(Na+, K+)- ATPase kinetics in Macrobrachium pantanalense: highlighting intra- and interspecific variation within the Macrobrachium amazonicum complex.

The Macrobrachium amazonicum complex is composed of at least the Macrobrachium amazonicum and Macrobrachium pantanalense species, with the latter described from specimens originally identified as part of an endemic M. amazonicum population in the Brazilian Pantanal region. While there may be a reproductive barrier between these two Macrobrachium species, both are phylogenetically close, with small genetic distance. However, there is currently no available biochemical information of Macrobrachium pantanalense (Na+, K+)-ATPase. Here, we report the kinetic characteristics of the gill (Na+, K+)-ATPase in two populations of M. pantanalense from Baiazinha Lagoon (Miranda, MS, Brazil) and Araguari River (Uberlândia, MG, Brazil), and compare them with Macrobrachium amazonicum populations from the Paraná-Paraguay River Basin. (Na+, K+)-ATPase activities were 67.9 ± 3.4 and 93.3 ± 4.1 nmol Pi min-1 mg-1 protein for the Baiazinha Lagoon and Araguari River populations, respectively. Two ATP hydrolyzing sites were observed for the Araguari River population while a single ATP site was observed for the Baiazinha Lagoon shrimps. Compared to the Araguari River population, a 3-fold greater apparent affinity for Mg2+ and Na+ was estimated for the Baiazinha Lagoon population, but no difference in K+ affinity and ouabain inhibition was seen. The kinetic differences observed in the gill (Na+, K+)-ATPase between the two populations of M. pantanalense, compared with those of various M. amazonicum populations, highlight interspecific divergence within the Macrobrachium genus, now examined from a biochemical perspective.

Differential effects of cobalt ions in vitro on gill (Na+, K+)-ATPase kinetics in the Blue crab Callinectes danae (Decapoda, Brachyura).

We used the gill (Na+, K+)-ATPase as a molecular marker to provide a comprehensive kinetic analysis of the effects of Co2+in vitro on the modulation of K+-phosphatase activity in the Blue crab Callinectes danae. Co2+ can stimulate or inhibit K+-phosphatase activity. With Mg2+, K+-phosphatase activity is almost completely inhibited by Co2+. Co2+ stimulates K+-phosphatase activity similarly to Mg2+ although with a ≈4.5-fold greater affinity. At saturating Mg2+ concentrations, Mg2+ displaces bound Co2+ from the Mg2+-binding site in a concentration dependent manner, but Co2+ cannot displace Mg2+ from its binding site even at millimolar concentrations. Saturation by Co2+ of the Mg2+ binding site does not affect pNPP recognition by the enzyme. Substitution of Mg2+ by Co2+ slightly increases enzyme affinity for K+ and NH4+. Independently of Mg2+, inhibition by ouabain or sodium ions is unaffected by Co2+. Investigation of gill (Na+, K+)-ATPase K+-phosphatase activity provides a reliable tool to examine the kinetic effects of Co2+ with and without Na+ and ATP. Given that the toxic effects of Co2+ at the molecular level are poorly understood, these findings advance our knowledge of the mechanism of action of Co2+ on the crustacean gill (Na+, K+)-ATPase.

A kinetic characterization of the gill V(H+)-ATPase from two hololimnetic populations of the Amazon River shrimp Macrobrachium amazonicum.

This investigation examines the kinetic characteristics and effect of acclimation to a brackish medium (21 ‰S) on gill V(H+)-ATPase activity in two hololimnetic populations of M. amazonicum. We also investigate the cellular immunolocalization of the enzyme. Immunofluorescence findings demonstrate that the V(H+)-ATPase c-subunit is distributed in the apical pillar cells of shrimps in fresh water but is absent after acclimation to 21 ‰S for 10 days. V(H+)-ATPase activity from the Tietê River population is ≈50% greater than the Grande River population, comparable to a wild population from the Santa Elisa Reservoir, but is 2-fold less than in cultivated shrimps. V(H+)-ATPase activity in the Tietê and the Grande River shrimps is abolished after 21 ‰S acclimation. The apparent affinities of the V(H+)-ATPase for ATP (0.27 ± 0.04 and 0.16 ± 0.03 mmol L-1, respectively) and Mg2+ (0.28 ± 0.05 and 0.14 ± 0.02 mmol L-1, respectively) are similar in both populations. The absence of V(H+)-ATPase activity in salinity-acclimated shrimps and its apical distribution in shrimps in fresh water underpins the importance of the crustacean V(H+)-ATPase for ion uptake in fresh water.

Xylose isomerase from Piromyces sp. E2 is a promiscuous enzyme with epimerase activity.

Xylose isomerase catalyzes the isomerization of D-xylose to D-xylulose with promiscuous activity for other saccharides including D-glucose, D-allose, and L-arabinose. The xylose isomerase from the fungus Piromyces sp. E2 (PirE2_XI) is used to engineer xylose usage by the fermenting yeast Saccharomyces cerevisiae, but its biochemical characterization is poorly understood with divergent catalytic parameters reported. We have measured the kinetic parameters of the PirE2_XI and analyzed its thermostability and pH-dependence towards different substrates. The PirE2_XI shows promiscuous activity towards D-xylose, D-glucose, D-ribose and L-arabinose with variable effects depending on different divalent ions and epimerizes D-xylose at C3 to produce D-ribulose in a substrate/product dependent ratio. The enzyme follows Michaelis-Menten kinetics for the substrates used and although KM values for D-xylose are comparable at 30 and 60 °C, the kcat/KM is three-fold greater at 60 °C. The purified PirE2_XI shows maximal activity at 65 °C in the pH range of 6.5-7.5 and is a thermostable enzyme, maintaining full activity over 48 h at 30 °C or 12 h at 60 °C. This is the first report demonstrating epimerase activity of the PirE2_XI and its ability to isomerize D-ribose and L-arabinose, and provides a comprehensive in vitro study of substrate specificity, effect of metal ions and temperature on enzyme activity and these findings advance the knowledge of the mechanism of action of this enzyme.

Salinity-dependent modulation by protein kinases and the FXYD2 peptide of gill (Na+, K+)-ATPase activity in the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

The geographical distribution of aquatic crustaceans is determined by ambient factors like salinity that modulate their biochemistry, physiology, behavior, reproduction, development and growth. We investigated the effects of exogenous pig FXYD2 peptide and endogenous protein kinases A and C on gill (Na+, K+)-ATPase activity, and characterized enzyme kinetic properties in a freshwater population of Macrobrachium amazonicum in fresh water (<0.5 ‰ salinity) or acclimated to 21 ‰S. Stimulation by FXYD2 peptide and inhibition by endogenous kinase phosphorylation are salinity-dependent. While without effect in shrimps in fresh water, the FXYD2 peptide stimulated activity in salinity-acclimated shrimps by ≈50 %. PKA-mediated phosphorylation inhibited gill (Na+, K+)-ATPase activity by 85 % in acclimated shrimps while PKC phosphorylation markedly inhibited enzyme activity in freshwater- and salinity-acclimated shrimps. The (Na+, K+)-ATPase in salinity-acclimated shrimp gills hydrolyzed ATP at a Vmax of 54.9 ± 1.8 nmol min-1 mg-1 protein, corresponding to ≈60 % that of freshwater shrimps. Mg2+ affinity increased with salinity acclimation while K+ affinity decreased. (Ca2+, Mg2+)-ATPase activity increased while V(H+)- and Na+- or K+-stimulated activities decreased on salinity acclimation. The 120-kDa immunoreactive band expressed in salinity-acclimated shrimps suggests nonspecific α-subunit phosphorylation by PKA and/or PKC. These alterations in (Na+, K+)-ATPase kinetics in salinity-acclimated M. amazonicum may result from regulatory mechanisms mediated by phosphorylation via protein kinases A and C and the FXYD2 peptide rather than through the expression of a different α-subunit isoform. This is the first demonstration of gill (Na+, K+)-ATPase regulation by protein kinases in freshwater shrimps during salinity challenge.

Effects of ammonia on gill (Na+, K+)-ATPase kinetics in a hololimnetic population of the Amazon River shrimp Macrobrachium amazonicum.

Water quality is essential for successful aquaculture. For freshwater shrimp farming, ammonia concentrations can increase considerably, even when culture water is renewed frequently, consequently increasing the risk of ammonia intoxication. We investigated ammonia lethality (LC50-96 h) in a hololimnetic population of the Amazon River shrimp Macrobrachium amazonicum from the Paraná/Paraguay River basin, including the effects of exposure to 4.93 mg L-1 total ammonia concentration on gill (Na+, K+)-ATPase activity. The mean LC50-96 h was 49.27 mg L-1 total ammonia, corresponding to 1.8 mg L-1 un-ionized ammonia. Except for NH4+ affinity that increased 2.5-fold, that of the gill (Na+, K+)-ATPase for ATP, Mg2+, Na+, K+ and ouabain was unchanged after ammonia exposure. Western blotting of gill microsomal preparations from fresh caught shrimps showed a single immunoreactive band of ≈110 kDa, corresponding to the gill (Na+, K+)-ATPase α-subunit. Ammonia exposure increased (Na+, K+)-ATPase activity by ≈25%, coincident with an additional 130 kDa α-subunit immunoreactive band, and increased K+-stimulated and V(H+)-ATPase activities by ≈2.5-fold. Macrobrachium amazonicum from the Paraná/Paraguay River basin is as tolerant to ammonia as are other Amazon River basins populations, showing toxicity comparable to that of marine crustaceans.

Osmotic and ionic regulation, and modulation by protein kinases, FXYD2 peptide and ATP of gill (Na+, K+)-ATPase activity, in the swamp ghost crab Ucides cordatus (Brachyura, Ocypodidae).

We analyzed the modulation by exogenous FXYD2 peptide and by endogenous protein kinases A and C, and Ca2+-calmodulin-dependent kinase, of gill (Na+, K+)-ATPase activity in the semi-terrestrial mangrove crab Ucides cordatus after 10-days acclimation to different salinities. Osmotic and ionic regulatory ability and gill (Na+, K+)-ATPase activity also were evaluated. (Na+, K+)-ATPase activity is stimulated by exogenous pig kidney FXYD2 peptide, while phosphorylation by endogenous protein kinases A and C and Ca2+/calmodulin-dependent kinase inhibits activity. Stimulation by FXYD2 and inhibition by protein kinase C and Ca2+/calmodulin-dependent kinase are salinity-dependent. This is the first demonstration of inhibitory phosphorylation of a crustacean (Na+, K+)-ATPase by Ca2+/calmodulin-dependent kinase. At low salinities, the (Na+, K+)-ATPase exhibited a single, low affinity ATP-binding site that showed Michaelis-Menten behavior. Above 18‰S, a second, cooperative, high affinity ATP-binding site appeared, corresponding to 10-20% of total (Na+, K+)-ATPase activity. Hemolymph osmolality was strongly hyper-/hypo-regulated in crabs acclimated at 2 to 35‰S. Cl- was well hyper-/hypo-regulated although Na+ much less so, becoming isonatremic at elevated salinity. (Na+, K+)-ATPase activity was greatest in isosmotic crabs (26‰S), decreasing notably at 35‰S and also diminishing progressively from 18to 2‰S. Hyper-osmoregulation in U. cordatus showed little dependence on gill (Na+, K+)-ATPase activity, suggesting a role for other ion transporters. These findings reveal that the salinity acclimation response in U. cordatus consists of a suite of enzymatic and osmoregulatory adjustments that maintain its osmotic homeostasis in a challenging, mangrove forest environment.

Biochemical Characterization and Allosteric Modulation by Magnesium of (Na+, K+)-ATPase Activity in the Gills of the Red Mangrove Crab Goniopsis cruentata (Brachyura, Grapsidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the grapsid crab Goniopsis cruentata. (Na+, K+)-ATPase activity constitutes 95% of total ATPase activity, and sucrose density centrifugation reveals an ATPase activity peak between 25 and 35% sucrose, distributed into two, partially separated protein fractions. The (Na+, K+)-ATPase α-subunit is localized throughout the ionocyte cytoplasm and has an Mr of ≈ 10 kDa and hydrolyzes ATP obeying cooperative kinetics. Low (VM = 186.0 ± 9.3 nmol Pi min-1 mg-1 protein and K0.5 = 0.085 ± 0.004 mmol L-1) and high (VM = 153.4 ± 7.7 nmol Pi min-1 mg-1 protein and K0.5 = 0.013 ± 0.0006 mmol L-1) affinity ATP binding sites were characterized. At low ATP concentrations, excess Mg2+ stimulates the enzyme, triggering exposure of a high-affinity binding site that accounts for 50% of (Na+, K+)-ATPase activity. Stimulation by Mg2+ (VM = 425.9 ± 25.5 nmol Pi min-1 mg-1 protein, K0.5 = 0.16 ± 0.01 mmol L-1), K+ (VM = 485.3 ± 24.3 nmol Pi min-1 mg-1 protein, K0.5 = 0.9 ± 0.05 mmol L-1), Na+ (VM = 425.0 ± 23.4 nmol Pi min-1 mg-1 protein, K0.5 = 5.1 ± 0.3 mmol L-1) and NH4+ (VM = 497.9 ± 24.9 nmol Pi min-1 mg-1 protein, K0.5 = 9.7 ± 0.5 mmol L-1) obeys cooperative kinetics. Ouabain inhibits up to 95% of ATPase activity with KI = 196.6 ± 9.8 µmol L-1. This first kinetic characterization of the gill (Na+, K+)-ATPase in Goniopsis cruentata enables better comprehension of the biochemical underpinnings of osmoregulatory ability in this semi-terrestrial mangrove crab.

Dopamine binding directly up-regulates (Na+, K+)-ATPase activity in the gills of the freshwater shrimp Macrobrachium amazonicum.

We examined the effects of exogenous dopamine on gill (Na+, K+)-ATPase activity in vitro in microsomal preparations from juvenile or adult freshwater shrimp, Macrobrachium amazonicum. Dopamine had no effect on enzyme activity in juveniles but stimulated activity in adult shrimp gills by ≈35%. Stimulation of the gill (Na+, K+)-ATPase in adult shrimps by 100 mmol L-1 dopamine was characterized kinetically by varying ATP, MgATP, and Na+ and K+ concentrations, together with inhibition by ouabain. Dopamine stimulated ATP hydrolysis by ≈40% obeying Michaelis-Menten kinetics, reaching VM = 190.5 ±â€¯15.7 nmol Pi min-1 mg-1 protein, KM remaining unaltered. Stimulation by Na+ (≈50%) and K+ (≈25%) revealed distinct kinetic profiles: although KM values were similar, Na+ stimulation followed cooperative kinetics, contrasting with the Michaelian kinetics seen for K+. Stimulation by MgATP increased activity by ≈30% with little change in KM. Similar saturation profiles were seen for ouabain inhibition with very similar calculated KI values. Our findings suggest that dopamine may be involved in hemolymph sodium homeostasis by directly binding to the gill (Na+, K+)-ATPase at a site different from ouabain, thus stimulating enzyme activity in an ontogenetic stage-specific manner. However, dopamine binding does not affect enzyme affinity for cations and ouabain. This is the first report of the direct action of dopamine in stimulating the crustacean gill (Na+, K+)-ATPase.

Kinetic characterization of the gill (Na+, K+)-ATPase in a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na+, K+)-ATPase with Mr of ≈110 kDa is present that hydrolyzes ATP with VM = 130.3 ±â€¯4.8 nmol Pi min-1 mg protein-1 and K0.5 = 0.065 ±â€¯0.00162 mmol L-1, exhibiting site-site interactions. Stimulation by Na+ (VM = 127.5 ±â€¯5.3 nmol Pi min-1 mg protein-1, K0.5 = 5.3 ±â€¯0.42 mmol L-1), Mg2+ (VM = 130.6 ±â€¯6.8 nmol Pi min-1 mg protein-1, K0.5 = 0.33 ±â€¯0.042 mmol L-1), K+ (VM = 126.7 ±â€¯7.7 nmol Pi min-1 mg protein-1, K0.5 = 0.65 ±â€¯0.0079 mmol L-1) and NH4+ (VM = 134.5 ±â€¯8.6 nmol Pi min-1 mg protein-1, K0.5 = 1.28 ±â€¯0.44 mmol L-1) also obeys cooperative kinetics. Ouabain (KI = 0.18 ±â€¯0.058 mmol L-1) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na+, K+)-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats.

Short- and long-term salinity challenge, osmoregulatory ability, and (Na+, K+)-ATPase kinetics and α-subunit mRNA expression in the gills of the thinstripe hermit crab Clibanarius symmetricus (Anomura, Diogenidae).

The evolutionary history of the Crustacea reveals ample adaptive radiation and the subsequent occupation of many osmotic niches resulting from physiological plasticity in their osmoregulatory mechanisms. We evaluate osmoregulatory ability in the intertidal, thinstripe hermit crab Clibanarius symmetricus after short-term exposure (6 h) or long-term acclimation (10 days) to a wide salinity range, also analyzing kinetic behavior and α-subunit mRNA expression of the gill (Na+, K+)-ATPase. The crab strongly hyper-regulates its hemolymph at 5 and 15‰S (Salinity, g L-1) but weakly hyper-regulates up to ≈27‰S. After 6 h exposure to 35‰S and 45‰S, C. symmetricus slightly hypo-regulates its hemolymph, becoming isosmotic after 10 days acclimation to these salinities. (Na+, K+)-ATPase specific activity decreases with increasing salinity for both exposure periods, reflecting physiological adjustment to isosmoticity. At low salinities, the gill enzyme exhibits a single, low affinity ATP binding site. However, at elevated salinities, a second, high affinity, ATP binding site appears, independently of exposure time. (Na+, K+)-ATPase α-subunit mRNA expression increases only after 10 days acclimation to 5‰S. Our findings suggest that hemolymph hyper-regulation is effected by alterations in enzyme activity during short-term exposure, but is sustained by increased mRNA expression during long-term acclimation. The decrease in gill (Na+, K+)-ATPase activity seen as a consequence of increasing salinity appears to underlie biochemical adjustments to hemolymph isosmoticity as hypo-regulatory ability diminishes.

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.

Polyamines regulate phosphorylation-dephosphorylation kinetics in a crustacean gill (Na+, K+)-ATPase.

Aiming to clarify the mechanism of inhibition of (Na+, K+)-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic behavior of phosphoenzyme-linked partial reactions using a microsomal gill (Na+, K+)-ATPase from juvenile and adult M. amazonicum, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 ± 0.006 s-1) in adults than in juveniles (0.053 ± 0.003 s-1) for spermidine, but similar to juveniles (0.059 ± 0.004 s-1) for putrescine. Maximum phosphointermediate formation for the (Na+, K+)-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult M. amazonicum gill (Na+, K+)-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.

Low salinity-induced alterations in epithelial ultrastructure, Na+/K+-ATPase immunolocalization and enzyme kinetic characteristics in the gills of the thinstripe hermit crab, Clibanarius vittatus (Anomura, Diogenidae).

Fresh caught Clibanarius vittatus [SW, 31‰ salinity (S)] were acclimated to a dilute medium (15‰ S) for 10 days, employing silver staining to locate gill ion transporting tissue, immunofluorescence to localize the Na+/K+-ATPase α-subunit in the lamellae, and electron microscopy to portray ultrastructural changes in the gill epithelia. Na+/K+-ATPase activity was characterized kinetically in a gill microsomal fraction, including synergistic stimulation by NH4+ plus K+. Silver staining revealed that all 26 phyllobranchiate arthro- and pleurobranchiae participate in ion transport. Na+/K+-ATPase α-subunit staining was weak in SW crabs and distributed exclusively and irregularly within the intralamellar septal cells, particularly at the septal-pillar cell body junctions, and septal cell cytoplasm facing the hemolymph space. In 15‰ S crabs, α-subunit localization was intense, occupying the entire thickened septum. Pillar cells and flanges did not stain. Mitochondria and membrane foldings increased in the pillar cell flanges and intralamellar septal cells, greatly amplifying surface area. Only a single ATP binding site (VM  =  130.8 ± 10.5 nmol min-1 mg protein-1; K0.5  =  55.3 ± 1.7 µmol l-1) obeying Michaelis-Menten kinetics was disclosed. Na+/K+-ATPase activity was modulated by Mg2+, Na+, and NH4+, exhibiting site-site interactions; K+ modulation showed Michaelis-Menten kinetics. K+ plus NH4+ synergistically stimulated activity ≈ 1.7-fold. Ouabain inhibited total ATPase activity by ≈ 70% (KI  =  220-300 µmol l-1), revealing phosphohydrolytic activities other than the Na+/K+-ATPase. Despite ample phylogenetic separation, the phyllobranchiate lamellae of the Anomura and Caridea share many ultrastructural features, that is, an intralamellar septum and opposed abutting pillar cells, similar Na+/K+-ATPase distribution, and comparable kinetic characteristics. These findings suggest either convergent evolution at the structural and biochemical levels, or preservation of traits present in a remote common ancestor.

[Current status and regulation of bioanalytical laboratories engaged in quantifying immunosuppressants for transplant patients in Argentina]. / Estado de situación y regulación de laboratorios bioanalíticos que cuantifican inmunosupresores para trasplantes en Argentina.

Objective Determine the status of analytical laboratories that quantify immunosuppressants in transplant patients who are under therapeutic drug monitoring (TDM) for these drugs in Argentina in order to identify potential perfectible areas for action. Methods A survey of the clinical and analytical TDM centers in Argentina was conducted between September 2013 and November 2014 under the direction of the Garrahan Hospital Clinical Pharmacokinetics Unit and the National Unified Central Institute for Ablation and Implant Coordination. Results A nationally representative sample of 27 clinical and analytical centers was identified, of which 45% were public hospitals. Most of these centers (95%) monitor ciclosporin and tacrolimus, and to a lesser extent, sirolimus and everolimus; a small number of them also monitor mycophenolic acid. The median number of samples of these five drugs analyzed per month was 251 (range: 10-2024). Nearly 60% of the samples were analyzed in private institutions. Only four of the respondents (17%) reported values within the therapeutic margin. Of all the centers, 92% use immunoassay as the analytical methodology. Of the bioanalytical installations that have their own facilities, 68% reported that they also have their own quality assurance program. Conclusions TDM of immunosuppressants is a recommended practice for transplant patients in Argentina. Initiatives need to be taken at the national level to develop uniform guidelines for analytical laboratories that include TDM-related quality assurance processes with regulatory force. There is also a need to train technical and professional personnel and to invite the participation of public and private organizations in the regulatory, public health, and research areas.

Estado de situación y regulación de laboratorios bioanalíticos que cuantifican inmunosupresores para trasplantes en Argentina / Current status and regulation of bioanalytical laboratories engaged in quantifying immunosuppressants for transplant patients in Argentina

RESUMEN Objetivo Establecer el estado de situación de los laboratorios analíticos que cuantifican inmunosupresores en pacientes con trasplantes que se encuentran bajo monitoreo terapéutico de drogas (TDM) de estos fármacos en Argentina, para establecer potenciales áreas de actuación perfectibles. Métodos Se realizó una encuesta en centros clínicos y analíticos de TDM de Argentina, coordinada por la Unidad de Farmacocinética Clínica del Hospital Garrahan y el Instituto Nacional Central Único Coordinador de Ablación e Implante, desde setiembre de 2013 hasta noviembre de 2014. Resultados Se incluyeron 27 centros clínicos y analíticos (muestra representativa nacional). El 45% fueron hospitales públicos. La mayoría (95%) monitorizan ciclosporina y tacrolimús; en menor medida, sirolimús y everolimús, y muy pocos el ácido micofenólico. La cantidad (mediana, rango) de muestras analizadas por mes para estos cinco fármacos fue de 251 (10 - 2024). Casi 60% de las muestras se analizaron en instituciones privadas. Solo cuatro (17%) de los encuestados informan valores del margen terapéutico. El 92% usa inmunoensayos como metodología analítica. El 68% de los encuestados que contaban con instalaciones bioanalíticas propias informaron poseer algún programa de garantía de calidad. Conclusiones El TDM de inmunosupresores es una práctica recomendada para pacientes con trasplante en Argentina. Se requiere generar iniciativas nacionales que desarrollen guías armonizadas para laboratorios analíticos que incluyan procesos de garantía de calidad con alcance regulatorio relacionados con el TDM. Por otra parte, también es necesario capacitar al personal técnico y profesional, e invitar a participar a organizaciones públicas y privadas del ámbito regulatorio, sanitario y de la investigación.

ABSTRACT Objective Determine the status of analytical laboratories that quantify immunosuppressants in transplant patients who are under therapeutic drug monitoring (TDM) for these drugs in Argentina in order to identify potential perfectible areas for action. Methods A survey of the clinical and analytical TDM centers in Argentina was conducted between September 2013 and November 2014 under the direction of the Garrahan Hospital Clinical Pharmacokinetics Unit and the National Unified Central Institute for Ablation and Implant Coordination. Results A nationally representative sample of 27 clinical and analytical centers was identified, of which 45% were public hospitals. Most of these centers (95%) monitor ciclosporin and tacrolimus, and to a lesser extent, sirolimus and everolimus; a small number of them also monitor mycophenolic acid. The median number of samples of these five drugs analyzed per month was 251 (range: 10-2024). Nearly 60% of the samples were analyzed in private institutions. Only four of the respondents (17%) reported values within the therapeutic margin. Of all the centers, 92% use immunoassay as the analytical methodology. Of the bioanalytical installations that have their own facilities, 68% reported that they also have their own quality assurance program. Conclusions TDM of immunosuppressants is a recommended practice for transplant patients in Argentina. Initiatives need to be taken at the national level to develop uniform guidelines for analytical laboratories that include TDM-related quality assurance processes with regulatory force. There is also a need to train technical and professional personnel and to invite the participation of public and private organizations in the regulatory, public health, and research areas.

Estado de situación y regulación de laboratorios bioanalíticos que cuantifican inmunosupresores para trasplantes en Argentina / Current status and regulation of bioanalytical laboratories engaged in quantifying immunosuppressants for transplant patients in Argentina

Objetivo. Establecer el estado de situación de los laboratorios analíticos que cuantifican inmunosupresores en pacientes con trasplantes que se encuentran bajo monitoreo terapéutico de drogas (TDM) de estos fármacos en Argentina, para establecer potenciales áreas de actuación perfectibles. Métodos. Se realizó una encuesta en centros clínicos y analíticos de TDM de Argentina, coordinada por la Unidad de Farmacocinética Clínica del Hospital Garrahan y el Instituto Nacional Central Único Coordinador de Ablación e Implante, desde setiembre de 2013 hasta noviembre de 2014. Resultados. Se incluyeron 27 centros clínicos y analíticos (muestra representativa nacional). El 45% fueron hospitales públicos. La mayoría (95%) monitorizan ciclosporina y tacrolimús; en menor medida, sirolimús y everolimús, y muy pocos el ácido micofenólico. La cantidad (mediana, rango) de muestras analizadas por mes para estos cinco fármacos fue de 251 (10 - 2024). Casi 60% de las muestras se analizaron en instituciones privadas. Solo cuatro (17%) de los encuestados informan valores del margen terapéutico. El 92% usa inmunoensayos como metodología analítica. El 68% de los encuestados que contaban con instalaciones bioanalíticas propias informaron poseer algún programa de garantía de calidad. Conclusiones. El TDM de inmunosupresores es una práctica recomendada para pacientes con trasplante en Argentina. Se requiere generar iniciativas nacionales que desarrollen guías armonizadas para laboratorios analíticos que incluyan procesos de garantía de calidad con alcance regulatorio relacionados con el TDM. Por otra parte, también es necesario capacitar al personal técnico y profesional, e invitar a participar a organizaciones públicas y privadas del ámbito regulatorio, sanitario y de la investigación.

Objective. Determine the status of analytical laboratories that quantify immunosuppressants in transplant patients who are under therapeutic drug monitoring (TDM) for these drugs in Argentina in order to identify potential perfectible areas for action. Methods. A survey of the clinical and analytical TDM centers in Argentina was conducted between September 2013 and November 2014 under the direction of the Garrahan Hospital Clinical Pharmacokinetics Unit and the National Unified Central Institute for Ablation and Implant Coordination. Results. A nationally representative sample of 27 clinical and analytical centers was identified, of which 45% were public hospitals. Most of these centers (95%) monitor ciclosporin and tacrolimus, and to a lesser extent, sirolimus and everolimus; a small number of them also monitor mycophenolic acid. The median number of samples of these five drugs analyzed per month was 251 (range: 10-2024). Nearly 60% of the samples were analyzed in private institutions. Only four of the respondents (17%) reported values within the therapeutic margin. Of all the centers, 92% use immunoassay as the analytical methodology. Of the bioanalytical installations that have their own facilities, 68% reported that they also have their own quality assurance program. Conclusions. TDM of immunosuppressants is a recommended practice for transplant patients in Argentina. Initiatives need to be taken at the national level to develop uniform guidelines for analytical laboratories that include TDM-related quality assurance processes with regulatory force. There is also a need to train technical and professional personnel and to invite the participation of public and private organizations in the regulatory, public health, and research areas.

Effects of ammonia stress in the Amazon river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We evaluate the effects of total ammonia nitrogen-N (TAN) exposure for 72h on (Na(+),K(+))- and V(H(+))-ATPase activities and on their subunit expressions in gills of the diadromous freshwater shrimp Macrobrachium amazonicum. Specific (Na(+),K(+))- and V(H(+))-ATPase activities increased roughly 1.5- to 2-fold, respectively, after exposure to 2.0mmolL(-1) TAN. Quantitative RT-PCR analyses revealed a 2.5-fold increase in V(H(+))-ATPase B subunit mRNA expression while (Na(+),K(+))-ATPase α-subunit expression was unchanged. Immunohistochemical analyses of the gill lamellae located the (Na(+),K(+))-ATPase throughout the intralamellar septal cells, independently of TAN concentration, while the V(H(+))-ATPase was located in both the apical pillar cell flanges and pillar cell bodies. Systemic stress parameters like total hemocyte count decreased by 30% after exposure to 2.0mmolL(-1) TAN, accompanied by increased activities of the oxidative stress enzymes superoxide dismutase, glutathione reductase and glucose-6-phosphate dehydrogenase in the gills. The stress responses of M. amazonicum to elevated TAN include increases in gill (Na(+),K(+))- and V(H(+))-ATPase activities that are accompanied by changes in oxidative stress enzyme activities, immune system effects and an increase in gill V(H(+))-ATPase gene expression. These findings likely underpin physiological effects in a crustacean like M. amazonicum that exploits multiple ecosystems during its life cycle, as well as under culture conditions that may significantly impact shrimp production by the aquaculture industry.

Gill-specific (Na(+), K(+))-ATPase activity and α-subunit mRNA expression during low-salinity acclimation of the ornate blue crab Callinectes ornatus (Decapoda, Brachyura).

We evaluate (Na(+), K(+))-ATPase activity, and protein and gene expression of the α-subunit in posterior gills 6 and 7 of Callinectes ornatus, a euryhaline crab, during a 10-day acclimation period from seawater (33‰ S) to low salinity (21‰ S). (Na(+), K(+))-ATPase activity decreased within 1h after transfer to 21‰ S, values recovering by 24h and attaining a maximum of ≈180 nmol Pi min(-1) mg(-1) after 10 days (≈2.5-fold increase). (Na(+), K(+))-ATPase activity is ≈1.5-fold greater in gill 6 than in gill 7, independently of salinity. Relative expression of (Na(+), K(+))-ATPase α-subunit mRNA increased in both gills within 1- to 2-h exposure to low salinity, reaching an ≈8-fold maximum after 24-h exposure, decreasing slightly by 10 days acclimation to low salinity. This increase in α-subunit mRNA expression may underpin the increased (Na(+), K(+))-ATPase activity seen after 10 days acclimation to low salinity. Enzyme affinity for ATP was greater in gill 6 than in gill 7, in contrast to ouabain affinity that was greater in gill 7. Western blotting analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈105 kDa, independently of gill number and low salinity acclimation. Despite these differences, gills 6 and 7 appear to perform similar functions in salt uptake from the dilute medium. The partial cDNA sequence obtained for the gill (Na(+), K(+))-ATPase of C. ornatus (GenBank deposit KF056804) showed 97 to 91% identities with similar sequences from other portunid crab gills. The regulation of gill (Na(+), K(+))-ATPase activity during acclimation to low salinity is discussed.

A kinetic characterization of the gill V(H(+))-ATPase in juvenile and adult Macrobrachium amazonicum, a diadromous palaemonid shrimp.

Novel kinetic properties of a microsomal gill V(H(+))-ATPase from juvenile and adult Amazon River shrimp, Macrobrachium amazonicum, are described. While protein expression patterns are markedly different, Western blot analysis reveals a sole immunoreactive band, suggesting a single V(H(+))-ATPase subunit isoform, distributed in membrane fractions of similar density in both ontogenetic stages. Immunofluorescence labeling locates the V(H(+))-ATPase in the apical regions of the lamellar pillar cells in both stages in which mRNA expression of the V(H(+))-ATPase B-subunit is identical. Juvenile (36.6±3.3 nmol Pi min(-1) mg(-1)) and adult (41.6±1.3 nmol Pi min(-1) mg(-1)) V(H(+))-ATPase activities are similar, the apparent affinity for ATP of the adult enzyme (K0.5=0.21±0.02 mmol L(-1)) being 3-fold greater than for juveniles (K0.5=0.61±0.01 mmol L(-1)). The K0.5 for Mg(2+) interaction with the juvenile V(H(+))-ATPase (1.40 ± 0.07 mmol L(-1)) is ≈6-fold greater than for adults (0.26±0.02 mmol L(-1)) while the bafilomycin A1 inhibition constant (KI) is 45.0±2.3 nmol L(-1) and 24.2±1.2 nmol L(-1), for juveniles and adults, respectively. Both stages exhibited residual bafilomycin-insensitive ATPase activity of ≈25 nmol Pi min(-1) mg(-1), suggesting the presence of ATPases other than the V(H(+))-ATPase. These differences may reflect a long-term regulatory mechanism of V(H(+))-ATPase activity, and suggest stage-specific enzyme modulation. This is the first kinetic analysis of V(H(+))-ATPase activity in different ontogenetic stages of a freshwater shrimp and allows better comprehension of the biochemical adaptations underpinning the establishment of palaemonid shrimps in fresh water.