Biochemical differentiation of lone star tick,Amblyomma americanum (L), salivary glands: Effects of attachment,feeding and mating

Salivary glands of female Amblyomma americanum (L.) are stimulated to differentiate by attachment to a host, subsequent feeding and mating. Incorporation of [³H]uridine into ribosomal and transfer RNAs as well as the synthesis of poly(A⁺)mRNA and protein parallel the pattern of increasing enzymatic activity and secretory ability of the glands. Unfed ticks contained 3.5 ± 0.47 ng poly(A⁺)mRNA/gland pr. By the second day of feeding this had increased more than 5-fold. The greatest amount of poly(A⁺)mRNA found in rapid-feeding phase females (body wt > 100 mg) was 370 ± 80 ng/gland pr. Poly(A⁺)mRNA mass doubles on the final day of feeding, just as the ticks exceeded 100 mg in wt. Ticks attached 1 to 10 days had increasingly greater amounts of salivary monosomes, 60 and 40S ribosomal subunits, and polysomes. Polysomal mass/gland pr also attained its maximum above 100 mg tick wt at the slow/rapid-feeding phase boundary; exceeding by 20 times that of unfed ticks. Degenerating glands from replete ticks continued to synthesize protein. In vitro incorporation of [³H]leucine was greatest within 24 h of attachment. Fluorographs of [³H]leucine labeled protein showed that mating caused a drop in incorporation after the 4th day of feeding. Glands from unmated females attached the same number of days continued to incorporate [³H]leucine at higher levels than those from mated females.     

Changes in dopamine-sensitive adenylate cyclase activity in salivary glands of female lone star ticks,Amblyomma americanum (L.), during feeding

The activity of dopamine-sensitive adenylate cyclase in feeding female ixodid tick salivary glands was dependent on the state of tick feeding. Activity was significantly greater than the “basal” activity in salivary glands from ticks at all stages of tick feeding. Enzyme activity was not detected in the glands of unfed females. Enzyme activity reached a peak in glands of ticks weighing approx. 200 mg then declined as ticks increased in weight beyond 200 mg to repletion. Replete ticks (detached from the host for 12–24 hr) had similar levels of basal and dopamine-stimulated adenylate cyclase activity as that measured in salivary glands of high weight (>200 mg) ticks. Enzyme activity was 19–62% less in glands from ticks feeding on hosts that had been parasitized 2–4 months earlier by lone star ticks.     

Cyclic nucleotide crosstalk in salivary glands from partially fed Dermacentor variabilis (Say)

Enzyme immunosorbent assays were used to measure cyclic nucleotide concentrations in homogenates of salivary glands from partially fed female Dermacentor variabilis. The adenylyl cyclase activator forskolin (100 μM) increased homogenate cGMP concentrations greater than three-fold over controls. Competitive inhibition of nitric oxide synthase with 1 mM l-NMMA, an l-arginine analog, demonstrated that crosstalk occurs downstream of nitric oxide synthesis. Forskolin-stimulated synthesis of cGMP was diminished 58% by the soluble guanylyl cyclase inhibitor ODQ (2 μM). The protein kinase A selective inhibitor Rp-cAMPS (50 μM) inhibited forskolin-stimulated cGMP by 49%. Whole glands treated with 10 μM dopamine increased cGMP levels two-fold in the presence of 1 mM IBMX. Treatment of whole salivary glands with equimolar concentrations of 8-Br-cAMP and 8-Br-cGMP produced no greater fluid uptake than in glands treated with 8-Br-cGMP alone, suggesting that cAMP and cGMP share a downstream target. The protein kinase G-selective inhibitor Rp-8-pCPT-cGMPS (100 μM) impeded 10 mM 8-Bromo-cGMP-stimulated gland weight increases. Pretreatment with verapamil, a Ca²⁺ channel blocker, attenuated cyclic nucleotide-stimulated fluid uptake indicating that whole gland fluid changes are dependent on extracellular Ca²⁺. Together, our data suggest that cGMP production is mediated in part by cAMP-dependent activation of soluble guanylyl cyclase. Experiments measuring changes in whole salivary gland weight support the hypothesis that cAMP and cGMP signaling cascades have a common target and that cyclic nucleotide-stimulated fluid movement is dependent on Ca²⁺ influx.     

Is there a “dopaminergic glial cell”?

Intracellular cAMP increased 9-fold in cerebral hemisphere primary cultures after incubation with dopamine (10^–4M). The effect was dose- and time-dependent (10^–6 M-10^–4M; 2–10 minutes). It was mimicked, to some extent, by the partial agonist apomorphine (10^–5 M-10^–4 M) and antagonized by fluphenazine (10^–5 M-10^–4 M). The elevation of cAMP caused by dopamine was incompletely antagonized by propanolol (10^–5 M-10^–4 M), obviating an interaction with -adrenergic receptors. A -adrenergic effect was antagonized by propranolol but only slightly by fluphenazine. The effect of dopamine on cAMP-level was more pronounced in a subpopulation of the hemisphere culture, i.e. in astroglial cultures from the striatum, 12-fold compared with controls at 10^–4 M. No dopamine stimulated formation of cAMP was found in primary cultures from brain-stem. The results demonstrated some heterogeneity among astroglial cells. The cultures used contained mainly astroglial-like cells, as judged from immnohistochemical localization mainly astroglial-like cells, as judged from immunohistochemical localization of the glial specific proteins S 100 and GFA (-albumin). No mature neurons or oligodendroglial cells have so far been demonstrated in the cultures.     

Isolation and characterization of an anticoagulant present in the salivary glands of the bont-legged tick,Hyalomma truncatum

A low molecular mass anticoagulant (17 kDa) was isolated from the salivary glands of prefed female Hyalomma truncatum ticks by means of reverse phase and anion-exchange HPLC. Trypsin digestion and amino acid analysis confirmed the protein nature of the anticoagulant. The inhibitor appears to be uncompetitive with a K_i of 6.9×10^–10M. The target of the anticoagulant is factor Xa at the junction of the extrinsic and intrinsic pathways. This may be crucial for the survival of the tick, making it feasible to investigate the possibility of vaccination with this antihaemostatic against tick feeding. In addition, tick anticoagulants may possibly have therapeutic application in controlling thrombosis.     

Interactions between cAMP- and cGMP-dependent protein kinase inhibitors and phosphodiesterase IV inhibitors on arachidonate release from human monocytes

The effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type IV inhibitors and of the cell permeable analogue of cAMP, db-cAMP, were investigated on fMLP-induced arachidonate release from human monocytes. When monocytes were preincubated with the combined PKA/PKG inhibitor H8 (10⁻⁶ to 10⁻⁴ M) or the selective PKG inhibitor Rp-8-cpt-cGMPs (10⁻⁶ to 10⁻⁴ M) a concentration-dependent reduction of the inhibitory effect of db-cAMP (10⁻ M), rolipram (10⁻⁵ M) and Ro 20-1724 (10⁻⁵ M) was noted. When monocytes were preincubated with the selective PKA inhibitor H89 (10⁻⁶ to 10⁻⁴ M), only a small inhibition of the effect of db-cAMP and no inhibition of the effects of rolipram and Ro 20–1724 were observed. The present data indicate that db-cAMP and PDE IV inhibitors elicit an in vitro anti-inflammatory activity by a PKA-independent mechanism, which do not appear to be mainly mediated via the PKG activation.     

Studies on the de novo synthesis of juvenile hormone III and methyl farnesoate by isolated corpora allata of adult female Gryllus bimaculatus

Activity of the corpora allata (CA) in vitro of adult female Gryllus bimaculatus was studied following incorporation of radioactivity from [2-¹⁴C]acetate and l-[methyl-³H]methionine into juvenile hormone III (JH III) and its immediate precursor methyl farnesoate (MF). Spontaneously active glands from females reared at 27°C utilized exogenous labelled acetate extensively for synthesis of MF and JH III (incorporation 80–84% at 2 mM acetate). 10⁻⁷ to 10⁻⁵ M exogenous JH III in the incubation medium had no effect on the rate of JH biosynthesis in spontaneously active glands. At 10⁻⁴ M JH III incorporation of acetate into JH III was reduced. The amount of MF was also lowered. JH III treatment (10⁻⁸–10⁻⁶ M) of spontaneously inactive glands led to an increase in the amount of MF. This increase was due to a de novo synthesis. Exogenous farnesol (20–200 μM) increased JH III biosynthesis and the amount of MF, but suppressed [2-¹⁴C]acetate incorporation. Dilution of the endogenous precursors is probably the most important cause of this suppression. As shown by the abnormally high MF levels in farnesol treated glands, epoxidation seems to be a rate-limiting step under certain experimental conditions.     

Cyclic AMP and calcium modulated ATPase activity in the salivary glands of the lone star tick Amblyomma americanum (L.)

Na⁺,K⁺-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na⁺,K⁺-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na⁺,K⁺-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na⁺,K⁺-ATPase activity exist in the plasma membrane-enriched fraction.ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg²⁺, Ca²⁺ and Na⁺. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg²⁺ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca²⁺-ATPase activity and oligomycin-sensitive Ca²⁺, Mg²⁺-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction.     

Effects of adrenergic drugs on the activity of tryptophan hydroxylase in midbrain raphe slices of the rat

The effects of α- and ß-adrenergic drugs on the activity of tryptophan hydroxylase were investigated in rat midbrain raphe slices. The tryptophan hydroxylase activity in slices was estimated by measuring the formation of 5-hydroxytryptophan (5-HTP) under inhibition of aromatic l-amino acid decarboxylase using 3-hydroxy-4-bromobenzyloxyamine (NSD 1055). Isoproterenol, a ß-adrenergic stimulant, significantly increased 5-HTP formation to 122% (P < 0.05) of control at 10⁻⁶ M and this effect was prevented by 10⁻⁶ M of propranolol, a ß-adrenergic blocker. 5-(1-Hydroxy-2-isopropylaminobutyl)-8-hydroxycarbostryril hydrochloride hemihydrate (OPC 2009), a ß-adrenergic stimulant which does not contain a catechol group, increased 5-HTP formation to 145% at 10⁻⁶ M. A-23187 at 5 × 10⁻⁷ M further enhanced the isoproterenol-stimulated 5-HTP formation to 156% of control. Dibutyryl cAMP at 10⁻² M, however, did not enhance it. 8-Bromo cAMP did not enhance the OPC 2009-stimulated 5-HTP formation, either. An α-adrenergic stimulant, clonidine, had no effect on 5-HTP formation. But an α-adrenergic blocker, yohimbine, reduced 5-HTP formation to 78% at 10⁻⁶ M. These results suggest that the activity of tryptophan hydroxylase can be controlled by a ß-adrenergic receptor coupled with adenylate cyclase via an intracellular cAMP-dependent process.     

Effect of nitrate feeding strategies on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana

Controlled nitrate feeding strategies for fed-batch cultures of microalgae were applied for the enhancement of lipid production and microalgal growth rates. In particular, in this study, the effect of nitrate feeding rates on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana were investigated using three feeding modes (i.e., pulse, continuous, and staged) and under two light variations on both lipid productivity and fatty acid compositions. Higher nitrate levels negatively affected lipid production in the study. Increasing the light intensity increased the lipid contents of the microalgae in all three fed-batch feeding modes. A maximum of 58.3% lipid- to dry weight ratio was achieved when using pulse-fed cultures at an illumination of 200 μmol photons m⁻² s⁻¹ and 10 mg/day of nitrate feeding. This condition also resulted in the maximum lipid productivity of 44.6 mg L⁻¹ day⁻¹. The fatty acid compositions of the lipids consisted predominantly of long-chain fatty acids (C:16 and C:18) and accounted for 70% of the overall fatty acid methyl esters. Pulse feeding mode was found to significantly enhance the biomass and lipid production. The other two feeding modes (continuous and staged) were not ideal for lipid and biomass production. This study demonstrates the applicability of pulse feeding strategies in fed-batch cultures as an appropriate cultivation strategy that can increase both lipid accumulation and biomass production.     

Spectral characteristics of human fetal adrenal microsomes.

Investigations of human fetal adrenal gland microsomes indicated that a carbon monoxide binding pigment had an absorption maximum of 446 to 448 nm. This pigment, upon heat treatment at 37°C was degraded to the form of cytochrome p-420. NADPH reduced cytochrome p-450 slowly and completely. Typical concentrations of 0.75 and 0.16 nmoles/mg protein cytochrome P-450 and b₅, respectively, were observed. Reduced ethylisocyanide spectra were similar to those of rat hepatic microsomes with absorption maxima at 430 as well as 454 nm. Typical type I spectral changes were observed with progesterone, 17-α-OH-progesterone, pregnenolone and androstenedione when these steroids were added to the sample cuvettes. Androstenedione exhibited an apparent spectral dissociation constant (K_S) of 5×10⁻⁶M pregnenolone and progesterone exhibited higher affinities with apparent dissociation constants of 1.1×10⁻⁷M and 1.8×10⁻⁷M, respectively. The maximal absorbance change induced by androstenedione was lower (Emax = 0.027 per mg protien) than the changes in absorbance maxima induced by pregnenolone or progesterone (Emax = 0.060 and 0.047 per mg protein, respectively) when saturating concentrations of these steroids were added to the sample cuvettes. Ethylmorphine and aminopyrine (10⁻³M final concentrations) did not exhibit observable spectral changes; however, type II spectra could be elicited with aniline and nicotinamide and apparent dissociation constants of 3.5×10⁻²M and 2.5×10⁻²M, respectively, were obtained.     

Salivary fluid secretion in the ixodid tick Rhipicephalus appendiculatus is inhibited by Thogoto virus infection

Adult Rhipicephalus appendiculatus ticks, infectedwith Thogoto (THO) virus or control, were fed on guinea pigs and removed atintervals throughout the feeding cycle. Salivary fluid secretion was measuredbyan in vitro technique. The salivary glandsof infected, partially-fed ticks secreted fluid in vitro at about 75% the rateof controls, but the difference between infected and controls among engorgedticks was not statistically significant. Basal and DA-stimulated levels ofcyclic AMP (cAMP) were determined in isolated glands and were significantlyaffected by THO virus infection. The differences in secretory rate amongcontroland infected ticks could not be explained in terms of altered cAMP levels.Haemolymph volume was measured by a tracer-dilution technique using³H-inulin. The mean haemolymph volume for both THO-infected andcontrol groups was between 23–24% body weight throughout the feedingcycle, indicating that infection by this arbovirus did not influence salivaryfluid secretion via altered haemolymph volume. The mechanism by which THO virusaffects secretory activity of its tick vector remains unknown.     

Determination of pioglitazone hydrochloride by flow injection chemiluminescence tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex system

A novel flow injection-chemiluminescence (FI–CL) approach is proposed for the assay of pioglitazone hydrochloride (PG-HCl) based on its enhancing influence on the tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex (Ru(bipy)₃²⁺-DPA) CL system in sulfuric acid medium. The possible CL reaction mechanism is discussed with CL and ultraviolet (UV) spectra. The optimum experimental conditions were found as: Ru(bipy)₃²⁺, 5.0 × 10⁻⁵ M; sulfuric acid, 1.0 × 10⁻³ M; diperiodatoargentate(III) (DPA), 1.0 × 10⁻⁴ M; potassium hydroxide, 1.0 × 10⁻³ M; flow rate 4.0 ml min⁻¹ for each flow stream and sample loop volume, 180 μl. The CL intensity of PG-HCl was linear in the range of 1.0 × 10⁻³ to 5.0 mg L⁻¹ (R² = 0.9998, n = 10) with limit of detection [LOD, signal-to-noise ratio (S/N) = 3] of 2.2 × 10⁻⁴ mg L⁻¹, limit of quantification (LOQ, S/N = 10) of 6.7 × 10⁻⁴ mg L⁻¹, relative standard deviation (RSD) of 1.0 to 3.3% and sampling rate of 106 h⁻¹. The methodology was satisfactorily used to quantify PG-HCl in pharmaceutical tablets with recoveries ranging from 93.17 to 102.77 and RSD from 1.9 to 2.8%.     

Evaluation of crystalline amino acids,betaine and AMP as food attractants of the giant tiger prawn (Penaeus monodon)

Laboratory observations of substrate probing by the chelate walking legs (chelipeds), antennular flicking rate and maxilliped activity of the prawn Penaeus monodon were used to evaluate various chemicals at seven different concentrations between 10⁻¹M and 10⁻⁷M as feeding stimulants. Exposure to amino acids (alanine, arginine, glutamine, glycine, isoleucine, serine and taurine) and betaine resulted in higher rates of substrate probing, antennular flicking and maxilliped activity in P. monodon at higher pipette concentrations (>10⁻²M) than at lower concentrations. Least response occurred in prawns which were exposed to nucleotide, adenosine 5′-monophosphate. Glutamine, betaine and taurine were the most effective single compounds tested, and stimulated significantly higher activities (p < 0.05) in prawns at concentrations above 10⁻⁶M than did controls (seawater only).An equimolar mixture of amino acids and betaine was also found to be an effective stimulant to P. monodon at concentrations above 10⁻⁶M and continued to elicit search responses in prawns at concentrations lower than that of any of the single chemicals. Such a strong response is consistent with synergistic interactions of the mixtures. All four molt stages tested (C, D₀, D₁, D₂) were equally responsive to food attractants.     

Effect of prostaglandin on luteinizing hormone-stimulated proliferation of theca externa cells from chicken prehierarchical follicles

The effect of prostaglandin (PG) on proliferation of chicken theca externa cells from prehierarchical small yellow follicles (SYF) was evaluated and involved signaling pathways as well as mRNA expression of cAMP response element binding protein (CREB1), cyclins (CCND1 and CCNE1) and cyclin-dependent kinases (CDKs) were investigated. Results showed that PGE₁ (1–100 ng/ml) manifested a similar proliferating effect as LH on theca externa cells, and this stimulating effect was restrained by the prostaglandin receptor antagonist SC19220 at 10⁻⁷ to 10⁻⁵ M. Moreover, prostaglandin synthase inhibitor indomethacin (10⁻⁷ to 10⁻⁵ M) suppressed LH-induced increase in the cell number. In addition, PGE₁-stimulated cell proliferation was also predominantly hindered by H₈₉ (PKA inhibitor) instead of H₇ (PKC inhibitor). Meanwhile, BrdU incorporation experiment displayed similar changes with the cell number. Furthermore, H₈₉, SC19220 and indomethacin abolished the PGE₁-stimulated increase in the expression of CREB1, CCND1/CDK6 and CCNE1/CDK2 mRNAs, indicating that cAMP/PKA/CREB1 signaling cascade was involved in PGE₁-stimulated DNA synthesis. In conclusion, PG could promote proliferation of theca externa cells from prehierarchical follicles through changes in cyclin D1/CDK6, cyclin E1/CDK2 and CREB1 mRNA expression via cAMP/PKA and CREB1 signaling cascade. These results suggest that PG may promote development of chicken prehierarchical follicles and is related to dominant follicle selection in laying hens.     

Ecdysone 20-hydroxylation in Manduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The K_m⁵ (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10⁻⁵ and 3.67 × 10⁻⁷ M, respectively. The V_max was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher V_max, at physiological ecdysone concentrations (10⁻⁷ − 10⁻⁸ M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

The effect of mercury on chloride secretion in the shark (Squalus acanthias) rectal gland

1. Mercuric chloride inhibited chloride secretion in a dose dependent way in isolated perfused rectal glands. The effect was readily apparent at a concentration of 10⁻⁶ M and profound and irreversible at 10⁻⁴ M.2. The dithiol dithiothreitol (DTT) 10⁻² M completely prevented the effect of 10⁻⁶ M mercuric chloride, reduced that at 10⁻⁵M and 10⁻⁴M, and made the inhibition at the latter concentration reversible.3. Two organic mercurials, mersalyl and meralluride, that are effective diuretics in the mammalian kidney, and p-chloromercuribenzoyl sulfonic acid (PCMBS), that has no diuretic activity, had no effect on chloride secretion by the rectal gland.4. The effect of mersalyl was not modified by lowering the pH of the solution perfusing the glands.5. These results indicate that inorganic mercury and organic mercurials do not share the same mechanism of action.6. The absence of an effect of organic mercurials on chloride transport in the rectal gland suggests that its effect on another chloride transporting epithelia, the thick ascending limb of the loop of Henle, is not mediated by inhibition of the chloride cotransporter or Na⁺, K⁺-ATPase, common to both epithelia.     

Failure to produce axon reflex sweating in the volar skin of Japanese monkey (Macaca Fuscata) and crab-eating monkey (Macaca Irus)

1. The functional properties of the sweat glands and their innervation in the volar skin of three Japanese monkeys and two crab-eating monkeys were investigated.2. The sweat glands responded to both cholinomimetic and adrenomimetic agents, the former being highly predominant in the sudorific effect.3. Spontaneous emotional sweating was strongly or completely inhibited by atropine at 10⁻⁸–10⁻⁷ g/ml, but not by dihydroergotamine at 10⁻⁵–10⁻⁴ g/ml.4. Axon reflex sweating could not be produced by nicotine at 10⁻⁵–10⁻⁴ g/ml in all of primates tested.5. The nerve fibers surrounding the sweat glands were histochemically confirmed to contain both acetyland butyrylcholinesterase.     

ACTH,cyclic nucleotides,and brain protein phosphorylation in vitro

Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated³²P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH_1–24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10^–10 to 10^–4 M. In a number of protein bands, a biphasic effect of ACTH_1–24 was observed: in concentrations of 10^–4–10^–5 M, a reduced amount of³²P was found; in concentrations of 10^–6–10^–7 M, hardly any effect could be detected, whereas consistently at concentrations around 10^–8 M, a significant decrease was again observed. The phosphoprotein bands affected by in vitro addition of ACTH_1–24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.