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ABSTRACT: A collection of EST clones from female tick Amblyomma americanum salivary glands was hybridized to RNA from different feeding stages of female tick salivary glands and from unfed or feeding adult male ticks. In the female ticks, the expression patterns changed dramatically upon starting feeding, then changed again towards the end of feeding. On beginning feeding, genes possibly involved in survival on the host increased in expression as did many housekeeping genes. As feeding progressed, some of the survival genes were downregulated, while others were upregulated. When the tick went into the rapid feeding phase, many of the survival genes were downregulated, while a number of transport-associated genes and genes possibly involved in organ degeneration increased. In the males, the presence of females during feeding made a small difference, but feeding made a larger difference. Males showed clear differences from females in expression, as well. Protein synthesis genes were expressed more in all male groups than in the partially fed females, while the putative secreted genes involved in avoiding host defenses were expressed less.
Archives of Insect Biochemistry and Physiology 07/2009; 71(4):236-53. · 1.36 Impact Factor
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ABSTRACT: Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins assemble in tight core complexes which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and distributed in distinct subcellular compartments. All vesicle transport mechanisms known to date have an essential requirement for a member of the Sec1 protein family, including the nSec1 in regulated exocytosis. A homolog of nSec1 was cloned and sequenced from the salivary glands of partially fed female ticks. Double-stranded RNA was used to specifically reduce the amount of nSec1 mRNA and protein in female adult tick salivary glands. This reduction was accompanied by a decrease in anticoagulant protein release by the glands and by abnormalities in feeding by dsRNA treated ticks. We report the efficacy of double-stranded RNA-mediated interference in "knocking down" nSec1 both in vivo and in vitro in tick salivary glands and the applicability of this technique for studying the mechanism of exocytosis in tick salivary glands.
Biochemical and Biophysical Research Communications 12/2004; 324(4):1256-63. · 2.48 Impact Factor
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ABSTRACT: Protein secretion into the saliva from the tick salivary glands is due to exocytosis of vesicular membrane bound granular material regulated by SNARE complex proteins after salivary gland stimulation by PGE2 [Insect Biochem. Mol. Biol. 32 (2002) 1711]. Proteins associated with vesicles (v-SNAREs) are essential components of the exocytotic process. Synaptobrevin is a key v-SNARE in all secreting cells studied to date. A vesicle-associated synaptobrevin cDNA fragment homologue from the salivary glands of partially fed lone star tick females was cloned and sequenced. Double-stranded (ds) RNA interference (RNAi) is an effective method to silence specific gene expression. The functional role of synaptobrevin in protein secretion in partially fed tick salivary glands was studied with an in vitro RNAi method. Incubation of isolated salivary glands with double-stranded RNA (dsRNA) transcribed from a tick salivary gland synaptobrevin cDNA fragment resulted in decreased expression of the transcript, a reduction in the level of synaptobrevin protein and inhibition of PGE2 stimulated anticoagulant protein secretion by isolated salivary glands. We demonstrate the applicability of RNAi for studying individual steps in the mechanism of PGE2 stimulated exocytosis in the salivary glands of ixodid ticks.
Insect Biochemistry and Molecular Biology 05/2004; 34(4):407-13. · 3.25 Impact Factor
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ABSTRACT: The salivary glands and saliva from the lone star tick Amblyomma americanum (L.) were analyzed for the presence of the two endogenous agonists of cannabinoid receptors, N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as of the anandamide congener, N-palmitoylethanolamine (PEA), an anti-inflammatory and analgesic mediator that is inactive at cannabinoid receptors. Two very sensitive mass-spectrometric techniques were used for this purpose. Both 2-AG and PEA, as well as other N-acylethanolamines (NAEs), were identified in salivary glands, but anandamide was below detection. The levels of 2-AG were considerably higher in the salivary glands of partially fed than replete females. Ex vivo gland stimulation with arachidonic acid increased the levels of 2-AG, but not of PEA or other NAEs, and caused the formation of anandamide and of the potent analgesic compound N-arachidonoylglycine. Instead, the amounts of anandamide, 2-AG and PEA were not influenced by treatment of salivary glands with dopamine, which stimulates saliva secretion. The possible biosynthetic precursors of anandamide, PEA and other NAEs were also detected in salivary glands, whereas only PEA was detected in tick saliva. These data demonstrate for the first time that the salivary glands of an obligate ectoparasite species can make endocannabinoids and/or related congeners with analgesic and anti-inflammatory activity, which possibly participate in the inhibition of the host defense reactions.
Biochimica et Biophysica Acta 08/2003; 1633(1):61-7. · 4.66 Impact Factor
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ABSTRACT: Previous studies identified a prostaglandin E2 (PGE2) receptor in the salivary glands of partially fed female lone star ticks, Amblyomma americanum (L.). In the present studies, protein secretion from dispersed salivary gland acini was shown to be specific for PGE2, as compared with PGF2α or the thromboxane analog U-46619, in accordance with their respective binding affinities for the PGE2 receptor. Furthermore, the selective PGE2 EP1 receptor agonist, 17-phenyl trinor PGE2, was as effective as PGE2 in stimulating secretion of anticoagulant protein. Calcium ionophore A-23187 (1 to 100 μM) stimulated secretion of anticoagulant protein in a dose-dependent manner but the voltage-gated Ca2+-channel blocker verapamil (1 to 1000 μM) and the receptor-mediated Ca2+-entry antagonist, SK&F 96365 (1 and 10 μM), and 5 mM ethylene glycol bis(β-aminoethyl ether)-N,NN′,N′-tetraacetic acid (EGTA) had no appreciable effect on inhibiting PGE2-stimulated secretion of anticoagulant protein. PGE2 (0.1 μM) and the non-hydrolyzable analog of guanosine triphosphate (GTP), GTPγS (10 μM), directly activated phospholipase C (PLC) in a membrane-enriched fraction of the salivary glands after PLC was first incubated with the PGE2 EP1 receptor antagonist AH-6809, which presumably antagonized endogenous PGE2 (0.3 μM) in the broken-cell-membrane-enriched fraction. TMB-8, an antagonist of intracellular inositol trisphosphate (IP3) receptors, inhibited PGE2-stimulated secretion. The results support the hypothesis that PGE2 stimulates secretion of tick salivary gland protein via a phosphoinositide signaling pathway and mobilization of intracellular Ca2+.
Insect Biochemistry and Molecular Biology.
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ABSTRACT: A factor from tick brain increases inositol phosphates in isolated, whole tick salivary glands. The factor is sensitive to trypsin and heat (5 min, 100°C) suggesting that it may be a neuropeptide or protein. The salivary glands undergo growth and differentiation accompanied by considerable proliferation of plasma membranes during tick feeding. Salivary glands from ticks in later stages of feeding produce higher levels of inositol phosphates than glands from ticks in early stages of feeding. Cyclic AMP modulates the formation of inositol phosphates suggesting interaction of salivary gland function by the transducing system that employs cyclic AMP as a “second messenger” and that which employs inositol phosphates.
Insect Biochemistry.
