Dispositius nanoplasmònics basats en polaritons anti-simètrics per apliacacions biosensores i nanofotòniques

Presentem un estudi teòric comparatiu de les sensibilitats en volum dels sensors d’índex de refracció basats en polaritó de plasmó superficial (SPP) i en ressonància localitzada de plasmó superficial (LSPR). Els resultats demostren que el sensor SPP, àmpliament conegut i comercialitzat, ofereix una major sensibilitat en volum comparada amb la del sensor LSPR, estant aquest últim basat en l’ús de nanorods d’or. A més, la figura de mèrit del sensor LSPR, emprada per a analitzar la seva capacitat sensora, exhibeix una regió sensora òptima quan la longitud d’ona de ressonància es troba propera a 700 nm.

Four new species of Oswaldocruzia (Nematoda: Trichostrongylina, Molineoidea) Parasitizing amphibians and lizards from Ecuador

Description of four new species of Oswaldocruzia parasitizing Iguanidae and Leptodactylidae from Ecuador, demonstrate that they are morphologically close to each other. Like most of the other neotropical and holarctic Oswaldocruzia , they are characterized by spicules with three main branches: blade, shoe and fork; the division of the fork within the distal third of the spicule length appears to be characteristic of the neotropical species. - Oswaldocruzia bainae n. sp. parasitizing Anolis chrysolepis and Anolis fuscoauratus possesses a synlophe visible only on transversal sections of the body. It is composed of rounded and not pointed ridges. - Oswaldocruzia tcheprakovae n. sp. parasitizing Eleutherodactylus altamazonicus is closely related to O. bainae , but the synlophe is present only in the anterior and posterior extremities of the body. - Oswaldocruzia cassonei n. sp. parasitizing Eleutherodactylus lanthanites is closely related to O. taranchoni, Ben Slimane and Durette-Desset, 1995, a parasite of Bufo marinus from Brazil. It is differentiated by the synlophe and the measurements. - Oswaldocruzia petterae n. sp. parasitizing Leptodactylus pentadactylus is closely related to O. chambrieri, Ben Slimane and Durette-Desset, 1993, parasitizing Bufo and Eleutherodactylus in the same region. It is differentiated since, for an equivalent length of the body, the ridges are almost two times fewer and the spicules smaller.

Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase.

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.

Hydrophobic labeling of (Na+,K+)-ATPase: further evidence that the beta subunit is embedded in the membrane bilayer.

O-Hexanoyl-3,5-diiodo-N-(4-azido-2-nitro-phenyl)tyramine has been used after photochemical conversion into the reactive nitrene to label (Na+,K+)-ATPase from Bufo marinus toad kidney. Immunochemical evidence indicates that the reagent labels both subunits of the enzyme in partially purified form as well as in microsomal membranes. These results support the view that the glycoprotein subunit, like the catalytic subunit, possesses hydrophobic domains by which it is integrated into the plasma membrane.

Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation.

Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.

Effects of thyromimetic drugs on aldosterone-dependent sodium transport in the toad bladder.

Aldosterone increases transepithelial Na+ transport in the urinary bladder of Bufo marinus. The response is characterized by 3 distinct phases: 1) a lag period of about 60 min, ii) an initial phase (early response) of about 2 hr during which Na+ transport increases rapidly and transepithelial electrical resistance falls, and iii) a late phase (late response) of about 4 to 6 hr during which Na+ transport still increases significantly but with very little change in resistance. Triiodothyronine (T3, 6 nM) added either 2 or 18 hr before aldosterone selectively antagonizes the late response. T3 per se (up to 6 nM) has no effect on base-line Na+ transport. The antagonist activity of T3 is only apparent after a latent period of about 6 to 8 hr. It is not rapidly reversible after a 4-hr washout of the hormone. The effects appear to be selective for thyromimetic drugs since reverse T3 (rT3) is inactive and isopropyldiiodothyronine (isoT2) is more active than T3. The relative activity of these analogs corresponds to their relative affinity for T3 nuclear binding sites which we have previously described. Our data suggest that T3 might control the expression of aldosterone by regulating gene expression, e.g. by the induction of specific proteins, which in turn will inhibit the late mineralocorticoid response, without interaction with the early response.

Testosterone: a specific competitive antagonist of aldosterone in the toad bladder.

Testosterone (100 nM to 40 microM) antagonized the effect of aldosterone (10 nM) on Na+ transport in the toad bladder measured in vitro as short-circuit current (SCC). Half-maximal inhibition occurred at an antagonist-agonist molar ratio of 150:1. The antagonist action of testosterone was reversed by addition of more aldosterone. The antagonism was specific in the sense that testosterone (20 microM) did not inhibit the response of the SCC to oxytocin (50 mU/ml). By itself, testosterone (up to 20 microM) had no agonist activity on base-line SCC. Finally, testosterone (500 nM to 20 microM) specifically displaced [3H]aldosterone (5 nm) from its cytoplasmic and nuclear binding sites in bladders incubated in vitro at 25 or 0 degrees C and labeled at steady state. There was a significant linear correlation between the effect of testosterone on the aldosterone-dependent SCC and its effect on [3H]aldosterone binding sites in the cytoplasm and in the nucleus. We conclude that 1) testosterone is a specific competitive antagonist of aldosterone, and 2) [3H]aldosterone nuclear and cytoplasmic binding sites could be mineralocorticoid receptors, mediating the action of aldosterone on Na+ transport.

Effects of thyroid hormones and aldosterone on mineralocorticoid binding sites in the toad bladder.

In the urinary bladder of the toad Bufo marinus triiodothyronine selectively inhibits the late effect of aldosterone on Na+ transport. We have investigated whether T3 might mediate its antimineralocorticoid action by controlling: i) the level of aldosterone binding sites in the soluble (cytosolic) pool isolated from tissues treated with T3 (60 nM) for up to 20 hr of incubation; ii) the kinetics of uptake of 3H-aldosterone into cytoplasmic and nuclear fractions after 2 or 20 hr of exposure to T3. The number and the affinity of Type I (high affinity, low capacity) and Type II (low affinity, high capacity) cytosolic binding sites (measured at 0 degrees C) did not vary significantly after 18 hr of exposure to T3, while aldosterone-dependent Na+ transport was significantly inhibited. In addition, T3 did not modify the kinetics of uptake (90 min) of 3H-aldosterone into cytoplasmic and nuclear fractions of toad bladder incubated in vitro at 25 degrees C. By contrast, aldosterone itself was able to down-regulate its cytosolic and nuclear binding sites after an 18-hr exposure to the steroid hormone (10 or 80 nM). T3 slightly (20%) but significantly potentiated the down regulation of nuclear binding sites. In conclusion, T3 does not appear to have major effects on the regulation of the aldosterone receptor, which could explain in a simple manner its antimineralocorticoid action.

Control of transepithelial Na+ transport and Na-K-ATPase by oxytocin and aldosterone.

Short- and long-term effect of oxytocin on Na+ transport and Na-K-ATPase biosynthesis in the toad bladder, and the potential interaction of this hormone with aldosterone have been studied, leading to the following observations. An early Na+ transport response (oxytocin, 50 mU/ml) peaked at 10-15 min of hormone addition. At maximal stimulation a three- to fourfold increase in Na+ transport was observed, a sustained Na+ transport response (about two-fold control base line) was observed as long as the hormone was present in the medium and for up to 20 h of incubation. Pretreatment for 30 min with actinomycin D (2 micrograms/ml) did not inhibit the early response, but significantly impaired the sustained response, suggesting that de novo protein synthesis was required. The simultaneous addition of the two hormones led within 60 min to a marked potentiation of the action on Na+ transport. This synergism could be mimicked by exogenous cyclic adenosine monophosphate (cAMP). Oxytocin alone (18 h exposure, 50 mU/ml) increased the relative rate of synthesis of both alpha and beta subunits of Na-K-ATPase (1.9- and 1.6-fold, respectively; P less than 0.05), whereas aldosterone (80 nM) increased the relative rate of synthesis of the same subunits (2.6- and 2.2-fold, respectively; P less than 0.02). Finally, in contrast to what was observed at the physiological level, the interaction of oxytocin and aldosterone did not lead to a similar potentiation at the biochemical level, i.e., induction of Na-K-ATPase biosynthesis (2.7- and 2.9-fold, for alpha and beta subunits, respectively; P less than 0.025).

Structural organization of alpha-subunit from purified and microsomal toad kidney (Na+ + K+)-ATPase as assessed by controlled trypsinolysis

The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (Jørgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).

Receptor occupancy vs. induction of Na+-K+-ATPase and Na+ transport by aldosterone.

In the urinary bladder of the toad Bufo marinus aldosterone (between 0.8 and 100 nM) stimulates Na+ transport [half-maximal induction concentration (K1/2) = 6.5 nM]. At low hormone concentrations (0.8-8 nM), the increase of Na+ transport between 0.75 and 2.5 h is accompanied by a fall in transepithelial resistance (R). Higher hormone concentrations (30-800 nM) induce an additional resistance-independent fraction of Na+ transport within 2.5-8 h. From 6 h on, aldosterone (between 0.2 and 20 nM) stimulates in the same tissue the biosynthesis rate of the alpha- and beta-subunits of Na+-K+-ATPase (K1/2 = 3 and 1.5 nM, respectively). New pump synthesis is thus not a prerequisite for the early mineralocorticoid response but might be linked to the late transport event. The mineralocorticoid response is usually ascribed to interaction with the higher affinity type 1 receptor. In the present study we show, however, that at least 55% of the overall Na+ transport response is linked to nuclear occupation of the lower affinity type 2 receptors [dissociation constant (Kd) = 50 nM, maximum number of binding sites (Nmax) = 315 fmol/mg protein]. Distinct aldosterone effects, such as the fall in R and the increase in Na+-K+-ATPase synthesis, are more closely related to occupation of type 1 receptors (Kd = 0.3 nM, Nmax = 23 fmol/mg protein). At maximal induction of these latter parameters, only about 20% of type 2 receptors are occupied. These results suggest that both types of aldosterone receptors are involved in the mediation of the full mineralocorticoid response: type 1 in the early and late and type 2 particularly in the late tissue response.

Hormonal regulation of (Na+,K+)-ATPase biosynthesis in the toad bladder. Effect of aldosterone and 3,5,3'-triiodo-L-thyronine.

Aldosterone stimulates transepithelial Na+ transport in the toad bladder, and thyroid hormone antagonizes this mineralocorticoid action. In the present study, we assessed the influence of these two hormones on the biosynthesis of (Na+,K+)ATPase, the major driving force of Na+ transport. Rates of enzyme synthesis were estimated by immunoprecipitation with monospecific alpha (96,000 daltons) and beta (60,000 daltons) subunit antibodies. After a 30-min pulse of intact tissue with [35S]methionine, the anti-alpha-serum recognized the 96,000-dalton alpha subunit and the anti-beta-serum, a 42,000-dalton protein, in total cell extracts. The biosynthesis rates of both these proteins were increased 2.8- and 2.4-fold respectively, over controls by 80 nM aldosterone after 18 h of hormone treatment. The hormonal effect was not apparent up to 3 h of incubation and was dose dependent between 0.2 and 20 nM aldosterone. The hormonal induction was antagonized by spironolactone (500-fold excess) but not by amiloride. The action of aldosterone thus seems to be a receptor-mediated process and a primary event independent of the Na+ permeability of the apical membrane. Thyroid hormone, on the other hand, had no effect on either basal or aldosterone-stimulated synthesis rates of both enzyme proteins. The results demonstrate a direct effect of aldosterone on gene expression of the (Na+,K+)-ATPase. Ultimately, this phenomenon could be linked to the late mineralocorticoid action of this hormone. On the other hand, thyroid hormone, in contrast to the situation in mammals, does not stimulate de novo enzyme synthesis in amphibia. Neither can the antimineralocorticoid action of thyroid hormone in the toad bladder be explained by an inhibition of the (Na+,K+)-ATPase synthesis.

The Effects of a Nematode Lungworm (Rhabdias hylae) on its Natural and Invasive Anuran Hosts.

Biological invasions can bring both the invader and native taxa into contact with novel parasites. As cane toads ( Rhinella marina ) have spread through Australia, they have encountered lungworms (Rhabdias hylae) that occur in native frogs. Field surveys suggest that these lungworms have not host-switched to toads. In our laboratory studies, R. hylae infected cane toads as readily as it infected native frogs, but failed to reach the lungs of the novel host (i.e., were killed by the toads' immune response). Plausibly, then, R. hylae might reduce the viability both of their native hosts (frogs, that can exhibit high parasite burdens) and cane toads (that must deal with infective larvae traveling through the host body). Our laboratory trials suggest, however, that the impacts of the parasite on infected anuran hosts (both frogs and toads) were minimal, with no significant decrements to host survival, activity, growth, or locomotor performance. Ironically, the lack of impact of the parasite on its native hosts appears to be an outcome of co-evolution (frogs tolerate the lungworm), whereas the lack of impact on the novel host is due to a lack of co-evolution (toads can recognize and eliminate the lungworm).

The dynamics of coexistence: habitat sharing versus segregation patterns among three sympatric montane vipers

Contact zones of closely related and ecologically similar species constitute rare opportunities to study the evolutionary consequences of past speciation processes. They represent natural laboratories in which strong competition could lead to the exclusion of one species, or the various species may switch into distinct ecological niches. Alternatively, if reproductive isolation has not yet been achieved, they may hybridize. We elucidate the degree of taxon integrity by comparing genetics and habitat use of three similar-sized congeneric viper species, Vipera ammodytes, Viperaaspis, and Viperaberus, of Nadiza Valley in western Slovenia. No hybridization was detected for either mitochondrial or nuclear genomes. Similarly, external intermediacy by a single prestudy viper (probably V.ammodytesxV. aspis) indicates that hybridization occasionally occurs, but should be very rare. Populations of the three related viperids are partially allopatric in Nadiza Valley, but they also coexist in a narrow contact zone in the montane grassland along the south-exposed slope of Mount Stol (1673m a.s.l.). Here, the three species that occupy areas in or near patches of rocky microhabitats (e.g. stone piles, slides, and walls) live in syntopy. However, fine-scale measurements of structural components show partial habitat segregation, in which V.berus becomes more dominant at elevations above 1400m and occupies mostly the mountain ridge and north-exposed slopes of Mount Stol, V.aspis occurs below 1300m and is the only species to inhabit stoneless patches of grass and bushes around 1000m and lower, and V.ammodytes occurs at all elevations up to 1500m, but is restricted to a rocky microhabitat. We suggest that a high degree of microstructure divergence, slightly different environmental niches, and a generally favourable habitat for all three viper species, keep the pressure for mis-mating and hybridization low, although mechanisms such as reduced hybrid inferiority and temporal mating segregation cannot yet be excluded.

Vitae

Albinus Andegavensis (251v).Alexander, Eventius et Theodulus (333v).Alexander episcopus Alexandrie (243).Amandus (194v).Amator Autissiodorensis (319v).Ambrosius (290v).Ananias (170v).Ancolianus (206v).Andeolus (314).Archadius (32).Athanasius (326v).Austregisilus Bituricensis (363).Baltildis (135).Baudelius (361).Bonitus (38v).Concordius (2).Crucis inventio (332).Decem millia mart. (239v).Eufrasia (213).Eutropius (313)Faustinus et Jobitta (229v).Felix, Fortunatus et Achilleus (296v).Felix Nolanus (33v).Felix Tubitanensis episcopus (36v).Fidolus (355).Fileas (189).Focas (255v).Fructuosus, Augurius et Eulogius (109v).Fulgencius (7).Gengulphus (343).Gennulphus (74v).Georgius (296).Grisogonus (269v).Honoratus Arelatensis (54).Hugo Gratianopolitanus (273v).lgnacius (178v).Jacobus minor (313v).Johannes Chrysostomus (145).Johannes presb. et conf. (175v).Juliana (236v).Julianus Cenomanensis (138v).Launomarus (101).Leobardus reclusus (98).Lucianus Antiochensis (24v).Lucianus Belvacensis (26v).Mapalicus (295).Marcellus papa (48v).Marciana (30v).Marcus ev. (305).Martina (2v).Maurus (174).Melanius Redonensis (21v).Pachomius (353v).Pancracius (342).Patroclus (111).Perpetus et Felicitas (260).Petrus et Andreas, Paulus et Dionysius (354).Petrus Balsamus (19v).Philemon, Choraula et Arrianus (262).Policarpus (128, 130).Poncius (347).Prejectus et Marinus (124).Quadraginta martyres (266v).Quiriacus episcopus (339).Richarius (309).Ricmarus (94v).Robertus abbas (299).Saturninus (223).Savinianus (122).Scolastica (207).Severinus (210).Sigismundus (318).Siviardus (254).Speusippus, Eleusippus et Meleusippus (67).Theodosia (285).Theogenes (20).Timotheus (114).Tirsus (161).Torpes (357v).Trifon (183).Victor et Corona (351v).Vincentius (115).Vincentius, Oruncius et Victor (118).