Glucocorticoid-inducible gene expression vectors for use in Drosophila melanogaster Schneider 2 cells.

Inducible, vector-based, expression systems that allow fine control of transgene expression are gaining more and more use in fundamental research as well as in therapeutic applications. In an effort to develop a tightly regulated heterologous expression system for Drosophila Schneider 2 cells, three different inducible reporter constructs were compared. These comprised six copies of the glucocorticoid response element fused to one of three distinct types of Drosophila gene promoters: (1) a TATA-box containing, (2) a TATA-less and (3) a bidirectional core sequence. These were fused to a luciferase reporter gene. The promoter constructs displayed different basal as well as agonist-induced activities. The implications of the observations made are discussed in the context of promoter properties and of induction of genes that may be studied in Drosophila.

Characterization of the ryanodine receptor-Ca2+ release channel from the thoracic tissues of the lepidopteran insect Heliothis virescens.

The existence of invertebrate forms of the RyR has recently been confirmed (Takeshima et al., 1994, Puente et al., 2000). However, information on the functional properties of this insect RyR is still limited. We report the functional characterization of a RyR from the thoracic muscle of H. virescens (Scott-Ward et al., 1997). A simple purification protocol produced membranes from homogenized prefrozen H. virescens thoracic muscle with a [3H]-ryanodine binding activity of 1.19 +/- 0.21 pmol/mg protein (mean +/- SE; n = 4). [3H]-Ryanodine binding to the H. virescens receptor was dependent on the ryanodine concentration in a hyperbolic fashion with a KD of 3.82 nM (n = 4). [3H]-ryanodine binding was dependent on [Ca2+] in a biphasic manner and was stimulated by 1 mM ATP. Millimolar caffeine did not stimulate [3H]-ryanodine binding to H. virescens membranes in the presence of either nanomolar or micromolar Ca2+. A protein of at least 400 KDa was recognized in H. virescens membrane proteins by a specific anti-H. virescens RyR antibody. Discontinuous density sucrose gradient fractionation of microsomal membranes produced vesicles suitable for single-channel studies. Ca2+-sensitive, Ca2+-permeable channels were successfully inserted into artificial lipid bilayers from H. virescens membrane vesicles. The H. virescens RyR-channel displayed a Ca2+ conductance of approximately 110 pS and underwent a persistent and characteristic modification of ion handling and gating following addition of 100 nM ryanodine. The gating of H. virescens channels was sensitive to ATP and ruthenium red in a manner similar to mammalian RyR. This is the first report to describe the single channel and [3H]-ryanodine binding properties of a native insect RyR.

Functional expression of a locust tyramine receptor in murine erythroleukaemia cells.

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.

Cloning and functional characterisation of two novel nicotinic acetylcholine receptor alpha subunits from the insect pest Myzus persicae.

Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor alpha subunits from a major insect pest, the peach-potato aphid Myzus persicae. These two subunits, termed Mp alpha1 and Mp alpha2, are respective structural homologues of the Drosophila D alpha2/Schistocerca gregaria alphaL1 alpha-subunit pair and the Drosophila ALS alpha subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine- or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick beta2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mp alpha2 and none with Mp alpha1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor alpha subunits from different species can exhibit distinctive physiological and pharmacological characteristics.

Pyrethroid resistance in German cockroaches (Dictyoptera: Blattelidae): resistance levels and underlying mechanisms.

Thirty strains of Blattella germanica (L.) reported to be pyrethroid resistant were collected from three continents. Greater than 2-fold resistance to the pyrethroids cyfluthrin, fenvalerate, cypermethrin, and lambda cyhalothrin appeared in 15 of these strains. Twelve of these strains were also resistant to chlorpyrifos and propoxur. All the field strains tested were heterogeneous with regard to resistance. Possible resistance mechanisms detected in these populations included elevated levels of cytochrome P450, general esterase and glutathione S-transferase, and nerve insensitivity (kdr-type resistance). The elevated esterases and oxidase-based resistance were the most prevalent; 11 and 10 strains, respectively, had evidence of these mechanisms. Resistance was synergized by piperonyl butoxide in some strains. In some strains, elevated esterases, although present, were primarily correlated with organophosphate resistance. Pyrethroid insecticides may still be effective against many of these populations because of the low levels of resistance detected. However, potential exists for more serious resistance problems to develop if only pyrethroids are used. Because many of these strains are already resistant to organophosphorus and carbamate insecticides, prospects for the future chemical control of these populations must be carefully considered.

Some neurobiological applications of the BBC Model B microcomputer and Unilab 8-bit interface.

Four computer programs written for the BBC Model B microcomputer (coupled to a Unilab 8-bit interface) are discussed. These programs enable the system to be used as (1) a transient recorder, (2) a rapid signal averager, (3) a spike-train analyser and (4) an instrument for measuring the amplitude of single channel currents. Flow-charts illustrating the operation of each program are given along with a detailed discussion of how the programs may be used in the laboratory. The discussion is illustrated using recordings taken from experiments conducted on a range of neurobiological preparations.

Single glutamate gated channels in insect visceral muscle.

Single glutamate-gated ion channels with a conductance of 135 pS are demonstrated in tonic muscle fibres of the locust hindgut. Channel kinetics closely resemble those of glutamatergic channels in locust skeletal muscles. Glutamate concentrations increasing within the range from 5 X 10(-5) to 1 X 10(-3) M result in an increase of the frequency of channel opening and a decrease in channel closed times. Delta-philanthotoxin, a toxin isolated from the venom of the digger wasp Philanthus triangulum, inhibits channel activity by blocking open channels and increasing channel closed times.

Is tyramine a specific inhibitor of proctolin?

Samples of tyramine purchased from six different manufacturers were tested for their effectiveness and specificity in blocking proctolin- and neurally-evoked contractions of the superior longitudinal muscles of the locust (Locusta migratoria) rectum. It was found that tyramine was neither specific (in that it also blocked glutamatergic responses) nor consistent in its action, as samples purchased from different manufacturers gave a range of different results. The venom of the wasp Philanthus triangulum was used to block glutamatergic responses to enable proctolinergic responses to be studied in isolation. Thin layer chromatography was performed to determine the purity of the tyramine samples but no correlation could be made between purity and efficacy or specificity of proctolinergic antagonism. It is concluded that, due to the inconsistency and non-specificity of its action, tyramine should not be used as an antagonist for proctolin.

The use of lanthanum as a tool to study calcium fluxes in an insect visceral muscle.

1. An ultrastructural study of the effect of lanthanum on an insect visceral muscle is presented. 2. Lanthanum was shown to penetrate the T-tubule system and the dyads, whilst little lanthanum was found bound to the outer sarcolemma. 3. X-ray microanalysis of lanthanum treated muscles showed that lanthanum was able to penetrate the cytoplasm. 4. Lanthanum was found bound to both the outer membranes of mitochondria and the sarcoplasmic reticulum. 5. Caution is therefore expressed in using lanthanum as a tool to study trans-sarcolemmal calcium fluxes in locust visceral muscle.