Determination of Cell Cycle Stage and Mitotic Exit Through the Quantification of the Protein Levels of Known Mitotic Regulators

2016 ◽  
pp. 45-57 ◽  
Author(s):  
Cristina Cepeda-García
Keyword(s):  
Cell Cycle ◽  
Mitotic Exit ◽  
Cell Cycle Stage ◽  
Protein Levels

scholarly journals Anaphase-Promoting Complex/Cyclosome Controls the Stability of TPX2 during Mitotic Exit

2005 ◽  
Vol 25 (23) ◽  
pp. 10516-10527 ◽  
Author(s):  
Scott Stewart ◽  
Guowei Fang
Keyword(s):  
Cell Cycle ◽  
Spindle Assembly ◽  
Mitotic Exit ◽  
Anaphase Promoting Complex ◽  
Discrete Elements ◽  
Protein Levels ◽  
Recognition Motifs ◽  
The Stability

ABSTRACT TPX2, a microtubule-associated protein, is required downstream of Ran-GTP to induce spindle assembly. TPX2 activity appears to be tightly regulated during the cell cycle, and we report here one molecular mechanism for this regulation. We found that TPX2 protein levels are cell cycle regulated, peaking in mitosis and declining sharply during mitotic exit. TPX2 is degraded in mitotic extracts, as well as in HeLa cells exiting from mitosis. This instability depends, both in vitro and in vivo, on the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. In a reconstituted system, TPX2 is efficiently ubiquitinated by APC/C that has been activated by Cdh1. Two discrete elements in TPX2 are required for recognition by APC/CCdh1: a KEN box and a novel element in amino acids 1 to 86. Interestingly, the latter element, which has no known APC/C recognition motifs, is required for the ubiquitination of TPX2 by APC/CCdh1 in vitro and for its degradation in vivo. We conclude that APC/CCdh1 controls the stability of TPX2, thereby ensuring accurate regulation of the spindle assembly in the cell cycle.

scholarly journals Isoflurane Causes Greater Neurodegeneration Than an Equivalent Exposure of Sevoflurane in the Developing Brain of Neonatal Mice

2010 ◽  
Vol 112 (6) ◽  
pp. 1325-1334 ◽  
Author(s):  
Ge Liang ◽  
Christopher Ward ◽  
Jun Peng ◽  
Yifan Zhao ◽  
Baosheng Huang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Brain Regions ◽  
Learning Ability ◽  
Memory And Learning ◽  
Protein Levels ◽  
Neonatal Mice ◽  
Cell Cycle Regulatory Proteins ◽  
Related Proteins ◽  
Old Mice

Background We hypothesized that isoflurane has a greater potency to induce neurodegeneration than sevoflurane in the developing brains of neonatal mice based on our previous studies in cell culture. Methods We treated 7-day-old mice with either 0.75% isoflurane or 1.1% sevoflurane ( approximately 0.5 minimum alveolar concentration) for 6 h and then obtained blood and brain samples at 2 h after the anesthesia treatment for determination of neuroapoptosis in different brain regions and the neurodegenerative biomarker S100beta in the blood. The mechanisms of neurodegeneration induced by isoflurane or sevoflurane were also compared by determining protein expressions of the cell cycle and apoptosis-related proteins. In separate groups, memory and learning ability were evaluated through the use of Morris Water Maze testing in mice at postnatal day 42 after anesthesia treatment at postnatal day 7. Results Isoflurane but not sevoflurane significantly increased the neurodegenerative biomarker S100beta in the blood. Isoflurane treatments significantly increased apoptosis indicated by the activation of caspase-3 and elevation of poly-(ADP-ribose) polymerase in different brain regions. An equipotent exposure of sevoflurane tended to increase apoptosis in hippocampal and cortex areas but was significantly less potent than isoflurane. Neither isoflurane nor sevoflurane significantly changed protein levels of glyceraldehyde-3-phosphate dehydrogenase, beta-site amyloid beta-precursor protein-cleaving enzyme, and cell cycle regulatory proteins (CDK4, cyclin D1). Isoflurane and sevoflurane at the selected exposures did not significantly alter memory and learning ability. Conclusion At equipotent exposures, isoflurane has a greater potency than sevoflurane to cause neurodegeneration in the developing brains of neonatal mice.

scholarly journals Systems Level Modeling of the Cell Cycle Using Budding Yeast

2007 ◽  
Vol 3 ◽  
pp. 117693510700300 ◽  
Author(s):  
B.P. Ingalls ◽  
B.P. Duncker ◽  
D.R. Kim ◽  
B.J. McConkey
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Human Cancer ◽  
Quality Data ◽  
Cell Cycle Gene ◽  
Mathematical Framework ◽  
Gene Transcript ◽  
Proteomics Data ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Levels

Proteins involved in the regulation of the cell cycle are highly conserved across all eukaryotes, and so a relatively simple eukaryote such as yeast can provide insight into a variety of cell cycle perturbations including those that occur in human cancer. To date, the budding yeast Saccharomyces cerevisiae has provided the largest amount of experimental and modeling data on the progression of the cell cycle, making it a logical choice for in-depth studies of this process. Moreover, the advent of methods for collection of high-throughput genome, transcriptome, and proteome data has provided a means to collect and precisely quantify simultaneous cell cycle gene transcript and protein levels, permitting modeling of the cell cycle on the systems level. With the appropriate mathematical framework and sufficient and accurate data on cell cycle components, it should be possible to create a model of the cell cycle that not only effectively describes its operation, but can also predict responses to perturbations such as variation in protein levels and responses to external stimuli including targeted inhibition by drugs. In this review, we summarize existing data on the yeast cell cycle, proteomics technologies for quantifying cell cycle proteins, and the mathematical frameworks that can integrate this data into representative and effective models. Systems level modeling of the cell cycle will require the integration of high-quality data with the appropriate mathematical framework, which can currently be attained through the combination of dynamic modeling based on proteomics data and using yeast as a model organism.

scholarly journals Genetic and Biochemical Evaluation of the Importance of Cdc6 in Regulating Mitotic Exit

2003 ◽  
Vol 14 (11) ◽  
pp. 4592-4604 ◽  
Author(s):  
Vincent Archambault ◽  
Caihong X. Li ◽  
Alan J. Tackett ◽  
Ralph Wäsch ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Exit ◽  
Terminal Region ◽  
Cyclin Dependent Kinase ◽  
Replication Control ◽  
Mitotic Cyclin ◽  
Biochemical Evaluation ◽  
Inhibitory Domain ◽  
Late Mitosis ◽  
Control Protein

We evaluated the hypothesis that the N-terminal region of the replication control protein Cdc6 acts as an inhibitor of cyclin-dependent kinase (Cdk) activity, promoting mitotic exit. Cdc6 accumulation is restricted to the period from mid-cell cycle until the succeeding G1, due to proteolytic control that requires the Cdc6 N-terminal region. During late mitosis, Cdc6 is present at levels comparable with Sic1 and binds specifically to the mitotic cyclin Clb2. Moderate overexpression of Cdc6 promotes viability of CLB2Δdb strains, which otherwise arrest at mitotic exit, and rescue is dependent on the N-terminal putative Cdk-inhibitory domain. These observations support the potential for Cdc6 to inhibit Clb2-Cdk, thus promoting mitotic exit. Consistent with this idea, we observed a cytokinesis defect in cdh1Δ sic1Δ cdc6Δ2–49 triple mutants. However, we were able to construct viable strains, in three different backgrounds, containing neither SIC1 nor the Cdc6 Cdk-inhibitory domain, in contradiction to previous work. We conclude, therefore, that although both Cdc6 and Sic1 have the potential to facilitate mitotic exit by inhibiting Clb2-Cdk, mitotic exit nevertheless does not require any identified stoichiometric inhibitor of Cdk activity.

Label-Free Determination of the Cell Cycle Phase in Human Embryonic Stem Cells by Raman Microspectroscopy

2013 ◽  
Vol 85 (19) ◽  
pp. 8996-9002 ◽  
Author(s):  
Stanislav O. Konorov ◽  
H. Georg Schulze ◽  
James M. Piret ◽  
Michael W. Blades ◽  
Robin F. B. Turner
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Raman Microspectroscopy ◽  
Cell Cycle Phase ◽  
Cycle Phase ◽  
Label Free

Simultaneous Determination of α-Fetoprotein and Immunoglobulin Levels From a Microhematocrit Capillary Tube

PEDIATRICS ◽  
1978 ◽  
Vol 61 (2) ◽  
pp. 301-303
Author(s):  
Alfried Kohlschütter
Keyword(s):  
Simultaneous Determination ◽  
Preterm Infants ◽  
Gestational Age ◽  
Capillary Tube ◽  
Long Island ◽  
Internal Diameter ◽  
Protein Levels ◽  
Immunoglobulin Levels ◽  
Blood Volumes

The α-fetoprotein (AFP) concentration in serum is an indicator of gestational age in preterm infants.1 IgM and IgA concentrations are helpful in the diagnosis of perinatal infections.2,3 Determination of all three protein levels can conveniently be made from one single microhematocrit capillary tube. The small blood volumes involved and the simplicity of the procedure make it a tool for routine use in newborn nurseries. The technique described can also be used for the determination of many other compounds. MATERIALS Heparinized microhematocrit capillary tubes (Propper Manufacturing Co., Inc., Long Island, N. Y.) are 75 mm long and have an internal diameter of 1.1 to 1.2 mm.

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