The association of Na,K-ATPase subunits studied by circular dichroism, surface tension and dilatational elasticity

Different stoichiometries are observed between alpha and beta subunits of Na,K-ATPase that depend on the method employed to solubilize and purify the enzyme. It is not known whether this variability is due to loss of protein-protein association, or is a result of the replacement of essential phospholipids by detergent molecules. With the aim of understanding the effect of enzyme/surfactant ratio on both the catalytic activity and the enzyme structure, we have investigated the bulk and surface properties of the enzyme. The circular dichroism (CD) spectra, surface tension and dilatational surface elasticity results were compared with the residual ATPase activity of the Na,K-ATPase in different surfactant and protein concentrations. Na,K-ATPase in the (alpha beta)(2) form dissociated to the alpha beta form on dilution, and associated to the (alpha beta)(4) form when concentrated. These different stoichiometries have similar ATPase activities and are in equilibrium at C(12)E(8) concentrations below the CIVIC (0.053 mg mL(-1)). At detergent concentrations above the CIVIC the ATPase activity of all forms was abolished, which is concomitant with the dissociation of the a and subunits. (C) 2008 Elsevier Inc. All rights reserved.

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.

Differential adjustment in gill Na(+)/K(+)- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

We evaluate osmotic and chloride (Cl(-)) regulatory capability in the diadromous shrimp Macrobrachium amazonicum, and the accompanying alterations in hemolymph osmolality and [Cl(-)], gill Na(+)/K(+)-ATPase activity, and expression of gill Na(+)/K(+)-ATPase alpha-subunit and V-ATPase B subunit mRNA during salinity (S) acclimation. We also characterize V-ATPase kinetics and the organization of transport-related membrane systems in the gill epithelium. Macrobrachium amazonicum strongly hyper-regulates hemolymph osmolality and [Cl(-)] in freshwater and in salinities up to 25 parts per thousand S. During a 10-day acclimation period to 25 parts per thousand S, hemolymph became isosmotic and hypo-chloremic after 5 days, [Cl(-)] alone remaining hyporegulated thereafter. Gill Na(+)/K(+)-ATPase alpha-subunit mRNA expression increased 6.5 times initial values after 1 h, then decreased to 3 to 4 times initial values by 24 h and to 1.5 times initial values after 10 days at 25 parts per thousand S. This increased expression was accompanied by a sharp decrease at 5 h then recovery of initial Na(+)/K(+)-ATPase activity within 24 h, declining again after 5 days, which suggests transient Cl(-) secretion. V-ATPase B-subunit mRNA expression increased 1.5-fold within 1 h, then reduced sharply to 0.3 times initial values by 5 h, and remained unchanged for the remainder of the 10-day period. V-ATPase activity dropped sharply and was negligible after a 10-day acclimation period to 21 parts per thousand S, revealing a marked downregulation of ion uptake mechanisms. The gill epithelium consists of thick, apical pillar cell flanges, the perikarya of which are coupled to an intralamellar septum. These two cell types respectively exhibit extensive apical evaginations and deep membrane invaginations, both of which are associated with numerous mitochondria, characterizing an ion transporting epithelium. These changes in Na(+)/K(+)- and V-ATPase activities and in mRNA expression during salinity acclimation appear to underpin ion uptake and Cl(-) secretion by the palaemonid shrimp gill.

Unraveling the Na,K-ATPase alpha(4) Subunit Assembling Induced by Large Amounts of C(12)E(8) by Means of Small-Angle X-ray Scattering

In the current work, we studied the effect of the nonionic detergent dodecyloctaethyleneglycol, C(12)E(8), on the structure and oligomeric form of the Na,K-ATPase membrane enzyme (sodium-potassium pump) in aqueous suspension, by means of small-angle X-ray scattering (SAXS). Samples composed of 2 mg/mL of Na,K-ATPase, extracted from rabbit kidney medulla, in the presence of a small amount of C(12)E(8) (0.005 mg/mL) and in larger concentrations ranging from 2.7 to 27 mg/mL did not present catalytic activity. Under this condition, an oligomerization of the alpha subunits is expected. SAXS data were analyzed by means of a global fitting procedure supposing that the scattering is due to two independent contributions: one coming from the enzyme and the other one from C(12)E(8) micelles. In the small detergent content (0.005 mg/mL), the SAXS results evidenced that Na,K-ATPase is associated into aggregates larger than (alpha beta)(2) form. When 2.7 mg/mL of C(12)E(8) is added, the data analysis revealed the presence of alpha(4) aggregates in the solution and some free micelles. Increasing the detergent amount up to 27 mg/mL does not disturb the alpha(4) aggregate: just more micelles of the same size and shape are proportionally formed in solution. We believe that our results shed light on a better understanding of how nonionic detergents induce subunit dissociation and reassembling to minimize the exposure of hydrophobic residues to the aqueous solvent.

Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase AN INTEGRATED, ANIMATED MODEL

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na(+) and K(+) translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P(2c)-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E(1)/E(2)-ATPase as it undergoes conformational changes between the E(1) and E(2) forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger`s scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na(+) and K(+) translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the ""core engine"" of the pump, with respect to ATP binding, cation transport, and ADP and P(i) release.

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.

Patterns of brain asymmetry in the perception of positive and negative facial expressions

The divided visual field technique was used to investigate the pattern of brain asymmetry in the perception of positive/approach and negative/withdrawal facial expressions. A total of 80 undergraduate students (65 female, 15 male) were distributed in five experimental groups in order to investigate separately the perception of expressions of happiness, surprise, fear, sadness, and the neutral face. In each trial a target and a distractor expression were presented simultaneously in a computer screen for 150 ms and participants had to determine the side (left or right) on which the target expression was presented. Results indicated that expressions of happiness and fear were identified faster when presented in the left visual field, suggesting an advantage of the right hemisphere in the perception of these expressions. Fewer judgement errors and faster reaction times were also observed for the matching condition in which emotional faces were presented in the left visual field and neutral faces in the right visual field. Other results indicated that positive expressions (happiness and surprise) were perceived faster and more accurately than negative ones (sadness and fear). Main results tend to support the right hemisphere hypothesis, which predicts a better performance of the right hemisphere to perceive emotions, as opposed to the approach-withdrawal hypothesis.

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 (approximate to 14 mu m(2) membrane per mu m(3) cytoplasm), deep invaginations that house the Na(+), K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 mu m(2) mu m(-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 mu m(2) mu m(-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 approximate to 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 alpha-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. J. Exp. Zool. 313A:508-523, 2010. (C) 2010 Wiley-Liss, Inc.

Species richness, abundance, and body size relationships from a neotropical chironomid assemblage: Looking for patterns

We investigated two of the most studied relationships in the macroecological research program (species richness vs. body size and abundance vs. body size) of a local chironomid assemblage from southeastern Brazil. Although numerous Studies have examined these relationships, few have investigated how they vary at different temporal scales. We used data from a forested stream to document and examine these patterns at monthly intervals. Both the species body size distribution and the abundance-body size relationship varied temporally. In some months the body size distribution was skewed to the right. whereas in others it approached normality. We Found both linear relationships (with different values of slopes). and a polygonal pattern in the abundance-body size relationship. This temporal variation was not related to environmental variables. Our results suggest that body size relationships are temporally instable properties of this chironomid assemblage. (C) 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

Phylogeny of the Limnophilinae (Limoniidae) and early evolution of the Tipulomorpha (Diptera)

Tipulomorpha (craneflies) comprise one of the largest subgroups of Diptera, but its phylogeny at different levels has been poorly explored. This study presents the most comprehensive cladistic analysis of the group ever made, with emphasis on the genera and subgenera of the subfamily Limnophilinae (Limoniidae), assumed to include some of the earliest lineages of Tipulomorpha sensu stricto and therefore important for the understanding of the early patterns in the evolution of the craneflies. Eighty-eight characters of the male imago were scored for 104 exemplar species. The most parsimonious trees were searched using implied weighting, in the framework of a sensitivity analysis with different values of k (2 to 6). The dataset based on the characters of adult male morphology showed high levels of homoplasy and yielded very incongruent and unstable phylogenetic results, which are very sensitive to changes in analytical parameters. In the preferred and most parsimonious phylogenetic hypothesis, the Pediciidae is the sister-group of all other Tipulomorpha sensu stricto. The results indicate the paraphyly of the Limoniidae with respect to the Cylindrotomidae and Tipulidae, which are considered sister-groups. The Limoniidae subfamilies Limnophilinae, Limoniinae and Chioneinae are considered non-monophyletic. The study allowed a reconstruction of the possible ground plan condition of selected features of the adult male morphology of craneflies. The genera/subgenera Epiphragma (Epiphragma), Acantholimnophila, Shannonomyia, Limnophila (Arctolimnophila), Eloeophila, Conosia, Polymera, Polymera (Polymerodes), Prionolabis, Eutonia, Phylidorea (Phylidorea), Metalimnophila, Gynoplistia (Cerozodia), Gynoplistia (Dirhipis), Nothophila, Pseudolimnophila (Pseudolimnophila), Pilaria and Ulomorpha are considered monophyletic, but in general are defined by combinations of very homoplastic character states. Two Temperate Gondwanan clades, (Tonnoirella + (Edwardsomyia + (Tinemyia + (Rhamphophila + (Nothophila))))) and ((Notholimnophila + Bergrothomyia) + (Mesolimnophila + (Chilelimnophila + Ctenolimnophila))) are recovered. The genera Limnophila, Neolimnomyia, Gynoplistia (sensu lato) and Hexatoma (sensu lato) are considered non-monophyletic. The systematic position and some morphological characters of `problematic` taxa, such as Dactylolabis, Elephantomyia, Helius and Atarba are discussed on the light of the proposed phylogeny and the analysis of the characters. Character states are richly illustrated. A detailed study of the morphology of the male genitalia is made, and several genera and species have the morphology of the male genitalia illustrated for the first time.

Bleeding patterns and menstrual-related symptoms with the continuous use of a contraceptive combination of ethinylestradiol and drospirenone: a randomized study

Background: The objective of this study was to compare bleeding patterns of women using a contraceptive combination of 30 mcg ethinylestradiol (EE) and 3 mg drospirenone (DRSP) continuously or cyclically. Menstrual-related symptoms were also evaluated. Study Design: This open, prospective, randomized study evaluated 78 women using the EE/DRSP combination continuously for 168 days or for six 28-day cycles, each followed by a 7-day hormone-free interval. A diary with pre-established scales was used to assess the frequency and intensity of bleeding and menstrual-related symptoms. Results: Amenorrhea increased with continuous use; 62.2% of women with continuous use were amenorrheic at the end of treatment (95% CI: 46.6-77.8%). Dysmenorrhea, headache, acne, nausea, edema and increased appetite improved significantly in the continuous-use group, and mastalgia and edema in the cyclic-use group (p<.05). Six subjects in the continuous-use group (15.4%) and three in the cyclic-use group (7.7%) discontinued due to adverse events. Conclusions: Continuous use was associated with amenorrhea and fewer menstrual-related symptoms compared to cyclic use. (C) 2010 Elsevier Inc. All rights reserved.

Ventilation Patterns Influence Airway Secretion Movement

BACKGROUND: Retention of airway secretions is a common and serious problem in ventilated patients. Treating or avoiding secretion retention with mucus thinning, patient-positioning, airway suctioning, or chest or airway vibration or percussion may provide short-term benefit. METHODS: In a series of laboratory experiments with a test-lung system we examined the role of ventilator settings and lung-impedance on secretion retention and expulsion. Known quantities of a synthetic dye-stained mucus simulant with clinically relevant properties were injected into a transparent tube the diameter of an adult trachea and exposed to various mechanical-ventilation conditions. Mucus-simulant movement was measured with a photodensitometric technique and examined with image-analysis software. We tested 2 mucus-simulant viscosities and various peak flows, inspiratory/ expiratory flow ratios, intrinsic positive end-expiratory pressures, ventilation waveforms, and impedance values. RESULTS: Ventilator settings that produced flow bias had a major effect on mucus movement. Expiratory How bias associated with intrinsic positive end-expiratory pressure generated by elevated minute ventilation moved mucus toward the airway opening, whereas intrinsic positive end-expiratory pressure generated by increased airway resistance moved the mucus toward the lungs. Inter-lung transfer of mucus simulant occurred rapidly across the ""carinal divider"" between interconnected test lungs set to radically different compliances; the mucus moved out of the low-compliance lung and into the high-compliance lung. CONCLUSIONS: The movement of mucus simulant was influenced by the ventilation pattern and lung impedance. Flow bias obtained with ventilator settings may clear or embed mucus during mechanical ventilation.

K/DOQI-recommended intact PTH levels do not prevent low-turnover bone disease in hemodialysis patients

The guidelines proposed by the Kidney Disease Outcomes Quality Initiative (K/DOQI) suggested that intact parathyroid hormone (iPTH) should be maintained in a target range between 150 and 300 pg ml(-1) for patients with stage 5 chronic kidney disease. Our study sought to verify the effectiveness of that range in preventing bone remodeling problems in hemodialysis patients. We measured serum ionized calcium and phosphorus while iPTH was measured by a second-generation assay. Transiliac bone biopsies were performed at the onset of the study and after completing 1 year follow-up. The PTH levels decreased within the target range in about one-fourth of the patients at baseline and at the end of the study. The bone biopsies of two-thirds of the patients were classified as showing low turnover and a one-fourth showed high turnover, the remainder having normal turnover. In the group achieving the target levels of iPTH 88% had low turnover. Intact PTH levels less than 150 pg ml(-1) for identifying low turnover and greater than 300 pg ml(-1) for high turnover presented a positive predictive value of 83 and 62%, respectively. Our study suggests that the iPTH target recommended by the K/DOQI guidelines was associated with a high incidence of low-turnover bone disease, suggesting that other biochemical markers may be required to accurately measure bone-remodeling status in hemodialysis patients.

Variation of Patterns of Malocclusion by Site of Pharyngeal Obstruction in Children

Objective: To correlate the type of dental occlusion and the type of pharyngeal lymphoid tissue obstruction in children. Design: Cross-sectional study. Setting: Ambulatory ear, nose, and throat clinic of Faculdade de Medicina da Universidade de Sao Paulo. Patients: One hundred fourteen children aged 3 to 12 years presenting with mouth breathing and snoring due to tonsil and/or adenoid enlargement. Interventions: Oroscopy and nasal fiber pharyngoscopy complemented by lateral head radiography to diagnose the type of obstruction, and clinical examination to evaluate the dental occlusion. Main Outcome Measures: Tonsil and adenoid obstruction (classified from grades 1-4) and sagittal, transverse, and vertical evaluation of dental occlusion. Results: Obstructive enlargement of both tonsils and adenoids was detected in 64.9% of the sample; isolated enlargement of the adenoids, in 21.9%; isolated enlargement of the palatine tonsils, in 7.0%; and nonobstructive tonsils and adenoids, in 6.1%. All types of pharyngeal obstruction were related to a high prevalence of posterior crossbite (36.8%). Statistically significant association was found between sagittal dental occlusion and the site of lymphoid tissue obstruction (P = .02). A higher rate of class II relationship (43.2%) was detected in the group with combined adenoid and tonsil obstructive enlargement. Isolated tonsil obstruction showed a higher rate of class III relationship (37.5%). Conclusions: Different sites of obstruction of the upper airway due to enlarged lymphoid tissue are associated with different types of dental malocclusion. Findings are relevant to orthodontic and surgical decision making in these mouth-breathing patients.