Lymphocyte Activation in Health and Disease.

Lymphocytes employ a complex assembly of signaling elements that have been organized on a spatiotemporal map to define their role in stimulating both proliferation and apoptosis. The antigen/major histocompatibility complex (MHC) initiates the sequence by organizing the assembly of an active T-cell receptor (TCR) complex responsible for transmitting information down various signaling cassettes (e.g.

A locust type 1 ADP-ribosylation factor (lARF1)* is 100% identical in amino acid sequence to Drosophila ARF1 despite obvious DNA sequence divergence.

The cDNA of a type 1 ADP-ribosylation factor (ARF) from the desert locust, Locusta migratoria was cloned, sequenced and compared to ARF1 genes of other species. The locust ARF1 protein is 100% identical with the ARF1 protein of the fruit fly Drosophila melanogaster even though the DNA sequences are only 79% identical. The significance of this finding in relation to the considerable evolutionary distance between hemimetabolous and holometabolous insects is discussed.

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines.

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization.

Cross-resistance with dieldrin of a novel tricyclic dinitrile GABA receptor antagonist.

A novel tricyclic dinitrile, KN244, blocked the wild-type (dieldrin-sensitive) homo-oligomeric gamma-aminobutyric acid (GABA)-gated chloride channel of Drosophila melanogaster expressed in Xenopus oocytes. Sensitivity to the block by KN244 of the response to 30 microM GABA (IC50=41.6 nM, wild-type RDLac) was reduced abut 100 fold (IC50=4.

Cultured insect mushroom body neurons express functional receptors for acetylcholine, GABA, glutamate, octopamine, and dopamine.

Fluorescence calcium imaging with fura-2 and whole cell, patch-clamp electrophysiology was applied to cultured Kenyon cells (interneurons) isolated from the mushroom bodies of adult crickets (Acheta domesticus) to demonstrate the presence of functional neurotransmitter receptors. In all cells investigated, 5 microM acetylcholine (ACh, n = 52) evoked an increase in intracellular free calcium ([Ca2+]i). Similar effects were observed in response to 10 microM nicotine.

A comparison of the release of a vasoactive-intestinal-peptide-like peptide and acetylcholine in the giant axon-Schwann cell preparation of the tropical squid Sepioteuthis sepioidea.

A vasoactive intestinal peptide (VIP)-like peptide is released by axonal stimulation in the giant axon-Schwann cell preparation from the tropical squid Sepioteuthis sepioidea. It is also released by direct application of l-glutamate, the giant axon-Schwann cell signalling molecule in this preparation. The release of the peptide parallels the release of acetylcholine from the Schwann cells themselves in this preparation in a number of different ways.

Antagonist profile and molecular dynamic simulation of a Drosophila melanogaster muscarinic acetylcholine receptor.

A stably-transfected, Drosophila cell line (S2-DMl-1) expressing the Drosophila DMl muscarinic acetylcholine receptor (mAChR) exhibits high-affinity, saturable, specific binding of the radiolabelled muscarinic antagonist [3H]-N-methyl scopolamine ([3H]-NMS) with an equilibrium dissociation constant (Kd) of 0.67 +/- 0.02 and a Bmax of 1.

Functional characterization of a mutated chicken alpha7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2.

1. Site-directed mutagenesis was used to create an altered form of the chicken alpha7 nicotinic acetylcholine (ACh) receptor subunit (alpha7x61) in which a leucine residue was inserted between residues Leu9' and Ser10' in transmembrane domain 2. The properties of alpha7x61 receptors are distinct from those of the wild-type receptor.

Thapsigargin and receptor-mediated activation of Drosophila TRPL channels stably expressed in a Drosophila S2 cell line.

The Drosophila melanogaster genes, transient receptor potential (trp) and transient receptor potential-like (trpl) encode putative plasma membrane cation channels TRP and TRPL, respectively. We have stably co-expressed Drosophila TRPL with a Drosophila muscarinic acetylcholine receptor (DM1) in a Drosophila cell line (S2 cells). Basal Ca2+ levels measured using Fura-2/AM in unstimulated S2-DM1-TRPL cells were low and indistinguishable from untransfected cells, indicating that the TRPL channels were not constitutively active in this expression system.

Effects of the alpha subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors.

1. Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (alpha4beta2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal alpha subunit (SAD) with the chicken beta2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs.

Ca2+ entry into PC12 cells initiated by ryanodine receptors or inositol 1,4,5-trisphosphate receptors.

Capacitative Ca2+ entry (CCE) is a universal mechanism for refilling intracellular Ca2+ stores in electrically non-excitable cells. The situation in excitable cells is less clear, however, since they may rely on other entry mechanisms for Ca2+-store refilling. In the present study we investigated CCE in intact PC12 cells, using acetylcholine to bring about activation of InsP3 receptors (InsP3Rs), caffeine to activate ryanodine receptors (RyRs) and thapsigargin to inhibit sarco/endoplasmic reticulum Ca2+-ATPase pumps.

The expression of a cloned Drosophila octopamine/tyramine receptor in Xenopus oocytes.

The expression of a cloned Drosophila octopamine/tyramine receptor (OctyR99AB) is described in Xenopus oocytes. Agonist stimulation of OctyR99AB receptors increased intracellular Ca2+ levels monitored as changes in the endogenous inward Ca2+-dependent chloride current. The receptor is preferentially sensitive to biogenic amines with a single hydroxyl on the aromatic ring.

Selective inhibition of adenylyl cyclase by octopamine via a human cloned alpha 2A-adrenoceptor.

1. In this study we have compared the abilities of the enantiomers of the structural isomers of the phenolamines, octopamine and synephrine, and the catecholamines, noradrenaline and adrenaline, to couple selectively a human cloned alpha 2A-adrenoceptor, stably expressed in a Chinese hamster ovary (CHO) cell line, to G-protein linked second messenger pathways mediating an increase and a decrease in cyclic AMP production. 2.

ACR-3, a Caenorhabditis elegans nicotinic acetylcholine receptor subunit. Molecular cloning and functional expression.

The molecular cloning and functional co-expression of a novel nicotinic acetylcholine receptor (nAChR) non-alpha subunit gene, acr-3, is described. Previously we determined the sequence and demonstrated the functional co-expression of acr-2, a nAChR non-alpha subunit gene from Caenorhabditis elegans. Analysis of the acr-2 genomic DNA revealed the existence of another potential nAChR subunit gene, acr-3, in the same orientation, only 281 bp downstream of acr-2.

Lymphocyte activation in health and disease.

Lymphocytes employ a complex assembly of signaling elements that have been organized on a spatiotemporal map to define their role in stimulating both proliferation and apoptosis. The antigen/major histocompatibility complex (MHC) initiates the sequence by organizing the assembly of an active T-cell receptor (TCR) complex responsible for transmitting information down various signaling cassettes (e.g.

The 1996 Massry Prize. Inositol trisphosphate and calcium: two interacting second messengers.

Inositol trisphosphate (InsP3) functions as a second messenger to control the release of internal calcium and the entry of external calcium. This InsP3/Ca2+ signalling pathway is based on a hierachical system with the release from individual channels being the fundamental event (Ca2+ blips). A collection of blips produces larger elementary events (Ca2+ puffs) which then fuse through a regenerative process of calcium-induced calcium release to give global events (Ca2+ waves).

Elementary and global aspects of calcium signalling.

Calcium is a ubiquitous second messenger used to regulate a wide range of cellular processes. This role in signalling has to be conducted against the rigid homeostatic mechanisms that ensure that the resting level of Ca2+ is kept low (i.e.

Genetic analysis of cholinergic nerve terminal function in invertebrates.

Genetic analysis of nerve terminal function is proving fruitful and studies on invertebrates are making a substantial impact. In this survey, particular emphasis has been placed on cholinergic chemical synaptic transmission. The advanced genetics of Drosophila melanogaster and Caenorhabditis elegans with their rich diversity of behavioural and biochemical mutants is providing new insights into the functions of key molecular components of synapses.

pH-dependent actions of THIP and ZAPA on an ionotropic Drosophila melanogaster GABA receptor.

The actions of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and ZAPA (Z-3-[(aminoiminomethyl)thio]prop-2-enoic acid) were tested on an ionotropic homo-oligomeric GABA receptor of Drosophila melanogaster. The amplitude of currents activated by THIP and ZAPA declined rapidly during agonist application and a rebound response was observed on washout. By correcting the pH shift induced by these acid salts, responses more typical of GABA agonists were seen.

Lophotoxin-insensitive nematode nicotinic acetylcholine receptors.

Nematode nicotinic acetylcholine receptors (nAChRs) are molecular targets of several anthelmintic drugs. Studies to date on Caenorhabditis elegans and Ascaris suum have demonstrated atypical pharmacology with respect to nAChR antagonists, including the finding that kappa-bungarotoxin is a more effective antagonist than alpha-bungarotoxin on Ascaris muscle nAChRs. Lophotoxin and its naturally occurring analogue bipinnatin B block all vertebrate and invertebrate nAChRs so far examined.

Subcellular Ca2+ signals underlying waves and graded responses in HeLa cells.

Background: Many agonist-evoked intracellular Ca2+ signals have a complex spatio-temporal arrangement, and are observed as repetitive Ca2+ spikes and Ca2+ waves. The key to revealing how these complex signals are generated lies in understanding the functional structure of the intracellular Ca2+ pool. Previous imaging studies, using relatively large cells such as oocytes and myocytes, have identified subcellular elementary Ca2+ signals, indicating that the intracellular Ca2+ pool releases Ca2+ from functionally discrete sites.

Capacitative calcium entry is colocalised with calcium release in Xenopus oocytes: evidence against a highly diffusible calcium influx factor.

Depletion of intracellular calcium stores activates the plasma membrane capacitative calcium entry pathway in many cell types. The nature of the signal that couples the depletion of the intracellular calcium stores to the activation of the plasma membrane calcium influx pathway is as yet unknown. It has recently been suggested that a highly diffusible calcium influx factor is involved in the activation of capacitative calcium entry, and that its action is potentiated by the protein phosphatase inhibitor okadaic acid.