The increase in intracellular pH associated with Xenopus egg activation is a Ca(2+)-dependent wave

1992 ◽  
Vol 101 (1) ◽  
pp. 55-67 ◽  
Author(s):  
N. Grandin ◽  
M. Charbonneau
Keyword(s):  
Cell Cycle ◽  
Intracellular Ph ◽  
Egg Activation ◽  
Free Calcium ◽  
Slowing Down ◽  
M Phase ◽  
Xenopus Egg ◽  
General Slowing ◽  
The Impact ◽  
Kinetics Of

In Xenopus eggs, the transient increase in intracellular free calcium ([Ca2+]i), or Ca2+ transient, which occurs 1–3 min after egg activation, is likely to be partly responsible for the release of the cell cycle blockade. In the present study, we have used microinjection of BAPTA or EGTA, two potent chelators of Ca2+, to buffer [Ca2+]i at various steps during Xenopus egg activation and evaluate the impact on some of the associated events. Microinjection of either one of the Ca2+ chelators into unactivated eggs prevented egg activation without, however, lowering [Ca2+]i, suggesting that only physiological [Ca2+]i changes, but not [Ca2+]i levels, were affected by the Ca2+ buffer. When BAPTA was microinjected around the time of occurrence of the Ca2+ transient, the egg activation-associated increase in intracellular pH (pHi) was clearly delayed. That delay was not due to a general slowing down of the cell cycle, since under the same conditions of microinjection of BAPTA the kinetics of MPF (a universal M-phase promoting factor) inactivation were unaffected. These results represent the first indication that the Ca2+ transient participates in determining the time of initiation of the pHi increase during Xenopus egg activation. The present results also demonstrate that the egg activation-associated pHi changes (a slight, transient decrease in pHi followed by a permanent increase in pHi) proceed as a wave propagating from the site of triggering of egg activation. Experiments of local microinjection of BAPTA support the view that the pH wave is a consequence of the Ca2+ wave, which it follows closely.

Intracellular pH and intracellular free calcium responses to protein kinase C activators and inhibitors in Xenopus eggs

Development ◽  
1991 ◽  
Vol 112 (2) ◽  
pp. 461-470 ◽  
Author(s):  
N. Grandin ◽  
M. Charbonneau
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Ph ◽  
Phorbol Esters ◽  
Intracellular Free Calcium ◽  
Egg Activation ◽  
Cortical Granule ◽  
Free Calcium ◽  
Kinase C

Cell activation during fertilization of the egg of Xenopus laevis is accompanied by various metabolic changes, including a permanent increase in intracellular pH (pHi) and a transient increase in intracellular free calcium activity ([Ca2+]i). Recently, it has been proposed that protein kinase C (PKC) is an integral component of the Xenopus fertilization pathway (Bement and Capco, J. Cell Biol. 108, 885–892, 1989). Indeed, activators of PKC trigger cortical granule exocytosis and cortical contraction, two events of egg activation, without, however, releasing the cell cycle arrest (blocked in second metaphase of meiosis). In the egg of Xenopus, exocytosis as well as cell cycle reinitiation are supposed to be triggered by the intracellular Ca2+ transient. We report here that PKC activators do not induce the intracellular Ca2+ transient, or the activation-associated increase in pHi. These results suggest that the ionic responses to egg activation in Xenopus do not appear to depend on the activation of PKC. In addition, in eggs already pretreated with phorbol esters, those artificial activators that act by releasing Ca2+ intracellularly, triggered a diminished increase in pHi. Finally, sphingosine and staurosporine, two potent inhibitors of PKC, were found to trigger egg activation, suggesting that a decrease in PKC activity might be an essential event in the release of the metaphase block, in agreement with recent findings on the release of the prophase block in Xenopus oocytes (Varnold and Smith, Development 109, 597–604, 1990).

scholarly journals Intracellular free calcium oscillates during cell division of Xenopus embryos.

1991 ◽  
Vol 112 (4) ◽  
pp. 711-718 ◽  
Author(s):  
N Grandin ◽  
M Charbonneau
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Volume ◽  
Intracellular Ph ◽  
Calcium Ions ◽  
Volume Ratio ◽  
Mean Amplitude ◽  
Free Calcium ◽  
Periodic Oscillations ◽  
Xenopus Embryos

In Xenopus embryos, previous results failed to detect changes in the activity of free calcium ions (Ca2+i) during cell division using Ca2(+)-selective microelectrodes, while experiments with aequorin yielded uncertain results complicated by the variation during cell division of the aequorin concentration to cell volume ratio. We now report, using Ca2(+)-selective microelectrodes, that cell division in Xenopus embryos is accompanied by periodic oscillations of the Ca2+i level, which occur with a periodicity of 30 min, equal to that of the cell cycle. These Ca2+i oscillations were detected in 24 out of 35 experiments, and had a mean amplitude of 70 nM, around a basal Ca2+i level of 0.40 microM. Ca2+i oscillations did not take place in the absence of cell division, either in artificially activated eggs or in cleavage-blocked embryos. Therefore, Ca2+i oscillations do not represent, unlike intracellular pH oscillations (Grandin, N., and M. Charbonneau. J. Cell Biol. 111:523-532. 1990), a component of the basic cell cycle ("cytoplasmic clock" or "master oscillator"), but appear to be more likely related to some events of mitosis.

Overexpressed miR-375-Loaded Restrains Development of Cervical Cancer Through Down-Regulation of Frizzled Class Receptor 4 (FZD4) with Liposome Nanoparticle as a Carrier

2021 ◽  
Vol 17 (9) ◽  
pp. 1882-1889
Author(s):  
Suqin Wang ◽  
Lina Xu ◽  
Zhiqiang Zhang ◽  
Ping Wang ◽  
Rong Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
M Phase ◽  
The Impact

Dysregulation expression of miR-375 is noted to correlate with progression of cervical cancer. This study attempted to investigate the impact of overexpressed miR-375-loaded liposome nanoparticles on proliferation of cervical cancer (CC), to provide an insight on pathogenesis of CC disorder. CC cells were co-cultured with pure liposome nanoparticles (empty vector group), miR-375 agonist-loaded liposome nanoparticles, or transfected with miR-375 antagonist. Besides, some cells were exposed to TGF-β/Smads signaling pathway inhibitor or activator whilst cell proliferation was assessed by MTT assay, and expressions of FZD4 and miR-375 were determined. Western blot analysis was carried out to detect the expression of TGF-β pathway factors (TGF-β, Smad2, Smad7, p-Smad2) and its downstream Smads pathway. The interaction between miR-375 and FZD4 was evaluated by dual-luciferase reporter gene assay. Overexpression of miR-375 induced arrest at the G0/G1 phase of cell cycle and elevation of Smad2 protein expression (P <0.05), with lower expressions of TGF-β, Smad7, p-Smad2, and FZD4, while transfection with miR-375 inhibitor exhibited opposite activity. Presence of miR-375 agonist-loaded liposome nanoparticles induced decreased cell proliferation. There was a targeting relationship between miR-375 and FZD4, and administration with TGF-β/Smads agonist resulted in increased miR-375 and Smad2 expressions, as well as decreased TGF-β, Smad7, p-Smad2, FZD4 protein expression, and the number of S phase and G2/M phase cells (P < 0.05). The signaling inhibitor oppositely suppressed cell proliferation decreasing miR-375 expression. miR-375-loaded liposome nanoparticles activated TGF-β/Smads signaling pathway to restrain cell cycle and suppress cell division, and proliferation through targeting FZD4 in CC. Its molecular mechanism is related to activation of TGF-β/Smads signaling pathway.

scholarly journals Cell Cycle Perturbations Induced by Infection with the Coronavirus Infectious Bronchitis Virus and Their Effect on Virus Replication

2006 ◽  
Vol 80 (8) ◽  
pp. 4147-4156 ◽  
Author(s):  
Brian Dove ◽  
Gavin Brooks ◽  
Katrina Bicknell ◽  
Torsten Wurm ◽  
Julian A. Hiscox
Keyword(s):  
Cell Cycle ◽  
Virus Replication ◽  
Infectious Bronchitis Virus ◽  
Rna Virus ◽  
Positive Strand ◽  
Infectious Bronchitis ◽  
M Phase ◽  
Infected Cells ◽  
Positive Strand Rna ◽  
The Impact

ABSTRACT In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G1 regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G2/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the G0 phase or asynchronously replicating cells. Our data suggested that IBV induces a G2/M phase arrest in infected cells to promote favorable conditions for viral replication.

Timing of calcium and protein synthesis requirements for the first mitotic cell cycle in fertilised Xenopus eggs

1999 ◽  
Vol 112 (22) ◽  
pp. 3975-3984
Author(s):  
C. Beckhelling ◽  
C. Penny ◽  
S. Clyde ◽  
C. Ford
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Calcium Concentration ◽  
Mitotic Cell ◽  
Sensitive Period ◽  
Free Calcium ◽  
Calcium Buffer ◽  
M Phase ◽  
Egg Extracts ◽  
Histone Kinase

Mitosis is governed by the activity of the M-phase promoting factor (MPF). In some systems, particularly early embryos, transient increases in calcium concentration have been shown to be necessary for mitosis and regulate its timing. By microinjection of the calcium buffer, dibromoBAPTA, into fertilised Xenopus eggs, we have assessed whether calcium events are required to initiate MPF activation and inactivation. Since initial experiments showed that this buffer inhibited protein synthesis, we measured when mitosis and cleavage became independent of translation. We found that, after a period of protein synthesis essential for cleavage, there was a phase during which continued translation affected the timing of cleavage, but was not essential for its occurrence. Measurement of MPF activity in single embryos injected with calcium buffer at different times in the first cell cycle, showed that there were two sensitive periods. The first period of sensitivity blocked MPF activation and coincided with the time at which cleavage became completely independent of protein synthesis. The second sensitive period occurred just before histone kinase activity peaked, and was necessary for kinase inactivation. Preventing inactivation in this way arrested egg extracts in mitosis. These results support the view that transient increases in free calcium concentration contribute to mitotic progression by first triggering MPF activation and subsequently, with elevated MPF activity, inducing its inactivation.

Phosphatidylcholine catabolism in the MCF-7 cell cycle

2006 ◽  
Vol 84 (5) ◽  
pp. 737-744 ◽  
Author(s):  
Weiyang Lin ◽  
Gilbert Arthur
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Specific Cell ◽  
Synchronized Cells ◽  
M Phase ◽  
G2 Phase ◽  
Cell Cycle Phases ◽  
Kinetics Of ◽  
Mcf 7

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.

scholarly journals Drosophila PLUTONIUM protein is a specialized cell cycle regulator required at the onset of embryogenesis.

1997 ◽  
Vol 8 (4) ◽  
pp. 583-593 ◽  
Author(s):  
L K Elfring ◽  
J M Axton ◽  
D D Fenger ◽  
A W Page ◽  
J L Carminati ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein A ◽  
Early Embryo ◽  
S Phase ◽  
Egg Activation ◽  
Direct Role ◽  
Eye Disc ◽  
Early Embryos ◽  
M Phase ◽  
Giant Nuclei

Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The PLU protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, PLU did not perturb the cell cycle, suggesting that PLU regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. We show that functional png is needed for the presence of PLU protein. By analyzing png mutations of differing severity, we find that the extent of the png mutant phenotype inversely reflects the level of PLU protein. Our data suggest that PLU protein is required at the time of egg activation and the completion of meiosis.

Negative Regulation of Cell Cycle Kinetics of Human Hematopoietic Cells by the Transcription Factor Dmtf1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3359-3359
Author(s):  
Michihiro Kobayashi ◽  
Edward F. Srour
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cord Blood ◽  
Hematopoietic Cells ◽  
Dominant Negative ◽  
Cell Cycle Kinetics ◽  
Cd34 Cells ◽  
The Impact ◽  
Kinetics Of ◽  
Time Point

Abstract Hematopoietic stem cells (HSCs) are predominantly quiescent with only a small number entering active phases of cell cycle at any time point. With such tightly regulated cell cycle kinetics, HSCs ensure preservation of the stem cell pool. Many cell cycle-related proteins, mainly tumor suppressor genes, are involved in maintenance of HSCs quiescence. Dmtf1 (Cyclin D-binding Myb-like Protein1) is a transcription factor that negatively regulates cell cycle by inducing Arf expression, and its deletion has been reported in some leukemias (Bodner SM et al, 1999). As there is no information regarding the role of Dmtf1 in hematopoiesis, we examined the impact of Dmtf1 on regulating cell cycle kinetics of hematopoietic progenitor cells. Dmtf1 mRNA was expressed in human granulocytes, lymphocytes, CD34+ cells, and in murine Sca1+lin-CD117+ (KSL) cells. Using retroviral vectors (MIEG3/IRES-GFP), we first investigated cell cycle progression in 293 cells transduced with four different constructs; GFP only control vector (−), wild type Dmtf1 (WT), and two dominant negative mutants expressing a point mutation (K319E) or a deleted myb-like repeat box (dMHR). A total of 36.0% of sorted and cultured GFP+ control (−) cells were in S/G2+M 24hr after initiation of culture. Whereas 30.8% of cells expressing (WT) were in S/G2+M at the same time point, expression of the two dominant negative mutants K319E and dMHR induced 46.6% and 45.5% of the cells into S/G2+M, respectively suggesting that loss of Dmtf1 activity results in rapid cell proliferation. Interestingly, a 2.5-fold increase in the ecxpression of Arf and p21 mesured by qPCR was detected in cells transduced with WT only whereas other transduced cells did not show any change in expression of both Arf and p21. The impact of these constructs was then evaluated in cord blood cells using CFU assays. Cord blood CD34+ cells were transduced with the four vectors mentioned above and GFP+ cells were subsequently sorted and cultured. Both K319E and dMHR induced a 25% increase in the number of clonogenic progenitors relative to (−) while a modest decrease of 10% in colony numbers was detected in the WT group. Cells cycle analysis of these cells is currently under investigation. These results demonstrate that Dmtf1 acts as a negative regulator of cell cycle control in hematopoietic cells and suggests that it may play a role in the maintenance of HSC quiescence.

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