A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+

Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeF_x) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeF_x is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, k_obs, for the formation of E2P decreases with [Pi] whereas that of E2BeF_x increases with [BeF_x]. This might wrongly be taken as evidence of a mechanism where the binding of BeF_x induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeF_x follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na⁺. Although E2P and E2BeF_x are able to form states with 2 occluded Rb⁺, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb⁺ is much lower in E2BeF_x than in E2P. The higher rates of Rb⁺ occlusion and deocclusion observed for E2BeF_x, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeF_x mimics the E2P ground state.     

Germination response of Arabian desert species to gibberellic acid and potassium nitrate seed treatment

Abstract

Desert plant species commonly use seed dormancy to prevent germination during unfavorable environmental conditions and thus increase the probability of seedling survival. Seed dormancy presents a challenge for restoration ecology, particularly in desert species for which our knowledge of dormancy regulation is limited. In the present study the effect of gibberellic acid (GA₃) and potassium nitrate (KNO₃) on seed dormancy release was investigated on eight Arabian desert species. Both treatments significantly enhanced the germination of most species tested. GA₃ was more effective than KNO₃ in enhancing germination percentage, reducing mean germination time and synchronizing the germination in most of the studied species. Light requirement during germination was species-specific, but in general the presence of light promoted germination more effectively when combined with KNO₃ and GA₃. The wide variation in dormancy and germination requirements among the tested species is indicative of distinct germination niches, which might assist their co-existence in similar habitat/environmental conditions. Seed pre-treatments that optimize germination in this habitat must therefore be assessed for individual species to improve the outcomes of ecological restoration.     

Helminths in the wolf, Canis lupus, from north-western Spain

Fifteen helminth species were collected from 47 wolves (Canis lupus ) which were surveyed from 1993 to 1999 in northwestern Spain. These included the trematode Alaria alata (2.1%); the cestodes Taenia hydatigena (44.7%), T. multiceps (29.8%), T. serialis (2.1%), Dipylidium caninum (6.4%) and Mesocestoides sp. aff. litteratus (4.2%); and the nematodes Pearsonema plica (7.4%), Trichuris vulpis (10.6%), Trichinella britovi (12.8%), Ancylostoma caninum (8.5%), Uncinaria stenocephala (51.1%), Toxocara canis (6.4%) Toxascaris leonina (4.2%), Angiostrongylus vasorum (2.1%) and Dirofilaria immitis (2.1%). Only two wolves were not infected. A single infection occurred in 28.9% of the cases, but the commonest infracommunity (31.1%) involved three species. The helminths Alaria alata, Taenia hydatigena, Mesocestoides sp. aff. litteratus, P. plica, Trichuris vulpis, and Ancylostoma caninum parasitizing C. lupus are reported for the first time in Spain. Taenia serialis and D. immitis are reported for the first time in wolves in Europe. Angiostrongylus vasorum represents a new host record for wolves. The helminth fauna of Spanish wolves is compared with that of other European wolf populations. Some epidemiological considerations of the helminth fauna of wolves in Spain and the health risk to humans are also discussed.     

Role of Glutamate Receptors and Glutamate Transporters in the Regulation of the Glutamate-Glutamine Cycle in the Awake Rat

In the present study we investigate the effects of a specific glutamate reuptake blocker, L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC), on extracellular concentrations of glutamine and glutamate in the striatum of the freely moving rat. Intracerebral infusions of PDC (1, 2 and 4 mM) produced a dose-related increase in extracellular concentrations of glutamate and a dose-related decrease in extracellular concentrations of glutamine. These increases in extracellular glutamate and decreases in extracellular glutamine were significantly correlated. To investigate the involvement of ionotropic glutamate receptors in the decreases of extracellular glutamine produced by PDC, N-methyl-D-aspartate (NMDA) receptor antagonist and -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist were used. Perfusion of the NMDA receptor antagonist blocked the decrease of extracellular glutamine but had no effect on the increase of extracellular glutamate, both produced by PDC. Perfusion of the AMPA/kainate receptor antagonist attenuated the increase of extracellular glutamate and not only blocked the decrease of extracellular glutamine but also produced a significant increase of extracellular glutamine. The results reported in this study suggest that both NMDA and AMPA/kainate glutamatergic receptors are involved in the regulation of extracellular glutamine.     

Inhibition of NHE-1 Na+/H+ exchanger by natriuretic peptides in ocular nonpigmented ciliary epithelium

The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering the intraocular pressure (IOP), a function that is mediated by the bilayer ocular ciliary epithelium (CE), in conjunction with the trabecular meshwork. ANP regulates Na⁺/H⁺ exchanger (NHE) activity, and inhibitors of NHE have been shown to lower IOP. We examined whether NPs influence the NHE activity of the CE, which is comprised of pigmented (PE) and nonpigmented (NPE) epithelial cells, by directly recording the rate of intracellular pH (pH_i) recovery from its inner NPE cell layer. NPs inhibited, in a dose-dependent manner (1–100 nM), the rate of pH_i recovery with the order of potency CNP > ANP > BNP, indicative that this inhibition is mediated by the presence of NPR type B receptors. 8-Bromo-cGMP (8-BrcGMP), a nonhydrolyzable analog of cGMP, mimicked NPs in inhibiting the rate of Na⁺-dependent pH_i recovery. In contrast, ethylisopropyl amiloride (EIPA, 100 nM) or amiloride (10 µM) completely abolished the pH_i recovery by NHE. 18-Glycyrrhetinic acid (18-GA), a gap junction blocker, attenuated the inhibitory effect of CNP on the rate of pH_i recovery, suggesting that NHE activity in both cell layers of the CE is coregulated. This interpretation was supported, in part, by the coexpression of NHE-1 isoform mRNA in both NPE and PE cells. The mechanism by which the inhibitory effect of NPs on NHE-1 activity might influence the net solute movement or fluid transport by the bilayer CE remains to be determined. Na⁺/H⁺ exchanger type 1; intracellular pH; aqueous humor     

Skeletons, noise and population growth: the end of an old debate?

Population dynamics models remain largely deterministic, although the presence of random fluctuations in nature is well recognized. This deterministic approach is based on the implicit assumption that systems can be separated into a deterministic part that captures the essential features of the system and a random part that can be neglected. But is it possible, in general, to understand population dynamics without the explicit consideration of random fluctuations? Here, we suggest perhaps not, and argue that the dynamics of many systems are a result of interactions between the deterministic nonlinear skeleton and noise.     

Conserved asparagine residue 54 of alpha-sarcin plays a role in protein stability and enzyme activity

Asparagine 54 of alpha-sarcin is a conserved residue within the proteins of the ribotoxin family of microbial ribonucleases. It is located in loop 2 of the protein, which lacks repetitive secondary structure elements but exhibits a well-defined conformation. Five mutant variants at this residue have been produced and characterized. The spectroscopic characterization of these proteins indicates that the overall conformation is not changed upon mutation. Activity and denaturation assays show that Asn-54 largely contributes to protein stability, and its presence is a requirement for the highly specific inhibitory activity of these ribotoxins on ribosomes.     

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.     

Dynamics of macrophage cell populations during murine pulmonary tuberculosis

The influx of macrophages into the lungs is the major component of the granulomatous response to infection with Mycobacterium tuberculosis. In this investigation we used flow cytometric analysis to define macrophage populations entering the airways and lung tissues of infected mice. We demonstrate that by the judicious use of cell surface markers, especially CD11b and CD11c, several cell populations can be distinguished, allowing cell sorting and morphological definition. Primary populations of CD11b(-)/CD11c(+/high) were defined as alveolar macrophages, CD11b(high)/CD11c(+/high) as dendritic cells, and CD11b(+/mid)/CD11c(+/mid) as small macrophages or monocytes, and changes in the activation phenotype of these populations were followed over the early course of the infection. In further studies, these cell populations were compared with cells harvested during the chronic stage of the disease. During the chronic stage of infection, Ag-presenting class II molecules and activation markers were poorly expressed on dendritic, small macrophage, and monocyte cell populations, which may have important implications for the breakdown of the lesions during reactivation disease. This analytical approach may facilitate the further characterization of macrophage populations entering into the lung tissues and their relative contributions to host resistance to tuberculosis infection.     

Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory-secretory antigens

Current methods for the serodiagnosis of sheep fascioliasis show suboptimal sensitivity, specificity, or both. With the aim of developing an improved method, we fractionated native Fasciola hepatica excretory-secretory antigens (ESAs) by size-exclusion FPLC (fast protein liquid chromatography) on a Superdex 75 HR 10/30 column and then tested the serodiagnostic value of the antigens contained in each one of the 4 peaks obtained (peaks I-IV). Serodiagnostic value was assessed using sera from sheep naturally infected with F. hepatica (group A); sera from the individuals of a fluke-free herd (most of which also had other intestinal nematodes, lung nematodes, Moniezia spp., and/or Cysticercus tenuicollis) sera from a fluke-free herd (group B); sera from lambs experimentally infected with 10-40 F. hepatica metacercariae (group C); and sera from uninfected control lambs (group D). Enzyme-linked immunosorbent assay (ELISA) with peak I or II as target antigens (and to a lesser extent with peak III as target) showed reactivity with negative sera, so that it was not possible to establish cutoff values discriminating infected and uninfected animals. In contrast, when peak IV was used as target, a low cutoff value of 0.235 optical density units (mean + 4 SD) discriminated infected and uninfected animals, with 100% sensitivity and 100% specificity. ELISA with peak IV as a target identified infected animals (even animals that had received only 10 metacercariae) within 3-5 wk of infection and subsequently throughout the rest of the 14-wk monitoring period. In Western blotting analysis, again only the antigens contained in peak IV (range 7-40 kDa, under reducing conditions) were specific for diagnosis of infected animals. These results indicate that molecular sieving of F. hepatica ESAs by this procedure is a fast, simple, reproducible way of obtaining antigens useful for serodiagnosis of sheep fascioliasis.