The LCR/MEL system (Locus Control Region/Murine Erythroleukaemia cells) was employed to express and characterize the Locusta migratoria tyramine receptor (TyrLoc), an insect G protein-coupled receptor. Functional agonist-dependent responses were recorded in stable, tyramine receptor expressing cell clones (MEL-TyrLoc). Tyramine elicited a dose-dependent increase of cytosolic Ca2+-ions and an attenuation of forskolin-induced cyclic adenosine monophosphate (AMP) production. Octopamine was shown to be a weak agonist for both responses. In addition, yohimbine proved to be a potent tyramine receptor antagonist. This study reports the first application of the LCR/MEL expression system in functional assays for G protein-coupled receptors and therefore expands the capabilities of this system by exploiting the functionality of the signal transduction pathways.
summary
We have used a pulsed u.v. laser to ablate the cell wall of the filamentous fungus Aspergillus niger van Tiegh., and expose the plasma membrane at defined points along the hypha. Using the patch clamp technique, giga ohm seals (up to 20 GΩ) and recordings of plasma membrane ion channels were obtained. We describe, for the first time in fungi, a 43 pS plasma membrane anion efflux channel. Its potential role in cell signalling and pH homeostasis is discussed.
: There are increasing opportunities for the development of highthroughput in-vitro screens to aid the discovery of fungicides with novel modes of action. In the past, such screens were developed when biochemical targets were validated by fungicides with deÐned modes of action. However, genetic information is beginning to have a major impact both on the way in-vitro targets are selected and on the speed at which mode-of-action information is gained on current fungicides having an, as yet, undeÐned mode of action.This paper discusses issues concerning target selection and high-throughput screening, using examples taken from the current literature and from investigations at Zeneca Agrochemicals, using inhibition of fungal respiration as an example.Saccharomyces cerevisiae is discussed as model for fungicide research, both in terms of its sensitivity to known fungicides and its well deÐned molecular genetics, which makes it amenable to such techniques as gene dosage for mode of action determination.1998 Society of Chemical Industry ( Pestic. Sci., 54, 338È344 (1998)
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