Bioassay techniques development

Stern, et al. (43,44) reported a bioassay technique based on fetal rat bone absorption of calcitriol. Parkes and Reynolds (45) developed an in-vitro bioassay using duodenal tissue from chicken embryos. 

The bioassay technique was developed to reduce the uncertainties associated with the use of native vegetation or cultivated crops. Plants can be started under controlled conditions and exposed under standardized conditions. Species and cultivars can be selected for oxidant sensitivity and symptom characteristics. The two studies just noted were the most closely controlled. Similar work has not been repeated. However, many investigators have grown plants under known cultural conditions and then transplanted them to field sites where they received special care. These plants can then be read for foliar symptoms throughout a given period, and the symptoms related to oxidant concentrations. The lack of apparent correlation in the two early studies could be due to the lack of specificity for the monitored oxidants, the presence of different concentrations of interacting oxidants at different times, or variations in cultural conditions between exposure times. 

Rahman, A., Choudhary, M. 1., Thomsen, W. J. Radioligand binding assays, in Bioassay Techniques for Drug Development, ed. Rahman, A., Choudhary, M. 1., Thomsen, W. J., Harwood Academic Publishers, Amsterdam, 2001, pp 179-218. 

There are literally hundreds of publications that, directly or indirectly, have contributed to the development, validation and refinement of bioassay techniques both for liquid and solid media assessment. These papers incorporate initiatives that... 

Many new microbial compounds with potential blocherbicldal activity have been Isolated, chemically characterized, and a portion of their biological activities determined using various bioassay techniques or by direct plant screening. Some of these compounds are virulence factors of plant pathogens, thus Information on these phytotoxins can benefit the development of microbes as herbicides and potentially provide new chemical herbicides. Data on some of these compounds and their structures are presented. Information on source, biological activity, and possible mode of action (when available) Is briefly summarized. 

Most synthetic methods for the generation of peptide libraries have been derived from various multiple parallel peptide synthesis techniques developed since 1984.bs-i l Consequently, the sohd supports used for hbrary synthesis are essentially the same as those used for multiple peptide synthesis. Standard divinylbenzene cross-linked polystyrene resins are typically used for hbraries that are cleaved from the resin and screened in solution.P Polyoxyethylene-grafted polystyrene resins,f l or acrylamide-polyoxyethylene copolymers, P on the other hand, are the sohd supports of choice for the synthesis of resin-bound peptide hbraries screened in sohd-phase binding assays.P Such resins are compatible with both organic solvents used for peptide synthesis, as well as aqueous buffers used in the bioassays. Various segmental supports previously employed for multiple peptide syntheses have also been utilized for the synthesis of peptide libraries, including polypropylene pins, PI cotton,t cellulose membrane,and glass shdes.P ... 

Two bioassay techniques, the capillary method [58], and the drop method [63], have already been developed to study the chemotaxis of phytopathogenic zoospores and gametes of marine brown algae. We have devised a new procedure (the "particle method") which is a qualitative, rather than a quantitative, method of detecting an attractant after it has been absorbed on to an inorganic particle. This method has been used to study attractants of A. cochlioides zoospores in the roots of both spinach and pigweed (Chenopodium album). 

Natural product chemistry has changed dramatically over the last 50 years. The advent of modern sophisticated instrumentation and new bioassay techniques has shifted the emphasis to the structure elucidation of minor natural products, particularly those which show bioactivity. The complex structures of many of these offer challenges to synthetic organic chemists to develop synthetic approaches to them, which often leads to the development of new synthetic methods in order to achieve specific transformations. 

Further development of quantitative methods is likely to be dominated by progress in radioimmunoassay and protein-binding methods. The rapidity with which these techniques have encouraged the demise of the classical isotope dilution and bioassay techniques implies that many drugs and their metabolites will eventually be measured by these methods. Progress along these lines, however, has been relatively slow and there are few instances. 

Until the recent development of appropriate HPLC techniques capable of detecting pmol amounts (see Flentge et al. 1997) ACh could only be measured chemically by relatively lengthy and expensive procedures (e.g. gas chromotography), which were not always very sensitive, or by bioassays. Although the latter, using muscle preparations that responded to ACh, such as the dorsal muscle of the leech, the rectus abdominus of the frog or certain clam hearts, were reasonably sensitive they were tiresome and not easily mastered. Thus studies on the release and turnover of ACh have not been as easy as for the monoamines. 

Improved Methods for Collection, Bioassay, Isolation, and Characterization of Compounds. Techniques used to characterize natural products are evolving rapidly as more sophisticated instrumentation is developed. Plant physiologists and chemists should work closely together on this aspect, since rapid and reproducable bioassays are essential at each step. There is no standard technique that will work effectively for every compound. Briefly, isolation of a compound involves extraction or collection in a appropriate solvent or adsorbant. Commonly used extraction solvents for plants are water or aqueous methanol in which either dried or live plant parts are soaked. After extracting the material for varying lengths of time, the exuded material is filtered or centrifuged before bioassay. Soil extraction is more difficult, since certain solvents (e.g. bases) may produce artifacts. 

Other kinds of bloassays have been used to detect the presence of specific allelochemical effects (8), effects on N2 fIxatlon (9), the presence of volatile compounds (10) and of Inhibitory substances produced by marine microalgae (11). Putnam and Duke (12) have summarized the extraction techniques and bioassay methods used In allelopathy research. Recent developments In high performance liquid chromatography (HPLC) separation of allelochemlcals from plant extracts dictates the need for bloassays with sensitivity to low concentrations of compounds contained In small volumes of eluent. Einhellig at al. (13) described a bloassay using Lemna minor L. growing In tissue culture cluster dish wells that maximizes sensitivity and minimizes sample requirements. 

In recent decades, the development of chemical, biochemical, and biological techniques has allowed the creation of analytical tools which can be used to facilitate the identification of the mechanisms involved in neoplastic transformation. Animal models remain, however, the most widely used approach of investigation. Cancer bioassays are usually conducted in rodents (rats and mice) and the experimental protocol takes 18-24 months and it is followed by extensive histopathological and statistical analysis. The procedure is time and... 

Continuous culture systems have been widely used to culture microorganisms for industrial and research purposes (Kubitschek 1970 Tempest 1970 Veldkamp 1976 Rhee 1980). In recent years, these culture techniques have found their way into the bioassay methods of ecotoxicology and allelopathy (Rhee 1980). The early development of a continuous culture system can be traced back to the work of Novik and Szilard (1950 a,b) who developed the first chemostat. In a continuous culture system, nutrients are supplied to the cell culture at a constant rate and to maintain a constant volume, an equal volume of cell culture is removed. This allows the cell population to reach a steady state, where the growth rate and the total number of cells/ml of culture remains constant. Two kind of continuous culture systems can be distinguished turbidostat and chemostat. ... 

Incubation periods in excess of 2 h were required before this activity was detected in cell-free supernatants. More recently, the use of cDNA probing of Northern transfers (to detect specific mRNA levels), the use of ELISA techniques (to detect protein levels immunologically) and the development of more specific bioassays (culture techniques in which a biomolecule stimulates proliferation in a particular cell line) have resulted in a more thorough analysis of IL-1 production by neutrophils. IL-1 is only poorly expressed in blood neutrophils because mRNA for this cytokine is detectable only at very low levels (if at all), and protein production is usually below the level of detection of most assays. However, exposure of neutrophils to lipopolysaccharide (LPS), or to cytokines such as GM-CSF, TNF or IL-1 itself, results in a rapid but transient increase in IL-1 expression. 

As first practiced by Geysen and Houghton, the preparation of combinatorial libraries produced discrete compounds of known identity through a technique known as "spatial separation," which simply means that individual compounds in the library are produced discretely and are not mixtures. Such spatially addressable compound sets are produced in such a way as to keep separate the reaction flasks or resin beads containing the individual components of the library and perform bioassays on the discrete compounds, one at a time. Thus, if the "history" of the reaction conditions performed in each flask or on each solid support, the identity of the compounds produced is known, without resort to structure elucidation techniques. Initially, this technique, after typically an extensive reaction development stage, allowed the preparation of between 10 and 1000 discrete combinatorial products. 

This report describes our results on analyses of antigens in cotton dust and bract by immunological techniques, and discusses the possibility of applying these results to the development of a bioassay for active agents which may be related to byssinosis. 

Analysis of the complex mixtures of gaseous and/or particulate POM in primary emissions or ambient air is a daunting task, given the huge numbers of species and the small concentrations. The development of the Salmonella typhimurium assay has helped to direct such analysis through the technique of bioassay-directed chemical analysis. 

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