Bioassay formats

Numerous bioassays format have been designed using derivatives of the mono-functionalized europium cryptate such as 47 (commercially available from CISbio, see http //www.htrf.com). Since the last reviews [127] some new applications Eu " C [BP.BP.BP] cryptate were developed through the HTRF technology, as caspase assay [128], telomerase assay [129], leukotriene b4 assay [130], inositol-1-phosphate assay [131], minisequencing [132], and MutS-DNA interaction [133]. Recently, the HTRF technology has been used in the study of protein-protein interactions and the authors demonstrated that measured values compare favorably with those calculated from independent experiments [134]. 

Acnegenic Response. Both 2,3,7,8-TCDD and HCDD produced chloracne in the rabbit ear bioassay as indicated by the formation of comedones. Solutions of 2,3,7,8-TCDD (sample c) in benzene ranging in concentration from 0.04 /xg/ml to 400 ju,g/ml produced a positive response with severity increasing with concentration. A negative response was obtained with a solution of 0.004 ju,g/ml. In contrast, a chloroform solution of 1,2,3,4-TCDD, 50 jug/ml, did not produce a positive response. With HCDD (samples a, b, c, and d), a response was produced by solutions of 10 to 50 ju,g/ml in chloroform and dimethoxyethane. Chloroform extracts from 10% suspensions of 2,7-DCDD or OCDD were negative, indicating that these have a low order or possibly no acnegenic activity. 

We have recently observed in our laboratory that water washes of undamaged leaves in a number of plants contained sterols and other lipids in sufficiently high concentration comparable with concentrations used in typical laboratory bioassays. These aqueous lipid solutions are frequently accompanied by long-chain (C-12 to C-18) fatty acids. We therefore suggest that micelle formation between the lipids and fatty acids may occur. By this mechanism the lipid solubility in the aqueous medium is significantly enhanced, thus allowing the release of otherwise water-insoluble plant constituents into the environment. Presently, experiments are in progress in our laboratory to provide further evidence for the "micelle-mechanism" of allelopathlc lipids. 

In another bioassay (Table III), an active compound (DAD) i solated from whole dwarf spikerush (Eleocharis coloradoensis) (6,7) appeared to induce floating leaf formation in American pondweed. The response to DAD was somewhat erratic and much less pronounced than that observed for ABA at even higher concentrations than ABA. (Previous work has shown that high concentrations of ABA, e. g. 5-13 ppm, inhibit sprouting of winterbuds). 

Grant WF (1994) The present status of higher plant bioassays for the detection of environmental mutagens. Mutat Res 310 175-185 Grant WF, Owens ET (2001) Chromosome aberrations in Pisum for the study of environmental mutagens. Mutat Res 488 93-118 Kalweit S, Utesch D, von der Hude W, Madle S (1999) Chemically induced micronucleus formation in V79 cells-comparison of three different test approaches. Mutat Res 439 183-190... 

Viral bioassays of various different formats have also been developed. One format entails incubation of the final product with cell lines sensitive to a range of viruses. The cells are subsequently monitored for cytopathic effects or other obvious signs of viral infection. 

Bioassays are especially useful inasmuch as they reflect the loss of biological activity of a molecule, but they are often replaced by chemical analysis showing the loss of the parent compound or the formation of products [128-135]. 

Numerous testing systems and protocols have been used to study 5-LO inhibitors in different laboratories, complicating attempts to compare compounds and series. In vitro, a variety of both cell-free and cellular preparations have been employed as primary screens. The most commonly used cell-free system is the crude cytosolic fraction from broken RBL-1 cells [25] various broken neutrophil preparations are also used, and more recently purified enzymes have occasionally been employed. The formation of 5-LO products is generally determined by radioimmunoassay or (in older work) HPLC or bioassay methods. 

Alternatively (or initially) the mixture is treated as a whole and tested in its crude state. The advantage of this strategy includes the relevancy of the tested sample to its environmental counterpart, decreased potential for artefact formation, and inclusion of combined effects of chemicals in the mixture. Moreover if the mixture is representative of others in its class (e.g., diesel emissions from different sources would share certain characteristics), it may be possible to extrapolate results across samples. This method also circumvents the labor-intensive process of individual testing of multiple chemicals. But sometimes a complex mixture is too cytotoxic to be tested directly in a bioassay. Furthermore, it may be incompatible with the test system because of the physical matrix. Other disadvantages include the inability to specify the constituent of the mixture responsible for the toxicity, as well as potential masking effects (e.g., the masking of mutagenicity by cytotoxicity). 

Guinea Pig Bioassay of GT-1 and GT-2. At less than nanogram concentrations, extracts 6T-1 and GT-2 produced an enhancement of histamine stimulation of the ileal preparation. At nanogram concentrations or larger, both caused an inhibition and hence a shift of the dose response curve (Figure 3). Replotting these data for GT-1 and GT-2 into a Michaelis-Menten format (Figure 4) indicates that the action of GT-1 and GT-2 fractions are... 

Several authors have obtained circumstantial evidence that allelopathic compounds reduce mycorrhizae formation (20-23). Kovacic and associates ( ) have shown that understory plants in a live ponderosa pine stand are largely nonmycorrhiza-forming species. They hypothesized that this was due to inhibition of the vesicular-arbuscular mycorrhiza necessary for the growth of herbaceous mycorrhizal plants, under living pines. They demonstrated that more mycorrhizal plants occurred under dead pines, bioassay plants formed mycorrhizae in soils beneath dead pines but not in soil beneath live pines, and mycorrhizal inoculum appeared to be absent from the live pine stand. 

A reduction in cutinase production should result in the PNB-1 mutant being less virulent. The pathogenesis of the two strains were evaluated in a pea stem bioassay developed by Kolattukudy and coworkers in which infection by Fusarium solani results in wound formation within three days on the epicotyl of pea sedlings (15). The virulence of T-8 had previously been shown to be reduced in this assay by the addition of inhibitors of cutinase or by rabbit anticutinase antibodies (15-18), indicating that cutinase played an important role in pathogenesis. When the cutinase-defective mutant was evaluated in the bioassay, the mutant exhibited a 55% reduction (p < 0.05) in virulence compared with the T-8 parental strain and the addition of purified cutinase at 1 mg/ml to the mutant enhanced wound formation to 80% of that of the parent (p > 0.5). These data further support the notion that the mutant was defective in cutinase. 

A novel bioassay for nystatin based on the use of a microbial sensor was recently reported. Nystatin is believed to bind to the steron present in the membranes of sensitive cells, leading to the formation of pores. The subsequent death of the microorganism is preceded by leakage of cellular materials. Microbial death can be detected by means of an oxygen electrode. 

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