Pre-incubation time

If protease inhibitors should be identified and characterized, the assay should be tested for signal stability and endpoint linearity in the next step. The progress curves recorded for substrate concentrations near or below the KM value should be linear for up to 2 h. Such signal stability is a prerequisite to run the assay with a pre-incubation time of 1 h for enzyme and inhibitor. This long pre-incubation is recommended to ensure also that the IC50 values for slowly binding inhibitors are correctly determined. An additional hour for the incubation of enzyme, inhibitor and substrate after this pre-incubation is recommended. 

After confirmation in the radiochemical assay, one compound was finally retained for further evaluation. It was identified as a known commercially available hydroquinone derivative (Salor S6.721-1, Table 1). The compound had an IC50 value of 2 pM, when preincubated with the enzyme for 10 min. Structures lacking either the hydroxyl or dimethylaminomethyl groups were not active (Table 1). No inhibition by S6,721-l was observed in the presence of 1 mg/ml ascorbate. Inhibition was time-dependent (Fig. 4) and could not be reversed by extensive dialysis. The presence of substrate in the reaction mixture protected the enzyme from inactivation (data not shown). This indicates that one or several active site residues are irreversibly modified by the hydroquinone derivative. A mutant enzyme, where Lys-36 in the active site had been replaced with glutamine (8) was not inactivated, suggesting that Lys-36 may be the modified residue. Interestingly, 56,721-1 was most effective after a short incubation time in pH 7.5 buffer alone, before addition of enzyme and finally substrate (Fig. 4). However, longer pre-incubation times under these conditions partially destroyed the inhibitory activity (Fig. 4) and after 1 hour at pH 7.5 the compound was completely inactive. 

Simultaneously suspend 2x10 cells in 10 ml of Soln. B and incubate at 37 °C for 60 min. Add Soln. E to a final concentration of 1 pM after this pre-incubation, vortex, and continue incubation. Take two aliquots of 50 pi each at to (time of nitrendipine addition) and 1,2,5,7, 10, 15,20,25, and 30 min after nitrendipine addition, precipitate with 2 ml of ice-cold Soln. D and suck off on glass fiber... 

To determine the mutagenic potential of nonaqueous liquids as measured by the Ames SaZmoneZ/a/mammalian-enzyme assay, the following protocol is recommended for the sample preparation. In step 1, the desiccator assay is performed on the neat material. The desiccator assay allows the detection of volatile mutagens (such as chlorinated solvents) that are often missed in the plate incorporation and pre-in-cubation assays (16, 17). In addition, a suspension of the neat material (20 mg/mL) is prepared by ultrasonication (5 min at room temperature) in high-purity DMSO (18, 19) and tested in the normal plate incorporation assay as well as in a pre-incubation Ames assay (20). The pre-in-cubation assay allows the detection of certain mutagens, such as dimethylnitrosamine, that require additional time for activation by mammalian or bacterial enzymes. A positive response in any of these three assays indicates the presence of mutagenic components, and the evaluation process is completed. 

In cell-based assays where the inhibitor is present for a long incubation time, such as with the BaF3 assay (discussed above), a slow allosteric inhibitor can be identified. However, with other faster readout cell-based assays, it may be necessary to include a long pre-incubation step to allow the compound to diffuse into the cell and bind to the target. 

Assaying the major component, SB-253514 (3b) against recombinant LpPLA2 confirmed that it possessed potent activity. Interestingly, the inhibition was time-dependent, as potency was improved by pre-incubation of the inhibitor with the enzyme (Table 1, Activity of SB-253514 (3b)). 

Apoptotic/necrotic transformation of excitable cells. Effects of dipeptides directed to support stability of cellular structures increase the reliability of cellular functions under normal conditions and especially during oxidative stress, which accompanies effect of several extreme factors. It was found in experiments on individual neurons that carnosine prevents cell death induced by excitotoxic compounds, N-methyl-D-aspartic acid (NMDA) or kainate [93-95] or experimental hypoxia/reoxigenation [96]. Apoptosis induced by exposure of cerebellum neurons to kainic acid (see Table 6), was arrested if the cells were pre-incubated with carnosine or anserine and simultaneously heavy necrotic processes were substitute by light (reversible) necrosis. At the same time, N-acetylcamosine or homocaraosine did not reveal protecting action [94,95]. 

The presence/absence procedure was based on the ISO method for the detection of Salmonella (ISO 6579) which is summarised as follows (1) pre-enrichment in Buffered Peptone water (incubation time (18 2) h at (37 1)°C) and (2) selective enrichment in broth of own choice (incubation time and temperature according to own procedure). The detailed procedure is described elsewhere [37]. Each laboratory determined the presence or absence of Salmonella in 50 capsules. Four of these individually identified capsules were negative control capsules. The numbers of these capsules were unknown to the laboratories at the time of analysis. For the presence/absence procedure, all capsules showing typical colonies on the isolation agar were subjected to a confirmation for Salmonella. At least two colonies per capsule were used for this confirmation. All colonies (>1000 colonies) tested by the laboratories gave a positive Salmonella identification. The type of confirmation test used is described elsewhere [37]. 

Assay duration/ time and type of measurement Seconds to months duration milliseconds to days measurements endpoint, multiple time points, or continuous real-time measurements Minutes to days duration seconds to minutes measurement typically endpoints preferred with incubation times long enough for automation (30-60 min) pre-reads possible... 

Plutonium dioxide microspheres (149 to 177-ym diam) were contacted with 10"3 M biocarbonate buffer (pH 7) for various periods of time. Some of the microspheres had been pre-incubated in pH 4 acetate buffer for two years prior to the bicarbonate experiments. Other microspheres were contacted with bicarbonate solution without any prior contact with water. The microspheres were primarily 239Pu (88.9% of Pu mass). The microspheres had been calcined at 1150°C and had a specific surface area of 0.012 m2/gram. 

Fig. 21. Rapid-scanning, stopped-flow spectra for the reaction of / -cysteine with CGS pre-incubated with i-allylglycine. Concentrations refer to conditions immediately after mixing [CGS] = 7.08 im, [i-allylglycine] = 28mM, [i-cysteine] = 10 mM, 0.1 M potassium phosphate, 5 mM DTE, 1 mM EDTA, pH 7.2 and 25°C. Initiation of scanning occurred 2.3 ms after flow stopped. Data shown are representative scans from a sequential 79-scan data set collected with a repetitive scan rate of 8.9 ms/scan. The spectra shown were acquired at 2.3, 11.2,20.1,29.0, 37.9, 55.7, 73.5,109.1,162.5, 289.6, and 696.5 ms after flow stopped. The inset to (A) represents single-wavelengths time courses taken from the entire set of 79 scans at (a) 300 nm and (b) 420 nm. The left and right ordinates correspond to the absorbance values at 300 and 420 nm, respectively. [Taken from Brzovic et al. (107) with permission.]...

Figure 1. Degradation of (a) iprodione and (b) vinclozolin in soil, t residues following sequential treatments at time 0, 50 and 100 days O treated once only after pre-incubation for 50 days A treated once only after pre-incubation for 100 days. (Reproduced with permission from Ref. 11. Copyright 1986 Society of Chemical Industry).

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