TRADE-OFF BETWEEN IMMUNE STIMULATION AND EXPRESSION OF STORAGE PROTEIN GENES

Proteins stored in insect hemolymph may serve (is a source of amino acids and energy for metabolism, and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCH and Western blot.. After ensuring that. the immune system had, been activated by measuring the ensuing expression (, the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (pro PO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein. were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLP-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLP-III transcripts. Our findings are consistent with a down-regulation, of the express and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon. represents a strategy to redirect resources to combat injury or infection. (C) 2009 Wiley Periodicals, Inc.

A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

in the Apis mellifera post-genomic era, RNAi protocols have been used in functional approaches. However, sample manipulation and invasive methods such as injection of double-stranded RNA (dsRNA) can compromise physiology and survival. To circumvent these problems, we developed a non-invasive method for honeybee gene knockdown, using a well-established vitellogenin RNAi system as a model. Second instar larvae received dsRNA for vitellogenin (dsVg-RNA) in their natural diet. For exogenous control, larvae received dsRNA for GFP (dsGFP-RNA). Untreated larvae formed another control group. Around 60% of the treated larvae naturally developed until adult emergence when 0.5 mu g of dsVg-RNA or dsGFP-RNA was offered while no larvae that received 3.0 mu g of dsRNA reached pupal stages. Diet dilution did not affect the removal rates. Viability depends not only on the delivered doses but also on the internal conditions of colonies. The weight of treated and untreated groups showed no statistical differences. This showed that RNAi ingestion did not elicit drastic collateral effects. Approximately 90% of vitellogenin transcripts from 7-day-old workers were silenced compared to controls. A large number of samples are handled in a relatively short time and smaller quantities of RNAi molecules are used compared to invasive methods. These advantages culminate in a versatile and a cost-effective approach. (c) 2008 Elsevier Ltd. All rights reserved.

Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21 parts per thousand salinity), as well as potential routes of Na(+) uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase alpha-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell inviginations, respectively. These findings ire discussed regarding the putative movement of Na(+) across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations. (C) 2008 Elsevier Inc. All rights reserved.

Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta: Liposomes fusion and/or lysis investigated by fluorescence and atomic force microscopy

Three dermaseptins, DS 01, DD K, and DD L, were compared with respect to their structural features and interactions with liposomes. Circular dichroic spectra at alcohols of different chain lengths revealed that DS 01 has the higher helicogenic potential in hydrophobic media. Binding of DS 01, DD K, and DD L to liposomes induced significant blue shifts of the emission spectra of the single tryptophan located at position 3 of all sequences indicating association of the peptides with bilayers. Kinetics evaluation of atomic force microscopy images evidenced the strong fusogenic activity of DS 01 whereas DD K and DD L showed increased lytic activities. (C) 2007 Elsevier Inc. All rights reserved.

Removal from the Membrane Affects the Interaction of Rat Osseous Plate Ecto-Nucleosidetriphosphate Diphosphohydrolase-1 with Substrates and Ions

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M(r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always < 2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae)

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine > spermidine > putrescine. Spermidine affected K-0.5 values for Na+ with minor alterations in K-0.5 values for K+ and N-H-4(+), causing a decrease in maximal velocities under saturating Na+, K+ and NH4+ concentrations. Phosphorylation measurements in the presence of 20 mu M ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na+, both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na+, the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na+ at the Na+-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments. (c) 2008 Elsevier Inc. All rights reserved.

Hemolymph ionic regulation and adjustments in gill (Na(+), K(+))-ATPase activity during salinity acclimation in the swimming crab Callinectes ornatus (Decapoda, Brachyura)

We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill microsomal (Na(+), K(+))-ATPase from the marine swimming crab, Callinectes ornatus, acclimated to 21 parts per thousand or 33 parts per thousand salinity. C ornatus is isosmotic after acclimation to 21 parts per thousand but is hyposmotic at 33 parts per thousand salinity; hemolymph ions do not recover initial levels on acclimation to 21 parts per thousand salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH(4)(+) modulates enzyme affinity for K(+), which increases 187-fold in crabs acclimated to 33%. salinity. The (Na(+), K(+))-ATPase redistributes into membrane fractions of different densities, suggesting that altered membrane composition results from salinity acclimation. ATP was hydrolyzed at maximum rates of 182.6 +/- 7.1 nmol Pi min(-1) mg(-1) (21 parts per thousand) and 76.2 +/- 3.5 nmol Pi min(-1) mg(-1) (33 parts per thousand), with little change in K(M) values (approximate to 50 mu mol L(-1)). K(+) together with NH(4)(+) synergistically stimulated activity to maximum rates of approximate to 240 nmol Pi min(-1) mg(-1). K, values for ouabain inhibition (approximate to 110 mu mol L(-1)) decreased to 44.9 +/- 1.0 mu mol L(-1) (21 parts per thousand) and 28.8 +/- 1.3 mu mol L(-1) (33 parts per thousand) in the presence of both K(+) and NH(4)(+). Assays employing various inhibitors suggest the presence of mitochondrial F(0)F(1)- and K(+)- and V-ATPase activities in the gill microsomes. (C) 2009 Elsevier Inc. All rights reserved.

Na(+), K(+)-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: Novel perspectives on ammonia excretion

This investigation provides an extensive characterization of the modulation by ATP, Mg(2+), Na(+), K(+) and NH(4)(+) of a gill microsomal (Na(+),K(+))-ATPase from Callinectes danae acclimated to 15 parts per thousand salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K(+) modulated by NH4+, discussed regarding NH4+ excretion in benthic marine crabs. The (Na(+),K(+))-ATPase hydrolyzed ATP at a maximum rate of 298.7 +/- 16.7 nmol Pi min(-1) mg(-1) and K(0.5) = 174.2 +/- 9.8 mmol L(-1) obeying cooperative kinetics (n(H) = 1.2). Stimulation by sodium (V = 308.9 +/- 15.7 nmol Pi min(-1) mg(-1), K(0.5) = 7.8 +/- 0.4 mmol L(-1)), magnesium (299.2 +/- 14.1 nmol Pi min(-1) mg(-1), K(0.5) = 767.3 +/- 36.1 mmol L(-1)), potassium (300.6 +/- 153 nmol Pi min(-1) mg(-1), K(0.5) = 1.6 +/- 0.08 mmol L(-1)) and ammonium (V = 345.1 +/- 19.0 nmol Pi min(-1) mg(-1), K(0.5) = 6.0 +/- 0.3 mmol L(-1)) ions showed site-site interactions. Ouabain inhibited (Na(+),K(+))-ATPase activity with K(1) = 45.1 +/- 2.5 mu mol L(-1), although affinity for the inhibitor increased (K(1) = 22.7 +/- 1.1 mu mol L(-1)) in 50 mmol L(-1) NH(4)(+). Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F(0)F(1)- and K(+)-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other. (C) 2009 Elsevier Inc. All rights reserved.

Anxiety-like symptoms induced by morphine withdrawal may be due to the sensitization of the dorsal periaqueductal grey

Withdrawal from morphine leads to the appearance of extreme anxiety accompanied of several physical disturbances, most of them linked to the activation of brainstem regions such as the locus coeruleus, ventral tegmental area, hypothalamic nuclei and periaqueductal grey (PAG). As anxiety remains one of the main components of morphine withdrawal the present study aimed to evaluating the influence of the dorsal aspects of the PAG on the production of this state, since this structure is well-known to be involved in defensive behaviour elicited by anxiety-evoking stimuli. Different groups of animals were submitted to 10 days of i.p. morphine injections, challenged 2 h after with an i.p. injection of naloxone (0.1 mg/kg), and submitted to the plus-maze, open-field and light-dark transition tests. The effects of morphine withdrawal on anxiety-induced Fos immunolabelling were evaluated in four animals that passed by the light-dark transition test randomly chosen for Fos-protein analysis. Besides the PAG, Fos neural expression was conducted in other brain regions involved in the expression of anxiety-related behaviours. Our results showed that morphine withdrawn rats presented enhanced anxiety accompanied of few somatic symptoms. Increased Fos immunolabelling was noted in brain regions well-known to modulate these states as the prelimbic cortex, nucleus accumbens, amygdala and paraventricular hypothalamus. Increased Fos labelling was also observed in the ventral and dorsal aspects of the PAG, a region involved in anxiety-related processes suggesting that this region could be a common neural substrate enlisted during anxiety evoked by dangerous stimuli as well as those elicited by opiate withdrawal. (c) 2008 Elsevier Inc. All rights reserved,