstream gene involved in embryonic development. The aim of the present study was to investigate the effect of the TCTN1 gene on the viability and migration of prostate cancer cells. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to silence the expression of TCTN1 in PC-3 and DU145 prostate cancer cells. Cell viability and proliferation were measured using MTT and colony formation assays, and the distribution of cells in phases of the cell cycle was determined using flow cytometry. Cell migration was detected using a Transwell assay. The results demonstrated that TCTN1 was widely expressed in several human prostate cancer cell lines. Knockdown of the TCTN1 gene by RNA interference markedly suppressed cell viability and colony formation in the PC-3 and DU145 cell lines. Cell cycle progression was also arrested by TCTN1 silencing. In addition, knockdown of the TCTN1 gene led to the inhibition of cell migration in the two cell lines. These findings confirmed the direct association between the TCTN1 gene and prostate cancer growth in vitro. With further understanding and clinical investigation, this indicates the potential for future development of a novel marker for early detection and gene therapy for prostate cancer. PMID:26310786

Long-term in vivo polychlorinated biphenyl 126 exposure induces oxidative stress and alters proteomic profile on islets of Langerhans

NASA Astrophysics Data System (ADS)

Loiola, Rodrigo Azevedo; Dos Anjos, Fabyana Maria; Shimada, Ana Lúcia; Cruz, Wesley Soares; Drewes, Carine Cristiane; Rodrigues, Stephen Fernandes; Cardozo, Karina Helena Morais; Carvalho, Valdemir Melechco; Pinto, Ernani; Farsky, Sandra Helena

2016-06-01

It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 μg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early β-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2.

Immuno-histochemical localization of LH-RH during different phases of estrus cycle of rat, with reference to the preoptic and arcuate neurons, and the ependymal cells.

PubMed

Naik, D V

1976-10-06

Immunohistochemical localization of luteinizing hormone-releasing hormone (LH-RH), during different phases of the estrus cycle, in the preoptic, suprachiasmatic and arcuate nuclei, and in the OVLT of rats, with special emphasis on the ependymal cells, was studied by light, fluorescent and electron microscopy, by using rabbit anti serum to synthetic LH-RH. The LH-RH neurons in the above mentioned areas, were very active during late diestrus and early proestrus phases. Specialized ependymal cells bordering the 3rd ventricle also showed varied LH-RH positive reaction during different phases of the estrus cycle. Immunofluorescent studies showed cyclic variations in the LH-RH material in the CSF of the preoptic and infundibular recesses, as well as in the 3rd ventricle near OVLT, in that, it was maximum during late diestrus and early proestrus phases. Immediately after this, the LH-RH late proestrus was reached. We have also observed that during the proestrus phase, as the LH-RH material started declining in the CSF, it had started building up in the specialized ependyma. Estrus, metaestrus and early diestrus phases showed very weak immunofluorescent LH-RH material in the lumen of the infundibular recess and in the specialized ependyma. Our immuno-electron microscopic observations showed pleomorphic LH-RH granules in the specialized ependyma during late kiestrus and proestrus phases. All these observations lead us to believe that LH-RH is not synthesized in the ependymal cells,but is phagocytosed from the CSF of the 3rd ventricle by the specialized ependyma, which transports it to the ME portal system. In males, the fluorescent LH-RH material did not show any noticeable changes. With the present and previous work,it is concluded that the neurons in differentnuclei synthesize LH-RH and transport it to the ME portal system,primarily through the nerve fibers and secondarily by the ventricular route. It is also suggested that the ependymal transport of LH-RH to the ME portal system is cyclic and thus controls the gonadotropin secretion.

LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit.

PubMed

Fowler, Sandy; Maguin, Pascal; Kalan, Sampada; Loayza, Diego

2018-06-22

DNA damage response pathways are essential for genome stability and cell survival. Specifically, the ATR kinase is activated by DNA replication stress. An early event in this activation is the recruitment and phosphorylation of RPA, a single stranded DNA binding complex composed of three subunits, RPA70, RPA32 and RPA14. We have previously shown that the LIM protein Ajuba associates with RPA, and that depletion of Ajuba leads to potent activation of ATR. In this study, we provide evidence that the Ajuba-RPA interaction occurs through direct protein contact with RPA70, and that their association is cell cycle-regulated and is reduced upon DNA replication stress. We propose a model in which Ajuba negatively regulates the ATR pathway by directly interacting with RPA70, thereby preventing inappropriate ATR activation. Our results provide a framework to further our understanding of the mechanism of ATR regulation in human cells in the context of cellular transformation.

The Endocrine Dyscrasia that Accompanies Menopause and Andropause Induces Aberrant Cell Cycle Signaling that Triggers Cell Cycle Reentry of Post-mitotic Neurons, Neurodysfunction, Neurodegeneration and Cognitive Disease

PubMed Central

Atwood, Craig S.; Bowen, Richard L.

2016-01-01

Sex hormones are the physiological factors that regulate neurogenesis during embryogenesis and continuing through adulthood. These hormones support the formation of brain structures such as dendritic spines, axons and synapses required for the capture of information (memories). Intriguingly, a recent animal study has demonstrated that induction of neurogenesis results in the loss of previously encoded memories in animals (e.g. infantile amnesia). In this connection, much evidence now indicates that Alzheimer’s disease (AD) also involves aberrant re-entry of post-mitotic neurons into the cell cycle. Cell cycle abnormalities appear very early in the disease, prior to the appearance of plaques and tangles, and explain the biochemical, neuropathological and cognitive changes observed with disease progression. Since sex hormones control when and how neurons proliferate and differentiate, the endocrine dyscrasia that accompanies menopause and andropause is a key signaling event that impacts neurogenesis and the acquisition, processing, storage and recall of memories. Here we review the biochemical, epidemiological and clinical evidence that alterations in endocrine signaling with menopause and andropause drive the aberrant re-entry of post-mitotic neurons into an abortive cell cycle with neurite retraction that leads to neuron dysfunction and death. When the reproductive axis is in balance, luteinizing hormone (LH), and its fetal homolog, human chorionic gonadotropin (hCG), promote pluripotent human and totipotent murine embryonic stem cell and neuron proliferation. However, strong evidence supports menopausal/andropausal elevations in the ratio of LH:sex steroids as driving aberrant mitotic events mediated by the upregulation of tumor necrosis factor, amyloid-β precursor protein processing towards the production of mitogenic Aβ, and the activation of Cdk5, a key regulator of cell cycle progression and tau phosphorylation (a cardinal feature of both neurogenesis and neurodegeneration). Cognitive studies also demonstrate the negative consequences of a high LH:sex steroid ratio on human cognitive performance. Prospective epidemiological and clinical evidence in humans supports lowering the ratio of circulating gonadotropins-GnRH to sex steroids in reducing the incidence of AD and halting cognitive decline. Together, these data support endocrine dyscrasia and the subsequent loss of cell cycle control as an important etiological event in the development of neurodegenerative diseases including AD, stroke and Parkinson’s disease. PMID:26188949

Cdk1 Activates Pre-mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem Cells.

PubMed

Baffet, Alexandre D; Hu, Daniel J; Vallee, Richard B

2015-06-22

Dynein recruitment to the nuclear envelope is required for pre-mitotic nucleus-centrosome interactions in nonneuronal cells and for apical nuclear migration in neural stem cells. In each case, dynein is recruited to the nuclear envelope (NE) specifically during G2 via two nuclear pore-mediated mechanisms involving RanBP2-BicD2 and Nup133-CENP-F. The mechanisms responsible for cell-cycle control of this behavior are unknown. We now find that Cdk1 serves as a direct master controller for NE dynein recruitment in neural stem cells and HeLa cells. Cdk1 phosphorylates conserved sites within RanBP2 and activates BicD2 binding and early dynein recruitment. Late recruitment is triggered by a Cdk1-induced export of CENP-F from the nucleus. Forced NE targeting of BicD2 overrides Cdk1 inhibition, fully rescuing dynein recruitment and nuclear migration in neural stem cells. These results reveal how NE dynein recruitment is cell-cycle regulated and identify the trigger mechanism for apical nuclear migration in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

Growth fraction in non-small cell lung cancer estimated by proliferating cell nuclear antigen and comparison with Ki-67 labeling and DNA flow cytometry data.

PubMed Central

Fontanini, G.; Pingitore, R.; Bigini, D.; Vignati, S.; Pepe, S.; Ruggiero, A.; Macchiarini, P.

1992-01-01

Results generated by the immunohistochemical staining with PC10, a new monoclonal antibody recognizing PCNA (a nuclear protein associated with cell proliferation) in formalin-fixed and paraffin-embedded tissue were compared with those of Ki-67 labeling and DNA flow cytometry in 47 consecutive non-small cell lung cancer (NSCLC). PCNA reactivity was observed in all samples and confined to the nuclei of cancer cells. Its frequency ranged from 0 to 80% (37.7 +/- 23.6) and larger sized, early-staged and DNA aneuploid tumors expressed a significant higher number of PCNA-reactive cells. The PCNA and Ki-67 labeling rates were closely correlated (r = 0.383, P = 0.009). By flow cytometry, we observed a good correlation among PCNA labeling and S-phase fraction (r = 0.422, P = .0093) and G1 phase (r = 0.303, P = .051) of the cell cycle. Results indicate that PCNA labeling with PC10 is a simple method for assessing the proliferative activity in formalin-fixed, paraffin-embedded tissue of NSCLC and correlates well with Ki-67 labeling and S-phase fraction of the cell cycle. Images Figure 2 PMID:1361306

Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi.

PubMed

de Jesus, Teresa Cristina Leandro; Nunes, Vinícius Santana; Lopes, Mariana de Camargo; Martil, Daiana Evelin; Iwai, Leo Kei; Moretti, Nilmar Silvio; Machado, Fabrício Castro; de Lima-Stein, Mariana L; Thiemann, Otavio Henrique; Elias, Maria Carolina; Janzen, Christian; Schenkman, Sergio; da Cunha, Julia Pinheiro Chagas

2016-06-03

Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

PubMed

Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

2017-11-15

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Ancient cellular structures and modern humans: change of survival strategies before prolonged low solar activity period

NASA Astrophysics Data System (ADS)

Ragulskaya, Mariya; Rudenchik, Evgeniy; Gromozova, Elena; Voychuk, Sergei; Kachur, Tatiana

The study of biotropic effects of modern space weather carries the information about the rhythms and features of adaptation of early biological systems to the outer space influence. The influence of cosmic rays, ultraviolet waves and geomagnetic field on early life has its signs in modern biosphere processes. These phenomena could be experimentally studied on present-day biological objects. Particularly inorganic polyphosphates, so-called "fossil molecules", attracts special attention as the most ancient molecules which arose in inanimate nature and have been accompanying biological objects at all stages of evolution. Polyphosphates-containing graves of yeast's cells of Saccharomyces cerevisiae strain Y-517, , from the Ukrainian Collection of Microorganisms was studied by daily measurements during 2000-2013 years. The IZMIRAN daily data base of physiological parameters dynamics during 2000-2013 years were analyzed simultaneously (25 people). The analysis showed significant simultaneous changes of the statistical parameters of the studied biological systems in 2004 -2006. The similarity of simultaneous changes of adaptation strategies of human organism and the cell structures of Saccharomyces cerevisiae during the 23-24 cycles of solar activity are discussed. This phenomenon could be due to a replacement of bio-effective parameters of space weather during the change from 23rd to 24th solar activity cycle and nonstandard geophysical peculiarities of the 24th solar activity cycle. It could be suggested that the observed similarity arose as the optimization of evolution selection of the living systems in expectation of probable prolonged period of low solar activity (4-6 cycles of solar activity).

Characterization of the Th Profile of the Bovine Endometrium during the Oestrous Cycle and Early Pregnancy

PubMed Central

Oliveira, Lilian J.; Mansourri-Attia, Nadéra; Fahey, Alan G.; Browne, John; Forde, Niamh; Roche, James F.; Lonergan, Patrick; Fair, Trudee

2013-01-01

Despite extensive research in the area of cow fertility, the extent to which the maternal immune system is modulated during pregnancy in cattle remains unclear. Therefore, the objective of the current study was to characterize the presence and response profile of B, T-helper (LTh), T- cytotoxic (LTc), gamma delta-T (γδT) and natural killer (NK) lymphocytes in terms of cell number, distribution and cytokine expression in bovine endometrial tissue to pregnancy. Endometrial tissue samples were collected from beef heifers on Days 5, 7, 13 and 16 of the estrous cycle or pregnancy. Samples were analysed by immunofluorescence to identify the presence and abundance of B-B7 (B-cells), CD4 (LTh), CD8 (LTc), γδT cell receptor (TCR) and CD335/NKp46 (NK cells) -positive immune cells. Quantitative real time PCR (QPCR) was carried out to analyse mRNA relative abundance of FOXP3 (a marker of regulatory T (Treg) cells) and a panel of immune factors, including MHC-I, LIF, Interleukins 1, 2, 6, 8, 10, 11,12A, IFNa and IFNG. Results indicate that B-B7+ cells are quite populous in bovine endometrial tissue, CD4+ and CD8+ -cells are present in moderate numbers and γδTCR+ and CD335+ cells are present in low numbers. Pregnancy affected the total number and distribution pattern of the NK cell population, with the most significant variation observed on Day 16 of pregnancy. Neither B lymphocytes nor T lymphocyte subsets were regulated temporally during the oestrous cycle or by pregnancy prior to implantation. mRNA transcript abundance of the immune factors LIF, IL1b, IL8 and IL12A, IFNa and IFNG, expression was regulated temporally during the estrous cycle and LIF, IL1b, IL-10, IL11, IL12A were also temporally regulated during pregnancy. In conclusion, the endometrial immune profile of the oestrous cycle favours a Th2 environment in anticipation of pregnancy and the presence of an embryo acts to fine tune this environment. PMID:24204576

Local homogeneity of cell cycle length in developing mouse cortex

NASA Technical Reports Server (NTRS)

Cai, L.; Hayes, N. L.; Nowakowski, R. S.

1997-01-01

We have measured the amount of variation in the length of the cell cycle for cells in the pseudostratified ventricular epithelium (PVE) of the developing cortex of mice on embryonic day 14. Our measurements were made in three cortical regions (i.e., the neocortex, archicortex, and periarchicortex) using three different methods: the cumulative labeling method (CLM), the percent labeled mitoses (PLM) method, and a comparison of the time needed for the PLM to ascend from 0 to 100% with the time needed for the PLM to descend from 100 to 0%. These 3 different techniques provide different perspectives on the cytokinetic parameters. Theoretically, CLM gives an estimate for a maximum value of the total length of the cell cycle (TC), whereas PLM gives an estimate of a minimum value of TC. The difference between these two estimates indicates that the range for TC is +/-1% of the mean TC for periarchicortex, +/-7% for neocortex, and +/-8% for archicortex. This was confirmed by a lengthening of the PLM descent time in comparison with its ascent time. The sharpness of the transitions and the flatness of the plateau of the PLM curves indicate that 99% of the proliferating cells are within this narrow estimated range for TC; hence, only approximately 1% deviate outside of a relatively restricted range from the average TC of the population. In the context of the possible existence within the cortical PVE of two populations with markedly dissimilar cell cycle kinetics from the mean, one such population must comprise approximately 99% of the total population, and the other, if it exists, is only approximately 1% of the total. This seems to be true for all three cortical regions. The narrow range of TC indicates a homogeneity in the cell cycle length for proliferating cells in three different cortical regions, despite the fact that progenitor cells of different lineages may be present. It further predicts the existence of almost synchronous interkinetic nuclear movements of the proliferating cells in the ventricular zone during early development of the cerebral cortex.

Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1

PubMed Central

Oda, Masako; Kanoh, Yutaka; Watanabe, Yoshihisa; Masai, Hisao

2012-01-01

Background Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as “replication domains”, and recent findings indicate that replication timing is under developmental and cell type-specific regulation. Methodology/Principal Findings We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. Conclusions Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome. PMID:22879953

Regulation of DNA replication timing on human chromosome by a cell-type specific DNA binding protein SATB1.

PubMed

Oda, Masako; Kanoh, Yutaka; Watanabe, Yoshihisa; Masai, Hisao

2012-01-01

Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as "replication domains", and recent findings indicate that replication timing is under developmental and cell type-specific regulation. We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome.

Transfection of Murine and Human Hematopoietic Progenitors with Rearranged Immunoglobulin Genes

DTIC Science & Technology

1991-01-01

fluorouracil (SFU) to eliminate most cycling progenitors. Previous studies have shown that 5FU -treatment enriches for one early progenitor with high...Table I shows a time course of SCA-I positive cell expression various times post- 5FU treatment. Table 1 clearly shows that 5FU treatment can increase...the percentage of SCA-l-positive cells to 6-7% by day 7 post- 5FU treatment. The level of SCA-I expression falls to approximately 1% of total nucleated

Corpora lutea in superovulated ewes fed different planes of nutrition.

PubMed

Kraisoon, A; Redmer, D A; Bass, C S; Navanukraw, C; Dorsam, S T; Valkov, V; Reyaz, A; Grazul-Bilska, A T

2018-01-01

The corpus luteum (CL) is an ovarian structure which is critical for the maintenance of reproductive cyclicity and pregnancy support. Diet and/or diet components may affect some luteal functions. FSH is widely used to induce multiple follicle development and superovulation. We hypothesized that FSH would affect luteal function in ewes fed different nutritional planes. Therefore, the aim of this study was to determine if FSH-treatment affects (1) ovulation rate; (2) CL weight; (3) cell proliferation; (4) vascularity; (5) expression of endothelial nitric oxide (eNOS) and soluble guanylate cyclase (sGC) proteins; and (6) luteal and serum progesterone (P4) concentration in control (C), overfed (O), and underfed (U) ewes at the early- and mid-luteal phases. In addition, data generated from this study were compared to data obtained from nonsuperovulated sheep and described by Bass et al. Ewes were categorized by weight and randomly assigned into nutrition groups: C (2.14 Mcal/kg; n = 11), O (2xC; n = 12), and U (0.6xC; n = 11). Nutritional treatment was initiated 60 d prior to day 0 of the estrous cycle. Ewes were injected with FSH on day 13-15 of the first estrous cycle, and blood samples and ovaries were collected at early- and mid-luteal phases of the second estrous cycle. The number of CL/ewe was determined, and CL was dissected and weighed. CL was fixed for evaluation of expression of Ki67 (a proliferating cell marker), CD31 (an endothelial cell marker), and eNOS and sGC proteins using immunohistochemistry and image analysis. From day 0 until tissue collection, C maintained, O gained, and U lost body weight. The CL number was greater (P < 0.03) in C and O than U. Weights of CL, cell proliferation, vascularity, and eNOS but not sGC expression were greater (P < 0.001), and serum, but not luteal tissue, P4 concentrations tended to be greater (P = 0.09) at the early- than mid-luteal phase. Comparisons of CL measurements demonstrated greater (P < 0.01) cell proliferation and serum P4 concentration, but less vascularity at the early and mid-luteal phases, and less CL weight at the mid-luteal phase in superovulated than nonsuperovulated ewes; however, concentration of P4 in luteal tissues was similar in both groups. Thus, in superovulated ewes, luteal cell proliferation and vascularity, expression of eNOS, and serum P4 concentration depend on the stage of luteal development, but not diet. Comparison to control ewes demonstrated several differences and some similarities in luteal functions after FSH-induced superovulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Sponge cell reaggregation: Cellular structure and morphogenetic potencies of multicellular aggregates.

PubMed

Lavrov, Andrey I; Kosevich, Igor A

2016-02-01

Sponges (phylum Porifera) are one of the most ancient extant multicellular animals and can provide valuable insights into origin and early evolution of Metazoa. High plasticity of cell differentiations and anatomical structure is characteristic feature of sponges. Present study deals with sponge cell reaggregation after dissociation as the most outstanding case of sponge plasticity. Dynamic of cell reaggregation and structure of multicellular aggregates of three demosponge species (Halichondria panicea (Pallas, 1766), Haliclona aquaeductus (Sсhmidt, 1862), and Halisarca dujardinii Johnston, 1842) were studied. Sponge tissue dissociation was performed mechanically. Resulting cell suspensions were cultured at 8-10°C for at least 5 days. Structure of multicellular aggregates was studied by light, transmission and scanning electron microscopy. Studied species share common stages of cell reaggregation-primary multicellular aggregates, early-stage primmorphs and primmorphs, but the rate of reaggregation varies considerably among species. Only cells of H. dujardinii are able to reconstruct functional and viable sponge after primmorphs formation. Sponge reconstruction in this species occurs due to active cell locomotion. Development of H. aquaeductus and H. panicea cells ceases at the stages of early primmorphs and primmorphs, respectively. Development of aggregates of these species is most likely arrested due to immobility of the majority of cells inside them. However, the inability of certain sponge species to reconstruct functional and viable individuals during cell reaggregation may be not a permanent species-specific characteristic, but depends on various factors, including the stage of the life cycle and experimental conditions. © 2016 Wiley Periodicals, Inc.

Raman Spectral Observation of a "New Phase" Observed in Nickel Electrodes Cycled to Failure

NASA Technical Reports Server (NTRS)

Loyselle, P. L.; Shan, X.; Cornilsen, B. C.; Reid, M. A.

1991-01-01

A "new phase" is reported in nickel electrodes from Ni/H boilerplate cells which were cycled to failure in electrolyte of variable kOH concentration. Raman spectra clearly show the presence of this phase, and these spectra have been used to quantify the amounts present in these electrodes (in the volume sampled by the laser beam) Raman spectroscopy has been found to be capable of differentiating the various phases which can be present in nickel .This differentiation is possible because of the structural variation observed for these phases. Ten of twelve electrodes examined contain at least some of this new phase. The presence of this "new phase" correlates with cell failure, and it is proposed that the presence of this phase may play a role in early electrode failure.

Qualification testing of general electric 50 A h nickel—cadmium cells with pellon 2536 separator and passivated positive plates

NASA Astrophysics Data System (ADS)

Morrow, George W.

Forty-two, 50 A h nickel—cadmium cells were delivered to the Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985 for the purpose of evaluating and qualifying a new, nonwoven nylon separator material, Pellon 2536, and the new GE positive plate passivation process. Testing began in May, 1985 at the Naval Weapons Support Center (NWSC) at Crane, Indiana with GSFC standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985 with approximately 6500 LEO cycles and three GEO eclipse seasons complete at this writing. After early problems in maintaining test pack temperature control, all packs were performing well but were exhibiting higher than normal charge voltage characteristics.

Qualification testing of General Electric 50 Ah nickel-cadmium cells with Pellon 2536 separator and passivated positive plates

NASA Technical Reports Server (NTRS)

Morrow, George W.

1987-01-01

Forty-two 50 Ah nickel-cadmium cells were delivered to the Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985 for the purpose of evaluating and qualifying a nonwoven nylon separator material, Pellon 2536, and the GE positive plate nickel attack control gas passivation process. Testing began May, 1985 at the Naval Weapons Support Center (NWSC) in Crane, Indiana with GSFC standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985 with approximately 6500 LEO cycles and three GEO eclipse seasons completed. After early problems in maintaining test pack temperature control, all packs are performing well but are exhibiting higher than normal charge voltage characteristics.

Qualification testing of General Electric 50 Ah nickel-cadmium cells with Pellon 2536 separator and passivated positive plates

NASA Astrophysics Data System (ADS)

Morrow, George W.

1987-09-01

Forty-two 50 Ah nickel-cadmium cells were delivered to the Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985 for the purpose of evaluating and qualifying a nonwoven nylon separator material, Pellon 2536, and the GE positive plate nickel attack control gas passivation process. Testing began May, 1985 at the Naval Weapons Support Center (NWSC) in Crane, Indiana with GSFC standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985 with approximately 6500 LEO cycles and three GEO eclipse seasons completed. After early problems in maintaining test pack temperature control, all packs are performing well but are exhibiting higher than normal charge voltage characteristics.

SMK-1/PPH-4.1–mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

PubMed Central

Kim, Seung-Hwan; Holway, Antonia H.; Wolff, Suzanne; Dillin, Andrew; Michael, W. Matthew

2007-01-01

During early embryogenesis in Caenorhabditis elegans, the ATL-1–CHK-1 (ataxia telangiectasia mutated and Rad3 related–Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1–PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context. PMID:17908915

Bm59 is an early gene, but is unessential for the propagation and assembly of Bombyx mori nucleopolyhedrovirus.

PubMed

Hu, Xiaolong; Shen, Yunwang; Zheng, Qin; Wang, Guobao; Wu, Xiaofeng; Gong, Chengliang

2016-02-01

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects the domestic silkworm and causes serious economic loss to sericulture around the world. The function of BmNPV Bm59 gene in the viral life cycle is inconclusive. To investigate the role of Bm59 during viral infection, the transcription initiation site and temporal expression of Bm59 were analyzed, and Bm59-knockout virus was generated through homologous recombination in Escherichia coli. The results showed that Bm59 is an early transcription gene with an atypia early transcriptional start motif. Budded virion (BV) production and DNA replication in the BmN cells transfected with the Bm59-knockout virus bacmid were similar to those in the cells transfected with the wild-type virus. Electron microscopy revealed that the occlusion-derived virus can be produced in cells infected with the Bm59-knockout virus. These results indicated that Bm59 is an early gene and is not essential for viral replication or assembly of BmNPV. These findings suggested that non-essential gene (Bm59) remained in the viral genome, which may interact with other viral/host genes in a certain situation.

Sites and Regulation of Polyamine Catabolism in the Tobacco Plant. Correlations with Cell Division/Expansion, Cell Cycle Progression, and Vascular Development1

PubMed Central

Paschalidis, Konstantinos A.; Roubelakis-Angelakis, Kalliopi A.

2005-01-01

We previously gave a picture of the homeostatic characteristics of polyamine (PA) biosynthesis and conjugation in tobacco (Nicotiana tabacum) plant organs during development. In this work, we present the sites and regulation of PA catabolism related to cell division/expansion, cell cycle progression, and vascular development in the tobacco plant. Diamine oxidase (DAO), PA oxidase (PAO), peroxidases (POXs), and putrescine N-methyltransferase expressions follow temporally and spatially discrete patterns in shoot apical cells, leaves (apical, peripheral, and central regions), acropetal and basipetal petiole regions, internodes, and young and old roots in developing plants. DAO and PAO produce hydrogen peroxide, a plant signal molecule and substrate for POXs. Gene expression and immunohistochemistry analyses reveal that amine oxidases in developing tobacco tissues precede and overlap with nascent nuclear DNA and also with POXs and lignification. In mature and old tissues, flow cytometry indicates that amine oxidase and POX activities, as well as pao gene and PAO protein levels, coincide with G2 nuclear phase and endoreduplication. In young versus the older roots, amine oxidases and POX expression decrease with parallel inhibition of G2 advance and endoreduplication, whereas putrescine N-methyltransferase dramatically increases. In both hypergeous and hypogeous tissues, DAO and PAO expression occurs in cells destined to undergo lignification, suggesting a different in situ localization. DNA synthesis early in development and the advance in cell cycle/endocycle are temporally and spatially related to PA catabolism and vascular development. PMID:16040649

The Biological Effects of Bilirubin Photoisomers

PubMed Central

Jasprova, Jana; Dal Ben, Matteo; Vianello, Eleonora; Goncharova, Iryna; Urbanova, Marie; Vyroubalova, Karolina; Gazzin, Silvia; Tiribelli, Claudio; Sticha, Martin; Cerna, Marcela; Vitek, Libor

2016-01-01

Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells. PMID:26829016

Changes in the expression of prostaglandin family members in bovine corpus luteum during the oestrous cycle and pregnancy.

PubMed

Berisha, Bajram; Schams, Dieter; Rodler, Daniela; Sinowatz, Fred; Pfaffl, Michael W

2018-06-06

The aim of this study was to characterize certain prostaglandin family members in the bovine corpus luteum (CL) during the oestrous cycle and pregnancy. The CL tissue was assigned to the following stages of the oestrous cycle: 1-2, 3-4, 5-7, 8-12, 13-16, >18 days (after regression) and of pregnancy: 1-2, 3-4, 6-7 and >8 months. In these samples, we investigated prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE) and their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS) and PTGE synthase (PTGES). The expression of mRNA was measured by RT-qPCR, hormones by EIA and localization by immunohistochemistry. The mRNA expression of COX-2, PTGFS and PTGES in CL during the early luteal phase was high followed by a continuous and significant downregulation afterwards, as well as during all phases of pregnancy. The concentration of PTGF in CL tissue was high during the early luteal phase, decreased significantly in the mid-luteal phase, and increased again afterwards. In contrast, the concentration of PTGE increased significantly during late luteal phase followed by a decrease during regression. The PTGE level increased again during late pregnancy. Immunohistochemically, the large granulose-luteal cells show strong staining for COX-2 and PTGES during the early luteal stage followed by lower activity afterwards. During pregnancy, most of the luteal cells were only weakly positive or negative. In conclusion, our results indicate that the examined prostaglandin family members are involved in the local mechanisms that regulate luteal function, specifically during CL formation, function and regression and during pregnancy in the cow. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

N-Myc down regulation induced differentiation, early cell cycle exit, and apoptosis in human malignant neuroblastoma cells having wild type or mutant p53.

PubMed

Janardhanan, Rajiv; Banik, Naren L; Ray, Swapan K

2009-11-01

Neuroblastomas, which mostly occur in children, are aggressive metastatic tumors of the sympathetic nervous system. The failure of the previous therapeutic regimens to target multiple components of N-Myc pathway resulted in poor prognosis. The present study investigated the efficacy of the combination of N-(4-hydroxyphenyl) retinamide (4-HPR, 0.5 microM) and genistein (GST, 25 microM) to control the growth of human neuroblastoma cells (SH-SY5Y and SK-N-BE2) harboring divergent molecular attributes. Combination of 4-HPR and GST down regulated N-Myc, Notch-1, and Id2 to induce neuronal differentiation. Transition to neuronal phenotype was accompanied by increase in expression of e-cadherin. Induction of neuronal differentiation was associated with decreased expression of hTERT, PCNA, survivin, and fibronectin. This is the first report that combination of 4-HPR and GST mediated reactivation of multiple tumor suppressors (p53, p21, Rb, and PTEN) for early cell cycle exit (due to G1/S phase arrest) in neuroblastoma cells. Reactivation of tumor suppressor(s) repressed N-Myc driven growth factor mediated angiogenic and invasive pathways (VEGF, b-FGF, MMP-2, and MMP-9) in neuroblastoma. Repression of angiogenic factors led to the blockade of components of mitogenic pathways [phospho-Akt (Thr 308), p65 NF-kappaB, and p42/44 Erk 1/2]. Taken together, the combination of 4-HPR and GST effectively blocked survival, mitogenic, and angiogenic pathways and activated proteases for apoptosis in neuroblastoma cells. These results suggested that combination of 4-HPR and GST could be effective for controlling the growth of heterogeneous human neuroblastoma cell populations.

INPP5E Preserves Genomic Stability through Regulation of Mitosis.

PubMed

Sierra Potchanant, Elizabeth A; Cerabona, Donna; Sater, Zahi Abdul; He, Ying; Sun, Zejin; Gehlhausen, Jeff; Nalepa, Grzegorz

2017-03-15

The partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of INPP5E in human and murine cells impairs the spindle assembly checkpoint, centrosome and spindle function, and maintenance of chromosomal integrity. Consistent with a cell cycle regulatory role, we found that INPP5E expression is cell cycle dependent, peaking at mitotic entry. INPP5E localizes to centrosomes, chromosomes, and kinetochores in early mitosis and shuttles to the midzone spindle at mitotic exit. Our findings identify the previously unknown, essential role of INPP5E in mitosis and prevention of aneuploidy, providing a new perspective on the function of this phosphoinositide phosphatase in health and development. Copyright © 2017 Sierra Potchanant et al.

Fasting induces a biphasic adaptive metabolic response in murine small intestine

PubMed Central

Sokolović, Milka; Wehkamp, Diederik; Sokolović, Aleksandar; Vermeulen, Jacqueline; Gilhuijs-Pederson, Lisa A; van Haaften, Rachel IM; Nikolsky, Yuri; Evelo, Chris TA; van Kampen, Antoine HC; Hakvoort, Theodorus BM; Lamers, Wouter H

2007-01-01

Background The gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting. Results Intestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gut's structural components, so that the microarrays' tissue base remained unaffected. Unsupervised hierarchical clustering of the microarrays revealed that the successive time points separated into distinct branches. Pathway analysis depicted a pronounced, but transient early response that peaked at 12 hours, and a late response that became progressively more pronounced with continued fasting. Early changes in gene expression were compatible with a cellular deficiency in glutamine, and metabolic adaptations directed at glutamine conservation, inhibition of pyruvate oxidation, stimulation of glutamate catabolism via aspartate and phosphoenolpyruvate to lactate, and enhanced fatty-acid oxidation and ketone-body synthesis. In addition, the expression of key genes involved in cell cycling and apoptosis was suppressed. At 24 hours of fasting, many of the early adaptive changes abated. Major changes upon continued fasting implied the production of glucose rather than lactate from carbohydrate backbones, a downregulation of fatty-acid oxidation and a very strong downregulation of the electron-transport chain. Cell cycling and apoptosis remained suppressed. Conclusion The changes in gene expression indicate that the small intestine rapidly looses mass during fasting to generate lactate or glucose and ketone bodies. Meanwhile, intestinal architecture is maintained by downregulation of cell turnover. PMID:17925015

Fasting induces a biphasic adaptive metabolic response in murine small intestine.

PubMed

Sokolović, Milka; Wehkamp, Diederik; Sokolović, Aleksandar; Vermeulen, Jacqueline; Gilhuijs-Pederson, Lisa A; van Haaften, Rachel I M; Nikolsky, Yuri; Evelo, Chris T A; van Kampen, Antoine H C; Hakvoort, Theodorus B M; Lamers, Wouter H

2007-10-09

The gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting. Intestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gut's structural components, so that the microarrays' tissue base remained unaffected. Unsupervised hierarchical clustering of the microarrays revealed that the successive time points separated into distinct branches. Pathway analysis depicted a pronounced, but transient early response that peaked at 12 hours, and a late response that became progressively more pronounced with continued fasting. Early changes in gene expression were compatible with a cellular deficiency in glutamine, and metabolic adaptations directed at glutamine conservation, inhibition of pyruvate oxidation, stimulation of glutamate catabolism via aspartate and phosphoenolpyruvate to lactate, and enhanced fatty-acid oxidation and ketone-body synthesis. In addition, the expression of key genes involved in cell cycling and apoptosis was suppressed. At 24 hours of fasting, many of the early adaptive changes abated. Major changes upon continued fasting implied the production of glucose rather than lactate from carbohydrate backbones, a downregulation of fatty-acid oxidation and a very strong downregulation of the electron-transport chain. Cell cycling and apoptosis remained suppressed. The changes in gene expression indicate that the small intestine rapidly looses mass during fasting to generate lactate or glucose and ketone bodies. Meanwhile, intestinal architecture is maintained by downregulation of cell turnover.

Renal denervation prevents long-term sequelae of ischemic renal injury

PubMed Central

Kim, Jinu; Padanilam, Babu J.

2014-01-01

Signals that drive interstitial fibrogenesis after renal ischemia reperfusion injury remain undefined. Sympathetic activation is manifest even in the early clinical stages of chronic kidney disease and is directly related to disease severity. A role for renal nerves in renal interstitial fibrogenesis in the setting of ischemia reperfusion injury has not been studied. In male 129S1/SvImJ mice, ischemia reperfusion injury induced tubulointerstitial fibrosis as indicated by collagen deposition and profibrotic protein expression 4 to 16 days after the injury.. Leukocyte influx, proinflammatory protein expression, oxidative stress, apoptosis, and cell cycle arrest at G2/M phase were enhanced after ischemia reperfusion injury. Renal denervation at the time of injury or up to 1 day post-injury improved histology, decreased proinflammatory/profibrotic responses and apoptosis, and prevented G2/M cell cycle arrest in the kidney. Treatment with afferent nerve-derived calcitonin gene-related peptide (CGRP) or efferent nerve-derived norepinephrine in denervated and ischemia reperfusion injury-induced kidneys mimicked innervation, restored inflammation and fibrosis, induced G2/M arrest, and enhanced TGF-β1 activation. Blocking norepinephrine or CGRP function using respective receptor blockers prevented these effects. Consistent with the in vivo study, treatment with either norepinephrine or CGRP induced G2/M cell cycle arrest in HK-2 proximal tubule cells, whereas antagonists against their respective receptors prevented G2/M arrest. Thus, renal nerve stimulation is a primary mechanism and renal nerve-derived factors drive epithelial cell cycle arrest and the inflammatory cascade causing interstitial fibrogenesis after ischemia reperfusion injury. PMID:25207878

The absence of p27Kip1, an inhibitor of G1 cyclin-dependent kinases, uncouples differentiation and growth arrest during the granulosa->luteal transition.

PubMed

Tong, W; Kiyokawa, H; Soos, T J; Park, M S; Soares, V C; Manova, K; Pollard, J W; Koff, A

1998-09-01

The involvement of cyclin-dependent kinase inhibitors in differentiation remains unclear: are the roles of cyclin-dependent kinase inhibitors restricted to cell cycle arrest; or also required for completion of the differentiation program; or both? Here, we report that differentiation of luteal cells can be uncoupled from growth arrest in p27-deficient mice. In these mice, female-specific infertility correlates with a failure of embryos to implant at embryonic day 4.5. We show by ovarian transplant and hormone reconstitution experiments that failure to regulate luteal cell estradiol is one physiological mechanism for infertility in these mice. This failure is not due to a failure of p27-deficient granulosa cells to differentiate after hormonal stimulation; P450scc, a marker for luteal progesterone biosynthesis, is expressed and granulosa cell-specific cyclin D2 expression is reduced. However, unlike their wild-type counterparts, p27-deficient luteal cells continue to proliferate for up to 3.5 days after hormonal stimulation. By day 5.5, however, these cells withdraw from the cell cycle, suggesting that p27 plays a role in the early events regulating withdrawal of cells from the cell cycle. We have further shown that in the absence of this timely withdrawal, estradiol regulation is perturbed, explaining in part how fertility is compromised at the level of implantation. These data support the interpretation of our previous observations on oligodendrocyte differentiation about a role for p27 in establishing the nonproliferative state, which in some cases (oligodendrocytes) is required for differentiation, whereas in other cases it is required for the proper functioning of a differentiated cell (luteal cell).

Phosphorylation of histone H3 on Ser-10 by Aurora B is essential for chromosome condensation in porcine embryos during the first mitotic division.

PubMed

Chen, Changchao; Zhang, Zixiao; Cui, Panpan; Liao, Yaya; Zhang, Yue; Yao, Lingyun; Rui, Rong; Ju, Shiqiang

2017-07-01

Phosphorylation of histone H3 on Ser-10 (H3S10ph) is involved in regulating mitotic chromosome condensation and decondensation, which plays an important regulatory role during mitotic cell cycle progression in mammalian cells. However, whether H3S10ph plays a similar role in early porcine embryos during the first mitotic division remains uncertain. In this study, the subcellular localization and possible roles of H3S10ph were evaluated in the first mitotic cell cycle progression of porcine embryos using western blot, indirect immunofluorescence and barasertib (H3S10ph upstream regulator Aurora-B inhibitor) treatments. H3S10ph exhibited a dynamic localization pattern and was localized to chromosomes from prometaphase to anaphase stages. Treatment of porcine embryos with barasertib inhibited mitotic division at the prophase stage and was associated with a defect in chromosome condensation accompanied by the reduction of H3S10ph. These results indicated that H3S10ph is involved in the first mitotic division in porcine embryos through its regulatory function in chromosome condensation, which further affects porcine embryo cell cycle progression during mitotic division.

The sow endosalpinx at different stages of the oestrous cycle and at anoestrus: studies on morphological changes and infiltration by cells of the immune system.

PubMed

Jiwakanon, J; Persson, E; Kaeoket, K; Dalin, A-M

2005-02-01

The aim of this study was to investigate the morphological changes of the sow endosalpinx and the distribution of leukocytes throughout the oestrous cycle and at anoestrus. Nineteen crossbred sows (Swedish Landrace x Swedish Yorkshire) at late dioestrus (three), prooestrus (three), oestrus (three), early dioestrus (three), dioestrus (three) and anoestrus (four) were used. Oviductal samples from three different parts (isthmus, ampulla and infundibulum), taken immediately after slaughter, were fixed, embedded in plastic resin and stained with toluidine blue or stored in a freezer at -70 degrees C until analysed by immunohistochemistry (prooestrus and anoestrus) with an avidin-biotin peroxidase method. Quantitative and qualitative examinations of oviductal epithelium and subepithelial connective tissue were performed by light microscopy. During all stages, a lower degree of morphological changes (pseudostratification, mitosis and secretory granules) was found in the isthmus compared with ampulla and infundibulum. In ampulla and infundibulum, pseudostratification, mitotic activity and secretory granules of the epithelium were high at prooestrus/oestrus. Cytoplasmic protrusions of epithelial cells with some extruded nuclei were prominent in ampulla and infundibulum at all stages except for oestrus and early dioestrus. Lymphocytes as well as CD2- and CD3-positive cells were the predominant immune cells in the epithelial layer. The numbers of lymphocytes and CD3-positive cells did not differ among segments and stages. Numbers of CD2-positive cells did not differ between prooestrus and anoestrus while the numbers were significantly higher in the infundibulum than in ampulla and isthmus. Neutrophils were only occasionally found and mainly in the infundibulum. In the subepithelial connective tissue layer, the two most commonly observed immune cell types were lymphocytes and plasma cells. The numbers of lymphocytes as well as CD2- and CD3-positive cells was lower in isthmus than in the other segments (p < or = 0.001). Higher numbers of plasma cells (p < or = 0.001) were found in infundibulum than in ampulla and isthmus. The numbers of lymphocytes and plasma cells were not significantly different between stages of the oestrous cycle. However, the number of neutrophils differed and were highest at prooestrus in ampulla and infundibulum. The numbers of CD2-, CD3- and CD79-positive cells did not differ between prooestrus and anoestrus whereas for CD14- and SWC3-positive cells, the numbers were higher at prooestrus (p < or = 0.05) than at anoestrus. In the oviduct, the morphology differed in ampulla and infundibulum with oestrous cycle stages, which indicates an effect by ovarian steroid hormones. The immune cell infiltration was less influenced by cyclic changes. However, the immune cell infiltration (in the connective tissue) in the upper part, especially infundibulum, differed significantly from the one in the lower part, isthmus, indicating different immune functions within various parts of the oviduct.

Dynamics of DNA replication during premeiosis and early meiosis in wheat.

PubMed

Rey, María-Dolores; Prieto, Pilar

2014-01-01

Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat.

Dynamics of DNA Replication during Premeiosis and Early Meiosis in Wheat

PubMed Central

Rey, María-Dolores; Prieto, Pilar

2014-01-01

Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat. PMID:25275307

Protein tyrosine phosphatase of liver regeneration-1 is required for normal timing of cell cycle progression during liver regeneration

PubMed Central

Jiao, Yang; Ye, Diana Z.; Li, Zhaoyu; Teta-Bissett, Monica; Peng, Yong; Taub, Rebecca; Greenbaum, Linda E.

2014-01-01

Protein tyrosine phosphatase of liver regeneration-1 (Prl-1) is an immediate-early gene that is significantly induced during liver regeneration. Several in vitro studies have suggested that Prl-1 is important for the regulation of cell cycle progression. To evaluate its function in liver regeneration, we ablated the Prl-1 gene specifically in mouse hepatocytes using the Cre-loxP system. Prl-1 mutant mice (Prl-1loxP/loxP;AlfpCre) appeared normal and fertile. Liver size and metabolic function in Prl-1 mutants were comparable to controls, indicating that Prl-1 is dispensable for liver development, postnatal growth, and hepatocyte differentiation. Mutant mice demonstrated a delay in DNA synthesis after 70% partial hepatectomy, although ultimate liver mass restoration was not affected. At 40 h posthepatectomy, reduced protein levels of the cell cycle regulators cyclin E, cyclin A2, cyclin B1, and cyclin-dependent kinase 1 were observed in Prl-1 mutant liver. Investigation of the major signaling pathways involved in liver regeneration demonstrated that phosphorylation of protein kinase B (AKT) and signal transducer and activator of transcription (STAT) 3 were significantly reduced at 40 h posthepatectomy in Prl-1 mutants. Taken together, this study provides evidence that Prl-1 is required for proper timing of liver regeneration after partial hepatectomy. Prl-1 promotes G1/S progression via modulating expression of several cell cycle regulators through activation of the AKT and STAT3 signaling pathway. PMID:25377314

A gradient in the duration of the G1 phase in the murine neocortical proliferative epithelium

NASA Technical Reports Server (NTRS)

Miyama, S.; Takahashi, T.; Nowakowski, R. S.; Caviness, V. S. Jr

1997-01-01

Neuronogenesis in the neocortical pseudostratified ventricular epithelium (PVE) is initiated rostrolaterally and progresses caudo-medially as development progresses. Here we have measured the cytokinetic parameters and the fractional neuronal output parameter, Q, of laterally located early-maturing regions over the principal embryonic days (E12-E15) of neocortical neuronogenesis in the mouse. These measures are compared with ones previously made of a medial, late-maturing portion of the PVE. Laterally, as medially, the duration of the neuronogenetic interval is 6 days and comprises 11 integer cell cycles. Also, in both lateral and medial areas the length of G1 phase (TG1) increases nearly 4-fold and is the only cell cycle parameter to change. Q progresses essentially identically laterally and medially with respect to the succession of integer cell cycles. Most importantly, from E12 to E13 there is a steeply declining lateral to medial gradient in TG1. The gradient is due both to the lateral to medial graded stage of neuronogenesis and to the stepwise increase in TG1 with each integer cycle during the neuronogenetic interval. To our knowledge this gradient in TG1 of the cerebral PVE is the first cell biological gradient to be demonstrated experimentally in such an extensive proliferative epithelial sheet. We suggest that this gradient in TG1 is the cellular mechanism for positionally encoding a protomap of the neocortex within the PVE.

Elucidating the functional role of endoreduplication in tomato fruit development

PubMed Central

Chevalier, Christian; Nafati, Mehdi; Mathieu-Rivet, Elodie; Bourdon, Matthieu; Frangne, Nathalie; Cheniclet, Catherine; Renaudin, Jean-Pierre; Gévaudant, Frédéric; Hernould, Michel

2011-01-01

Background Endoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague,although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination. Scope Here recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described. Conclusions The cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits. PMID:21199834

Dichlorodiphenyltrichloroethane technical mixture regulates cell cycle and apoptosis genes through the activation of CAR and ERα in mouse livers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O., E-mail: pustylnyak@ngs.ru

Dichlorodiphenyltrichloroethane (DDT) is a widely used organochlorine pesticide and a xenoestrogen that promotes rodent hepatomegaly and tumours. A recent study has shown significant correlation between DDT serum concentration and liver cancer incidence in humans, but the underlying mechanisms remain elusive. We hypothesised that a mixture of DDT isomers could exert effects on the liver through pathways instead of classical ERs. The acute effects of a DDT mixture containing the two major isomers p,p′-DDT (85%) and o,p′-DDT (15%) on CAR and ERα receptors and their cell cycle and apoptosis target genes were studied in mouse livers. ChIP results demonstrated increased CARmore » and ERα recruitment to their specific target gene binding sites in response to the DDT mixture. The results of real-time RT-PCR were consistent with the ChIP data and demonstrated that the DDT was able to activate both CAR and ERα in mouse livers, leading to target gene transcriptional increases including Cyp2b10, Gadd45β, cMyc, Mdm2, Ccnd1, cFos and E2f1. Western blot analysis demonstrated increases in cell cycle progression proteins cMyc, Cyclin D1, CDK4 and E2f1 and anti-apoptosis proteins Mdm2 and Gadd45β. In addition, DDT exposure led to Rb phosphorylation. Increases in cell cycle progression and anti-apoptosis proteins were accompanied by a decrease in p53 content and its transcriptional activity. However, the DDT was unable to stimulate the β-catenin signalling pathway, which can play an important role in hepatocyte proliferation. Thus, our results indicate that DDT treatment may result in cell cycle progression and apoptosis inhibition through CAR- and ERα-mediated gene activation in mouse livers. These findings suggest that the proliferative and anti-apoptotic conditions induced by CAR and ERα activation may be important contributors to the early stages of hepatocarcinogenesis as produced by DDT in rodent livers. - Highlights: • DDT activated both CAR and ERα and their cell cycle and apoptosis target genes. • DDT produced increases in cell cycle and anti-apoptosis proteins and decrease in p53. • DDT mixture was unable to stimulate the β-catenin signalling pathway in mouse livers.« less

Kinase-dead ATM protein causes genomic instability and early embryonic lethality in mice.

PubMed

Yamamoto, Kenta; Wang, Yunyue; Jiang, Wenxia; Liu, Xiangyu; Dubois, Richard L; Lin, Chyuan-Sheng; Ludwig, Thomas; Bakkenist, Christopher J; Zha, Shan

2012-08-06

Ataxia telangiectasia (A-T) mutated (ATM) kinase orchestrates deoxyribonucleic acid (DNA) damage responses by phosphorylating numerous substrates implicated in DNA repair and cell cycle checkpoint activation. A-T patients and mouse models that express no ATM protein undergo normal embryonic development but exhibit pleiotropic DNA repair defects. In this paper, we report that mice carrying homozygous kinase-dead mutations in Atm (Atm(KD/KD)) died during early embryonic development. Atm(KD/-) cells exhibited proliferation defects and genomic instability, especially chromatid breaks, at levels higher than Atm(-/-) cells. Despite this increased genomic instability, Atm(KD/-) lymphocytes progressed through variable, diversity, and joining recombination and immunoglobulin class switch recombination, two events requiring nonhomologous end joining, at levels comparable to Atm(-/-) lymphocytes. Together, these results reveal an essential function of ATM during embryogenesis and an important function of catalytically inactive ATM protein in DNA repair.

14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression.

PubMed

Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S; Balyan, Renu; Borde, Lalit; Kundu, Tapas K; Dalal, Sorab N

2016-06-02

More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering.

Synchronization of Mammalian Cells and Nuclei by Centrifugal Elutriation.

PubMed

Banfalvi, Gaspar

2017-01-01

Synchronized populations of large numbers of cells can be obtained by centrifugal elutriation on the basis of sedimentation properties of small round particles, with minimal perturbation of cellular functions. The physical characteristics of cell size and sedimentation velocity are operative in the technique of centrifugal elutriation also known as counterstreaming centrifugation. The elutriator is an advanced device for increasing the sedimentation rate to yield enhanced resolution of cell separation. A random population of cells is introduced into the elutriation chamber of an elutriator rotor running in a specially designed centrifuge. By increasing step-by-step the flow rate of the elutriation fluid, successive populations of relatively homogeneous cell size can be removed from the elutriation chamber and used as synchronized subpopulations. For cell synchronization by centrifugal elutriation, early log S phase cell populations are most suitable where most of the cells are in G1 and S phase (>80 %). Apoptotic cells can be found in the early elutriation fractions belonging to the sub-Go window. Protocols for the synchronization of nuclei of murine pre-B cells and high-resolution centrifugal elutriation of CHO cells are given. The verification of purity and cell cycle positions of cells in elutriated fractions includes the measurement of DNA synthesis by [ 3 H]-thymidine incorporation and DNA content by propidium iodide flow cytometry.

Genome‐wide gene expression dynamics of the fungal pathogen Dothistroma septosporum throughout its infection cycle of the gymnosperm host Pinus radiata

PubMed Central

Guo, Yanan; Sim, Andre D.; Kabir, M. Shahjahan; Chettri, Pranav; Ozturk, Ibrahim K.; Hunziker, Lukas; Ganley, Rebecca J.; Cox, Murray P.

2015-01-01

Summary We present genome‐wide gene expression patterns as a time series through the infection cycle of the fungal pine needle blight pathogen, Dothistroma septosporum, as it invades its gymnosperm host, Pinus radiata. We determined the molecular changes at three stages of the disease cycle: epiphytic/biotrophic (early), initial necrosis (mid) and mature sporulating lesion (late). Over 1.7 billion combined plant and fungal reads were sequenced to obtain 3.2 million fungal‐specific reads, which comprised as little as 0.1% of the sample reads early in infection. This enriched dataset shows that the initial biotrophic stage is characterized by the up‐regulation of genes encoding fungal cell wall‐modifying enzymes and signalling proteins. Later necrotrophic stages show the up‐regulation of genes for secondary metabolism, putative effectors, oxidoreductases, transporters and starch degradation. This in‐depth through‐time transcriptomic study provides our first snapshot of the gene expression dynamics that characterize infection by this fungal pathogen in its gymnosperm host. PMID:25919703

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

PubMed

Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

2001-09-18

It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

Mitochondrial DNA polymerase editing mutation, PolgD257A, disturbs stem-progenitor cell cycling in the small intestine and restricts excess fat absorption.

PubMed

Fox, Raymond G; Magness, Scott; Kujoth, Gregory C; Prolla, Tomas A; Maeda, Nobuyo

2012-05-01

Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg(D257A), that exhibit accelerated aging phenotype. As early as 3-7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.

Early onset of puberty and early ovarian failure in CYP7B1 knockout mice.

PubMed

Omoto, Yoko; Lathe, Richard; Warner, Margaret; Gustafsson, Jan-Ake

2005-02-22

CYP7B1 is the enzyme responsible for hydroxylation and termination of the estrogenic actions of the androgen metabolite, 5alpha-androstane-3beta, 17beta-diol (3betaAdiol). 3betaAdiol is estrogenic in ERalpha or ERbeta positive cells only if they do not express CYP7B1. In this study we show that female CYP7B1(-/-) mice experience early onset of growth of the uterus and mammary glands and commence estrus cycles 2 days earlier than their wild-type littermates. Adult mammary glands and uteri appear to be under continuous estrogenic stimulation. We conclude that, by cell-specific regulation of the estrogenicity of 3betaAdiol, CYP7B1 performs two major tasks: (i) it allows 3betaAdiol to have growth inhibitory effects through ERbeta and (ii) it permits estradiol-specific activation of estrogen receptors by protection of certain cells from the estrogenic effects of 3betaAdiol. When CYP7B1 is inactivated, 3betaAdiol activates estrogen receptors indiscriminately, and the overall effect is prolonged and inappropriate exposure to estrogen.

Role of early growth response 1 in arteriogenesis: impact on vascular cell proliferation and leukocyte recruitment in vivo.

PubMed

Pagel, Judith-Irina; Ziegelhoeffer, Tibor; Heil, Matthias; Fischer, Silvia; Fernández, Borja; Schaper, Wolfgang; Preissner, Klaus T; Deindl, Elisabeth

2012-03-01

Based on previous findings that early growth response 1 (Egr-1) participates in leukocyte recruitment and cell proliferation in vitro, this study was designed to investigate its mode of action during arteriogenesis in vivo. In a model of peripheral arteriogenesis, Egr-1 was significantly upregulated in growing collaterals of wild-type (WT) mice, both on mRNA and protein level. Egr-1(-/-) mice demonstrated delayed arteriogenesis after femoral artery ligation. They further showed increased levels of monocytes and granulocytes in the circulation, but reduced levels in adductor muscles under baseline conditions. After femoral artery ligation, elevated numbers of macrophages were detected in the perivascular zone of collaterals in Egr-1(-/-) mice and mRNA of leukocyte recruitment mediators was upregulated. Other Egr family members (Egr-2 to -4) were significantly upregulated only in Egr-1(-/-) mice, suggesting a mechanism of counterbalancing Egr-1 deficiency. Moreover, splicing factor-1, downregulated in WT mice after femoral artery ligation in the process of increased vascular cell proliferation, was upregulated in Egr-1(-/-) mice. αSM-actin on the other hand, significantly downregulated in WT mice, showed no differential expression in Egr-1(-/-) mice. While cell cycle regulator cyclin E and cdc20 were upregulated in Egr-1(-/-) mice, cyclin D1 expression decreased below the detection limit in collaterals, and the proliferation marker ki67 was not differentially expressed. In conclusion, compensation for deficiency in Egr-1 function in leukocyte recruitment can presumably be mediated by other transcription factors; however, Egr-1 is indispensable for effective vascular cell cycle progression in arteriogenesis.

Changes in insulin-like growth factor-binding protein-3 messenger ribonucleic acid in endothelial cells of the human corpus luteum: a possible role in luteal development and rescue.

PubMed

Fraser, H M; Lunn, S F; Kim, H; Duncan, W C; Rodger, F E; Illingworth, P J; Erickson, G F

2000-04-01

In the human menstrual cycle, extensive angiogenesis accompanies luteinization; and the process is physiologically important for corpus luteum (CL) function. During luteolysis, the vasculature collapses, and the endothelial cells die. In a conceptual cycle, the CL persists both functionally and structurally beyond the luteoplacental shift. Although luteal rescue is not associated with increased angiogenesis, endothelial survival is extended. Despite the central role of the luteal vasculature in fertility, the mechanisms regulating its development and demise are poorly understood. There is increasing evidence that insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) may be important effectors of luteal function. Here, we have found that IGFBP-3 messenger RNA is expressed in the endothelium of the human CL and that the levels of message change during luteal development and rescue by human CG. The signal was strong during the early luteal phase, but it showed significant reduction during the mid- and late luteal phases. Interestingly, administration of human CG caused a marked increase in the levels of IGFBP-3 messenger RNA in luteal endothelial cells that was comparable to that observed during the early luteal phase. We conclude that endothelial cell IGFBP-3 expression is a physiological property of the CL of menstruation and pregnancy. These observations raise the intriguing possibility that the regulated expression of endothelial IGFBP-3 may play a role in controlling angiogenesis and cell responses in the human CL by autocrine/paracrine mechanisms.

Spatiotemporal Dynamics of Adenovirus Membrane Rupture and Endosomal Escape

PubMed Central

Maier, Oana; Marvin, Shauna A.; Wodrich, Harald; Campbell, Edward M.

2012-01-01

A key step in adenovirus cell entry is viral penetration of cellular membranes to gain access to the cytoplasm and deliver the genome to the nucleus. Yet little is known about this important event in the adenoviral life cycle. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture with both live- and fixed-cell imaging, we demonstrate that in the majority of instances, exposure of pVI and recruitment of gal3 to ruptured membranes occur early at or near the cell surface and occur minimally in EEA-1-positive (EEA-1+) early endosomes or LAMP-1+ late endosomes/lysosomes. Live-cell imaging of Ad5 egress from gal3+ endosomes occurs most frequently from perinuclear locations. While the Ad5 capsid is observed escaping from gal3+ endosomes, pVI appears to remain associated with the gal3+ ruptured endosomes. Thus, Ad5 membrane rupture and endosomal escape appear to be both spatially and temporally distinct events. PMID:22855481

Nascent life cycles and the emergence of higher-level individuality.

PubMed

Ratcliff, William C; Herron, Matthew; Conlin, Peter L; Libby, Eric

2017-12-05

Evolutionary transitions in individuality (ETIs) occur when formerly autonomous organisms evolve to become parts of a new, 'higher-level' organism. One of the first major hurdles that must be overcome during an ETI is the emergence of Darwinian evolvability in the higher-level entity (e.g. a multicellular group), and the loss of Darwinian autonomy in the lower-level units (e.g. individual cells). Here, we examine how simple higher-level life cycles are a key innovation during an ETI, allowing this transfer of fitness to occur 'for free'. Specifically, we show how novel life cycles can arise and lead to the origin of higher-level individuals by (i) mitigating conflicts between levels of selection, (ii) engendering the expression of heritable higher-level traits and (iii) allowing selection to efficiently act on these emergent higher-level traits. Further, we compute how canonical early life cycles vary in their ability to fix beneficial mutations via mathematical modelling. Life cycles that lack a persistent lower-level stage and develop clonally are far more likely to fix 'ratcheting' mutations that limit evolutionary reversion to the pre-ETI state. By stabilizing the fragile first steps of an evolutionary transition in individuality, nascent higher-level life cycles may play a crucial role in the origin of complex life.This article is part of the themed issue 'Process and pattern in innovations from cells to societies'. © 2017 The Author(s).

Casein kinase 2 and the cell response to growth factors.

PubMed

Filhol-Cochet, O; Loue-Mackenbach, P; Cochet, C; Chambaz, E M

1994-01-01

Different approaches have been followed with the aim of delineating a possible role of casein kinase 2 (CK2) in the mitogenic signalling in response to cell growth factors. (a) Immunocytochemical detection of CK2 showed that while the kinase is evenly distributed throughout cycle arrested cells, it becomes preferentially associated with the nuclear compartment in activity growing cells; (b) CK2 biosynthesis is activated as an early response of quiescent cells to growth factors. The newly synthesized CK2 steadily accumulates as the cells progress through the G1 phase. This growth factor-induced CK2 biosynthesis involves in parallel the two alpha and beta subunits of the kinase, with no detectable preferential subcellular localization of the newly synthesized enzyme; and (c) In addition to substrate phosphorylation, CK2 may form molecular complexes with cell components of functional significance. Such is the case with the protein p53, a major negative regulator of the cell cycle. CK2 forms a high affinity association (Kd 70 nM) with p53, through its beta subunit. The complex dissociates in the presence of adenosine triphosphate (ATP). These observations suggest that CK2 and p53 may play a coordinated regulatory role in the cell response to growth factors.

A systematic analysis of the early transcribed membrane protein family throughout the life cycle of Plasmodium yoelii.

PubMed

MacKellar, Drew C; Vaughan, Ashley M; Aly, Ahmed S I; DeLeon, Sasha; Kappe, Stefan H I

2011-11-01

The early transcribed membrane proteins (ETRAMPs) are a family of small, highly charged transmembrane proteins unique to malaria parasites. Some members of the ETRAMP family have been localized to the parasitophorous vacuole membrane that separates the intracellular parasite from the host cell and thus presumably have a role in host-parasite interactions. Although it was previously shown that two ETRAMPs are critical for rodent malaria parasite liver-stage development, the importance of most ETRAMPs during the parasite life cycle remains unknown. Here, we comprehensively identify nine new etramps in the genome of the rodent malaria parasite Plasmodium yoelii, and elucidate their conservation in other malaria parasites. etramp expression profiles are diverse throughout the parasite life cycle as measured by RT-PCR. Epitope tagging of two ETRAMPs demonstrates protein expression in blood and liver stages, and reveals differences in both their timing of expression and their subcellular localization. Gene targeting studies of each of the nine uncharacterized etramps show that two are refractory to deletion and thus likely essential for blood-stage replication. Seven etramps are not essential for any life cycle stage. Systematic characterization of the members of the ETRAMP family reveals the diversity in importance of each family member at the interface between host and parasite throughout the developmental cycle of the malaria parasite. © 2011 Blackwell Publishing Ltd.

Indicators of replicative damage in equine tendon fibroblast monolayers

PubMed Central

2013-01-01

Background Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it. Results We found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents. Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress. Conclusions Researchers using tendon fibroblast monolayers should assess their “health” using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled. PMID:24025445

Biologically based multistage modeling of radiation effects

DOE Office of Scientific and Technical Information (OSTI.GOV)

William Hazelton; Suresh Moolgavkar; E. Georg Luebeck

2005-08-30

This past year we have made substantial progress in modeling the contribution of homeostatic regulation to low-dose radiation effects and carcinogenesis. We have worked to refine and apply our multistage carcinogenesis models to explicitly incorporate cell cycle states, simple and complex damage, checkpoint delay, slow and fast repair, differentiation, and apoptosis to study the effects of low-dose ionizing radiation in mouse intestinal crypts, as well as in other tissues. We have one paper accepted for publication in ''Advances in Space Research'', and another manuscript in preparation describing this work. I also wrote a chapter describing our combined cell-cycle and multistagemore » carcinogenesis model that will be published in a book on stochastic carcinogenesis models edited by Wei-Yuan Tan. In addition, we organized and held a workshop on ''Biologically Based Modeling of Human Health Effects of Low dose Ionizing Radiation'', July 28-29, 2005 at Fred Hutchinson Cancer Research Center in Seattle, Washington. We had over 20 participants, including Mary Helen Barcellos-Hoff as keynote speaker, talks by most of the low-dose modelers in the DOE low-dose program, experimentalists including Les Redpath (and Mary Helen), Noelle Metting from DOE, and Tony Brooks. It appears that homeostatic regulation may be central to understanding low-dose radiation phenomena. The primary effects of ionizing radiation (IR) are cell killing, delayed cell cycling, and induction of mutations. However, homeostatic regulation causes cells that are killed or damaged by IR to eventually be replaced. Cells with an initiating mutation may have a replacement advantage, leading to clonal expansion of these initiated cells. Thus we have focused particularly on modeling effects that disturb homeostatic regulation as early steps in the carcinogenic process. There are two primary considerations that support our focus on homeostatic regulation. First, a number of epidemiologic studies using multistage carcinogenesis models that incorporate the ''initiation, promotion, and malignant conversion'' paradigm of carcinogenesis are indicating that promotion of initiated cells is the most important cellular mechanism driving the shape of the age specific hazard for many types of cancer. Second, we have realized that many of the genes that are modified in early stages of the carcinogenic process contribute to one or more of four general cellular pathways that confer a promotional advantage to cells when these pathways are disrupted.« less

GATA-3 is required for early T lineage progenitor development

PubMed Central

Hosoya, Tomonori; Kuroha, Takashi; Moriguchi, Takashi; Cummings, Dustin; Maillard, Ivan; Lim, Kim-Chew

2009-01-01

Most T lymphocytes appear to arise from very rare early T lineage progenitors (ETPs) in the thymus, but the transcriptional programs that specify ETP generation are not completely known. The transcription factor GATA-3 is required for the development of T lymphocytes at multiple late differentiation steps as well as for the development of thymic natural killer cells. However, a role for GATA-3 before the double-negative (DN) 3 stage of T cell development has to date been obscured both by the developmental heterogeneity of DN1 thymocytes and the paucity of ETPs. We provide multiple lines of in vivo evidence through the analysis of T cell development in Gata3 hypomorphic mutant embryos, in irradiated mice reconstituted with Gata3 mutant hematopoietic cells, and in mice conditionally ablated for the Gata3 gene to show that GATA-3 is required for ETP generation. We further show that Gata3 loss does not affect hematopoietic stem cells or multipotent hematopoietic progenitors. Finally, we demonstrate that Gata3 mutant lymphoid progenitors exhibit neither increased apoptosis nor diminished cell-cycle progression. Thus, GATA-3 is required for the cell-autonomous development of the earliest characterized thymic T cell progenitors. PMID:19934022

Development of a lead-acid battery for a hybrid electric vehicle

NASA Astrophysics Data System (ADS)

Cooper, A.

In September 2000, a project reliable, highly optimized lead-acid battery (RHOLAB) started under the UK Foresight Vehicle Programme with the objective of developing an optimized lead-acid battery solution for hybrid electric vehicles. The work is based on a novel, individual, spirally-wound valve-regulated lead-acid 2 V cell optimized for HEV use and low variability. This cell is being used as a building block for the development of a complete battery pack that is managed at the cell level. Following bench testing, this battery pack is to be thoroughly evaluated by substituting it for the Ni-MH pack in a Honda Insight. The RHOLAB cell is based on the 8 Ah Hawker Cyclon cell which has been modified to have current take-off at both ends—the dual-tab design. In addition, a variant has been produced with modified cell chemistry to help deal with problems that can occur when these valve-regulated lead-acid battery (VRLA) cells operate in a partial-state-of-charge condition. The cells have been cycled to a specially formulated test cycle based on real vehicle data derived from testing the Honda Insight on the various test tracks at the Millbrook Proving Grounds in the UK. These cycling tests have shown that the lead-acid pack can be successfully cycled when subjected to the high current demands from the vehicle, which have been measured at up to 15 C on discharge and 8 C during regenerative recharging, and cycle life is looking very promising under this arduous test regime. Concurrent with this work, battery development has been taking place. It was decided early on to develop the 144 V battery as four 36 V modules. Data collection and control has been built-in and special steps taken to minimize the problems of interconnect in this complex system. Development of the battery modules is now at an advanced stage. The project plan then allows for extensive testing of the vehicle with its lead-acid battery at Millbrook so it can be compared with the benchmark tests which have already been carried out on the vehicle with its Ni-MH batteries.

R-CHOP with or without bevacizumab in patients with previously untreated diffuse large B-cell lymphoma: final MAIN study outcomes

PubMed Central

Seymour, John F.; Pfreundschuh, Michael; Trnĕný, Marek; Sehn, Laurie H.; Catalano, John; Csinady, Eva; Moore, Nicola; Coiffier, Bertrand

2014-01-01

Vascular endothelial growth factor is involved in lymphoma growth, suggesting a potential role for anti-vascular endothelial growth factor therapies in hematologic malignancies. In this phase III study, patients with CD20-positive diffuse large B-cell lymphoma were randomized to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone plus either placebo (R-CHOP) or bevacizumab (RA-CHOP). Treatment was administered every 21 (8 cycles) or 14 days (6 cycles plus 2 rituximab cycles) as per institutional practice. An early analysis of risk/benefit by the Data and Safety Monitoring Board showed that RA-CHOP increased cardiotoxicity without prolonging progression-free survival compared with R-CHOP, and the trial was stopped early. The study protocol was amended to allow for 12 additional months of follow up to evaluate safety. With 787 patients enrolled, median follow up was 23.7 and 23.6 months for R-CHOP and RA-CHOP, respectively. Median progression-free survival for R-CHOP and RA CHOP was 42.9 and 40.2 months, respectively (hazard ratio=1.09; P=0.49). The proportion of deaths was identical for R-CHOP (83 of 387, 21%) and RA-CHOP (82 of 390, 21%). Relative to R-CHOP, RA-CHOP had a higher rate of left ventricular ejection fraction perturbation (18% vs. 8%; odds ratio=2.51; 95% confidence interval (CI): 1.60–3.93) and congestive heart failure (16% vs. 7%; odds ratio=2.79; 95%CI: 1.72–4.54). Bevacizumab added to R-CHOP increased cardiac events, without increasing efficacy, arguing against further evaluation of RA-CHOP in patients with diffuse large B-cell lymphoma. The MAIN study is registered at clinicaltrials.gov identifier:00486759. PMID:24895339

R-CHOP with or without bevacizumab in patients with previously untreated diffuse large B-cell lymphoma: final MAIN study outcomes.

PubMed

Seymour, John F; Pfreundschuh, Michael; Trnĕný, Marek; Sehn, Laurie H; Catalano, John; Csinady, Eva; Moore, Nicola; Coiffier, Bertrand

2014-08-01

Vascular endothelial growth factor is involved in lymphoma growth, suggesting a potential role for anti-vascular endothelial growth factor therapies in hematologic malignancies. In this phase III study, patients with CD20-positive diffuse large B-cell lymphoma were randomized to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone plus either placebo (R-CHOP) or bevacizumab (RA-CHOP). Treatment was administered every 21 (8 cycles) or 14 days (6 cycles plus 2 rituximab cycles) as per institutional practice. An early analysis of risk/benefit by the Data and Safety Monitoring Board showed that RA-CHOP increased cardiotoxicity without prolonging progression-free survival compared with R-CHOP, and the trial was stopped early. The study protocol was amended to allow for 12 additional months of follow up to evaluate safety. With 787 patients enrolled, median follow up was 23.7 and 23.6 months for R-CHOP and RA-CHOP, respectively. Median progression-free survival for R-CHOP and RA CHOP was 42.9 and 40.2 months, respectively (hazard ratio=1.09; P=0.49). The proportion of deaths was identical for R-CHOP (83 of 387, 21%) and RA-CHOP (82 of 390, 21%). Relative to R-CHOP, RA-CHOP had a higher rate of left ventricular ejection fraction perturbation (18% vs. 8%; odds ratio=2.51; 95% confidence interval (CI): 1.60-3.93) and congestive heart failure (16% vs. 7%; odds ratio=2.79; 95%CI: 1.72-4.54). Bevacizumab added to R-CHOP increased cardiac events, without increasing efficacy, arguing against further evaluation of RA-CHOP in patients with diffuse large B-cell lymphoma. The MAIN study is registered at clinicaltrials.gov identifier:00486759. Copyright© Ferrata Storti Foundation.

Mitotic waves in the early embryogenesis of Drosophila: Bistability traded for speed.

PubMed

Vergassola, Massimo; Deneke, Victoria E; Di Talia, Stefano

2018-03-06

Early embryogenesis of most metazoans is characterized by rapid and synchronous cleavage divisions. Chemical waves of Cdk1 activity were previously shown to spread across Drosophila embryos, and the underlying molecular processes were dissected. Here, we present the theory of the physical mechanisms that control Cdk1 waves in Drosophila The in vivo dynamics of Cdk1 are captured by a transiently bistable reaction-diffusion model, where time-dependent reaction terms account for the growing level of cyclins and Cdk1 activation across the cell cycle. We identify two distinct regimes. The first one is observed in mutants of the mitotic switch. There, waves are triggered by the classical mechanism of a stable state invading a metastable one. Conversely, waves in wild type reflect a transient phase that preserves the Cdk1 spatial gradients while the overall level of Cdk1 activity is swept upward by the time-dependent reaction terms. This unique mechanism generates a wave-like spreading that differs from bistable waves for its dependence on dynamic parameters and its faster speed. Namely, the speed of "sweep" waves strikingly decreases as the strength of the reaction terms increases and scales as the powers 3/4, -1/2, and 7/12 of Cdk1 molecular diffusivity, noise amplitude, and rate of increase of Cdk1 activity in the cell-cycle S phase, respectively. Theoretical predictions are supported by numerical simulations and experiments that couple quantitative measurements of Cdk1 activity and genetic perturbations of the accumulation rate of cyclins. Finally, our analysis bears upon the inhibition required to suppress Cdk1 waves at the cell-cycle pause for the maternal-to-zygotic transition.

Raman spectrum reveals the cell cycle arrest of Triptolide-induced leukemic T-lymphocytes apoptosis

NASA Astrophysics Data System (ADS)

Zhang, Daosen; Feng, Yanyan; Zhang, Qinnan; Su, Xin; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

2015-04-01

Triptolide (TPL), a traditional Chinese medicine extract, possesses anti-inflammatory and anti-tumor properties. Though some research results have implicated that Triptolide (TPL) can be utilized in the treatment of leukemia, it remains controversial about the mechanism of TPL-induced leukemic T-lymphocytes apoptosis. In this study, combining Raman spectroscopic data, principal component analysis (PCA) and atomic force microscopy (AFM) imaging, both the biochemical changes and morphological changes during TPL-induced cell apoptosis were presented. In contrast, the corresponding data during Daunorubicin (DNR)-induced cell apoptosis was also exhibited. The obtained results showed that Raman spectral changes during TPL-induced cell apoptosis were greatly different from DNR-induced cell apoptosis in the early stage of apoptosis but revealed the high similarity in the late stage of apoptosis. Moreover, above Raman spectral changes were respectively consistent with the morphological changes of different stages during TPL-induced apoptosis or DNR-induced apoptosis, including membrane shrinkage and blebbing, chromatin condensation and the formation of apoptotic bodies. Importantly, it was found that Raman spectral changes with TPL-induced apoptosis or DNR-induced apoptosis were respectively related with the cell cycle G1 phase arrest or G1 and S phase arrest.

LIN9, a Subunit of the DREAM Complex, Regulates Mitotic Gene Expression and Proliferation of Embryonic Stem Cells

PubMed Central

Esterlechner, Jasmina; Reichert, Nina; Iltzsche, Fabian; Krause, Michael; Finkernagel, Florian; Gaubatz, Stefan

2013-01-01

The DREAM complex plays an important role in regulation of gene expression during the cell cycle. We have previously shown that the DREAM subunit LIN9 is required for early embryonic development and for the maintenance of the inner cell mass in vitro. In this study we examined the effect of knocking down LIN9 on ESCs. We demonstrate that depletion of LIN9 alters the cell cycle distribution of ESCs and results in an accumulation of cells in G2 and M and in an increase of polyploid cells. Genome-wide expression studies showed that the depletion of LIN9 results in downregulation of mitotic genes and in upregulation of differentiation-specific genes. ChIP-on chip experiments showed that mitotic genes are direct targets of LIN9 while lineage specific markers are regulated indirectly. Importantly, depletion of LIN9 does not alter the expression of pluripotency markers SOX2, OCT4 and Nanog and LIN9 depleted ESCs retain alkaline phosphatase activity. We conclude that LIN9 is essential for proliferation and genome stability of ESCs by activating genes with important functions in mitosis and cytokinesis. PMID:23667535

Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis

PubMed Central

Parajuli, Keshab R; Zhang, Qiuyang; Liu, Sen; Patel, Neil K; Lu, Hua; Zeng, Shelya X; Wang, Guangdi; Zhang, Changde; You, Zongbing

2014-01-01

Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production of consumer products and pharmaceutical products. MAA causes embryo malformation and spermatocyte death through inhibition of histone deacetylases (HDACs). Little is known about MAA’s effects on cancer cells. In this study, two immortalized human normal prostatic epithelial cell lines (RWPE-1 and pRNS-1-1) and four human prostate cancer cell lines (LNCaP, C4-2B, PC-3, and DU-145) were treated with MAA at different doses and for different time periods. Cell viability, apoptosis, and cell cycle analysis were performed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR, Western blot, and chromatin immunoprecipitation analyses. We found that MAA dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. MAA-induced apoptosis was due to down-regulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, also named cIAP1), leading to activation of caspases 7 and 3 and turning on the downstream apoptotic events. MAA-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and CDK2 expression at the late time. MAA up-regulated p21 expression through inhibition of HDAC activities, independently of p53/p63/p73. These findings demonstrate that MAA suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which suggests that MAA could be used as a potential therapeutic drug for prostate cancer. PMID:25606576

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells.

PubMed

Wei, Ren-Jie; Lin, Su-Shuan; Wu, Wen-Ren; Chen, Lih-Ren; Li, Chien-Feng; Chen, Han-De; Chou, Chien-Ting; Chen, Ya-Chun; Liang, Shih-Shin; Chien, Shang-Tao; Shiue, Yow-Ling

2016-11-15

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G 2 /M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G 2 /M cell cycle regulators, ABT-751 induced G 2 /M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. Copyright © 2016 Elsevier Inc. All rights reserved.

Arctigenin anti-tumor activity in bladder cancer T24 cell line through induction of cell-cycle arrest and apoptosis.

PubMed

Yang, Shucai; Ma, Jing; Xiao, Jianbing; Lv, Xiaohong; Li, Xinlei; Yang, Huike; Liu, Ying; Feng, Sijia; Zhang, Yafang

2012-08-01

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti-tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell-cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose- and time-dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 μmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin-V-FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32-7.01% and 3.07-7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose-dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the anti-tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer. Copyright © 2012 Wiley Periodicals, Inc.

The life cycle of Phaeocystis (Prymnesiophycaea): evidence and hypotheses

NASA Astrophysics Data System (ADS)

Rousseau, V.; Vaulot, D.; Casotti, R.; Cariou, V.; Lenz, J.; Gunkel, J.; Baumann, M.

1994-04-01

The present paper reviews the literature related to the life cycle of the prymnesiophyte Phaeocystis and its controlling factors and proposes novel hypotheses based on unpublished observations in culture and in the field. We chiefly refer to P. globosa Scherffel as most of the observations concern this species. P. globosa exhibits a complex alternation between several types of free-living cells (non-motile, flagellates, microzoopores and possibly macrozoospores) and colonies for which neither forms nor pathways have been completely identified and described. The different types of Phaeocystis cells were reappraised on the basis of existing microscopic descriptions complemented by unpublished flow cytometric investigations. This analysis revealed the existence of at least three different types of free-living cells identified on the basis of a combination of size, motility and ploidy characteristics: non-motile cells, flagellates and microzoospores. Their respective function within Phaeocystis life cycle, and in particular their involvement in colony formation is not completely understood. Observational evidence shows that Phaeocystis colonies are initiated at the early stage of their bloom each by one free-living cell. The mechanisms controlling this cellular transformation are still uncertain due to the lack of information on the overwintering Phaeocystis fomms and on the cell type responsible for colony induction. The existence of haploid microzoospores released from senescent colonies gives however some support to sexuality involvement at some stages of colony formation. Once colonies are formed, at least two mechanisms were identified as responsible of the spreading of colony form: colony multiplication by colonial division or budding and induction of new colony from colonial cells released in the external medium after colony disruption. The latter mechanism was clearly identified, involving at least two successive cell differentiations in the following sequence: motility development, subsequent flagella loss and settlement to a surface, mucus secretion and colony formation, colonial cell division and colony growth. Aggregate formation, cell motility development and subsequent emigration from the colonies, release of non-motile cells after colony lysis on the other hand, were identified as characteristics for termination of Phaeocystis colony development. These pathways were shown to occur similarly in natural environments. In the early stages of the bloom however, many recently-formed colonies were found on the setae of Chaetoceros spp, suggesting this diatom could play a key-rôle in Phaeocystis bloom inception. Analysis of the possible environmental factors regulating the transition between the different phases of the life cycle, suggested that nutrient status and requirement of a substrate for attachment of free-living cells would be essential for initiation of the colonial form. Physical constraints obviously would be important in determining colony shape and fragmentation although autogenic factors cannot be excluded. Some evidence exists that nutrients regulate colony division, while temperature and nutrient stress would stimulate cell emigration from the colonies.

Application of the Maximum Amplitude-Early Rise Correlation to Cycle 23

NASA Technical Reports Server (NTRS)

Willson, Robert M.; Hathaway, David H.

2004-01-01

On the basis of the maximum amplitude-early rise correlation, cycle 23 could have been predicted to be about the size of the mean cycle as early as 12 mo following cycle minimum. Indeed, estimates for the size of cycle 23 throughout its rise consistently suggested a maximum amplitude that would not differ appreciably from the mean cycle, contrary to predictions based on precursor information. Because cycle 23 s average slope during the rising portion of the solar cycle measured 2.4, computed as the difference between the conventional maximum (120.8) and minimum (8) amplitudes divided by the ascent duration in months (47), statistically speaking, it should be a cycle of shorter period. Hence, conventional sunspot minimum for cycle 24 should occur before December 2006, probably near July 2006 (+/-4 mo). However, if cycle 23 proves to be a statistical outlier, then conventional sunspot minimum for cycle 24 would be delayed until after July 2007, probably near December 2007 (+/-4 mo). In anticipation of cycle 24, a chart and table are provided for easy monitoring of the nearness and size of its maximum amplitude once onset has occurred (with respect to the mean cycle and using the updated maximum amplitude-early rise relationship).

Temporal variations in early developmental decisions: an engine of forebrain evolution.

PubMed

Bielen, H; Pal, S; Tole, S; Houart, C

2017-02-01

Tight control of developmental timing is pivotal to many major processes in developmental biology, such as patterning, fate specification, cell cycle dynamics, cell migration and connectivity. Temporal change in these ontogenetic sequences is known as heterochrony, a major force in the evolution of body plans and organogenesis. In the last 5 years, studies in fish and rodents indicate that heterochrony in signaling during early development generates diversity in forebrain size and complexity. Here, we summarize these findings and propose that, additionally to spatio-temporal tuning of neurogenesis, temporal and quantitative modulation of signaling events drive pivotal changes in shape, size and complexity of the forebrain across evolution, participating to the generation of diversity in animal behavior and emergence of cognition. Copyright © 2017 Elsevier Ltd. All rights reserved.

p21, an important mediator of quiescence during pituitary tumor formation, is dispensable for normal pituitary development during embryogenesis.

PubMed Central

Monahan, Pamela; Himes, Ashley D.; Parfieniuk, Agata; Raetzman, Lori T.

2011-01-01

A delicate balance between proliferation and differentiation must be maintained in the developing pituitary to ensure the formation of the appropriate number of hormone producing cells. In the adult, proliferation is actively restrained to prevent tumor formation. The cyclin dependent kinase inhibitors (CDKIs) of the CIP/KIP family, p21, p27 and p57, mediate cell cycle inhibition. Although p21 is induced in the pituitary upon loss of Notch signaling or initiation of tumor formation to halt cell cycle progression, its role in normal pituitary organogenesis has not been explored. In wildtype pituitaries, expression of p21 is limited to a subset of cells embryonically as well as during the postnatal proliferative phase. Mice lacking p21 do not have altered cell proliferation during early embryogenesis, but do show a slight delay in separation of proliferating progenitors from the oral ectoderm. By embryonic day 16.5, p21 mutants have an alteration in the spatial distribution of proliferating pituitary progenitors, however there is no overall change in proliferation. At postnatal day 21, there appears to be no change in proliferation, as assessed by cells expressing Ki67 protein. However, p21 mutant pituitaries have significantly less mRNA of Myc and the cyclins Ccnb1, Ccnd1, Ccnd2 and Ccne1 than wildtype pituitaries. Interestingly, unlike the redundant role in cell cycle inhibition uncovered in p27/p57 double mutants, the pituitary of p21/p27 double mutants has a similar proliferation profile to p27 single mutants at the time points examined. Taken together, these studies demonstrate that unlike p27 or p57, p21 does not play a major role in control of progenitor proliferation in the developing pituitary. However, p21 may be required to maintain normal levels of cell cycle components. PMID:22154697

Degradation of the human mitotic checkpoint kinase Mps1 is cell cycle-regulated by APC-cCdc20 and APC-cCdh1 ubiquitin ligases.

PubMed

Cui, Yongping; Cheng, Xiaolong; Zhang, Ce; Zhang, Yanyan; Li, Shujing; Wang, Chuangui; Guadagno, Thomas M

2010-10-22

Mps1 is a dual specificity protein kinase with key roles in regulating the spindle assembly checkpoint and chromosome-microtubule attachments. Consistent with these mitotic functions, Mps1 protein levels fluctuate during the cell cycle, peaking at early mitosis and abruptly declining during mitotic exit and progression into the G(1) phase. Although evidence in budding yeast indicates that Mps1 is targeted for degradation at anaphase by the anaphase-promoting complex (APC)-c(Cdc20) complex, little is known about the regulatory mechanisms that govern Mps1 protein levels in human cells. Here, we provide evidence for the ubiquitin ligase/proteosome pathway in regulating human Mps1 levels during late mitosis through G(1) phase. First, we showed that treatment of HEK 293T cells with the proteosome inhibitor MG132 resulted in an increase in both the polyubiquitination and the accumulation of Mps1 protein levels. Next, Mps1 was shown to co-precipitate with APC and its activators Cdc20 and Cdh1 in a cell cycle-dependent manner. Consistent with this, overexpression of Cdc20 or Cdh1 led to a marked reduction of endogenous Mps1 levels during anaphase or G(1) phase, respectively. In contrast, depletion of Cdc20 or Cdh1 by RNAi treatment both led to the stabilization of Mps1 protein during mitosis or G(1) phase, respectively. Finally, we identified a single D-box motif in human Mps1 that is required for its ubiquitination and degradation. Failure to appropriately degrade Mps1 is sufficient to trigger centrosome amplification and mitotic abnormalities in human cells. Thus, our results suggest that the sequential actions of the APC-c(Cdc20) and APC-c(Cdh1) ubiquitin ligases regulate the clearance of Mps1 levels and are critical for Mps1 functions during the cell cycle in human cells.

Interferon-induced Sus scrofa Mx1 blocks endocytic traffic of incoming influenza A virus particles.

PubMed

Palm, Mélanie; Garigliany, Mutien-Marie; Cornet, François; Desmecht, Daniel

2010-01-01

The interferon-induced Mx proteins of vertebrates are dynamin-like GTPases, some isoforms of which can additionally inhibit the life cycle of certain RNA viruses. Here we show that the porcine Mx1 protein (poMx1) inhibits replication of influenza A virus and we attempt to identify the step at which the viral life cycle is blocked. In infected cells expressing poMx1, the level of transcripts encoding the viral nucleoprotein is significantly lower than normal, even when secondary transcription is prevented by exposure to cycloheximide. This reveals that a pretranscriptional block participates to the anti-influenza activity. Binding and internalization of incoming virus particles are normal in the presence of poMx1 but centripetal traffic to the late endosomes is interrupted. Surprisingly but decisively, poMx1 significantly alters binding of early endosome autoantigen 1 to early endosomes and/or early endosome size and spatial distribution. This is compatible with impairment of traffic of the endocytic vesicles to the late endosomes. INRA, EDP Sciences, 2010.

Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies

PubMed Central

Gonsalves, Wilson I.; Hitosugi, Taro; Ghosh, Toshi; Jevremovic, Dragan; Petterson, Xuan-Mai; Wellik, Linda; Kumar, Shaji K.; Nair, K. Sreekumaran

2018-01-01

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention. PMID:29321378

Developmental stage-specific regulation of the circadian clock by temperature in zebrafish.

PubMed

Lahiri, Kajori; Froehlich, Nadine; Heyd, Andreas; Foulkes, Nicholas S; Vallone, Daniela

2014-01-01

The circadian clock enables animals to adapt their physiology and behaviour in anticipation of the day-night cycle. Light and temperature represent two key environmental timing cues (zeitgebers) able to reset this mechanism and so maintain its synchronization with the environmental cycle. One key challenge is to unravel how the regulation of the clock by zeitgebers matures during early development. The zebrafish is an ideal model for studying circadian clock ontogeny since the process of development occurs ex utero in an optically transparent chorion and many tools are available for genetic analysis. However, the role played by temperature in regulating the clock during zebrafish development is poorly understood. Here, we have established a clock-regulated luciferase reporter transgenic zebrafish line (Tg (-3.1) per1b::luc) to study the effects of temperature on clock entrainment. We reveal that under complete darkness, from an early developmental stage onwards (48 to 72 hpf), exposure to temperature cycles is a prerequisite for the establishment of self-sustaining rhythms of zfper1b, zfaanat2, and zfirbp expression and also for circadian cell cycle rhythms. Furthermore, we show that following the 5-9 somite stage, the expression of zfper1b is regulated by acute temperature shifts.

Dynamics of Pertussis Transmission in the United States

PubMed Central

Magpantay, F. M. G.; Rohani, P.

2015-01-01

Past patterns of infectious disease transmission set the stage on which modern epidemiologic dynamics are played out. Here, we present a comprehensive account of pertussis (whooping cough) transmission in the United States during the early vaccine era. We analyzed recently digitized weekly incidence records from Morbidity and Mortality Weekly Reports from 1938 to 1955, when the whole-cell pertussis vaccine was rolled out, and related them to contemporary patterns of transmission and resurgence documented in monthly incidence data from the National Notifiable Diseases Surveillance System. We found that, during the early vaccine era, pertussis epidemics in US states could be categorized as 1) annual, 2) initially annual and later multiennial, or 3) multiennial. States with predominantly annual cycles tended to have higher per capita birth rates, more household crowding, more children per family, and lower rates of school attendance than the states with multiennial cycles. Additionally, states that exhibited annual epidemics during 1938–1955 have had the highest recent (2001–2010) incidence, while those states that transitioned from annual cycles to multiennial cycles have had relatively low recent incidence. Our study provides an extensive picture of pertussis epidemiology in the United States dating back to the onset of vaccination, a back-story that could aid epidemiologists in understanding contemporary transmission patterns. PMID:26022662

TRIM28, a new molecular marker predicting metastasis and survival in early-stage non-small cell lung cancer.

PubMed

Liu, Lei; Zhao, Enhong; Li, Chunhui; Huang, Liang; Xiao, Lijun; Cheng, Luyang; Huang, Xu; Song, Youxin; Xu, Dawei

2013-02-01

TRIM28 is a universal corepressor for Kruppel-associated box zinc finger proteins. In this study, we demonstrated the expression of TRIM28 gene was significantly higher in cancerous tissues than in noncancerous tissues (P < 0.001). TRIM28 knockdown resulted in a decrease in cell proliferation in liquid media as well as in soft agar. The proliferation rate was impaired and the cell cycle progression was inhibited after knockdown of TRIM28 in non-small cell lung cancer cell lines PAa and SK-MES-1. We used real-time polymerase chain reaction to detect circulating cancer cells in 138 non-small cell lung cancer patients. The overall positive detection rate was 30.4% (42 of 138) in peripheral blood of NSCLC patients and was 29.9% (29 of 97) in early-stage patients. In a 70-month follow-up study, 20 of 29 patients (69.0%) in TRIM28 positive group had recurrence and/or metastasis, significantly higher (P = 0.004) than in the TRIM28 negative group (25 of 68, 36.8%). In addition, non-small cell lung cancer patients whose circulating cancer cells expressed TRIM28 suffered shorter tumor-specific survival compared with those with absent TRIM28 expression (P < 0.001). Results of our study showed that TRIM28 provides a survival advantage to lung cancer cells and may be a new marker to predict metastasis and prognosis in early-stage non-small cell lung cancer patients. Copyright © 2012 Elsevier Ltd. All rights reserved.

Conditional ablation of the Notch2 receptor in the ocular lens

PubMed Central

Saravanamuthu, Senthil S.; Le, Tien T.; Gao, Chun Y.; Cojocaru, Radu I.; Pandiyan, Pushpa; Liu, Chunqiao; Zhang, Jun; Zelenka, Peggy S.; Brown, Nadean L.

2011-01-01

Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation. PMID:22173065

Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy.

PubMed Central

Preuss, D; Mulholland, J; Franzusoff, A; Segev, N; Botstein, D

1992-01-01

The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth. Images PMID:1381247

Inhibin may be involved in negative feedback in the prairie dog (Cynomys ludovicianus).

PubMed

Foreman, Darhl

2007-02-01

The changes in inhibin immunostaining in the gonads during the annual reproductive cycle of both sexes of the prairie dog are described. No inhibin immunostaining was found in primary or secondary follicles of the ovary. Theca and granulosa cells of preovulatory Graafian follicles found in January and February stained for inhibin. Corpora lutea of both pregnant and non-pregnant females stain more densely for inhibin than follicles. Inhibin staining is present in luteal cells for at least 4 months during regression, longer than detectable progesterone is secreted. Sertoli cells in the testes do not have inhibin immunostaining during recrudescence. These cells show light immunostain for inhibin during peak spermatogenic activity in January and February but stain more deeply during early regression of the testis. Stain is gradually lost in the next 4-5 months as the tubules close. Leydig cells and germ cells do not stain for inhibin at any stage of the annual cycle but interstitial cells and tunic cells stain during the breeding phase. The presence of immunochemical staining for inhibin in prairie dog gonads during regression suggests that inhibin is part of a negative feedback complex that includes progesterone in the female and testosterone or another androgen in the male. Negative feedback during regression may also cause gonadal inactivity.

Cell cycle phase dependent emergence of thymidylate synthase studied by monoclonal antibody (M-TS-4).

PubMed

Shibui, S; Hoshino, T; Iwasaki, K; Nomura, K; Jastreboff, M M

1989-05-01

A method of identifying thymidylate synthase (TS) at the cellular level was developed using anti-TS monoclonal antibody (M-TS-4), a monoclonal antibody created against purified TS from a HeLa cell line. In HeLa cells and four human glioma cell lines (U-251, U-87, 343-MGA, and SF-188), TS was identified primarily in the cytoplasm. Autoradiographic and flow cytometric studies showed that TS appeared mainly in the G1 phase and subsided early in the S phase; thus, the G1 phase can be divided into TS-positive and -negative fractions. Nuclear TS was not demonstrated unequivocally with M-TS-4, and the relationship between nuclear TS and DNA synthesis could not be determined. Although the percentage of TS-positive cells was larger than the S-phase fraction measured by autoradiography after a pulse of tritiated thymidine or by the immunoperoxidase method using BUdR, the ratios were within a similar range (1.2-1.4) in all cell lines studied. Therefore, the S-phase fraction can be estimated indirectly from the percentage of TS-positive cells measured by M-TS-4. Because the emergence of TS detected by our method is cell cycle dependent, M-TS-4 may be useful for biochemical studies of TS and for cytokinetic analysis.

The endocrine dyscrasia that accompanies menopause and andropause induces aberrant cell cycle signaling that triggers re-entry of post-mitotic neurons into the cell cycle, neurodysfunction, neurodegeneration and cognitive disease.

PubMed

Atwood, Craig S; Bowen, Richard L

2015-11-01

This article is part of a Special Issue "SBN 2014". Sex hormones are physiological factors that promote neurogenesis during embryonic and fetal development. During childhood and adulthood these hormones support the maintenance of brain structure and function via neurogenesis and the formation of dendritic spines, axons and synapses required for the capture, processing and retrieval of information (memories). Not surprisingly, changes in these reproductive hormones that occur with menopause and during andropause are strongly correlated with neurodegeneration and cognitive decline. In this connection, much evidence now indicates that Alzheimer's disease (AD) involves aberrant re-entry of post-mitotic neurons into the cell cycle. Cell cycle abnormalities appear very early in the disease, prior to the appearance of plaques and tangles, and explain the biochemical, neuropathological and cognitive changes observed with disease progression. Intriguingly, a recent animal study has demonstrated that induction of adult neurogenesis results in the loss of previously encoded memories while decreasing neurogenesis after memory formation during infancy mitigated forgetting. Here we review the biochemical, epidemiological and clinical evidence that alterations in sex hormone signaling associated with menopause and andropause drive the aberrant re-entry of post-mitotic neurons into an abortive cell cycle that leads to neurite retraction, neuron dysfunction and neuron death. When the reproductive axis is in balance, gonadotropins such as luteinizing hormone (LH), and its fetal homolog, human chorionic gonadotropin (hCG), promote pluripotent human and totipotent murine embryonic stem cell and neuron proliferation. However, strong evidence supports menopausal/andropausal elevations in the LH:sex steroid ratio as driving aberrant mitotic events. These include the upregulation of tumor necrosis factor; amyloid-β precursor protein processing towards the production of mitogenic Aβ; and the activation of Cdk5, a key regulator of cell cycle progression and tau phosphorylation (a cardinal feature of both neurogenesis and neurodegeneration). Cognitive and biochemical studies confirm the negative consequences of a high LH:sex steroid ratio on dendritic spine density and human cognitive performance. Prospective epidemiological and clinical evidence in humans supports the premise that rebalancing the ratio of circulating gonadotropins:sex steroids reduces the incidence of AD. Together, these data support endocrine dyscrasia and the subsequent loss of cell cycle control as an important etiological event in the development of neurodegenerative diseases including AD, stroke and Parkinson's disease. Published by Elsevier Inc.

Small G Rac1 is involved in replication cycle of dengue serotype 2 virus in EAhy926 cells via the regulation of actin cytoskeleton.

PubMed

Zhang, Jing; Wu, Na; Gao, Na; Yan, Wenli; Sheng, Ziyang; Fan, Dongying; An, Jing

2016-05-01

Bleeding is a clinical characteristic of severe dengue and may be due to increased vascular permeability. However, the pathogenesis of severe dengue remains unclear. In this study, we showed that the Rac1-microfilament signal pathway was involved in the process of DENV serotype 2 (DENV2) infection in EAhy926 cells. DENV2 infection induced dynamic changes in actin organization, and treatment with Cytochalasin D or Jasplakinolide disrupted microfilament dynamics, reduced DENV2 entry, and inhibited DENV2 assembly and maturation. Rac1 activities decreased during the early phase and gradually increased by the late phase of infection. Expression of the dominant-negative form of Rac1 promoted DENV2 entry but inhibited viral assembly, maturation and release. Our findings demonstrated that Rac1 plays an important role in the DENV2 life cycle by regulating actin reorganization in EAhy926 cells. This finding provides further insight into the pathogenesis of severe dengue.

Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent.

PubMed

Zhang, Duo; Chen, Hui-Peng; Duan, Hai-Feng; Gao, Li-Hua; Shao, Yong; Chen, Ke-Yan; Wang, You-Liang; Lan, Feng-Hua; Hu, Xian-Wen

2016-07-01

Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Gonadotropin binding sites in human ovarian follicles and corpora lutea during the menstrual cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shima, K.; Kitayama, S.; Nakano, R.

Gonadotropin binding sites were localized by autoradiography after incubation of human ovarian sections with /sup 125/I-labeled gonadotropins. The binding sites for /sup 125/I-labeled human follicle-stimulating hormone (/sup 125/I-hFSH) were identified in the granulosa cells and in the newly formed corpora lutea. The /sup 125/I-labeled human luteinizing hormone (/sup 125/I-hLH) binding to the thecal cells increased during follicular maturation, and a dramatic increase was preferentially observed in the granulosa cells of the large preovulatory follicle. In the corpora lutea, the binding of /sup 125/I-hLH increased from the early luteal phase and decreased toward the late luteal phase. The changes in 3more » beta-hydroxysteroid dehydrogenase activity in the corpora lutea corresponded to the /sup 125/I-hLH binding. Thus, the changes in gonadotropin binding sites in the follicles and corpora lutea during the menstrual cycle may help in some important way to regulate human ovarian function.« less

Deep cytoplasmic rearrangements in ventralized Xenopus embryos

NASA Technical Reports Server (NTRS)

Brown, E. E.; Denegre, J. M.; Danilchik, M. V.

1993-01-01

Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos, which have not undergone vegetal rotation, most of this labeled material remains more equatorial.

Prolonged early G1 arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle–coupled loss of IRF4

PubMed Central

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L.; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C.; Staudt, Louis M.; Niesvizky, Ruben; Moore, Malcolm A. S.

2012-01-01

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G1 block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy. PMID:22718837

Butyrate and deoxycholic acid play common and distinct roles in HCT116 human colon cell proliferation.

PubMed

Zeng, Huawei; Claycombe, Kate J; Reindl, Katie M

2015-10-01

Consumption of a high-fat diet causes an increase in bile acid deoxycholic acid (DCA) in colon lumen and colon cancer risk, while butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit colon cancer-preventive effects. To distinguish these opposing effects of DCA and butyrate (two major metabolites in colon lumen), we examined the effects of physiologically relevant doses of butyrate (0.5-2 mmol/l) and DCA (0.05-0.3 mmol/l) on colon cell proliferation. We hypothesize that butyrate and DCA each modulates the cell cycle and apoptosis via common and distinct cellular signaling targets. In this study, we demonstrated that both butyrate and DCA inhibited cell proliferation by up to 89% and 92% and increased cell apoptosis rate by up to 3.1- and 4.5-fold, respectively. Cell cycle analyses revealed that butyrate led to an increase in G1 and G2 fractions with a concomitant drop in the S-phase fraction, but DCA induced an increase in only G1 fraction with a concomitant drop in the S-phase fraction when compared with the untreated cells. The examination of early cellular signaling revealed that DCA but not butyrate increased intracellular reactive oxygen species, genomic DNA breakage, the activation of ERK1/2, caspase-3 and PARP. In contrast, DCA decreased activated Rb protein level, and butyrate but not DCA increased p21 expression. Collectively, although both butyrate and DCA inhibit colonic cell proliferation, butyrate increases tumor suppressor gene expression, whereas DCA decreases tumor suppressor activation in cell cycle and apoptosis pathways. Published by Elsevier Inc.

BAP1 and Cancer

PubMed Central

Carbone, Michele; Yang, Haining; Pass, Harvey I.; Krausz, Thomas; Testa, Joseph R.; Gaudino, Giovanni

2013-01-01

Preface BAP1 is a deubiquitylase that is found associated with multi-protein complexes that regulate key cellular pathways, including the cell cycle, cellular differentiation, cell death, gluconeogenesis and the DNA damage response (DDR). Recent findings indicate that germline BAP1 mutations cause a novel cancer syndrome, characterized, at least in the affected families studied so far, by the onset at an early age of benign melanocytic skin tumours with mutated BAP1, and later in life by a high incidence of mesothelioma, uveal melanoma, cutaneous melanoma and possibly additional cancers. PMID:23550303

c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines.

PubMed Central

Harrington, E A; Bennett, M R; Fanidi, A; Evan, G I

1994-01-01

We have investigated the mechanism by which deregulated expression of c-Myc induces death by apoptosis in serum-deprived fibroblasts. We demonstrate that Myc-induced apoptosis in low serum is inhibited by a restricted group of cytokines, principally the insulin-like growth factors and PDGF. Cytokine-mediated protection from apoptosis is not linked to the cytokines' abilities to promote growth. Protection from apoptosis is evident in the post-commitment (mitogen-independent) S/G2/M phases of the cell cycle and also in cells that are profoundly blocked in cell cycle progression by drugs. Moreover, IGF-I inhibition of apoptosis occurs in the absence of protein synthesis, and so does not require immediate early gene expression. We conclude that c-Myc-induced apoptosis does not result from a conflict of growth signals but appears to be a normal physiological aspect of c-Myc function whose execution is regulated by the availability of survival factors. We discuss the possible implications of these findings for models of mammalian cell growth in vivo. Images PMID:8045259

Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon.

PubMed

Filone, Claire Marie; Hanna, Sheri L; Caino, M Cecilia; Bambina, Shelly; Doms, Robert W; Cherry, Sara

2010-11-24

As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.

Arrest of cell cycle by inhibition of ribonucleotide reductase induces accumulation of NAD+ by Mn2+-supplemented growth of Corynebacterium ammoniagenes.

PubMed

Abbouni, Bouziane; Elhariry, Hesham M; Auling, Georg

2003-01-01

Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 microM Mn2+ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn2+-supplemented fermentation medium at an appropriate time. Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria. The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD+ by a salvage biosynthetic pathway. An assay of nucleotide-permeable cells for ribonucleotide reductase activity using [3H-CDP] as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control. Overproduction of NAD+ is an alternative to the conventional fermentation process using Mn2+ deficiency. A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain.

Mitotic UV Irradiation Induces a DNA Replication-Licensing Defect that Potentiates G1 Arrest Response

PubMed Central

Morino, Masayuki; Nukina, Kohei; Sakaguchi, Hiroki; Maeda, Takeshi; Takahara, Michiyo; Shiomi, Yasushi; Nishitani, Hideo

2015-01-01

Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis. PMID:25798850

The Role of Cellular Proliferation in Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

PubMed

Marquez, Maribel P; Alencastro, Frances; Madrigal, Alma; Jimenez, Jossue Loya; Blanco, Giselle; Gureghian, Alex; Keagy, Laura; Lee, Cecilia; Liu, Robert; Tan, Lun; Deignan, Kristen; Armstrong, Brian; Zhao, Yuanxiang

2017-11-01

Mitotic clonal expansion has been suggested as a prerequisite for adipogenesis in murine preadipocytes, but the precise role of cell proliferation during human adipogenesis is unclear. Using adipose tissue-derived human mesenchymal stem cells as an in vitro cell model for adipogenic study, a group of cell cycle regulators, including Cdk1 and CCND1, were found to be downregulated as early as 24 h after adipogenic initiation and consistently, cell proliferation activity was restricted to the first 48 h of adipogenic induction. Cell proliferation was either further inhibited using siRNAs targeting cell cycle genes or enhanced by supplementing exogenous growth factor, basic fibroblast growth factor (bFGF), at specific time intervals during adipogenesis. Expression knockdown of Cdk1 at the initiation of adipogenic induction resulted in significantly increased adipocytes, even though total number of cells was significantly reduced compared to siControl-treated cells. bFGF stimulated proliferation throughout adipogenic differentiation, but exerted differential effect on adipogenic outcome at different phases, promoting adipogenesis during mitotic phase (first 48 h), but significantly inhibiting adipogenesis during adipogenic commitment phase (days 3-6). Our results demonstrate that cellular proliferation is counteractive to adipogenic commitment in human adipogenesis. However, cellular proliferation stimulation can be beneficial for adipogenesis during the mitotic phase by increasing the population of cells capable of committing to adipocytes before adipogenic commitment.

Cellular injury evidenced by impedance technology and infrared microspectroscopy

NASA Astrophysics Data System (ADS)

le Roux, K.; Prinsloo, L. C.; Meyer, D.

2015-03-01

Fourier Transform Infrared (FTIR) spectroscopy is finding increasing biological application, for example in the analysis of diseased tissues and cells, cell cycle studies and investigating the mechanisms of action of anticancer drugs. Cancer treatment studies routinely define the types of cell-drug responses as either total cell destruction by the drug (all cells die), moderate damage (cell deterioration where some cells survive) or reversible cell cycle arrest (cytostasis). In this study the loss of viability and related chemical stress experienced by cells treated with the medicinal plant, Plectranthus ciliatus, was investigated using real time cell electronic sensing (RT-CES) technology and FTIR microspectroscopy. The use of plants as medicines is well established and ethnobotany has proven that crude extracts can serve as treatments against various ailments. The aim of this study was to determine whether FTIR microspectroscopy would successfully distinguish between different types of cellular injury induced by a potentially anticancerous plant extract. Cervical adenocarcinoma (HeLa) cells were treated with a crude extract of Pciliatus and cells monitored using RT-CES to characterize the type of cellular responses induced. Cell populations were then investigated using FTIR microspectroscopy and statistically analysed using One-way Analysis of Variance (ANOVA) and Principal Component Analysis (PCA). The plant extract and a cancer drug control (actinomycin D) induced concentration dependent cellular responses ranging from nontoxic, cytostatic or cytotoxic. Thirteen spectral peaks (915 cm-1, 933 cm-1, 989 cm-1, 1192 cm-1, 1369 cm-1, 1437 cm-1, 1450 cm-1, 1546 cm-1, 1634 cm-1, 1679 cm-1 1772 cm-1, 2874 cm-1 and 2962 cm-1) associated with cytotoxicity were significantly (p value < 0.05, one way ANOVA, Tukey test, Bonferroni) altered, while two of the bands were also indicative of early stress related responses. In PCA, poor separation between nontoxic and cytostatic responses was evident while clear separation was linked to cytotoxicity. RT-CES detected morphological changes as indicators of cell injury and could distinguish between viable, cytostatic and cytotoxic responses. FTIR microspectroscopy confirmed that cytostatic cells were viable and could still recover while also describing early cellular stress related responses on a molecular level.

Gigantism in a bacterium, Epulopiscium fishelsoni, correlates with complex patterns in arrangement, quantity, and segregation of DNA.

PubMed

Bresler, V; Montgomery, W L; Fishelson, L; Pollak, P E

1998-11-01

Epulopiscium fishelsoni, gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus) in the Red Sea, attains a larger size than any other eubacterium, varies 10- to 20-fold in length (and >2, 000-fold in volume), and undergoes a complex daily life cycle. In early morning, nucleoids contain highly condensed DNA in elongate, chromosome-like structures which are physically separated from the general cytoplasm. Cell division involves production of two (rarely three) nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell. Fluorescence measurements of DNA, RNA, and other cell components indicate the following. DNA quantity is proportional to cell volume over cell lengths of approximately 30 micrometers to >500 micrometers. For cells of a given size, nucleoids of cells with two nucleoids (binucleoid) contain approximately equal amounts of DNA. And each nucleoid of a binucleoid cell contains one-half the DNA of the single nucleoid in a uninucleoid cell of the same size. The life cycle involves approximately equal subdivision of DNA among daughter cells, formation of apical caps of condensed DNA from previously decondensed and diffusely distributed DNA, and "pinching" of DNA near the middle of the cell in the absence of new wall formation. Mechanisms underlying these patterns remain unclear, but formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and bacterial cell growth and during nocturnal periods of host inactivity when mean bacterial cell size declines.

An Epstein-Barr virus-associated superantigen

PubMed Central

1996-01-01

More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II- dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta- restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis. PMID:9064357

A systems biology approach to Down syndrome: identification of Notch/Wnt dysregulation in a model of stem cells aging.

PubMed

Cairney, C J; Sanguinetti, G; Ranghini, E; Chantry, A D; Nostro, M C; Bhattacharyya, A; Svendsen, C N; Keith, W N; Bellantuono, I

2009-04-01

Stem cells are central to the development and maintenance of many tissues. This is due to their capacity for extensive proliferation and differentiation into effector cells. More recently it has been shown that the proliferative and differentiative ability of stem cells decreases with age, suggesting that this may play a role in tissue aging. Down syndrome (DS), is associated with many of the signs of premature tissue aging including T-cell deficiency, increased incidence of early Alzheimer-type, Myelodysplastic-type disease and leukaemia. Previously we have shown that both hematopoietic (HSC) and neural stem cells (NSC) in patients affected by DS showed signs of accelerated aging. In this study we tested the hypothesis that changes in gene expression in HSC and NSC of patients affected by DS reflect changes occurring in stem cells with age. The profiles of genes expressed in HSC and NSC from DS patients highlight pathways associated with cellular aging including a downregulation of DNA repair genes and increases in proapoptotic genes, s-phase cell cycle genes, inflammation and angiogenesis genes. Interestingly, Notch signaling was identified as a potential hub, which when deregulated may drive stem cell aging. These data suggests that DS is a valuable model to study early events in stem cell aging.

β1-integrin controls cell fate specification in early lens development

PubMed Central

Pathania, Mallika; Wang, Yan; Simirskii, Vladimir N.; Duncan, Melinda K.

2016-01-01

Integrins are heterodimeric cell surface molecules that mediate cell-extracellular matrix (ECM) adhesion, ECM assembly, and regulation of both ECM and growth factor induced signaling. However, the developmental context of these diverse functions is not clear. Loss of β1-integrin from the lens vesicle (mouse E10.5) results in abnormal exit of anterior lens epithelial cells (LECs) from the cell cycle and their aberrant elongation toward the presumptive cornea by E12.5. These cells lose expression of LEC markers and initiate expression of the Maf (also known as c-Maf) and Prox1 transcription factors as well as other lens fiber cell markers, β1-integrin null LECs also upregulate the ERK, AKT and Smad1/5/8 phosphorylation indicative of BMP and FGF signaling. By E14.5, β1-integrin null lenses have undergone a complete conversion of all lens epithelial cells into fiber cells. These data suggest that shortly after lens vesicle closure, β1-integrin blocks inappropriate differentiation of the lens epithelium into fibers, potentially by inhibiting BMP and/or FGF receptor activation. Thus, β1-integrin has an important role in fine-tuning the response of the early lens to the gradient of growth factors that regulate lens fiber cell differentiation. PMID:27596755

The onset of homologous chromosome pairing during Drosophila melanogaster embryogenesis.

PubMed

Hiraoka, Y; Dernburg, A F; Parmelee, S J; Rykowski, M C; Agard, D A; Sedat, J W

1993-02-01

We have determined the position within the nucleus of homologous sites of the histone gene cluster in Drosophila melanogaster using in situ hybridization and high-resolution, three-dimensional wide field fluorescence microscopy. A 4.8-kb biotinylated probe for the histone gene repeat, located approximately midway along the short arm of chromosome 2, was hybridized to whole-mount embryos in late syncytial and early cellular blastoderm stages. Our results show that the two homologous histone loci are distinct and separate through all stages of the cell cycle up to nuclear cycle 13. By dramatic contrast, the two homologous clusters were found to colocalize with high frequency during interphase of cycle 14. Concomitant with homolog pairing at cycle 14, both histone loci were also found to move from their position near the midline of the nucleus toward the apical side. This result suggests that coincident with the initiation of zygotic transcription, there is dramatic chromosome and nuclear reorganization between nuclear cycles 13 and 14.

Nodular lymphocyte-predominant Hodgkin lymphoma: a unique disease deserving unique management.

PubMed

Eichenauer, Dennis A; Engert, Andreas

2017-12-08

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma entity with an incidence of 0.1 to 0.2/100 000/y. Compared with the more common subtypes of classical Hodgkin lymphoma, NLPHL is characterized by distinct pathological and clinical features. Histologically, the disease-defining lymphocyte predominant cells consistently express CD20 but lack CD30. Clinically, NLPHL mostly has a rather indolent course, and patients usually are diagnosed in early stages. The prognosis of early-stage NLPHL is excellent, with progression-free survival and overall survival rates exceeding 90% after involved-field radiotherapy (IF-RT) alone (stage IA) or combined modality treatment consisting of a brief chemotherapy with 2 cycles of ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) chemotherapy followed by IF-RT (early stages other than stage IA). In contrast, patients with advanced disease at diagnosis tend to relapse either with NLPHL histology or with histological transformation into aggressive B-cell non-Hodgkin lymphoma despite more aggressive first-line treatment with 6 to 8 cycles of multiagent chemotherapy. However, even NLPHL patients with multiple relapses successfully respond to salvage therapy in many cases. Salvage therapies range from single-agent anti-CD20 antibody treatment to high-dose chemotherapy followed by autologous stem cell transplantation. Treatment at disease recurrence should be chosen on the basis of various factors, including histology at relapse, time to relapse, extent of disease at relapse, and prior treatment. Because death among NLPHL patients is more often caused by therapy-related late effects than lymphoma-related complications, optimizing the risk-benefit ratio of treatment by decreasing toxicity whenever possible is the major goal of clinical research in this disease. © 2016 by The American Society of Hematology. All rights reserved.

MYC and gastric adenocarcinoma carcinogenesis

PubMed Central

Calcagno, Danielle Queiroz; Leal, Mariana Ferreira; Assumpção, Paulo Pimentel; Smith, Marília de Arruda Cardoso; Burbano, Rommel Rodríguez

2008-01-01

MYC is an oncogene involved in cell cycle regulation, cell growth arrest, cell adhesion, metabolism, ribosome biogenesis, protein synthesis, and mitochondrial function. It has been described as a key element of several carcinogenesis processes in humans. Many studies have shown an association between MYC deregulation and gastric cancer. MYC deregulation is also seen in gastric preneoplastic lesions and thus it may have a role in early gastric carcinogenesis. Several studies have suggested that amplification is the main mechanism of MYC deregulation in gastric cancer. In the present review, we focus on the deregulation of the MYC oncogene in gastric adenocarcinoma carcinogenesis, including its association with Helicobacter pylori (H pylori) and clinical applications. PMID:18932273

Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by Inhibiting BAD-Mediated caspase-3-Dependent Apoptosis

PubMed Central

Kim, Hyoji; Choi, Hoyun

2015-01-01

ABSTRACT Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and the in vivo triggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genes BRLF1 and BZLF1 as well as Bcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels of BRLF1 and BZLF1 were suppressed in cells following BAD knockdown and increased after BAD overexpression. Progeny virus production was also downregulated by specific knockdown of BAD. Our results demonstrated that caspase-3-dependent apoptosis is a prerequisite for BAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting BAD-mediated caspase-3-dependent apoptosis, which would trigger immediate early gene expression. IMPORTANCE EBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressing BAD-induced caspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis. PMID:26581978

Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy.

PubMed

Wu, Feng-Hua; Mu, Lei; Li, Xiao-Lan; Hu, Yi-Bing; Liu, Hui; Han, Lin-Tao; Gong, Jian-Ping

2017-10-03

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo . Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo . Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro . Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.

Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy

PubMed Central

Wu, Feng-Hua; Mu, Lei; Li, Xiao-Lan; Hu, Yi-Bing; Liu, Hui; Han, Lin-Tao; Gong, Jian-Ping

2017-01-01

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo. Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo. Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro. Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence. PMID:29108242

ATM-Mediated Transcriptional and Developmental Responses to γ-rays in Arabidopsis

PubMed Central

Renou, Jean-Pierre; Pichon, Olivier; Fochesato, Sylvain; Ortet, Philippe; Montané, Marie-Hélène

2007-01-01

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of γ-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed. PMID:17487278

ATM-mediated transcriptional and developmental responses to gamma-rays in Arabidopsis.

PubMed

Ricaud, Lilian; Proux, Caroline; Renou, Jean-Pierre; Pichon, Olivier; Fochesato, Sylvain; Ortet, Philippe; Montané, Marie-Hélène

2007-05-09

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of gamma-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed.

Fetal bovine serum simultaneously stimulates apoptosis and DNA synthesis in premeiotic stages of spermatogenesis in spiny dogfish (Squalus acanthias) in vitro: modulation by androgen and spermatogenic activity status.

PubMed

McClusky, Leon Mendel

2008-05-01

Using the simple cystic spermatogenesis in the shark testis as a model, we previously reported the relative resistance of immature spermatogonia (stem cell and early-stage spermatogonia) to apoptosis in the normal testis and after spermatoxicant exposure in vivo. Apoptosis was monitored by fluorescence image analysis of living cysts, using the validated acridine orange (AO) vital staining technique. Findings show that FBS simultaneously stimulates both apoptosis and [(3)H]thymidine incorporation in immature spermatogonial clones in a concentration-dependent manner in vitro. Furthermore, androgen inhibits apoptosis and increases cyst viability, more so with 10% FBS than with 1% FBS. All the effects were as a function of spermatogenic activity status but were distinct in early-stage spermatogonial cysts isolated from testes awakening from the previous winter spermatogenic arrest period. Results are discussed in the context of the alternating germ-Sertoli cell population kinetics of early-stage spermatogonial cysts in Squalus acanthias's protracted testicular cycle.

The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes.

PubMed

Divakaruni, Arun V; Baida, Cyril; White, Courtney L; Gober, James W

2007-10-01

MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.

X box binding protein XBP-1s transactivates the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF50 promoter, linking plasma cell differentiation to KSHV reactivation from latency.

PubMed

Wilson, Sam J; Tsao, Edward H; Webb, Benjamin L J; Ye, Hongtao; Dalton-Griffin, Lucy; Tsantoulas, Christoforos; Gale, Catherine V; Du, Ming-Qing; Whitehouse, Adrian; Kellam, Paul

2007-12-01

Reactivation of lytic replication from viral latency is a defining property of all herpesviruses. Despite this, the authentic physiological cues for the latent-lytic switch are unclear. Such cues should ensure that viral lytic replication occurs under physiological conditions, predominantly in sites which facilitate transmission to permissive uninfected cells and new susceptible hosts. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with the B-cell neoplasm primary effusion lymphoma (PEL), in which the virus remains latent. We have previously shown that PEL cells have the gene expression profile and immunophenotype of cycling preplasma cells (plasmablasts). Here, we show that the highly active spliced isoform of plasma cell transcription factor X box binding protein 1 (XBP-1s) is a lytic switch for KSHV. XBP-1s is normally absent in PEL, but the induction of endoplasmic reticulum stress leads to XBP-1s generation, plasma cell-like differentiation, and lytic reactivation of KSHV. XBP-1s binds to and activates the KSHV immediate-early gene ORF50 and synergizes with the ORF50 gene product RTA to induce a full lytic cycle. These data suggest that KSHV remains latent until B-cell terminal differentiation into plasma cells, the transcriptional environment of which provides the physiological "lytic switch" through XBP-1s. This links B-cell terminal differentiation to KSHV lytic reactivation.

Cytometry of DNA Replication and RNA Synthesis: Historical Perspective and Recent Advances Based on “Click Chemistry”

PubMed Central

Darzynkiewicz, Zbigniew; Traganos, Frank; Zhao, Hong; Halicka, H. Dorota; Li, Jiangwei

2011-01-01

This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of 3H- or 14C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G1, S, G2, and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of 3H-uridine incorporation and RNA content provided the means to distinguish quiescent G0 from cycling G1 cells. Subsequent progress in analysis of DNA replication was based on the use of BrdU as a DNA precursor and its detection by the quenching of the fluorescence intensity of DNA-bound fluorochromes such as Hoechst 33358 or acridine orange as measured by flow cytometry. Several variants of this methodology have been designed and used in studies to detect anticancer drug-induced perturbations of cell cycle kinetics. The next phase of method development, which was particularly useful in studies of the cell cycle in vivo, including clinical applications, relied on immunocytochemical detection of incorporated halogenated DNA or RNA precursors. This approach however was hampered by the need for DNA denaturation, which made it difficult to concurrently detect other cell constituents for multiparametric analysis. The recently introduced “click chemistry” approach has no such limitation and is the method of choice for analysis of DNA replication and RNA synthesis. This method is based on the use of 5-ethynyl-2′deoxyuridine (EdU) as a DNA precursor or 5-ethynyluridine (EU) as an RNA precursor and their detection with fluorochrome-tagged azides utilizing a copper (I) catalyzed [3+2] cycloaddition. Several examples are presented that illustrate incorporation of EdU or EU in cells subjected to DNA damage detected as histone H2AX phosphorylation that have been analyzed by flow or laser scanning cytometry. PMID:21425239

The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells

PubMed Central

Badia, Roger; Pujantell, Maria; Riveira-Muñoz, Eva; Puig, Teresa; Torres-Torronteras, Javier; Martí, Ramón; Clotet, Bonaventura; Ampudia, Rosa M.; Ballana, Ester

2016-01-01

Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages. PMID:27541004

Alpha Interferon Restricts Human T-Lymphotropic Virus Type 1 and 2 De Novo Infection through PKR Activation

PubMed Central

Cachat, Anne; Chevalier, Sébastien Alain; Alais, Sandrine; Ko, Nga Ling; Ratner, Lee; Journo, Chloé; Dutartre, Hélène

2013-01-01

Type I interferon (IFN-I) inhibits the replication of different viruses. However, the effect of IFN-I on the human T-lymphotropic virus type 1 (HTLV-1) viral cycle is controversial. Here, we investigated the consequences of IFN-α addition for different steps of HTLV-1 and HTLV-2 infection. We first show that alpha interferon (IFN-α) efficiently impairs HTLV-1 and HTLV-2 de novo infection in a T cell line and in primary lymphocytes. Using pseudotyped viruses expressing HTLV-1 envelope, we then show that cell-free infection is insensitive to IFN-α, demonstrating that the cytokine does not affect the early stages of the viral cycle. In contrast, intracellular levels of Gag, Env, or Tax protein are affected by IFN-α treatment in T cells, primary lymphocytes, or 293T cells transfected with HTLV-1 or HTLV-2 molecular clones, demonstrating that IFN-α acts during the late stages of infection. We show that IFN-α does not affect Tax-mediated transcription and acts at a posttranscriptional level. Using either small interfering RNA (siRNA) directed against PKR or a PKR inhibitor, we demonstrate that PKR, whose expression is induced by interferon, plays a major role in IFN-α-induced HTLV-1/2 inhibition. These results indicate that IFN-α has a strong repressive effect on the HTLV-1 and HTLV-2 viral cycle during de novo infection of cells that are natural targets of the viruses. PMID:24089560

Phase 1/2 study of cyclin-dependent kinase (CDK)4/6 inhibitor palbociclib (PD-0332991) with bortezomib and dexamethasone in relapsed/refractory multiple myeloma.

PubMed

Niesvizky, Ruben; Badros, Ashraf Z; Costa, Luciano J; Ely, Scott A; Singhal, Seema B; Stadtmauer, Edward A; Haideri, Nisreen A; Yacoub, Abdulraheem; Hess, Georg; Lentzsch, Suzanne; Spicka, Ivan; Chanan-Khan, Asher A; Raab, Marc S; Tarantolo, Stefano; Vij, Ravi; Zonder, Jeffrey A; Huang, Xiangao; Jayabalan, David; Di Liberto, Maurizio; Huang, Xin; Jiang, Yuqiu; Kim, Sindy T; Randolph, Sophia; Chen-Kiang, Selina

2015-01-01

This phase 1/2 study was the first to evaluate the safety and efficacy of the cyclin-dependent kinase (CDK) 4/6-specific inhibitor palbociclib (PD-0332991) in sequential combination with bortezomib and dexamethasone in relapsed/refractory multiple myeloma. The recommended phase 2 dose was palbociclib 100 mg orally once daily on days 1-12 of a 21-day cycle with bortezomib 1.0 mg/m2 (intravenous) and dexamethasone 20 mg (orally 30 min pre-bortezomib dosing) on days 8 and 11 (early G1 arrest) and days 15 and 18 (cell cycle resumed). Dose-limiting toxicities were primarily cytopenias; most other treatment-related adverse events were grade≤3. At a bortezomib dose lower than that in other combination therapy studies, antitumor activity was observed (phase 1). In phase 2, objective responses were achieved in 5 (20%) patients; 11 (44%) achieved stable disease. Biomarker and pharmacodynamic assessments demonstrated that palbociclib inhibited CDK4/6 and the cell cycle initially in most patients.

Stage and season effects on cell cycle and apoptotic activities of germ cells and Sertoli cells during spermatogenesis in the spiny dogfish (Squalus acanthias).

PubMed

McClusky, L M

2005-01-01

To understand the processes involved in the spatial and temporal maturation of testicular cells in Squalus acanthias, we used standard morphometry, proliferating-cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) immunohistochemistry. Except for immature spermatocysts (germinal zone, GZ; early-stage pre-meiotic, E-PrM), the number of cysts in all subsequent stages and the total number of cysts in the spermatogenic progression varied seasonally. The spermatogenic cycle spans about 2 years and is interrupted by germcell clone deletion via apoptosis at the mitosis-meiosis transition in April/May, manifesting as a zone of degeneration (ZD). Rate of displacement of the ZD across the testis diameter indicates that late-stage premeiotic (L-PrM) generations 12-13 require 9-10 months to reach the mature-spermatid stage. Also, the number of cysts completing spermatogenesis is approximately 4-5-fold less than the number that entered spermatogenesis proper 2 years earlier. Pronounced gonocytogenesis in the germinal ridge was coincident with ZD formation in April/May, but it was absent in the fall when mature spermatogonial and meiotic activities had resumed. Whereas strong Sertoli cell PCNA immunoreactivity dominated the GZ cyst cell-cycle activities throughout the year, except during the spring/summer months, the spermatogonial- and Sertoli-cell PCNA indices in E-PrM cysts were inversely related. PCNA immunoreactivity in spermatocytes was seasonal and dependent on the stage of meiosis. TUNEL labelling was limited to spermatogonia and increased stage-dependently in the PrM region (L-PrM = mid-stage PrM >E-PrM >GZ), correlating with ZD formation, in a season-dependent manner. Results imply that effects of normal regulatory factors in Squalus are stage- and process-specific.

Constant light during lactation programs circadian and metabolic systems.

PubMed

Madahi, Palma-Gómez; Ivan, Osnaya; Adriana, Balderas; Diana, Ortega; Carolina, Escobar

2018-04-24

Exposure to light at night is a disruptive condition for the adult circadian system, leading to arrhythmicity in nocturnal rodents. Circadian disruption is a risk factor for developing physiological and behavioral alterations, including weight gain and metabolic disease. During early stages of development, the circadian system undergoes a critical period of adjustment, and it is especially vulnerable to altered lighting conditions that may program its function, leading to long-term effects. We hypothesized that during lactation a disrupted light-dark cycle due to light at night may disrupt the circadian system and in the long term induce metabolic disorders. Here we explored in pups, short- and long-term effects of constant light (LL) during lactation. In the short term, LL caused a loss of rhythmicity and a reduction in the immunopositive cells of VIP, AVP, and PER1 in the suprachiasmatic nucleus (SCN). In the short term, the affection on the circadian clock in the pups resulted in body weight gain, loss of daily rhythms in general activity, plasma glucose and triglycerides (TG). Importantly, the DD conditions during development also induced altered daily rhythms in general activity and in the SCN. Exposure to LD conditions after lactation did not restore rhythmicity in the SCN, and the number of immunopositve cells to VIP, AVP, and PER1 remained reduced. In the long term, daily rhythmicity in general activity was restored; however, daily rhythms in glucose and TG remained disrupted, and daily mean levels of TG were significantly increased. Present results point out the programming role played by the LD cycle during early development in the function of the circadian system and on metabolism. This study points out the risk represented by exposure to an altered light-dark cycle during early stages of development. AVP: arginine vasopressin peptide; CRY: cryptochrome; DD: constant darkness; DM: dorsomedial; LD: light-dark cycle; LL: constant light; NICUs: neonatal intensive care units; P: postnatal days; PER: period; S.E.M.: standard error of the mean; SCN: suprachiasmatic nucleus; TG: triglycerides; VIP: vasointestinal peptide; VL: ventrolateral; ZT: zeitgeber time.

The apical complex couples cell fate and cell survival to cerebral cortical development

PubMed Central

Kim, Seonhee; Lehtinen, Maria K.; Sessa, Alessandro; Zappaterra, Mauro; Cho, Seo-Hee; Gonzalez, Dilenny; Boggan, Brigid; Austin, Christina A.; Wijnholds, Jan; Gambello, Michael J.; Malicki, Jarema; LaMantia, Anthony S.; Broccoli, Vania; Walsh, Christopher A.

2010-01-01

Cortical development depends upon tightly controlled cell fate and cell survival decisions that generate a functional neuronal population, but the coordination of these two processes is poorly understood. Here we show that conditional removal of a key apical complex protein, Pals1, causes premature withdrawal from the cell cycle, inducing excessive generation of early-born postmitotic neurons followed by surprisingly massive and rapid cell death, leading to the abrogation of virtually the entire cortical structure. Pals1 loss shows exquisite dosage sensitivity, so that heterozygote mutants show an intermediate phenotype on cell fate and cell death. Loss of Pals1 blocks essential cell survival signals, including the mammalian target of rapamycin (mTOR) pathway, while mTORC1 activation partially rescues Pals1 deficiency. These data highlight unexpected roles of the apical complex protein Pals1 in cell survival through interactions with mTOR signaling. PMID:20399730

Association of pKi-67 with satellite DNA of the human genome in early G1 cells.

PubMed

Bridger, J M; Kill, I R; Lichter, P

1998-01-01

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matthews, Q; Lum, JJ; Isabelle, M

Purpose: To use label-free Raman spectroscopy (RS) for early treatment monitoring of tumour cell radioresistance. Methods: Three human tumour cell lines, two radioresistant (H460, SF{sub 2} = 0.57 and MCF7, SF{sub 2} = 0.70) and one radiosensitive (LNCaP, SF{sub 2} = 0.36), were irradiated with single fractions of 2, 4, 6, 8 or 10 Gy. In additional experiments, H460 and MCF7 cells were irradiated under co-treatment with the anti-diabetic drug metformin, a known radiosensitizing agent. Treated and control cultures were analyzed with RS daily for 3 days post-treatment. Single-cell Raman spectra were acquired from 20 live cells per sample, andmore » experiments were repeated in triplicate. The combined data sets were analyzed with principal component analysis using standard algorithms. Cells from each culture were also subjected to standard assays for viability, proliferation, cell cycle, and radiation clonogenic survival. Results: The radioresistant cells (H460, MCF7) exhibited a RS molecular radiation response signature, detectable as early as 1 day post-treatment, of which radiation-induced glycogen synthesis is a significant contributor. The radiosensitive cells (LNCaP) exhibited negligible glycogen synthesis. Co-treatment with metformin in MCF7 cells blocked glycogen synthesis, reduced viability and proliferation, and increased radiosensitivity. Conversely, metformin co-treatment in H460 cells did not produce these same effects; importantly, both radiation-induced synthesis of glycogen and radiosensitivity were unaffected. Conclusions: Label-free RS can detect early glycogen synthesis post-irradiation, a previously undocumented metabolic mechanism associated with tumour cell radioresistance that can be targeted to increase radiosensitivity. RS monitoring of intratumoral glycogen may provide new opportunities for personalized combined modality radiotherapy treatments.« less

Endogenous circadian system and circadian misalignment impact glucose tolerance via separate mechanisms in humans

PubMed Central

Morris, Christopher J.; Yang, Jessica N.; Garcia, Joanna I.; Myers, Samantha; Bozzi, Isadora; Wang, Wei; Buxton, Orfeu M.; Shea, Steven A.; Scheer, Frank A. J. L.

2015-01-01

Glucose tolerance is lower in the evening and at night than in the morning. However, the relative contribution of the circadian system vs. the behavioral cycle (including the sleep/wake and fasting/feeding cycles) is unclear. Furthermore, although shift work is a diabetes risk factor, the separate impact on glucose tolerance of the behavioral cycle, circadian phase, and circadian disruption (i.e., misalignment between the central circadian pacemaker and the behavioral cycle) has not been systematically studied. Here we show—by using two 8-d laboratory protocols—in healthy adults that the circadian system and circadian misalignment have distinct influences on glucose tolerance, both separate from the behavioral cycle. First, postprandial glucose was 17% higher (i.e., lower glucose tolerance) in the biological evening (8:00 PM) than morning (8:00 AM; i.e., a circadian phase effect), independent of the behavioral cycle effect. Second, circadian misalignment itself (12-h behavioral cycle inversion) increased postprandial glucose by 6%. Third, these variations in glucose tolerance appeared to be explained, at least in part, by different mechanisms: during the biological evening by decreased pancreatic β-cell function (27% lower early-phase insulin) and during circadian misalignment presumably by decreased insulin sensitivity (elevated postprandial glucose despite 14% higher late-phase insulin) without change in early-phase insulin. We explored possible contributing factors, including changes in polysomnographic sleep and 24-h hormonal profiles. We demonstrate that the circadian system importantly contributes to the reduced glucose tolerance observed in the evening compared with the morning. Separately, circadian misalignment reduces glucose tolerance, providing a mechanism to help explain the increased diabetes risk in shift workers. PMID:25870289

P276-00, a cyclin-dependent kinase inhibitor, modulates cell cycle and induces apoptosis in vitro and in vivo in mantle cell lymphoma cell lines

PubMed Central

2012-01-01

Background Mantle cell lymphoma (MCL) is a well-defined aggressive lymphoid neoplasm characterized by proliferation of mature B-lymphocytes that have a remarkable tendency to disseminate. This tumor is considered as one of the most aggressive lymphoid neoplasms with poor responses to conventional chemotherapy and relatively short survival. Since cyclin D1 and cell cycle control appears as a natural target, small-molecule inhibitors of cyclin-dependent kinases (Cdks) and cyclins may play important role in the therapy of this disorder. We explored P276-00, a novel selective potent Cdk4-D1, Cdk1-B and Cdk9-T1 inhibitor discovered by us against MCL and elucidated its potential mechanism of action. Methods The cytotoxic effect of P276-00 in three human MCL cell lines was evaluated in vitro. The effect of P276-00 on the regulation of cell cycle, apoptosis and transcription was assessed, which are implied in the pathogenesis of MCL. Flow cytometry, western blot, immunoflourescence and siRNA studies were performed. The in vivo efficacy and effect on survival of P276-00 was evaluated in a Jeko-1 xenograft model developed in SCID mice. PK/PD analysis of tumors were performed using LC-MS and western blot analysis. Results P276-00 showed a potent cytotoxic effect against MCL cell lines. Mechanistic studies confirmed down regulation of cell cycle regulatory proteins with apoptosis. P276-00 causes time and dose dependent increase in the sub G1 population as early as from 24 h. Reverse transcription PCR studies provide evidence that P276-00 treatment down regulated transcription of antiapoptotic protein Mcl-1 which is a potential pathogenic protein for MCL. Most importantly, in vivo studies have revealed significant efficacy as a single agent with increased survival period compared to vehicle treated. Further, preliminary combination studies of P276-00 with doxorubicin and bortezomib showed in vitro synergism. Conclusion Our studies thus provide evidence and rational that P276-00 alone or in combination is a potential therapeutic molecule to improve patients’ outcome in mantle cell lymphoma. PMID:23075291

Dynamics of venom composition across a complex life cycle

PubMed Central

Macrander, Jason; Fridrich, Arie; Modepalli, Vengamanaidu; Reitzel, Adam M; Sunagar, Kartik

2018-01-01

Little is known about venom in young developmental stages of animals. The appearance of toxins and stinging cells during early embryonic stages in the sea anemone Nematostella vectensis suggests that venom is already expressed in eggs and larvae of this species. Here, we harness transcriptomic, biochemical and transgenic tools to study venom production dynamics in Nematostella. We find that venom composition and arsenal of toxin-producing cells change dramatically between developmental stages of this species. These findings can be explained by the vastly different interspecific interactions of each life stage, as individuals develop from a miniature non-feeding mobile planula to a larger sessile polyp that predates on other animals and interact differently with predators. Indeed, behavioral assays involving prey, predators and Nematostella are consistent with this hypothesis. Further, the results of this work suggest a much wider and dynamic venom landscape than initially appreciated in animals with a complex life cycle. PMID:29424690

Mechanisms involved in the cytotoxic action of Brazilian propolis and caffeic acid against HEp-2 cells and modulation of P-glycoprotein activity.

PubMed

da Silva, Lívia M; Frión-Herrera, Yahima; Bartolomeu, Ariane R; Gorgulho, Carolina Mendonça; Sforcin, José M

2017-11-01

The effects of propolis and phenolic compounds (caffeic acid - Caf; dihydrocinnamic acid - Cin; p-coumaric acid - Cou) in the same quantity found in our propolis sample were investigated on human laryngeal epidermoid carcinoma (HEp-2) cells. Cell viability, apoptosis/necrosis and cell cycle arrest, P53 and CASPASE-3 gene expression, generation of reactive oxygen species (ROS) and the ability of propolis to induce doxorubicin (DOX) efflux using a P-glycoprotein (P-gp) inhibitor (verapamil) were assayed. Propolis exerted a cytotoxic effect on HEp-2 cells, whereas isolated compounds had no effect on cell viability. Higher concentrations were tested and Caf induced late apoptosis or necrosis in HEp-2 cells, while propolis induced apoptosis, both probably due to ROS generation. P53 expression was downregulated by propolis but not by Caf. CASPASE-3 expression was correlated with induction of both early and late apoptosis, with both propolis and Caf alone upregulating its expression. Propolis induced cell cycle arrest at G2/M phase and Caf at S phase. Propolis but not Caf may act as a P-gp inhibitor by modulating P-gp activity and inhibiting DOX efflux. Propolis exerted cytotoxic effects on HEp-2 cells, and the mechanisms are discussed, showing its potential as an antitumour drug. © 2017 Royal Pharmaceutical Society.

Common Motifs in the Response of Cereal Primary Metabolism to Fungal Pathogens are not Based on Similar Transcriptional Reprogramming

PubMed Central

Voll, Lars Matthias; Horst, Robin Jonathan; Voitsik, Anna-Maria; Zajic, Doreen; Samans, Birgit; Pons-Kühnemann, Jörn; Doehlemann, Gunther; Münch, Steffen; Wahl, Ramon; Molitor, Alexandra; Hofmann, Jörg; Schmiedl, Alfred; Waller, Frank; Deising, Holger Bruno; Kahmann, Regine; Kämper, Jörg; Kogel, Karl-Heinz; Sonnewald, Uwe

2011-01-01

During compatible interactions with their host plants, biotrophic plant–pathogens subvert host metabolism to ensure the sustained provision of nutrient assimilates by the colonized host cells. To investigate, whether common motifs can be revealed in the response of primary carbon and nitrogen metabolism toward colonization with biotrophic fungi in cereal leaves, we have conducted a combined metabolome and transcriptome study of three quite divergent pathosystems, the barley powdery mildew fungus (Blumeria graminis f.sp. hordei), the corn smut fungus Ustilago maydis, and the maize anthracnose fungus Colletotrichum graminicola, the latter being a hemibiotroph that only exhibits an initial biotrophic phase during its establishment. Based on the analysis of 42 water-soluble metabolites, we were able to separate early biotrophic from late biotrophic interactions by hierarchical cluster analysis and principal component analysis, irrespective of the plant host. Interestingly, the corresponding transcriptome dataset could not discriminate between these stages of biotrophy, irrespective, of whether transcript data for genes of central metabolism or the entire transcriptome dataset was used. Strong differences in the transcriptional regulation of photosynthesis, glycolysis, the TCA cycle, lipid biosynthesis, and cell wall metabolism were observed between the pathosystems. However, increased contents of Gln, Asn, and glucose as well as diminished contents of PEP and 3-PGA were common to early post-penetration stages of all interactions. On the transcriptional level, genes of the TCA cycle, nucleotide energy metabolism and amino acid biosynthesis exhibited consistent trends among the compared biotrophic interactions, identifying the requirement for metabolic energy and the rearrangement of amino acid pools as common transcriptional motifs during early biotrophy. Both metabolome and transcript data were employed to generate models of leaf primary metabolism during early biotrophy for the three investigated interactions. PMID:22645534

Pretreatment with oleic acid accelerates the entrance into the mitotic cycle of EGF-stimulated fibroblasts.

PubMed

Zugaza, J L; Casabiell, X A; Bokser, L; Eiras, A; Beiras, A; Casanueva, F F

1995-07-01

We have previously demonstrated that pretreatment of several cell lines with cis-unsaturated fatty acids, like oleic acid, blocks epidermal growth factor (EGF)-induced early ionic signals, and in particular the [Ca2+]i rise. In the present work we show that this blockade does not alter EGF-stimulated cellular proliferation evaluated by direct cell counting, but induces a powerful enhancement in the pulsed thymidine incorporation assay. The lack of effect of oleic acid on EGF-stimulated cellular proliferation was confirmed by repeated cell counts, cumulative thymidine incorporation, and protein synthesis, but a clear synergistic effect between oleic acid and EGF was again obtained by means of time course experiments with pulsed thymidine. Combined flow cytometry analysis and cell counts at earlier times in EGF-stimulated cells showed that oleic acids accelerates the entrance of cells into the replicative cycle leading to an earlier cell division. Afterward, these oleic acid-pretreated cells became delayed by an unknown compensatory mechanism in such a way that at 48 h post-EGF, the cell count in control and oleic acid-pretreated cells was equal. In conclusion (a) oleic acid accelerates or enhances the EGF mitogenic action and (b) in the long term cells compensate the initial perturbation with respect to untreated cells. As a side observation, the widely employed pulsed thymidine incorporation method as a measure of cell division could be extremely misleading unless experimental conditions are well controlled.

HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes.

PubMed

Domínguez-Catzín, Victoria; Reveles-Espinoza, Alicia-María; Sánchez-Ramos, Janet; Cruz-Cadena, Raúl; Lemus-Hernández, Diana; Garrido, Efraín

2017-04-03

Cervical cancer is the fourth cause of death worldwide by cancer in women and is a disease associated to persistent infection with human papillomavirus (HPV), particularly from two high-risk types HPV16 and 18. The virus initiates its replicative cycle infecting cells located in the basal layer of the epithelium, where a small population of epithelial stem cells is located performing important functions of renewal and maintenance of the tissue. Viral E2 gene is one of the first expressed after infection and plays relevant roles in the replicative cycle of the virus, modifying fundamental processes in the infected cells. Thus, the aim of the present study was to demonstrate the presence of hierarchic subpopulations in HaCaT cell line and evaluate the effect of HPV16-E2 expression, on their biological processes. HaCaT-HPV16-E2 cells were generated by transduction of HaCaT cell line with a lentiviral vector. The α6-integrin-CD71 expression profile was established by immunostaining and flow cytometric analysis. After sorting, cell subpopulations were analyzed in biological assays for self-renewal, clonogenicity and expression of stemness factors (RT-qPCR). We identified in HaCaT cell line three different subpopulations that correspond to early differentiated cells (α6-integrin dim ), transitory amplifying cells (α6-integrin bri /CD71 bri ) and progenitor cells (α6-integrin bri /CD71 dim ). The last subpopulation showed stem cell characteristics, such as self-renewal ability, clonogenicity and expression of the well-known stem cell factors SOX2, OCT4 and NANOG, suggesting they are stem-like cells. Interestingly, the expression of HPV16-E2 in HaCaT cells changed its α6-integrin-CD71 immunophenotype modifying the relative abundance of the cell subpopulations, reducing significantly the percentage of α6-integrin bri /CD71 dim cells. Moreover, the expression of the stem cell markers was also modified, increasing the expression of SOX2 and NANOG, but decreasing notably the expression of OCT4. Our data demonstrated the presence of a small subpopulation with epithelial "progenitor cells" characteristics in the HaCaT cell line, and that HPV16-E2 expression on these cells induces early differentiation.

PP2A: more than a reset switch to activate pRB proteins during the cell cycle and in response to signaling cues

PubMed Central

Kurimchak, Alison; Graña, Xavier

2015-01-01

In their active hypophosphorylated state, members of the retinoblastoma family of pocket proteins negatively regulate cell cycle progression at least in part by repressing expression of E2F-dependent genes. Mitogen-dependent activation of G1 and G1/S Cyclin Dependent Kinases (CDKs) results in coordinated hyperphosphorylation and inactivation of these proteins, which no longer bind and repress E2Fs. S and G2/M CDKs maintain pocket protein hyperphosphorylated through the end of mitosis. The inactivating action of inducible CDKs is opposed by the Ser/Thr protein phosphatases PP2A and PP1. Various trimeric PP2A holoenzymes have been implicated in dephosphorylation of pocket proteins in response to specific cellular signals and stresses or as part of an equilibrium with CDKs throughout the cell cycle. PP1 has specifically been implicated in dephosphorylation of pRB in late mitosis and early G1. This review is particularly focused on the emerging role of PP2A as a major hub for integration of growth suppressor signals that require rapid inactivation of pocket proteins. Of note, activation of particular PP2A holoenzymes triggers differential activation of pocket proteins in the presence of active CDKs.

Life-history stages of natural bloom populations and the bloom dynamics of a tropical Asian ribotype of Alexandrium minutum.

PubMed

Lau, Winnie Lik Sing; Law, Ing Kuo; Liow, Guat Ru; Hii, Kieng Soon; Usup, Gires; Lim, Po Teen; Leaw, Chui Pin

2017-12-01

In 2015, a remarkably high density bloom of Alexandrium minutum occurred in Sungai Geting, a semi-enclosed lagoon situated in the northeast of Peninsular Malaysia, causing severe discoloration and contaminated the benthic clams (Polymesoda). Plankton and water samples were collected to investigate the mechanisms of bloom development of this toxic species. Analysis of bloom samples using flow cytometry indicated that the bloom was initiated by the process of active excystment, as planomycetes (>4C cells) were observed in the early stage of the bloom. Increase in planozygotes (2C cells) was evident during the middle stage of the bloom, coinciding with an abrupt decrease in salinity and increase of temperature. The bloom was sustained through the combination of binary division of vegetative cells, division of planozygotes, and cyst germination through continuous excystment. Nutrient depletion followed by precipitation subsequently caused the bloom to terminate. This study provides the first continuous record of in situ life-cycle stages of a natural bloom population of A. minutum through a complete bloom cycle. The event has provided a fundamental understanding of the pelagic life-cycle stages of this tropical dinoflagellate, and demonstrated a unique bloom development characteristic shared among toxic Alexandrium species in coastal embayments. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning and expression analysis of vasa during the reproductive cycle of Korean rockfish, Sebastes schlegeli

NASA Astrophysics Data System (ADS)

Mu, Weijie; Wen, Haishen; He, Feng; Li, Jifang; Liu, Miao; Ma, Ruiqin; Zhang, Yuanqing; Hu, Jian; Qi, Baoxia

2013-03-01

Vasa, which is a conserved member of the DEAD-box protein family, plays an indispensable role in primordial germ cell proliferation. However, the expression of vasa gene during the reproductive cycle in ovoviviparous fish has not been documented. In this study, the full-length sequence of vasa was obtained from the ovary of Korean rockfish ( Sebastes schlegeli) using reverse transcription-PCR and rapid amplification of cDNA ends. The Vasa with a mature protein of 650 amino acids showed greatest homology (84%) with giant gourami ( Osphronemus goramy) and Pacific bluefin tuna ( Thunnus orientalis). The expression of vasa mRNA in Korean rockfish was detected in gonads only, suggesting its specific role in gonadal development. In addition, seasonal changes in the vasa expression levels were examined in gonads by quantitative real-time PCR. The vasa transcript levels in adult testis were found higher during spermatogenesis than during spermiation. The vasa transcript levels remained relatively high at the early ovary stage but declined during ovary maturation in adult female fish. These results suggest that the vasa gene play an important role in spermatogenesis and early oogenesis during the reproductive cycle of Korean rockfish.

Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies.

PubMed

Friedman, Jay; Morisada, Megan; Sun, Lillian; Moore, Ellen C; Padget, Michelle; Hodge, James W; Schlom, Jeffrey; Gameiro, Sofia R; Allen, Clint T

2018-06-21

Natural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined. Tumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed. Cultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab. Tumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.

Long non-coding RNA NNT-AS1 contributes to cell proliferation, metastasis and apoptosis in human ovarian cancer.

PubMed

Huang, Yaqing; Shi, Junyu; Xu, Yun

2018-06-01

Ovarian cancer is a markedly heterogeneous malignancy characterized by various histological subtypes. Molecular biomarkers have been indicated to serve significant functions in the early diagnosis and treatment of early-stage ovarian cancer. However, the detailed mechanism underlying the tumorigenesis of ovarian cancer remains unclear. The present study aimed to identify a novel long non-coding RNA in patients with ovarian cancer. Nicotinamide nucleotide transhydrogenase-antisense 1 (NNT-AS1) was markedly downregulated in patients with ovarian cancer and in cultured human ovarian cancer cells. Knockdown of NNT-AS1 in the human ovarian cancer cell lines HO-8910 and SK-OV-3 promoted colony formation and arrested the cell cycle at G 0 /G 1 phase. Furthermore, Transwell demonstrated that the downregulation of NNT-AS1 increased cell migration and invasion by ~60 and 70%, respectively, in HO-8910 and SK-OV-3 cells. Furthermore, cell apoptosis was inhibited by the transfection of siNNT-AS1 in the two cell lines, whereas the relative activities of caspase-3 and caspase-9 were decreased. These results indicated a protective function of NNT-AS1 in human ovarian cancer, providing novel insights into the diagnosis and treatment of ovarian cancer in clinical settings.

Early onset of puberty and early ovarian failure in CYP7B1 knockout mice

PubMed Central

Omoto, Yoko; Lathe, Richard; Warner, Margaret; Gustafsson, Jan-Åke

2005-01-01

CYP7B1 is the enzyme responsible for hydroxylation and termination of the estrogenic actions of the androgen metabolite, 5α-androstane-3β, 17β-diol (3βAdiol). 3βAdiol is estrogenic in ERα or ERβ positive cells only if they do not express CYP7B1. In this study we show that female CYP7B1–/– mice experience early onset of growth of the uterus and mammary glands and commence estrus cycles 2 days earlier than their wild-type littermates. Adult mammary glands and uteri appear to be under continuous estrogenic stimulation. We conclude that, by cell-specific regulation of the estrogenicity of 3βAdiol, CYP7B1 performs two major tasks: (i) it allows 3βAdiol to have growth inhibitory effects through ERβ and (ii) it permits estradiol-specific activation of estrogen receptors by protection of certain cells from the estrogenic effects of 3βAdiol. When CYP7B1 is inactivated, 3βAdiol activates estrogen receptors indiscriminately, and the overall effect is prolonged and inappropriate exposure to estrogen. PMID:15710898

Polo-like kinase 1 is essential for early embryonic development and tumor suppression.

PubMed

Lu, Lin-Yu; Wood, Jamie L; Minter-Dykhouse, Katherine; Ye, Lin; Saunders, Thomas L; Yu, Xiaochun; Chen, Junjie

2008-11-01

Polo-like kinases (Plks) are serine/threonine kinases that are highly conserved in organisms from yeasts to humans. Previous reports have shown that Plk1 is critical for all stages of mitosis and may play a role in DNA replication during S phase. While much work has focused on Plk1, little is known about the physiological function of Plk1 in vivo. To address this question, we generated Plk1 knockout mice. Plk1 homozygous null mice were embryonic lethal, and early Plk1(-/-) embryos failed to survive after the eight-cell stage. Immunocytochemistry studies revealed that Plk1-null embryos were arrested outside the mitotic phase, suggesting that Plk1 is important for proper cell cycle progression. It has been postulated that Plk1 is a potential oncogene, due to its overexpression in a variety of tumors and tumor cell lines. While the Plk1 heterozygotes were healthy at birth, the incidence of tumors in these animals was threefold greater than that in their wild-type counterparts, demonstrating that the loss of one Plk1 allele accelerates tumor formation. Collectively, our data support that Plk1 is important for early embryonic development and may function as a haploinsufficient tumor suppressor.

Mitotic effects of monochromatic ultraviolet radiation at 225, 265, and 280 nm on eleven stages of the cell cycle of the grasshopper neuroblast in culture. II. Changes in progression rate and cell sequence between the stage irradiated and nuclear membrane breakdown

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlson, J.G.

1976-10-01

Portions of embryos of the grasshopper, Chortophaga viridifasciata (DeGeer), were cultured in hanging drops under quartz cover slips. Immediately after exposure to 225, 265, or 280 nm radiation, microscope observations at 38/sup 0/C were begun. The morphologically identified stage and the time after treatment of selected neuroblasts were recorded at short-time intervals until prometaphase was reached. Mitotic retardation induced by irradiation of prereplication stages (metaphase, anaphase, or early telophase) or S phase (middle or late telophase, interphase, or very early prophase) is greatest in postreplication stages (early, middle, and late prophase) and absent or minimal in stages morphologically identified asmore » parts of S phase. Ultraviolet irradiation superimposes on the normal diversity of progression rates an additional variation factor, so that cells do not necessarily reach prometaphase in the order of their sequence at the time of treatment. This suggests the need for caution in ascribing particular radiosensitivities to substages of limited duration on the basis of the order in which they attain a subsequent stage.« less

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process.

PubMed

Laranjeiro, Ricardo; Tamai, T Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-04-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process

PubMed Central

Tamai, T. Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-01-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles. PMID:29444612

NeoPalAna: Neoadjuvant palbociclib, a cyclin-dependent kinase 4/6 inhibitor, and anastrozole for clinical stage 2 or 3 estrogen receptor positive breast cancer

PubMed Central

Ma, Cynthia X.; Gao, Feng; Luo, Jingqin; Northfelt, Donald W.; Goetz, Matthew; Forero, Andres; Hoog, Jeremy; Naughton, Michael; Ademuyiwa, Foluso; Suresh, Rama; Anderson, Karen S.; Margenthaler, Julie; Aft, Rebecca; Hobday, Timothy; Moynihan, Timothy; Gillanders, William; Cyr, Amy; Eberlein, Timothy J.; Hieken, Tina; Krontiras, Helen; Guo, Zhanfang; Lee, Michelle V.; Spies, Nicholas C.; Skidmore, Zachary L.; Griffith, Obi L.; Griffith, Malachi; Thomas, Shana; Bumb, Caroline; Vij, Kiran; Bartlett, Cynthia Huang; Koehler, Maria; Al-Kateb, Hussam; Sanati, Souzan; Ellis, Matthew J.

2017-01-01

Purpose Cyclin-dependent kinase (CDK) 4/6 drives cell proliferation in estrogen receptor positive (ER+) breast cancer. This single-arm phase II neoadjuvant trial (NeoPalAna) assessed the anti-proliferative activity of the CDK4/6 inhibitor palbociclib in primary breast cancer as a prelude to adjuvant studies. Experimental Design Eligible patients with clinical stage II/III ER+/HER2- breast cancer received anastrozole 1mg daily for 4 weeks (cycle 0) (with goserelin if premenopausal), followed by adding palbociclib (125mg daily on days 1-21) on cycle 1 day 1 (C1D1) for four 28-day cycles unless C1D15 Ki67>10%, in which case patients went off study due to inadequately response. Anastrozole was continued until surgery, which occurred 3-5 weeks post palbociclib exposure. Later patients received additional 10-12 days of palbociclib (Cycle 5) immediately before surgery. Serial biopsies at baseline, C1D1, C1D15, and surgery were analyzed for Ki67, gene expression and mutation profiles. The primary endpoint was Complete Cell Cycle Arrest (CCCA: central Ki67<2.7%). Results Fifty patients enrolled. The CCCA rate was significantly higher after adding palbociclib to anastrozole (C1D15 87% vs C1D1 26%, p<0.001). Palbociclib enhanced cell cycle control over anastrozole monotherapy regardless of luminal subtype (A vs B) and PIK3CA status with activity observed across a broad range of clinicopathological and mutation profiles. Ki67 recovery at surgery following palbociclib washout was suppressed by cycle 5 palbociclib. Resistance was associated with non-luminal subtypes and persistent E2F-target gene expression. Conclusions Palbociclib is an active anti-proliferative agent for early-stage breast cancer resistant to anastrozole, however, prolonged administration may be necessary to maintain its effect. PMID:28270497

Investigating the Flow and Biomechanics of the Embryonic Zebrafish Heart

NASA Astrophysics Data System (ADS)

Johnson, Brennan; Garrity, Deborah; Dasi, Lakshmi

2010-11-01

Understanding flow and kinematic characteristics of the embryonic heart is a prerequisite to devise early intervention or detection methods in the context of congenital heart defects. In this study, the kinematics and fluid dynamics of the embryonic zebrafish heart were analyzed through the early stages of cardiac development (24-48 hours post-fertilization) in vivo using optical microscopy and high-speed video. Endocardial walls and individual blood cells were segmented from raw images and were tracked through the cardiac cycle. Particle tracking velocimetry analysis yielded quantitative blood cell velocity field, chamber volume, and flow rate information. It was seen that the pumping mechanism starts as a combined peristaltic and suction pump while the heart is in the tube configuration and transforms into a positive displacement pump after cardiac looping. Strong two-phase nature of the fluid is evident. This work provides us new understanding of the spatio-temporal characteristics of kinematics and blood cell velocity field inside the developing heart.

Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

PubMed Central

Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

2011-01-01

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507

Expression of {beta}{sub 1} integrins in human endometrial stromal and decidual cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiokawa, Shigetatsu; Yoshimura, Yasunori; Nakamura, Yukio

The present study was undertaken to investigate the expression of {beta}{sub 1} integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, and {alpha}{sub 5} subunits of the {beta}{sub 1} integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E{sub 2}) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of {beta}{sub 1} integrins in vitro. Flow cytometry also demonstrated the expression of themore » {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, {alpha}{sub 3}, {alpha}{sub 5}, and {alpha}{sub 6} subunits of {beta}{sub 1} integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of {beta}{sub 1} integrins expression. Immunohistochemistry confirmed the {beta}{sub 1} integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed {beta}{sub 1} integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, {beta}{sub 1} integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation. 43 refs., 7 figs., 2 tabs.« less

p53 independent epigenetic-differentiation treatment in xenotransplant models of acute myeloid leukemia

PubMed Central

Ng, Kwok Peng; Ebrahem, Quteba; Negrotto, Soledad; Mahfouz, Reda Z.; Link, Kevin A.; Hu, Zhenbo; Gu, Xiaorong; Advani, Anjali; Kalaycio, Matt; Sobecks, Ronald; Sekeres, Mikkael; Copelan, Edward; Radivoyevitch, Tomas; Maciejewski, Jaroslaw; Mulloy, James C.; Saunthararajah, Yogen

2013-01-01

Suppression of apoptosis by TP53 mutation contributes to resistance of acute myeloid leukemia (AML) to conventional cytotoxic treatment. Using differentiation to induce irreversible cell cycle exit in AML cells could be a p53-independent treatment alternative, however, this possibility requires evaluation. In vitro and in vivo regimens of the deoxycytidine analogue decitabine that deplete the chromatin modifying enzyme DNA methyl-transferase 1 (DNMT1) without phosphorylating p53 or inducing early apoptosis were determined. These decitabine regimens but not equimolar DNA-damaging cytarabine up regulated the key late differentiation factors CEBPε and p27/CDKN1B, induced cellular differentiation, and terminated AML cell-cycle, even in cytarabine-resistant p53- and p16/CDKN2A-null AML cells. Leukemia initiation by xeno-transplanted AML cells was abrogated but normal hematopoietic stem cell (HSC) engraftment was preserved. In vivo, the low toxicity allowed frequent drug administration to increase exposure, an important consideration for S-phase specific decitabine therapy. In xeno-transplant models of p53-null and relapsed/refractory AML, the non-cytotoxic regimen significantly extended survival compared to conventional cytotoxic cytarabine. Modifying in vivo dose and schedule to emphasize this pathway of decitabine action can bypass a mechanism of resistance to standard therapy. PMID:21701495

Essential Roles for Caenorhabditis elegans Lamin Gene in Nuclear Organization, Cell Cycle Progression, and Spatial Organization of Nuclear Pore Complexes

PubMed Central

Liu, Jun; Ben-Shahar, Tom Rolef; Riemer, Dieter; Treinin, Millet; Spann, Perah; Weber, Klaus; Fire, Andrew; Gruenbaum, Yosef

2000-01-01

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs. PMID:11071918

On the dynamics of acute EBV infection and the pathogenesis of infectious mononucleosis

PubMed Central

Hadinoto, Vey; Shapiro, Michael; Greenough, Thomas C.; Sullivan, John L.; Luzuriaga, Katherine

2008-01-01

Memory B cells latently infected with Epstein-Barr virus (mBLats) in the blood disappear rapidly on presentation with acute symptomatic primary infection (acute infectious mononucleosis [AIM]). They undergo a simple exponential decay (average half-life: 7.5 ± 3.7 days) similar to that of normal memory B cells. The cytotoxic T lymphocyte (CTL) response to immediate early (IE) lytic antigens (CTLIEs) also decays over this time period, but no such correlation was observed for the CTL response to lytic or latent antigens or to the levels of virions shed into saliva. We have estimated the average half-life of CTLIEs to be 73 (± 23) days. We propose that cycles of infection and reactivation occur in the initial stages of infection that produce high levels of mBLats in the circulation. Eventually the immune response arises and minimizes these cycles leaving the high levels of mBLats in the blood to decay through simple memory B-cell homeostasis mechanisms. This triggers the cells to reactivate the virus whereupon most are killed by CTLIEs before they can release virus and infect new cells. The release of antigens caused by this large-scale destruction of infected cells may trigger the symptoms of AIM and be a cofactor in other AIM-associated diseases. PMID:17991806

Interkinetic and migratory behavior of a cohort of neocortical neurons arising in the early embryonic murine cerebral wall

NASA Technical Reports Server (NTRS)

Takahashi, T.; Nowakowski, R. S.; Caviness, V. S. Jr

1996-01-01

Neocortical neuronogenesis occurs in the pseudostratified ventricular epithelium (PVE) where nuclei of proliferative cells undergo interkinetic nuclear movement. A fraction of daughter cells exits the cell cycle as neurons (the quiescent, or Q, fraction), whereas a complementary fraction remains in the cell cycle (the proliferative, or P, fraction). By means of sequential thymidine and bromodeoxyuridine injections in mouse on embryonic day 14, we have monitored the proliferative and post-mitotic migratory behaviors of 1 and 2 hr cohorts of PVE cells defined by the injection protocols. Soon after mitosis, the Q fraction partitions into a rapidly exiting (up to 50 microns/hr) subpopulation (Qr) and a more slowly exiting (6 microns/hr) subpopulation (Qs). Qr and Qs are separated as two distributions on exit from the ventricular zone with an interpeak distance of approximately 40 microns. Cells in Qr and Qs migrate through the intermediate zone with no significant change in the interpeak distance, suggesting that they migrate at approximately the same velocities. The rate of migration increases with ascent through the intermediate zone (average 2-6.4 microns/hr) slowing only transiently on entry into the developing cortex. Within the cortex, Qr and Qs merge to form a single distribution most concentrated over layer V.

The metastasis suppressor SOX11 is an independent prognostic factor for improved survival in gastric cancer

PubMed Central

QU, YING; ZHOU, CHENFEI; ZHANG, JIANIAN; CAI, QU; LI, JIANFANG; DU, TAO; ZHU, ZHENGGANG; CUI, XIAOJIANG; LIU, BINGYA

2014-01-01

SOX11 is involved in gastrulation and in malignant diseases. The aim of this study was to investigate the role of SOX11 in gastric cancer and its expression pattern and clinical significance. SOX11 overexpression cell model was used to examine in vitro and in vivo the role of SOX11 in cell growth and metastasis. Cell cycle analysis and Annexin V/PI double staining were used to investigate the effect of SOX11 on cell cycle progression and apoptosis. The expression of SOX11 in human gastric cancer was examined by immunohistochemistry. The correlation of SOX11 expression with clinicopathological characteristics and survival of patients was analyzed by Pearson’s χ2 and Kaplan-Meier analyses, respectively. Cox’s proportional hazard model was employed in multivariate analysis. SOX11 overexpression did not inhibit cell growth but strongly suppressed cell migration/invasion in vitro and in vivo. We found a significant correlation between high SOX11 protein levels and Lauren’s classification (intestinal type), differentiation status (high and medium), and early TNM stage. SOX11 is an independent prognostic factor for improved survival in gastric cancer patients. SOX11 was a potential tumor-suppressor and an independent positive prognostic factor in gastric cancer patients with less advanced clinicopathological features. PMID:24604109

Cloning of nascent monkey DNA synthesized early in the cell cycle.

PubMed

Kaufmann, G; Zannis-Hadjopoulos, M; Martin, R G

1985-04-01

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.

Limb-bud and Heart Overexpression Inhibits the Proliferation and Migration of PC3M Cells.

PubMed

Liu, Qicai; Li, Ermao; Huang, Long; Cheng, Minsheng; Li, Li

2018-01-01

Background: The limb-bud and heart gene ( LBH ) was discovered in the early 21st century and is specifically expressed in the mouse embryonic limb and heart development. Increasing evidences have indicated that LBH not only plays an important role in embryo development, it is also closely correlated with the occurance and progression of many tumors. However, its function in prostate cancer (PCa) is still not well understood. Here, we explored the effects of LBH on the proliferation and migration of the PCa cell line PC3M. Methods: LBH expression in tissues and cell lines of PCa was detected by immunohistochemistry and Western blotting. Lentivirus was used to transduct the LBH gene into the PC3M cells. Stable LBH-overexpressing PC3M-LBH cells and PC3M-NC control cells were obtained via puromycin screening. Cell proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution and apoptosis rate were investigated using flow cytometry. Cell migration was studied using the Transwell assay. Results: LBH expression level was down-regulated in 3 different PCa cell lines, especially in PC3M cells, compared with the normal prostate epithelial cells(RWPE-1). Cell lines of LBH-upregulated PC3M-LBH and PC3M-NC control were successfully constructed. Significantly increased LBH expression level and decreased cyclin D1 and cyclin E2 expression level was found in PC3M-LBH cells as compared to the PC3M-NC cells. The overexpression of LBH significantly inhibited PC3M cell proliferation in vitro and tumor growth in nude mice. LBH overexpression in PC3M cell, also induced cell cycle G0/G1 phase arrest and decreased the migration of PC3M cells. Conclusions : Our results reveal that LBH expression is down-regulated in the tissue and cell lines of PCa. LBH overexpression inhibits PC3M cell proliferation and tumor growth by inducing cell cycle arrest through down-regulating cyclin D1and cyclin E2 expression. LBH might be a therapeutic target and potential diagnostic marker in PCa.

Beta-mangostin from Cratoxylum arborescens activates the intrinsic apoptosis pathway through reactive oxygen species with downregulation of the HSP70 gene in the HL60 cells associated with a G0/G1 cell-cycle arrest.

PubMed

Omer, Fatima Abdelmutaal Ahmed; Hashim, Najihah Binti Mohd; Ibrahim, Mohamed Yousif; Dehghan, Firouzeh; Yahayu, Maizatulakmal; Karimian, Hamed; Salim, Landa Zeenelabdin Ali; Mohan, Syam

2017-11-01

Xanthones are phytochemical compounds found in a number of fruits and vegetables. Characteristically, they are noted to be made of diverse properties based on their biological, biochemical, and pharmacological actions. Accordingly, the apoptosis mechanisms induced by beta-mangostin, a xanthone compound isolated from Cratoxylum arborescens in the human promyelocytic leukemia cell line (HL60) in vitro, were examined in this study. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done to estimate the cytotoxicity effect of β-mangostin on the HL60 cell line. Acridine orange/propidium iodide and Hoechst 33342 dyes and Annexin V tests were conducted to detect the apoptosis features. Caspase-3 and caspase-9 activities; reactive oxygen species; real-time polymerase chain reaction for Bcl-2, Bax, caspase-3, and caspase-9 Hsp70 genes; and western blot for p53, cytochrome c, and pro- and cleavage-caspase-3 and caspase-9 were assessed to examine the apoptosis mechanism. Cell-cycle analysis conducted revealed that β-mangostin inhibited the growth of HL60 at 58 µM in 24 h. The administration of β-mangostin with HL60 caused cell morphological changes related to apoptosis which increased the number of early and late apoptotic cells. The β-mangostin-catalyzed apoptosis action through caspase-3, caspase-7, and caspase-9 activation overproduced reactive oxygen species which downregulated the expression of antiapoptotic genes Bcl-2 and HSP70. Conversely, the expression of the apoptotic genes Bax, caspase-3, and caspase-9 were upregulated. Meanwhile, at the protein level, β-mangostin activated the formation of cleaved caspase-3 and caspase-9 and also upregulated the p53. β-mangostin arrested the cell cycle at the G 0 /G 1 phase. Overall, the results for β-mangostin showed an antiproliferative effect in HL60 via stopping the cell cycle at the G 0 /G 1 phase and prompted the intrinsic apoptosis pathway.

Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Mitotic Cycles

PubMed Central

Su, Tin Tin; O'Farrell, Patrick H.

1997-01-01

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM–chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM–chromatin association. PMID:9314525

Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Peng-Yeh; Tsai, Chong-Bin; Department of Ophthalmology, Chiayi Christian Hospital, Chiayi 600, Taiwan, ROC

2013-01-18

Highlights: ► Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ► Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ► Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ► Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCRmore » analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBPδ and PPARγ was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.« less

Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

PubMed

Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

2013-03-01

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

Low-dose cisplatin protects human neuroblastoma SH-SY5Y cells from paclitaxel-induced apoptosis.

PubMed

Villa, Daniela; Miloso, Mariarosaria; Nicolini, Gabriella; Rigolio, Roberta; Villa, Antonello; Cavaletti, Guido; Tredici, Giovanni

2005-09-01

Combined anticancer therapy using platinum compounds and antitubulins has increased the risk of neurotoxicity. However, the combination of low-dose cisplatin (CDDP) with toxic doses of paclitaxel significantly reduces cellular death in a human neuroblastoma SH-SY5Y cell line. To analyze the mechanisms of this protection, we evaluated various signaling molecules possibly involved in apoptosis and some relevant cell cycle regulatory proteins. CDDP does not interfere with the tubulin-stabilizing action of paclitaxel. The evaluation of molecular pathways involved in apoptosis indicates that the Bcl-2 but not the caspases may be involved in the CDDP protection of paclitaxel-induced apoptosis. The increase in p53 protein and its nuclear accumulation suggests a possible involvement of p53 in CDDP protection. The use of the chemical inhibitor of p53, pifithrin alpha, excluded this possibility. The study of cyclins and the flow cytometric analysis (fluorescence-activated cell sorting) suggest that CDDP exerts a protective action by blocking cells early in the cell cycle. The determination of the mitotic index indicates that CDDP prevents cells from reaching the mitosis. We concluded that low doses of CDDP are protective against toxic doses of paclitaxel and that the possible mechanism of this protection is that the CDDP prevents human neuroblastoma SH-SY5Y cells from achieving mitosis.

Thermal Cycling of Mir Cooperative Solar Array (MCSA) Test Panels

NASA Technical Reports Server (NTRS)

Hoffman, David J.; Scheiman, David A.

1997-01-01

The Mir Cooperative Solar Array (MCSA) project was a joint US/Russian effort to build a photovoltaic (PV) solar array and deliver it to the Russian space station Mir. The MCSA is currently being used to increase the electrical power on Mir and provide PV array performance data in support of Phase 1 of the International Space Station (ISS), which will use arrays based on the same solar cells used in the MCSA. The US supplied the photovoltaic power modules (PPMs) and provided technical and programmatic oversight while Russia provided the array support structures and deployment mechanism and built and tested the array. In order to ensure that there would be no problems with the interface between US and Russian hardware, an accelerated thermal life cycle test was performed at NASA Lewis Research Center on two representative samples of the MCSA. Over an eight-month period (August 1994 - March 1995), two 15-cell MCSA solar array 'mini' panel test articles were simultaneously put through 24,000 thermal cycles (+80 C to -100 C), equivalent to four years on-orbit. The test objectives, facility, procedure and results are described in this paper. Post-test inspection and evaluation revealed no significant degradation in the structural integrity of the test articles and no electrical degradation, not including one cell damaged early as an artifact of the test and removed from consideration. The interesting nature of the performance degradation caused by this one cell, which only occurred at elevated temperatures, is discussed. As a result of this test, changes were made to improve some aspects of the solar cell coupon-to-support frame interface on the flight unit. It was concluded from the results that the integration of the US solar cell modules with the Russian support structure would be able to withstand at least 24,000 thermal cycles (4 years on-orbit).

Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Fangke; Chen, Yun; Shi, Jintong

Duck hepatitis A virus type 1 (DHAV-1) is an important agent of duck viral hepatitis. Until recently, the replication cycle of DHAV-1 is still unknown. Here duck embryonic hepatocytes infected with DHAV-1 were collected at different time points, and dynamic changes of the relative DHAV-1 gene expression during replication were detected by real-time PCR. And the morphology of hepatocytes infected with DHAV was evaluated by electron microscope. The result suggested that the adsorption of DHAV-1 saturated at 90 min post-infection, and the virus particles with size of about 50 nm including more than 20 nm of vacuum drying gold weremore » observed on the infected cells surface. What's more, the replication lasted around 13 h after the early protein synthesis for about 5 h, and the release of DHAV-1 was in steady state after 32 h. The replication cycle will enrich the data for DVH control and provide the foundation for future studies. - Highlights: • This is the first description of the replication cycle of DHAV-1. • Firstly find that DHAV-1 adsorption saturated at 90 min post-infection. • The replication lasted around 13 h after early protein synthesis for about 5 h. • The release of DHAV-1 was in steady state after 32 h.« less

Characterizing Bacteriophage PR772 as a Potential Surrogate for Adenovirus in Water Disinfection: A Comparative Analysis of Inactivation Kinetics and Replication Cycle Inhibition by Free Chlorine.

PubMed

Gall, Aimee M; Shisler, Joanna L; Mariñas, Benito J

2016-03-01

Elucidating mechanisms by which pathogenic waterborne viruses become inactivated by drinking water disinfectants would facilitate the development of sensors to detect infectious viruses and novel disinfection strategies to provide safe water. Using bacteriophages as surrogates for human pathogenic viruses could assist in elucidating these mechanisms; however, an appropriate viral surrogate for human adenovirus (HAdV), a medium-sized virus with a double-stranded DNA genome, needs to be identified. Here, we characterized the inactivation kinetics of bacteriophage PR772, a member of the Tectiviridae family with many similarities in structure and replication to HAdV. The inactivation of PR772 and HAdV by free chlorine had similar kinetics that could be represented with a model previously developed for HAdV type 2 (HAdV-2). We developed and tested a quantitative assay to analyze several steps in the PR772 replication cycle to determine if both viruses being inactivated at similar rates resulted from similar replication cycle events being inhibited. Like HAdV-2, we observed that PR772 inactivated by free chlorine still attached to host cells, and viral DNA synthesis and early and late gene transcription were inhibited. Consequently, free chlorine exposure inhibited a replication cycle event that was post-binding but took place prior to early gene synthesis for both PR772 and HAdV-2.

Gigantism in a Bacterium, Epulopiscium fishelsoni, Correlates with Complex Patterns in Arrangement, Quantity, and Segregation of DNA

PubMed Central

Bresler, V.; Montgomery, W. L.; Fishelson, L.; Pollak, P. E.

1998-01-01

Epulopiscium fishelsoni, gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus) in the Red Sea, attains a larger size than any other eubacterium, varies 10- to 20-fold in length (and >2,000-fold in volume), and undergoes a complex daily life cycle. In early morning, nucleoids contain highly condensed DNA in elongate, chromosome-like structures which are physically separated from the general cytoplasm. Cell division involves production of two (rarely three) nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell. Fluorescence measurements of DNA, RNA, and other cell components indicate the following. DNA quantity is proportional to cell volume over cell lengths of ∼30 μm to >500 μm. For cells of a given size, nucleoids of cells with two nucleoids (binucleoid) contain approximately equal amounts of DNA. And each nucleoid of a binucleoid cell contains one-half the DNA of the single nucleoid in a uninucleoid cell of the same size. The life cycle involves approximately equal subdivision of DNA among daughter cells, formation of apical caps of condensed DNA from previously decondensed and diffusely distributed DNA, and “pinching” of DNA near the middle of the cell in the absence of new wall formation. Mechanisms underlying these patterns remain unclear, but formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and bacterial cell growth and during nocturnal periods of host inactivity when mean bacterial cell size declines. PMID:9791108

Expression of Idh1R132H in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis.

PubMed

Bardella, Chiara; Al-Dalahmah, Osama; Krell, Daniel; Brazauskas, Pijus; Al-Qahtani, Khalid; Tomkova, Marketa; Adam, Julie; Serres, Sébastien; Lockstone, Helen; Freeman-Mills, Luke; Pfeffer, Inga; Sibson, Nicola; Goldin, Robert; Schuster-Böeckler, Benjamin; Pollard, Patrick J; Soga, Tomoyoshi; McCullagh, James S; Schofield, Christopher J; Mulholland, Paul; Ansorge, Olaf; Kriaucionis, Skirmantas; Ratcliffe, Peter J; Szele, Francis G; Tomlinson, Ian

2016-10-10

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1 R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1 R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The Us3 Protein of Herpes Simplex Virus 1 Inhibits T Cell Signaling by Confining Linker for Activation of T Cells (LAT) Activation via TRAF6 Protein*

PubMed Central

Yang, Yin; Wu, Songfang; Wang, Yu; Pan, Shuang; Lan, Bei; Liu, Yaohui; Zhang, Liming; Leng, Qianli; Chen, Da; Zhang, Cuizhu; He, Bin; Cao, Youjia

2015-01-01

Herpes simplex virus 1 (HSV-1) is the most prevalent human virus and causes global morbidity because the virus is able to infect multiple cell types. Remarkably, HSV infection switches between lytic and latent cycles, where T cells play a critical role. However, the precise way of virus-host interactions is incompletely understood. Here we report that HSV-1 productively infected Jurkat T-cells and inhibited antigen-induced T cell receptor activation. We discovered that HSV-1-encoded Us3 protein interrupted TCR signaling and interleukin-2 production by inactivation of the linker for activation of T cells. This study unveils a mechanism by which HSV-1 intrudes into early events of TCR-mediated cell signaling and may provide novel insights into HSV infection, during which the virus escapes from host immune surveillance. PMID:25907557

Disparities in reproductive outcomes according to the endometrial preparation protocol in frozen embryo transfer : The risk of early pregnancy loss in frozen embryo transfer cycles.

PubMed

Hatoum, I; Bellon, L; Swierkowski, N; Ouazana, M; Bouba, S; Fathallah, K; Paillusson, B; Bailly, M; Boitrelle, F; Alter, L; Bergère, M; Selva, J; Wainer, R

2018-03-01

The purpose of this study was to determine the effect of stimulated and artificial endometrial preparation protocols on reproductive outcomes in frozen embryo transfer (FET) cycles. We performed a retrospective study of 1926 FET cycles over a 3.5-year period in the Fertility Unit at a University Hospital. Stimulated and artificial protocols were used for endometrial preparation. The embryos for FET were obtained from either in vitro fertilization or intracytoplasmic sperm injection cycles. Live birth rate and early pregnancy loss rates were retrospectively compared. In artificial protocols, oral or vaginal administration of oestradiol 2 mg two or three times a day was followed by vaginal supplementation with progesterone 200 mg two or three times a day. In stimulated protocols, recombinant follicle-stimulating hormone was administered from day 4 onward. Vaginal ultrasound was used for endometrial and ovarian monitoring. A pregnancy test was performed 14 days after FET. If it was positive, oestradiol and progesterone were administered up until the 12th week of gestation in artificial cycles. We defined early pregnancy losses as biochemical pregnancies (preclinical losses) and miscarriages. Data on 865 artificial cycles (45% of the total) and 1061 stimulated cycles (55%) were collected. Early pregnancy loss rate was significantly lower for stimulated cycles (34.2%) than for artificial cycles (56.9%), and the live birth rate was significantly higher for stimulated cycles (59.7%) than for artificial cycles (29.1%). In frozen embryo transfer, artificial cycles were associated with more early pregnancy loss and lower live birth rate than stimulated cycles.

Virus replication cycle of white spot syndrome virus in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei.

PubMed

Li, Wenfeng; Desmarets, Lowiese M B; De Gryse, Gaëtan M A; Theuns, Sebastiaan; Van Tuan, Vo; Van Thuong, Khuong; Bossier, Peter; Nauwynck, Hans J

2015-09-01

The replication cycle of white spot syndrome virus (WSSV) was investigated in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei. The secondary cells formed a confluent monolayer at 24 h post-reseeding, and this monolayer could be maintained for 10 days with a viability of 90 %. Binding of WSSV to cells reached a maximum (73 ± 3 % of cells and 4.84 ± 0.2 virus particles per virus-binding cell) at 120 min at 4 °C. WSSV entered cells by endocytosis. The co-localization of WSSV and early endosomes was observed starting from 30 min post-inoculation (p.i.). Double indirect immunofluorescence staining showed that all cell-bound WSSV particles entered these cells in the period between 0 and 60 min p.i. and that the uncoating of WSSV occurred in the same period. After 1 h inoculation at 27 °C, the WSSV nucleocapsid protein VP664 and envelope protein VP28 started to be synthesized in the cytoplasm from 1 and 3 h p.i., and were transported into nuclei from 3 and 6 h p.i., respectively. The percentage of cells that were VP664- and VP28-positive in their nuclei peaked (50 ± 4 %) at 12 h p.i. Quantitative PCR showed that WSSV DNA started to be synthesized from 6 h p.i. In vivo titration of the supernatants showed that the progeny WSSV were released from 12 h p.i. and peaked at 18 h p.i. In conclusion, the secondary cell cultures from the lymphoid organ were proven to be ideal for examination of the replication cycle of WSSV.

Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

USGS Publications Warehouse

Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

2008-01-01

The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had ???8 cells at 84 hpa, while 32% of the bovine NT embryos had ???8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had ???8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

miR-181a and miR-630 regulate cisplatin-induced cancer cell death.

PubMed

Galluzzi, Lorenzo; Morselli, Eugenia; Vitale, Ilio; Kepp, Oliver; Senovilla, Laura; Criollo, Alfredo; Servant, Nicolas; Paccard, Caroline; Hupé, Philippe; Robert, Thomas; Ripoche, Hugues; Lazar, Vladimir; Harel-Bellan, Annick; Dessen, Philippe; Barillot, Emmanuel; Kroemer, Guido

2010-03-01

MicroRNAs (miRNA) are noncoding RNAs that regulate multiple cellular processes, including proliferation and apoptosis. We used microarray technology to identify miRNAs that were upregulated by non-small cell lung cancer (NSCLC) A549 cells in response to cisplatin (CDDP). The corresponding synthetic miRNA precursors (pre-miRNAs) per se were not lethal when transfected into A549 cells yet affected cell death induction by CDDP, C2-ceramide, cadmium, etoposide, and mitoxantrone in an inducer-specific fashion. Whereas synthetic miRNA inhibitors (anti-miRNAs) targeting miR-181a and miR-630 failed to modulate the response of A549 to CDDP, pre-miR-181a and pre-miR-630 enhanced and reduced CDDP-triggered cell death, respectively. Pre-miR-181a and pre-miR-630 consistently modulated mitochondrial/postmitochondrial steps of the intrinsic pathway of apoptosis, including Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3. In addition, pre-miR-630 blocked early manifestations of the DNA damage response, including the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and of two ATM substrates, histone H2AX and p53. Pharmacologic and genetic inhibition of p53 corroborated the hypothesis that pre-miR-630 (but not pre-miR-181a) blocks the upstream signaling pathways that are ignited by DNA damage and converge on p53 activation. Pre-miR-630 arrested A549 cells in the G0-G1 phase of the cell cycle, correlating with increased levels of the cell cycle inhibitor p27(Kip1) as well as with reduced proliferation rates and resulting in greatly diminished sensitivity of A549 cells to the late S-G2-M cell cycle arrest mediated by CDDP. Altogether, these results identify miR-181a and miR-630 as novel modulators of the CDDP response in NSCLC.

Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

PubMed

Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

2015-01-01

We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

A map of protein dynamics during cell-cycle progression and cell-cycle exit

PubMed Central

Gookin, Sara; Min, Mingwei; Phadke, Harsha; Chung, Mingyu; Moser, Justin; Miller, Iain; Carter, Dylan

2017-01-01

The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence. PMID:28892491