Cell cycle analysis kinetics

Cell Cycle Analysis and Kinetics Apoptosis Calcium Flux Chromosome Analysis Micronuclei Analysis Intracellular pH Intracellular Glutathione Oxidative Burst Cell Viability... 

Fig. 8.7. Reprinted (A,D) from Dean PN (1987). Data analysis in cell kinetics. Gray JW and Darzynkiewicz Z (eds). Techniques in Cell Cycle Analysis. Clifton, NJ Humana Press, pp 207-253 and (B,C) from Dean PN (1985). Methods of data analysis in flow cytometry. Van Dilla MA, et al. (eds). Flow Cytometry Instrumentation and Data Analysis. London Academic Press, pp 195-221.

Boehmer, R. M. How cytometric cell cycle analysis using the quenching of Hoechst 33258 fluorescence by bromodeoxyuridine incorporation. Cell Tissue Kinet. 1979,12,101-110. 

Flow cytometry (FCM) is widely used for exploring mechanism of action of compounds that compromise proliferation since it is rapid, accurate and usable for any cellular context [5], In this chapter we want to point out technical and strategic aspects of use of FCM for cell cycle studies of a putative anticancer agent. As an example we used Edotecarin, a topi inhibitor, firstly evaluating proliferation outcome and classical DNA content analysis by propidium iodide, and then since the compound treatment produced cell cycle perturbation difficult to interprete, a two-parametric analysis by 5-bromo-deoxyuridine (BrdU) was applied for separating cell cycle phases. Moreover we put our efforts into identifing specific cell cycle arrest not easily demonstrable by previously described methods, through the use of in vitro kinetics ( pulse and chase ). Finally, in vivo assessment of efficacy and biomarkers modulation after treatment was analyzed. 

Terry NH, White RA. Cell cycle kinetics estimated by analysis of bromodeoxyuridine incorporation. Methods Cell Biol. 2001 63 355-74. 

Gong J, Traganos F, Darzynkiewicz Z. 1993. Simultaneous analysis of cell cycle kinetics at two different DNA ploidy levels based on DNA content and cyclin B measurements. 

Temin (1970) and Todaro et al. (1965) showed similar effects for chicken fibroblasts and 3T3 mouse fibroblasts. The low level of serum is important for survival as well as for the subsequent stimulation of DNA synthesis (Cherrington, 1984). A kinetic analysis using time lapse cinematography (Zetterberg and Larsson, 1985) showed that Swiss 3T3 cells were only susceptible to cell cycle arrest in a short period (3-4 h) following mitosis. Even a 1-h exposure to serum-free medium during this time forced the cells into GO from which they required 8 h to return to Gl. The length of the postmitotic sensitive phase was very constant at between 3 and 4 h but considerable intercellular variability existed in the duration of the pre S-phase Gl period consistent with a transition probability event ( 10.4). 

K.C. Chen, A. Csikasz-Nagy, B. Gyorffy, J. Val, B. Novak, J.J. Tyson. Kinetic analysis of a molecular model of the budding yeast cell cycle. Mol Biol Cell 11(1), 369-391 (2000)... 

Cell Division Disruption. If a cell cycle kinetic analysis reveals the herbicide effect is not caused by inhibition, the effect may be on mitosis. In this instance, the kinetic analysis will identify mitotic stages not recognizable as prophase, metaphase, anaphase, or telophase. 

Some tours deforce of these methods have been presented in several publications, (see [6,7] and references therein). The studies of Tyson and coworkers are focused on the kinetic analysis of the budding yeast cell cycle. The molecular mechanism of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. Many experiments have been done on this system over many years there are about 125 references cited in [6]. The biological details are second to stressing the enormity of this task. The model has nearly twenty variables and that many kinetic equations, and there are about fifty parameters (rate coefficients, binding constants, thresholds, relative efficiencies). A fair number of assumptions need to be made in the cases of absence of any substantiating experimental evidence, and a fair number of approximations need to be made to simplify the kinetic equations. The complexity of this system is indicated in fig. 13.3 and its caption. 

There is almost no biochemical reaction in a cell that is not catalyzed by an enzyme. (An enzyme is a specialized protein that increases the flux of a biochemical reaction by facilitating a mechanism [or mechanisms] for the reaction to proceed more rapidly than it would without the enzyme.) While the concept of an enzyme-mediated kinetic mechanism for a biochemical reaction was introduced in the previous chapter, this chapter explores the action of enzymes in greater detail than we have seen so far. Specifically, catalytic cycles associated with enzyme mechanisms are examined non-equilibrium steady state and transient kinetics of enzyme-mediated reactions are studied an asymptotic analysis of the fast and slow timescales of the Michaelis-Menten mechanism is presented and the concepts of cooperativity and hysteresis in enzyme kinetics are introduced. 

The specific system that we use to explore these questions is the conventional myosin, also termed myosin 11, which plays key physiological functions in muscle contraction and cell division. It is an ideal system for in-depth theoretical and computational analysis because its structural and kinetic properties have been characterized by a large body of diverse experimental techniques." For example, at the time our research was initiated, myosin 11 was one of the few motor systems for which high-resolution x-ray structures are available for multiple functional states " since then, multiple high-resolution x-ray structures have also been obtained for myosin V and VI, two other widely studied members of the myosin superfamily that are more processive in nature compared to myosin 11. The functional cycle of myosin 11 is best described by the celebrated Lymn-Taylor schane (Figure 2.1a)," in which... 

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