Time-course experiments

Principle Phytotoxic effects of the aqueous leachate of an allelopathic plant can be tested in vitro bioassays. Test or target plants are placed in contact with 0.5 % aqueous leachate from the allelopathic plant. Germination and radicle growth can be monitored during time-course experiments (i.e. after 24,48 and 72 h of treatment), but in this chapter we will include only the results obtained after 72 h of treatment. 

The ninhydrin reaction (see Basic Protocol 1), the TNBS reaction (see Alternate Protocol 1), the fluorescamine reaction (see Alternate Protocol 2), and formol titration (see Alternate Protocol 3) all evaluate released amino groups by comparing the amounts of free amino groups before and after hydrolysis. The first three methods are spectro-photometric techniques, whereas the fourth is a potentiometric technique. The first and second are chromogenic techniques, whereas the third is fluorometric. These techniques are usually performed as time-course experiments. As the hydrolysis reaction proceeds, aliquots (samples) of the reaction are taken periodically and treated with a test reagent. Products of this reaction are proportional to the amount of free amino groups at each time point. 

The depicted time-course experiment would be appropriate if identifying differentially expressed genes at any time-point compared to vehicle and temporal expression changes within treatment (including vehicle) compared to an adjacent time-point Population comparisons can be carried out between parent tumor samples (Tn) and metastases (Vn), as well as between all parent and metastases... 

The system chosen to conduct CYP inhibition studies should be well characterized. This procedure requires initial time-course experiments and determination of linearity of metabolite formation with the chosen incubation time and enzyme concentration. After these experiments, the kinetic parameters (i.e., Km and Vmax) for each substrate used with six or more concentrations spanning from 1/3 to 3 A), and inhibition potencies (i.e., IC50 or K,) of typical inhibitors should be determined. This characterization does not need to be repeated for each batch or lot of test system. 

Identification of the 5-HT2C receptor as a 60-kDa N-glycosylated protein in the choroid plexus and hippocampus has also been carried out in another study (145). When rat choroid plexus and hippocampal membrane lysates were treated with PNGase F, 5-HT2C receptor polypeptides from both tissues were converted to a sharp 38-kDa band. In a time-course experiment performed in the choroid plexus,... 

In the murine model of septic shock, DS-96 afforded significantly better protection than PMB at 4 mg/kg (p<0.01, Ch-square test (see manuscript)) (Fig. 12.18a). We confirmed that the protection afforded by DS-96 was attributable to attenuated LPS-induced cytokine production. DS-96 was without any effect on lethality induced by 100 ng/animal of recombinant murine TNF-a. Furthermore, an in vivo Schild-type profile was observed with multiples of LD100 doses of LPS requiring escalating doses of DS-96 for protection which clearly show the specificity of the compound as an LPS-sequestrant (Fig. 12.18b). In time-course experiments, DS-96 was maximally effective when administered concurrent to, or up to 4 h prior to LPS administration, partial protection persisted even up to 8 h prior to LPS challenge implying a favorable half-life (Fig. 12.19). This has been confirmed by pharmacokinetic (PK) experiments (see below). 

Incubate at room temperature, agitating every minute or so. It is advisable to perform time course experiments to investigate linearity of the reaction, but 15-20 min incubation periods are usually sufficient. 

Time course experiments can be performed using different classes of control toxins to determine the length of exposure necessary to induce apoptosis or result in necrotic cell death. A toxin with properties that disrupt cell membranes will result in rapid necrotic cell death. Other chemicals may not become toxic until after conversion by modifying enzymes in the cytoplasm. In many cases, subpopulations of cells at different stages of the cell cycle may undergo cell death at different times. 

The final or equilibrium adsorption levels in time course experiments are listed. For Silastic, adsorption values obtained after 1 to 25 hrs were averaged to obtain the listed value. For poly (HEM A)/Silastic and poly (N VP)/Silastic, adsorption after 8 to 45 hrs were averaged to obtain the listed value. 

Pellet cells and wash once with 2 mL PBS. Resuspend the pellet in 300 pL ice-cold PBS. Add 700 pL ice-cold ethanol, again while the cells are being vortexed. The cells can now be stored in the refrigerator for up to 2 wk if the cells are to be used immediately, they must be in this ethanol fixative for at least 30 min on ice. The ability to keep the fixed cells for a period of time means that time-course experiments can be undertaken and the samples then processed for end-labeling at the same time. 

The length of the post-treatment time is dependent on the type of crosslinking agent used and the peak of ISC formation. A time-course experiment should be performed to ascertain this. For example, following a 1-h treatment the peak of ISC for chlorambucil is reached following a 3-h post-incubation, while cisplatin requires a post-incubation of 6-12 h. For repair experiments, the post-treatment time can be further extended. 

The HCV helicase assay is linearly dependent on enzyme concentration up to 60 fmol with 250 fmol of dsRNA substrate (Fig. 3). The optimal time of incubation for the assay was determined by a time course experiment. Capture oligomer-coated FlashPlatePLUS wells containing 250 fmol of dsRNA and 10 fmol of helicase per reaction were incubated at 37°C, with stop solution being added at the time points shown in Fig. 4. The formation of ssRNA product as measured by cpm or graphed as percent of displaced ssRNA product was linearly dependent on time up to 60 min. Based on these data, 250 fmol of dsRNA... 

We recommend performing two different immunoblot experiments First, a dose response experiment to determine an optimal dose of inhibitor, i.e., the lowest concentration that yields maximal response and second, a time course experiment that captures the effects of inhibitors (used at optimal dose) at various time points after TCR stimulation. Efficacious PTP inhibitors not only are expected to augment phosphorylation levels of substrates, but also to induce a sustained effect on these molecules, compared to the vehicle control. For instance, treatment of Jmkat T cells with the chemical probe MLl 19 [21 ] resulted in augmented and sustained phosphorylation of the MAP kinases ERKl/2 and p38 [22]. This effect was due to direct inhibition of HePTP PTPN7), a classical PTP that specifically dephosphorylates the phosphotyrosine residue in the activation loop of ERKl/2 andp38. 

FIG U RE 4.8 A demonstration of the speed of RapidFire system. The 24 samples (in triplicates) for the 0-60min time course experiment were analyzed under 2.5min. (Courtesy of BioTrove, Woburn, MA, http //www.biotrove.com. With permission.)... 

Time course experiments demonstrated a time dependent increase in insulin-like material extracted from both cells and medium (17). 

Fig. 5.15 Inhibitor experiment for the demonstration of substrates used by sulfate reducing bacteria in a coastal marine sediment. The concentrations of volatile fatty acids, hydrogen and methane are followed during a time-course experiment over 8 hours. At 3.5 hours (arrow) molybdate was added and the substrates accumulate at a rate corresponding to their rate of consumption before inhibition. The formation of methane shows the release of competition for the common substrates for methanogenesis and sulfate reduction (H and acetate). Data from Sorensen et al. (1981).

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