Identification of arginine kinase as an allergen of brown crab, Callinectes bellicosus, and in silico analysis of IgE-binding epitopes.

In recent years there has been an increase in the prevalence of allergic reactions to contact with/or consumption of crustaceans by immune responses mediated by IgE antibodies. Arginine kinase (AK) is considered one of the main allergens present in marine invertebrates. Currently, the allergenic potential of the brown crab (Callinectes bellicosus), which is a crustacean of great economic importance, has not been studied. Therefore, the aim of this work was to identify C. bellicosus AK as an allergen and to predict IgE-binding epitopes through immunobioinformatic analysis. AK was purified by precipitation with ammonium sulfate and ion- exchange chromatography. AK allergenicity was evaluated by IgE reactivity against sera from crustacean-allergic and non-allergic patients in both native and denaturing conditions. Additionally, a homology model was built based on the deduced amino acid sequence. A single band (~40 kDa) was found in SDS-PAGE, which was identified as an AK by mass spectrometry. AK showed immunoreactivity against crab-allergenic sera in both native and denaturing conditions with 70% and 80% positive reactions, respectively. Additionally, a 1073 bp ORF was obtained which codes for a deduced sequence of 357 amino acids corresponding to AK with > 90% identity with other AKs. Structural homology model of AK showed two main domains with conserved / folding of phospho-guanidine kinases. BediPred and Discotope were used for epitope prediction analysis, which suggests eight possible linear epitopes and seven conformational epitopes, respectively; and shows to be similar to other crustaceans AKs. C. bellicosus AK was identified as an allergenic protein by IgE reactivity and immunobioinformatic analysis indicates that both linear and conformational epitopes could be located in the surface of C. bellicosus AK structure.

Combined effects of waterborne copper exposure and salinity on enzymes related to osmoregulation and ammonia excretion by blue crab Callinectes sapidus.

Copper is essential, but can be toxic to aquatic organisms when present in high concentrations. In freshwater crustaceans, copper inhibits enzymes related to ionic and osmoregulation and to the ammonia efflux, that leads to Na+ imbalance and inhibition of ammonia excretion. In the animals inhabiting estuarine or seawater, mechanisms of copper toxicity is not clear, but had been described as disruption of ionregulation and metabolism. To clarify the mechanism of copper toxicity in crustaceans inhabiting variable salinity, this work investigated whether copper affects ammonia excretion and enzymes used for ammonia balance and osmoregulation in the blue crab Callintectes sapidus acclimated to salinity 2 and 30 ppt. To achieve this, juveniles of the blue crab were exposed to 63.5 µg/L of copper at both salinities for 96 h. This is an environmentally realistic copper concentration. Results of ammonia efflux, free amino acids and Na+ concentrations in hemolymph, Na+/K+-ATPase, H+-ATPase and, carbonic anhydrase (CA) activities in gills were consistent with the osmoregulatory pattern adopted by the blue crab, which hyperosmoregulates at salinity 2 ppt and osmoconforms at 30 ppt. At 30 ppt copper reduced free amino acid in hemolymph of crabs, suggesting an effect of the metal on osmotic performance. At 2 ppt, copper significantly increased the H+-ATPase activity involved in ammonia excretion. This may be a compensatory response of crabs to maintain low levels of ammonia in their hemolymph; which can be increased by copper exposure. Results presented here are useful for the improvement of the Biotic Ligand Model (BLM) to predict copper toxicity for saltwater environments.

A Kinetic Characterization of the Gill (Na+, K+)-ATPase from the Semi-terrestrial Mangrove Crab Cardisoma guanhumi Latreille, 1825 (Decapoda, Brachyura).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the semi-terrestrial mangrove crab Cardisoma guanhumi. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)-ATPase activity, but also containing other microsomal ATPases. The (Na+, K+)-ATPase, notably immuno-localized to the apical region of the epithelial pillar cells, and throughout the pillar cell bodies, has an M r of around 110 kDa and hydrolyzes ATP with V M = 146.8 ± 6.3 nmol Pi min-1 mg protein-1 and K M = 0.05 ± 0.003 mmol L-1 obeying Michaelis-Menten kinetics. While stimulation by Na+ (V M = 139.4 ± 6.9 nmol Pi min-1 mg protein-1, K M = 4.50 ± 0.22 mmol L-1) also follows Michaelis-Menten kinetics, modulation of (Na+, K+)-ATPase activity by MgATP (V M = 136.8 ± 6.5 nmol Pi min-1 mg protein-1, K 0.5 = 0.27 ± 0.04 mmol L-1), K+ (V M = 140.2 ± 7.0 nmol Pi min-1 mg protein-1, K 0.5 = 0.17 ± 0.008 mmol L-1), and NH4+ (V M = 149.1 ± 7.4 nmol Pi min-1 mg protein-1, K 0.5 = 0.60 ± 0.03 mmol L-1) shows cooperative kinetics. Ouabain (K I = 52.0 ± 2.6 µmol L-1) and orthovanadate (K I = 1.0 ± 0.05 µmol L-1) inhibit total ATPase activity by around 75%. At low Mg2+ concentrations, ATP is an allosteric modulator of the enzyme. This is the first study to provide a kinetic characterization of the gill (Na+, K+)-ATPase in C. guanhumi, and will be useful in better comprehending the biochemical underpinnings of osmoregulatory ability in a semi-terrestrial mangrove crab.

Biochemical characteristics and modulation by external and internal factors of aminopeptidase-N activity in the hepatopancreas of a euryhaline burrowing crab.

Strikingly, in spite of its physiological importance, information about occurrence, biochemical characteristics and mechanisms of regulation of aminopeptidase-N (APN) in the hepatopancreas of intertidal euryhaline crabs is still lacking. In this work, we determined the occurrence, biochemical characteristics, response to environmental salinity and dopamine of APN in the hepatopancreas of the euryhaline crab Neohelice granulata (Dana 1851) from the open mudflat of Mar Chiquita coastal lagoon (Buenos Aires province, Argentina). APN activity was maximal at pH and temperature range of 7.6-9.0 and 37-45 °C, respectively. APN activity exhibited Michaelis-Menten kinetics (apparent Km = 0.19 ± 0.10 mM) (pH 7.6, 37 °C) and appeared to be sensitive to bestatin (I 50 = 15 mM) and EDTA (I 50 = 9 mM). In crabs acclimated to 10 psu (hyper-regulation conditions) and 37 psu (hypo-regulation conditions), APN activity was about 45 and 160% higher, respectively, than in 35 psu (osmoconformation). APN activity in the hepatopancreas was stimulated in vitro (about 137%) by 10(-4) M dopamine. Higher dopamine concentrations produced a similar extent of increase. The responses of APN activity to salinity and dopamine in vitro suggest the role of APN in digestive adjustments upon hyper and hypo-regulatory conditions and its modulation via direct mechanisms on hepatopancreas by dopamine.

Molecular characterization of a cytosolic manganese superoxide dismutase from the Chinese mitten crab, Eriocheir sinensis.

A cytosolic manganese superoxide dismutase gene (Es-cMnSOD) was cloned from the Chinese mitten crab Eriocheir sinensis, using reverse transcription-polymerase chain reaction and the rapid amplification of cDNA ends. The open reading frame of Es-cMnSOD is 867 bp in length and encodes a 288-amino acid protein without a signal peptide. The calculated molecular mass of the translated protein of Es-cMnSOD is 31.43 kDa, with an estimated isoelectric point of 6.30. The deduced amino acid sequence of Es-cMnSOD has similarities of 90, 89, 84, 87, and 81% to those of white shrimp Litopenaeus vannamei MnSOD, black tiger shrimp Penaeus monodon MnSOD, giant freshwater prawn Macrobrachium rosenbergii MnSOD, blue crab Callinectes sapidus MnSOD, and red swamp crayfish Procambarus clarkii MnSOD, respectively. Es-cMnSOD contains a manganese superoxide dismutase domain (DVWEHAYY) and 4 conserved amino acids responsible for binding manganese. Es-cMnSOD was expressed in the hemocytes, eyestalk, muscle, intestine, gill, and hepatopancreas. Es-cMnSOD transcripts in hemocytes of E. sinensis increased at 1.5 and 48 h after injection of Aeromonas hydrophila, indicating that the induction of the SOD system response occurred within a short period of time. This study suggests that MnSOD may play a critical role in crab immunity, allowing efficient activation of an early innate immune response in the crab.

The effects of copper on Na(+)/K (+)-ATPase and aquaporin expression in two euryhaline invertebrates.

We used immunocytochemical and fluorometric techniques to show that gill cells of two marine invertebrates, the crab Neohelice granulata (osmoregulator) and the clam Mesodesma mactroides (osmoconformer), increase the expression of membrane transporters [Na(+)/K(+)-ATPase and aquaporin (AQP1)] after whole-animals exposure (96 h) to sublethal concentrations of copper in water of salinity 30 ppt, when both clams and crabs are isosmotic with respect to the environmental medium. A plausible interpretation of our findings is that this increased expression in membrane transporters may serve as an attempt to ameliorate the deleterious effects of copper on the mechanisms involved in ion and volume regulation in gill cells.

Synergistic stimulation by potassium and ammonium of K(+)-phosphatase activity in gill microsomes from the crab Callinectes ornatus acclimated to low salinity: novel property of a primordial pump.

We provide an extensive characterization of the modulation by p-nitrophenylphosphate, Mg²âº, Na⁺, K(+), Rb⁺, NH(4)(+) and pH of gill microsomal K⁺-phosphatase activity in the posterior gills of Callinectes ornatus acclimated to low salinity (21‰). The synergistic stimulation by K⁺ and NH(4)(+) of the K⁺-phosphatase activity is a novel finding, and may constitute a species-specific feature of K(+)/NH(4)(+) interplay that regulates crustacean gill (Na⁺, K⁺)-ATPase activity. p-Nitrophenylphosphate was hydrolyzed at a maximum rate (V) of 69.2 ± 2.8nmolPimin⁻¹mg⁻¹ with K(0.5)=2.3 ± 0.1mmolL(-1), obeying cooperative kinetics (n(H)=1.7). Stimulation by Mg²âº (V=70.1 ± 3.0nmolPimin⁻¹mg⁻¹, K(0.5)=0.88 ± 0.04mmolL⁻¹), K⁺ (V=69.6 ± 2.7nmolPimin⁻¹mg⁻¹, K(0.5)=1.60 ± 0.07mmolL⁻¹) and NH(4)(+) (V=90.8 ± 4.0nmolPimin⁻¹mg⁻¹, K(0.5)=9.2 ± 0.3mmol L⁻¹) all displayed site-site interaction kinetics. In the presence of NH(4)(+), enzyme affinity for K⁺ unexpectedly increased by 7-fold, while affinity for NH(4)(+) was 28-fold greater in the presence than absence of K⁺. Ouabain partially inhibited K⁺-phosphatase activity (K(I)=320 ± 14.0µmolL⁻¹), more effectively when NH(4)(+) was present (K(I)=240 ± 12.0µmolL⁻¹). We propose a model for the synergistic stimulation by K⁺ and NH(4)(+) of the K⁺-phosphatase activity of the (Na⁺, K⁺)-ATPase from C. ornatus posterior gill tissue.

Copper effects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at different salinities.

The estuarine crab Neohelice granulata was exposed (96 h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2 ppt salinity, 1 mg Cu/L; isosmotic crabs: 30 ppt salinity, 5 mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2 ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, increased lactate dehydrogenase activity, and reduced the mitochondrial membrane potential. In posterior gills, copper inhibited hexokinase and pyruvate kinase activity, and increased citrate synthase activity. In anterior gills of crabs acclimated to 30 ppt salinity, copper exposure inhibited phosphofructokinase and citrate synthase activity, and increased hexokinase activity. In posterior gills, copper inhibited phosphofructokinase and pyruvate kinase activity, and increased hexokinase and lactate dehydrogenase activity. Copper did not affect cytochrome c oxidase activity in either anterior or posterior gills of crabs acclimated to 2 and 30 ppt salinity. These findings indicate that exposure to a sublethal copper concentration affects the activity of enzymes involved in glycolysis and Krebs cycle, especially in anterior (respiratory) gills of hyperosmoregulating crabs. Changes observed indicate a switch from aerobic to anaerobic metabolism, characterizing a situation of functional hypoxia. In this case, reduced mitochondrial membrane potential would suggest a decrease in ATP production. Although gills of isosmotic crabs were also affected by copper exposure, changes observed suggest no impact in the overall tissue ATP production. Also, findings suggest that copper exposure would stimulate the pentose phosphate pathway to support the antioxidant system requirements. Although N. granulata is very tolerant to copper, acute exposure to this metal can disrupt the energy balance by affecting biochemical systems involved in carbohydrate metabolism.

Na⁺,K⁺-ATPase activity in the posterior gills of the blue crab, Callinectes ornatus (Decapoda, Brachyura): modulation of ATP hydrolysis by the biogenic amines spermidine and spermine.

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K⁺, Na⁺, NH4⁺ and Mg²âº and on inhibition by ouabain of posterior gill microsomal Na⁺,K⁺-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na⁺,K⁺-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na⁺,K⁺-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis-Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K⁺, Na⁺, NH4⁺ and Mg²âº varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na⁺,K⁺-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.

Short- and long-term, salinity-induced modulation of V-ATPase activity in the posterior gills of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae).

To better understand the biochemical mechanisms underlying anisosmotic extracellular regulation in the freshwater Brachyura, we kinetically characterized the V-ATPase from the posterior gills of Dilocarcinus pagei, acclimated for 10days to salinities up to 21‰. Specific activity was highest in fresh water (26.5±2.1U mg(-1)), decreasing in 5‰ to 21‰, attaining 3-fold less at 15‰. Apparent affinities for ATP and Mg(2+) respectively increased 3.2- and 2-fold at 10‰, suggesting expression of different isoenzymes. In a 240-h time-course study of exposure to 21‰, maximum specific activity decreased 2.5- to 4-fold within 1 to 24h while apparent affinities for ATP and Mg(2+) respectively increased by 12-fold within 24h and 2.4-fold after 1h, unchanged thereafter. K(I) for bafilomycin A(1) decreased 150-fold after 1h, remaining constant up to 120h. This is the first kinetic analysis of V-ATPase specific activity in crustacean gills during salinity acclimation. Our findings indicate active gill Cl(-) uptake by D. pagei in fresh water, and short- and long-term down-regulation of V-ATPase-driven ion uptake processes during salinity exposure, aiding in comprehension of the biochemical adaptations underpinning the establishment of the Brachyura in fresh water.

Between-habitat comparison of digestive enzymes activities and energy reserves in the SW Atlantic euryhaline burrowing crab Neohelice granulata.

The digestive and metabolic characteristics at the biochemical level underlying between-habitat dietary shift of the SW Atlantic euryhaline burrowing crab Neohelice granulata under natural conditions are unknown. We made studies on adult males of N. granulata from the open mudflat and the vegetated saltmarsh in a SW Atlantic costal lagoon (Mar Chiquita, 37°32'-37°45'S; 57°19'-57°26'W, Argentina). We determined and compared amylase, maltase, sucrase, proteolytic, lipase and alkaline phosphatases activities in the hepatopancreas; glycemia, and glycogen, free glucose, triglycerides and protein concentrations in hepatopancreas, chela muscle, and anterior and posterior gills. The results show that N. granulata exhibits characteristics and between-habitat differences at the biochemical level (i.e. high amylase and disaccharidase activities, differences in total proteolytic, lipase and levamisole-insensitive AP activities in the hepatopancreas, and in the concentrations of glycogen in the gills, triglycerides in the hepatopancreas and of protein in the chela muscle) which could represent adaptive digestive and metabolic strategies to face the differences in environmental conditions (i.e. food availability). The possible relationship between digestive and metabolic characteristics and feeding patterns, type of food available and environmental conditions in each habitat is discussed.

Effects of hypo- or hyperosmotic stress on lipid synthesis and gluconeogenic activity in tissues of the crab Neohelice granulata.

The present study assesses the effects of osmotic stress on phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) activities and (14)C-total lipid synthesis from (14)C-glycine in the anterior and posterior gills, jaw muscle, and hepatopancreas of Neohelice granulata. In posterior gills, 24-h exposure to hyperosmotic stress increased PEPCK, FBPase and G6Pase activities. Increase in (14)C-lipid synthesis was associated to the decrease in PEPCK activity after 72-h exposure to hyperosmotic stress. Hypo-osmotic stress decreased PEPCK and G6Pase activities in posterior gills; however, (14)C-lipids increased after 72-h exposure to stress. In anterior gills, decreases in the G6Pase activity after 72-h of hyperosmotic stress and in (14)C-lipogenesis after 144-h were observed, while PEPCK activity increased after 144 h. Exposure to hypo-osmotic stress increased (14)C-lipid synthesis and PEPCK activity in anterior gills. Muscle G6Pase activity increased after 72-h exposure to hypo-osmotic stress; however, no significant change was observed in the lipogenesis. PEPCK decreased in muscle after 144-h exposure to hyperosmotic, coinciding with increased (14)C-lipid synthesis. In the hepatopancreas, a decrease in the (14)C-lipogenesis occurred after 24-h exposure to hyperosmotic stress, accompanied by increase in (14)C-lipid synthesis. Additionally, PEPCK activity returned to control levels. The hepatopancreatic lipogenesis from amino acids was not involved in the metabolic adjustment during hypo-osmotic stress. However, gluconeogenesis is one of the pathways involved in the adjustment of the intracellular concentration of nitrogenated compounds.

Structural and biochemical correlates of Na+,K+-ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae).

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+),K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (≈14 µm² membrane per µm³cytoplasm), deep invaginations that house the Na(+),K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 µm² µm⁻²)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 µm² µm⁻²), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+),K(+)-ATPase specific activity resembles marine crabs but is ≈5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two α-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4) (+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water.

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 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 NH(4)(+), discussed regarding NH(4)(+) 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+/-15.3 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(I)=45.1+/-2.5 micromol L(-1), although affinity for the inhibitor increased (K(I)=22.7+/-1.1 micromol 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.

The crustacean gill (Na+,K+)-ATPase: allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium.

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na+,K+)-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na+ and K+ of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na+,K+)-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na+ and K+. However, in contrast to Na+, a threshold K+ concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations.Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na+,K+)-ATPase compared to the vertebrate enzyme.

Long-term memory consolidation depends on proteasome activity in the crab Chasmagnathus.

Long-term memory formation depends on protein and mRNA synthesis that subserves synaptic reorganization. The removal of pre-existing inhibitory proteins by the ubiquitin-proteasome system (UPS) is proposed as a crucial step to support these modifications. The activation of the constitutive transcription factor nuclear factor kappaB (NF-kappaB) depends on the degradation of the inhibitor of NF-kappaB (IkappaB) by the UPS. Here we study the effect of a UPS inhibitor, MG132, on long-term memory consolidation and NF-kappaB activation in the learning paradigm of the crab Chasmagnathus, a model in which this transcription factor plays a key role. Here we found that administration of MG132 interferes with long-term memory but not with short-term memory, and no facilitatory effects were found. Then we studied the effect of the UPS inhibitor on NF-kappaB pathway, finding that MG132 blocks the activation of NF-kappaB induced by training. These results suggest that the UPS is necessary for long-term memory consolidation, allowing for the activation of NF-kappaB as one of the target molecular pathways.

K+ and NH4(+) modulate gill (Na+, K+)-ATPase activity in the blue crab, Callinectes ornatus: fine tuning of ammonia excretion.

To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na+, K+, NH4(+) and ATP of the (Na+, K+)-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis-Menten kinetics with KM=0.61+/-0.03 mmol L(-1) and maximal rate of V=116.3+/-5.4 U mg(-1). Stimulation by Na+ (V=110.6+/-6.1 U mg(-1); K0.5=6.3+/-0.2 mmol L(-1)), Mg2+ (V=111.0+/-4.7 U mg(-1); K0.5=0.53+/-0.03 mmol L(-1)), NH4(+) (V=173.3+/-6.9 U mg(-1); K0.5=5.4+/-0.2 mmol L(-1)) and K+ (V=116.0+/-4.9 U mg(-1); K0.5=1.5+/-0.1 mmol L(-1)) followed a single saturation curve, although revealing site-site interactions. In the absence of NH4(+), ouabain (K(I)=74.5+/-1.2 micromol L(-1)) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F0F1 and K+-ATPases but not Na+-, V- or Ca2+-ATPase as contaminants. (Na+, K+)-ATPase activity was synergistically modulated by K+ and NH4(+). At 10 mmol L(-1) K+, increasing NH4(+) concentrations stimulated maximum activity to V=185.9+/-7.4 U mg(-1). However, at saturating NH4(+) (50 mmol L(-1)), increasing K+ concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na+, K+)-ATPase may be particularly well suited for extremely efficient active NH4(+) excretion. At elevated NH4(+) concentrations, the enzyme is fully active, regardless of hemolymph K+ concentration, and K+ cannot displace NH4(+) from its exclusive binding sites. Further, the binding of NH4(+) to its specific sites induces an increase in enzyme apparent affinity for K+, which may contribute to maintaining K+ transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na+, K+)-ATPase, and should further our understanding of NH4(+) excretion in benthic crabs.

Biochemical and physiological responses after exposure to microcystins in the crab Chasmagnathus granulatus (Decapoda, Brachyura).

Microcystins are usually the predominant cyanotoxins present in both drinking and recreational waters after cyanobacterial blooms. Their classic toxic effect is hepatotoxicity through inhibition of serine/threonine phosphatases. However, recent studies also reported oxidative stress generation and disruption of ion regulation in aquatic organisms after microcystins exposure. In the present study, aqueous extracts of Microcystis aeruginosa were administered to the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) by gavage in variable doses (from 34 to 860 microg kg(-1)) and exposure times (6, 12, and 72 h). A control group was exposed to saline solution. Analyzed variables included oxygen consumption, lipid peroxidation (LPO), enzyme activities (glutathione S-transferases or GST; alanine aminotransferase or ALT; aspartate aminotransferase or AST; and lactate dehydrogenase or LDH), glycogen, and microcystins content. Oxygen consumption increased in organisms exposed for 12h to 860 microg kg(-1) of microcystins and a similar result was observed after 72 h at doses equal to or higher than 34 microg kg(-1). LPO levels increased in doses equal to or higher than 34 microg kg(-1) after 72 h. GST and LDH activities increased after 12 h (at a dose of 860 microg kg(-1)), but ALT and AST activities remained unaltered in all experimental conditions. Glycogen content decreased after 72 h exposure at doses equal to or higher than 172 microg kg(-1). After 12h of exposure to 860 microg kg(-1) of microcystins, the concentration found in the hepatopancreas of C. granulatus was 13.17+/-0.56 microg kg(-1). In crabs exposed to doses higher than 172 microg kg(-1) during 72 h this value raised to 32.14+/-4.12 microg kg(-1). The obtained results indicated that microcystins exposure led the tissue to an oxidative stress condition (high LPO levels), at least in part favored by the augment of oxygen consumption, altering the glycogen metabolism. GST responses were only observed in the short-term experiment (12 h) and no effect on classical markers of vertebrate liver damage (ALT and AST) was observed. Although the hepatopancreas from C. granulatus accumulated a relatively low concentration of toxins, it was enough to induce physiological and biochemical disturbances.

Gill microsomal (Na+,K+)-ATPase from the blue crab Callinectes danae: Interactions at cationic sites.

Euryhaline crustaceans tolerate exposure to a wide range of dilute media, using compensatory, ion regulatory mechanisms. However, data on molecular interactions occurring at cationic sites on the crustacean gill (Na+,K+)-ATPase, a key enzyme in this hyperosmoregulatory process, are unavailable. We report that Na+ binding at the activating site leads to cooperative, heterotropic interactions that are insensitive to K+. The binding of K+ ions to their high affinity sites displaces Na+ ions from their sites. The increase in Na+ ion concentrations increases heterotropic interactions with the K+ ions, with no changes in K0.5 for K+ ion activation at the extracellular sites. Differently from mammalian (Na+,K+)-ATPases, that from C. danae exhibits additional NH4+ ion binding sites that synergistically activate the enzyme at saturating concentrations of Na+ and K+ ions. NH4+ binding is cooperative, and heterotropic NH4+ ion interactions are insensitive to Na+ ions, but Na+ ions displace NH4+ ions from their sites. NH4+ ions also displace Na+ ions from their sites. Mg2+ ions modulate enzyme stimulation by NH4+ ions, displacing NH4+ ion from its sites. These interactions may modulate NH4+ ion excretion and Na+ ion uptake by the gill epithelium in euryhaline crustaceans that confront hyposmotic media.

Differential activity profile of cAMP-dependent protein kinase isoforms during long-term memory consolidation in the crab Chasmagnathus.

The isoforms of cAMP-dependent protein kinase (PKA) show distinct biochemical properties and subcellular localization, suggesting different physiological functions, and conferring the fine-tuning between the activation of cAMP-PKA cascade and the cellular response. The critical role of PKA in memory and synaptic plasticity has been extensively demonstrated both in vertebrates and invertebrates, but the role of PKA isoforms is a matter of debate. Here we present experimental data showing differential PKA activation profiles after two different experiences: an instance of associative contextual learning (context-signal learning) and a single exposure to a novel context, both in the learning and memory model of the crab Chasmagnathus. Differences were found in the temporal course of activation and in the involvement of PKA isoforms. We found increased PKA activity immediately and 6 h after context-signal training correlating with the critical periods during which pharmacological inhibition of PKA disrupts memory formation. In contrast, PKA activity increased immediately but not 6 h after single exposure to a novel context. The amounts of PKA I and PKA II holoenzymes were analyzed to determine changes in holoenzyme levels and/or differential activation induced by both experiences. Results indicate that context-induced PKA activation is at least in part due to PKA II, and that PKA activation 6 h after context-signal learning coincides with an increase in the total level of PKA I. Considering the higher sensitivity of PKA I to cAMP, its increment can account for the PKA activation found 6 h after training and is proposed as a novel mechanism providing the prolonged PKA activation during memory consolidation.