Cellular activities kinetic analysis

The reversibility of QM adducts also creates numerous challenges. For example, measuring the full burden of DNA alkylation by a QM can be obscured by the loss of its labile products during or before chemical identification can be completed. Results from a deoxynucleotide model system indicated that only a small fraction of the possible adducts could be measured after the interval required for analysis of DNA. Perhaps the kinetic products of QMs also contribute to the cellular activity of these intermediates although this has yet to be explored. QM equivalents can be envisioned to migrate from one reversible nucleophile such as the N1 of adenine in such cofactors as ATP to another until quenched by a compound such as glutathione that is present in cells as a defense against undesirable electrophiles. 

Majmudar, J.D., et al. (2011). Amide-modified prenylcysteine based Icmt inhibitors structure-activity relationships, kinetic analysis and cellular characterization. Bioorg Med Chem. (in press). 

As one of its major interests, a model represents an efficient tool for the kinetic analysis of cellular processes. It is able to account for the main phenomena that may simultaneously control the activities of cells. As such, depending on the culture conditions, composition of the medium and whether there is batch or continuous mode of operation, it can be used first to identify the rate-limiting factors and then to characterize quantitatively their relative importance. For instance, with a model it is possible to evaluate the kinetic effect of a depletion of glucose, glutamine and other amino acids or of an accumulation of ammonia and lactate on the rates of cell growth and death. 

Infection of cultured cells with many lytic viruses results in a marked decrease in the rate of cellular protein synthesis. Usually, this decrease is accompanied by increasing rates of viral protein synthesis, marked cytopathic effects, and ultimately cell death. In most cases, it is not known whether the shut-off of host cell protein synthesis results from an active process induced by the virus evolved for that (or some other) purpose, or whether it is merely a passive result of another viral function, such as production of large quantities of viral mRNA which compete effectively with their cellular counterparts. In the case of poliovirus, however, three types of studies suggested that the former, active type of mechanism was at work. Kinetic analysis of the rate of protein synthesis in cells synchronously infected with high multiplicities of virus showed that cellular protein synthesis could be virtually completely inhibited prior to the synthesis of significant quantities of viral RNA and protein (Summers et ai, 1965). In addition, infection in the presence of 1-3 mM guanidine, which prevents detectable replication of viral RNA, nevertheless results in viral inhibition of host cell protein synthesis (Holland, 1%4 ... 

Instruments of this type may also be used quite effectively to evaluate kinetics of time-dependent changes in foods, be they enzymatic or reactive changes of other types. The computerized data-acquisition capabilities of these instruments allow precise measurement of absorbance or fluorescence changes, often over very brief time periods ( milliseconds). This is particularly useful for analysis of fluorescence decay rates, and in measurement of enzymatic activity in situ. A number of enzyme substrates is available commercially which, although non-fluorescent initially, release fluorescent reaction products after hydrolysis by appropriate enzymes. This kinetic approach is a relatively underused capability of computerized microspectrophotometers, but one which has considerable capability for comparing activities in individual cells or cellular components. Fluorescein diacetate, for example, is a non-fluorescent compound which releases intensely fluorescent fluorescein on hydrolysis. This product is readily quantified in individual cells which have high levels of esterase [50]. Changes in surface or internal color of foods may also be evaluated over time by these methods. 

In vitro experiments demonstrated that reduced cellular uptake of [ Tc]MIBI is also related with overexpression of MRPl. Utsunomiya et al. studied the uptake and efflux kinetics of [ Tc]MIBI in a nasopharyngeal carcinoma cell line (CNE-1) [68]. Western blot and RT-PCR analysis indicated that these cells express moderate levels of both MRPl and MRP2 but not Pgp. The maximal accumulation [ Tc]MIBI, calculated as the ratio of radioactivity concentration inside the cell to that found in the supernatant (C n/Cout), was 6.3 and increased significantly after inhibition with verapamil and CsA when compared with control. The efflux rate of [ Tc]MIBI diminished significantly after addition of verapamil or CsA that are both not selective for Pgp and exert inhibitor effects on MRPl activity. Additionally, PSC833 and GG918 did not elicit significant alterations on the efflux rate [ Tc]MIBI due to their specificity for Pgp. 

---