Whole cell activity assays

Peters B, Mientus M, Kostner D, Junker A, Liebl W, Ehrenreich A (2013) Characterization of membrane-bound dehydrogenases from Gluconobacter oxydans 62IH via whole-cell activity assays using multideletion strains. Appl Microbiol Biotechnol 97(14) 6397-6412... 

Analog ent-(+)-8 is 20-fold more potent than racemic acifran and is threefold more potent than niacin in the cAMP whole-cell assay [55]. The selectivity was also improved with respect to acifran with 10-fold selectivity for GPR109A over GPR109B. The importance of the absolute configuration of ent-(+)-8, which is tentatively assigned S, is demonstrated by the 70-fold better cAMP whole-cell activity of the eutomer compared to that of the distomer. 

Paclitaxel, camptothecin, vincristine, vinblastine, and other compounds currently under development as potential anticancer drugs (i.e., the bryostatins isolated from marine dinoflagellates) were discovered through a broad-based screening program to identify, using a whole-cell inhibition assay, natural products that are active against a battery of representative cancer cell lines. 

On the other hand, it is possible to underline an estrogenie aetivity of PAHs and their metabolites. One of the best described modes of action is the erosstalk between aetivated AhR and ER (Safe and Wormke, 2003). PAHs have been shown to be aetive in various ER-mediated systems that measure either the displacement of 3H-Estradiol-17 beta from the ER in a whole-cell binding assay (Arcaro et al., 1999) or the induetion of ER-mediated reporter activity (Charles et al., 2000 Fertuek et al., 2001a, 2001b Tran et al., 1996 Vondracek, 2002), thus indicating the potential role for estrogenieity. 

Whole cell OPH activity was measured by following the increase in absorbancy of p-nitrophenol from the hydrolysis of substrate (0.1 mM Paraoxon) at 400 nm (sm = 17,000 M cm ). Samples of culture (1 ml) were centrifuged at 10,000 g and 4 C for 5 min. The cells were washed, resuspended with distilled water, and 100 pi was added to an assay mixture containing 400 pi 250 mM CHES [2-(N-cyclohexylamino)ethane-sulfonic acid] buffer, pH 9.0, 100 pi 1 mM Paraoxon, and 400 pi distilled water. One unit of OPH activity was defined as pmoles Paraoxon hydrolyzed per min. Each value and error bar represents the mean of two independent experiments and its standard deviation. 

Therefore, mRNA capping represents a potentially useful target for inhibitors with antifungal or antiviral activity indeed, whole-yeast cell-based assays developed to identify and characterize inhibitors of fungal mRNA capping allowed the identification and characterization of... 

Whole-cell assays (allowing measurement of phosphoinositide hydrolysis, adenylate cyclase activity, etc.)... 

Primary assays are devised to incorporate physiological or enzymatic targets for screening biological activity of potential drug compounds, ilie biological assays are then reconfirmed in specific biochemical and whole cell assays to characterize the target-compound interaction. 

A variety of assays have been developed to quantify phagocytic activity. These include direct microscopic visualization (2,3), spectrophotometric evaluation of phagocytized paraffin droplets containing dye (4), scintillation counting of radiolabeled bacteria (5), fluorometric (6), and flow cytometric analysis of fluorescent particles (7-13). The flow cytometric assay offers the advantage of rapid analysis of thousands of cells and quantification of the internalized particle density for each analyzed cell. The assay may be performed with purified leukocyte preparations (7-13) or anficoagulated whole blood (14,15). 

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