Cell-free assays systems

Several cell-free assay systems have been used to dissect the mechanisms of DNA damage sensing in vitro. The most extensively applied system is the one using Xenopus extracts. As DNA replication can occur efficiently in Xenopus extracts, this system has been used to analyze how replication interference is sensed by checkpoint sensors (60, 61) and how activated checkpoint regulates DNA replication. The checkpoint can also be elicited in a replication-independent manner by various synthetic DNA structures in Xenopus extracts (9, 62). The checkpoint response in Xenopus extracts is often monitored by the phosphorylation of various checkpoint proteins. The association of checkpoint proteins with damaged chromatin or DNA is used to monitor the recognition of DNA damage in Xenopus extracts. 

FELCs were cultured for 36 hr at these increasing NAm concentrations, DNA and crade nuclear extracts were isolated, and DNA methylase was assayed as previously described (17). DNA from these cells exhibited a dose-dependent hypomethylation, measured by increased methyl accepting ability, which peaked with 20 mM NAm treatment (Fig. 1). DNA isolated from cells treated with 40 mM NAm was not hypomethylated and was presumed to be due to lack of DNA synthesis and cell division, caused by cytotoxic effects of this compound. DNA methylase activity (Fig. 1) was found to be normal at non-cytotoxic doses of NAm. NAm added to the cell-free assay system was unable to significantly inhibit the DNA methylase activity. These results suggest that the DNA from FELCs grown in NAm is hypomethylated and that flie activity of the DNA methylase enzyme is not directly altered by NAm. 

There was no evidence from cell-free assay systems that carvedilol is a scavenger for 02 or NO (Asbrink et al. 2000). Carvedilol did not affect other reactions dependent on NO, e.g. spontaneous of formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leucocyte migration or hpoxin A4-, fMLP-, or A23187-induced neutrophil cytotoxicity for human umbiUcal vein endothehal cells. Thus, these effects point to the possibility that carvedilol modulates the NADPH oxidase of polymorphonuclear leucocytes but leaves the nitric oxide synthase of phagocytes intact. Carvedilol exerted poor reactivity toward phenoxyl, alkoxyl, and per-oxyl radicals in acetonitrile solution nor did it show an appreciable antioxidant effect against either the peroxyl radical-induced oxidation of methyl Unole-... 

Purification of the membrane-bound lactose carrier protein is a very different problem from the purification of the soluble OMP synthase. Both the approach to purification and the assays for the protein during purification are quite novel. The assay involves reconstituting a transport system with membranes that are free of lactose carrier protein, then adding the partially purified carrier protein and radioactively labeled lactose. The activity in this assay system is proportional to the transport of radioactive lactose across the membrane in the cell-free reconstituted system. 

Plants are not different from other natural product samples in that they too tend to interfere with various screening formats in nonspecific ways as nuisance compounds displaying unwanted color, inherent fluorescence, promiscuous or aggregate behaviors, detergent-like activities, or toxicity (Feng et al., 2005 Appleton, Buss, and Butler, 2007). Biochemical assays (cell-free defined systems) are notoriously sensitive to such interference by natural product extracts. Cell-based reporter assays and cell-based so-called phenotypic screens always require parental cell controls to determine extract toxicity. However, plants contain their own sets of components that are problematic to screening assays and compound identification. 

Analyzing the minimal inhibition concentration (MIC) data for nine different strains, resulted in two significant PCs being obtained, accounting for 77.1% and 16.1%, respectively, of the data variance. Thus, the intrinsic dimensionality of the data matrix of nine assays was only two. The loading plot, that is, a plot of the calculated PCs with respect to the descriptors, shows that the first component is mainly related to the seven cell-free test systems, while the second component represents the two whole-cell test results. Thus, there was much correlation between the results of the seven cell-free assays and the two whole-cell assays were correlated with each other but not with the cell-free assays. In other words, much redundant information was obtained... 

For each BoNT serotype, the dichain form constimtes the active configuration of the neurotoxin the isolated LC and HC are devoid of systemic toxicity. The absence of toxicity is consistent with findings that the LC cannot gain access to the cytosol unless it is coupled to the HC and that the HC lacks the ability to inhibit neurotransmitter release (Stecher et al., 1989 Goodnough et al., 2002). The isolated LC does, however, remain enzymatically active as evidenced by its ability to inhibit exocytosis from permeabilized chromaffin cells (Stecher et al., 1989), by its ability to cleave SNARE proteins in cell-free assays (Adler et al., 1998), and by its capacity to inhibit ACh release in skeletal muscle when delivered by liposomes (de Paiva and Dolly, 1990). It is not clear whether any portion of the HC is translocated along with the LC, and if so, whether it exerts a role in enhancing the catalytic activity or stability of the LC. 

A cell-free system from photosynthetically active species has to be developed that is able to produce substantial amounts of intermediates and desaturated carotenes. Noteworthy a recent report using isolated chromoplasts from daffodil flowers. Narcissus pseudonarcissus > and isopentenyl pyrophosphate as substrate noted increased levels of phytoene and geranylgeraniol vs. control in the presence of high concentration (50 j,M) of SAN 6706 (27). These cell-free assays should be investigated further with regard to possible different sensitivity against inhibitors because of the species used. 

The development of a cell-free transcription system, which faithfully transcribes a synthetic rDNA template, was first described about two decades ago (Grummt, 1981b Haglund and Rothblum, 1987 Learned and Tjian, 1982 Mishima et al., 1981 Wilkinson and Sollner-Webb, 1982). This accomplishment encouraged a large number of biochemical studies on the dissection of the RNA Pol I transcription system. In vitro transcription assays coupled to column fractionation analyses of cell extracts from a... 

Na also appears to play a role in palytoxin action in some systems. To determine if there is a Na requirement for palytoxin action on the EGF receptor, Swiss 3T3 cells were assayed for palytoxin activity in Na containing medium versus Na deficient medium. When NaCl is replac by cholineCl, palytoxin can no longer inhibit EGF binding in Swiss 3T3 cells (Figure 5). By contrast, PDBu is equipotent in both Na containing and Na free media (data not shown). 

The second major breakthrough in understanding the defect in CGD neutrophils came through the development of assays in which the NADPH oxidase can be activated in a cell-free system in vitro ( 5.3.2.3). In these systems, activation of the oxidase can be achieved by the addition of cytoplasm to plasma membranes in the presence of NADPH and arachidonic acid (or SDS or related substances). Interestingly, the oxidase cannot be activated in these cell-free systems using extracts from CGD neutrophils however, cytosol and plasma membranes from normal and CGD neutrophils may be mixed, and in most cases activity is restored if the correct mixing pattern is used. For example, as may be predicted, in X-linked CGD it is the membranes that are defective (because the cytochrome b is deficient), whereas in autosomal recessive CGD the cytosol is defective in the cell-free system. 

This review describes our unique assay system to investigate the role of cell-free hydrogenases in anaerobic biocorrosion, which resulted in proposing a new biocorrosion model. 

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