The application of energy-filtering electron microscopy for the cytochemical localization of Ca(2+)-ATPase activity in synaptic terminals.

The energy-filtering electron microscopical modes of electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) have been applied to the cytochemical detection of Ca(2+)-ATPase activity in synaptic terminals in the brain of a cichlid fish. Using a recently developed modification of an enzyme-histochemical method, cerium phosphate was precipitated as a marker of high-affinity Ca(2+)-ATPase activity. This is considered to be a marker for the plasmalemma-bound calcium pump, an enzyme which plays a crucial role in the regulation of the cytoplasmic calcium concentrations and therefore of the reactivity of nerve cells.

Cloning of genomic and complementary DNA encoding insect pheromone binding proteins: evidence for microdiversity.

Genomic DNA from the silk moth Antheraea pernyi bearing the gene of a pheromone binding protein has been isolated from a partial genomic library using specific cDNA probes. The DNA spans 3.5 kilobases, contains three exons and two intervening sequences that interrupt the protein coding region of the gene.

Rapid kinetics of second messenger formation in olfactory transduction.

Olfactory transduction is thought to be mediated by a membrane-bound receptor protein initiating a multistep reaction cascade which ultimately leads to a depolarizing generator current. There is considerable evidence for the involvement of adenylate cyclase in vertebrate olfactory transduction, and some data indicate that phospholipase C may have a central role in insect olfaction. However, one must show that odorants not only stimulate enzyme activity but also induce changes in concentrations of relevant second messengers.

Differential stimulation of second messenger pathways by distinct classes of odorants.

Using ciliary preparations from rat olfactory epithelia, representative compounds of different odorant classes were assayed for the capability of stimulating the formation of second messengers in a subsecond time range. Fruity, floral and minty odors were found to induce a transient accumulation of cyclic adenosine monophosphate, whereas the herbaceous and putrid compounds under test caused a rise in inositol trisphosphate concentration.

Odorant-sensitive phospholipase C in insect antennae.

Exogenous tritiated phosphatidylinositol bisphosphate added to antennal preparations from locust and cockroach was hydrolysed releasing inositol trisphosphate. High activity of phospholipase C was detected in the soluble as well as in the membrane fraction. At low free calcium concentrations hydrolysis of the labelled lipid was stimulated by odorants and pheromones in a GTP-dependent manner.

Molecular cloning, sequencing and expression of cDNA encoding a G0-protein from insect.

A locust cDNA clone encoding the complete sequence of a guanine nucleotide-binding protein was isolated and its nucleotide sequence determined. Comparing the deduced amino acid sequence with primary structures of other G-proteins revealed striking homologies with the vertebrate G0-protein. The cloned cDNA was expressed and the translation product detected by specific antibodies.

Molecular cloning of an insect pheromone-binding protein.

Clones coding for the pheromone binding protein precursor have been selected from a cDNA library derived from antennae of the male moth, Antheraea polyphemus. The deduced protein sequence consists of a signal peptide of 20 amino acid residues and a mature binding protein of 142 amino acid residues. RNA blot hybridization indicated that the mRNA is selectively expressed in the antennae of the male moth.

Immunocytochemical localization of glutamate decarboxylase in the mesothoracic ganglion of Locusta migratoria.

Glutamate decarboxylase immunoreactivity has been located in the thoracic ganglia of the locust, Locusta migratoria, using an antiserum raised from rat brain. At the light microscopic level clusters of nerve cell somata as well as nerve fibres were positively labelled by the antiserum. Electron microscopy showed that glutamic acid decarboxylase was localized in numerous synaptic terminals.

Comparison of acetylcholine and alpha-bungarotoxin binding sites in insects and vertebrates.

1. The nervous tissue of locusts contains high affinity as well as low affinity binding sites for acetylcholine which display a similar nicotinic pharmacology. 2.