Regulation of nicotinic receptor trafficking by the transmembrane Golgi protein UNC-50

Nicotinic acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels that mediate fast synaptic transmission at the neuromuscular junction (NMJ). After assembly in the endoplasmic reticulum (ER), AChRs must be transported to the plasma membrane through the secretory apparatus. Little is known about specific molecules that mediate this transport. Here we identify a gene that is required for subtype-specific trafficking of assembled nicotinic AChRs in Caenorhabditis elegans. unc-50 encodes an evolutionarily conserved integral membrane protein that localizes to the Golgi apparatus. In the absence of UNC-50, a subset of AChRs present in body-wall muscle are sorted to the lysosomal system and degraded. However, the trafficking of a second AChR type and of GABA ionotropic receptors expressed in the same muscle cells is not affected in unc-50 mutants. These results suggest that, in addition to ER quality control, assembled AChRs are sorted within the Golgi system by a mechanism that controls the amount of cell-surface AChRs in a subtype-specific way.     

Messinian palaeoenvironments and hydrology in Sicily (Italy): The dinoflagellate cyst record

Hydrological conditions prevailing before, during and after the Messinian salinity crisis in Sicily have been approached using dinoflagellate cyst records. The synthetic sequence considered is based on five classical sections from the Caltanissetta Basin. Our interpretations are based on the recognition of autochthonous, allochthonous and reworked population among the dinocyst assemblages. For the first time, sea-surface temperatures and seasonal salinity contrasts were tentatively reconstructed using a “Mutual Climatic Range Method”. Sicilian late Tortonian deposits correspond to marine environment with significant terrestrial inputs favourable to eutrophic dinocyst species. Immediately after the beginning of the Messinian Stage, euryhaline assemblages took place, followed by meso-hyperhaline taxa, within a general trend to shallowing. At the end of the Tripoli diatomitic Formation, environment appears confined, with regular oceanic inflows. Such marine inflows remain persistent during the deposition of the salt Member, witnessing the probable persistence of nearby normal marine sea-surface water conditions in the Mediterranean Sea but with possible reduced hydrological circulation and/or low nutrient component. Such inflows are slightly decreasing up to the top of the Sicilian Upper Evaporites. At the same time, river inputs appear weak during the salt deposition, as the consequence of a rather dry climatic context. As shown by reworking activity, terrestrial inputs increase progressively from the base of the Upper Evaporites. During the Lago Mare period, while climate remains rather dry (absence of freshwater algae inputs, very low amount in trees requiring humid conditions), local deposition environment is confined with mesohaline to hypohaline sea-surface waters, in a context with relatively high seasonal sea-surface salinity contrast (up to 6‰). The very high reworking observed in the Arenazzolo silts, in addition to slightly increasing water depth, led us to consider the presence of a discontinuity between the Lago Mare and the Arenazzolo Fms. We consider the Arenazzolo Formation as a transgressive facies following a deep downcutting period. The Arenazzolo Formation presents a two-step development. The first one, correlative with the presence of a G. etrusca (a species with Paratethyan affinities), corresponds to a relative high-stand sea-level with oceanic influxes, low seasonal sea-surface salinity contrast and probable more humid context, as revealed by the important freshwater algal inputs. During the second step, salinity becomes much more variable, with a clear increase of seasonal sea-surface salinity contrast, a possible slight mean sea-surface temperature increase (only few degrees) and a clear weakening of the river inputs. Taking the proposed Zanclean position of the Arenazzolo Fm. into consideration, we state that mean sea-surface temperature did not change significantly from the base of the Messinian to the earliest Zanclean. At 5.33 Ma, the suddenly achieved flooding restored a fair, deep oceanic environment characterized, at the beginning, by a clear mean sea-surface temperature cooling (up to 6-7 °C) and a nutrient depletion, associated with the basins starvation. Sea-surface salinities were normal, with very low seasonal contrast. Hydrodynamics then nutrient supply became then quite normal from c.a. 5.08 Ma. The status of the Sicilian Caltanissetta Basin as a marginal basin although fastly deepening and the stratigraphical location of the Messinian discontinuity at the base of the Arenazzolo is the scenario that best matches our dinocyst record.     

Carbonic anhydrase inhibitors: the X-ray crystal structure of the adduct of N-hydroxysulfamide with isozyme II explains why this new zinc binding function is effective in the design of potent inhibitors

N-Hydroxysulfamide is a 2000-fold more potent inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared to sulfamide. It also inhibits other physiologically relevant isoforms, such as the tumor-associated CA IX and XII (K(I)s in the range of 0.865-1.34microM). In order to understand the binding of this inhibitor to the enzyme active site, the X-ray crystal structure of the human hCA II-N-hydroxysulfamide adduct was resolved. The inhibitor coordinates to the active site zinc ion by the ionized primary amino group, participating in an extended network of hydrogen bonds with amino acid residues Thr199, Thr200 and two water molecules. The additional two hydrogen bonds in which N-hydroxysulfamide bound to hCA II is involved as compared to the corresponding adduct of sulfamide may explain its higher affinity for the enzyme, also providing hints for the design of tight-binding CA inhibitors possessing an organic moiety substituting the NH group in the N-hydroxysulfamide structure.     

Carbonic anhydrase inhibitors. Inhibition of isoforms I, II, IV, VA, VII, IX, and XIV with sulfonamides incorporating fructopyranose-thioureido tails

A series of aromatic/heterocyclic sulfonamides incorporating 2,3:4,5-bis-O-(isopropylidene)-beta-d-fructopyranosyl-thioureido moieties has been synthesized and assayed for the inhibition of seven human isoforms of the zinc enzyme carbonic anhydrase (hCA, EC 4.2.1.1). The new derivatives behaved as weak hCA I inhibitors (K(I)s of 9.4 -13.3microM), were efficient hCA II inhibitors (K(I)s of 6-750nM), and slightly inhibited isoforms hCA IV and hCA VA. Only the sulfanilamide derivative showed efficient and selective inhibition of hCA IV (K(I) of 10nM). These derivatives also showed excellent hCA VII inhibitory activity (K(I)s of 10-79nM), being less efficient as inhibitors of the transmembrane isoforms hCA IX (K(I)s of 10-4500nM) and hCA XIV (K(I)s of 21-3500nM). Two of the new compounds showed anticonvulsant action in a maximal electroshock seizure test in mice, with the fluorosulfanilamide derivative being a more efficient anticonvulsant than the antiepileptic drug topiramate.     

Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+ -ATPase by combining X-ray crystallography and electron cryomicroscopy

Regulatory 14-3-3 proteins activate the plant plasma membrane H(+)-ATPase by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal, mode III, recognition motif as well as an adjacent span of approximately 50 amino acids. Here we report the X-ray crystal structure of 14-3-3 in complex with the entire binding motif, revealing a previously unidentified mode of interaction. A 14-3-3 dimer simultaneously binds two H(+)-ATPase peptides, each of which forms a loop within the typical 14-3-3 binding groove and therefore exits from the center of the dimer. Several H(+)-ATPase mutants support this structure determination. Accordingly, 14-3-3 binding could result in H(+)-ATPase oligomerization. Indeed, by using single-particle electron cryomicroscopy, the 3D reconstruction of the purified H(+)-ATPase/14-3-3 complex demonstrates a hexameric arrangement. Fitting of 14-3-3 and H(+)-ATPase atomic structures into the 3D reconstruction map suggests the spatial arrangement of the holocomplex.     

Impairment of thioredoxin reductase activity by oxidative stress in human rheumatoid synoviocytes

The thioredoxin/thioredoxin reductase system is strongly induced in patients with rheumatoid arthritis (RA). We have investigated the impact on TR activity of doses of superoxide anion generated by the hypoxanthine (HX)/xanthine oxidase (XO) system and by hydrogen peroxide, H(2)O(2), for various times and compared the findings with synoviocytes obtained from osteoarthritis (OA) patients. At baseline, TR activity in RA cells was significantly higher than in OA cells (2.31 +/- 0.65 versus 0.74 +/- 0.43 mUnit/mg protein, p < 0.01). HX/XO and H(2)O(2) in RA cells decreased TR activity, which was found to be unchanged in OA cells. H(2)O(2) and superoxide anion caused a time-dependent accumulation of oxidized TR and induced the formation of carbonyl groups in TR protein in RA cells rather than OA cells, and oxidized the selenocysteine of the active site. The oxidation in TR protein was irreversible in RA cells but not in OA cells. In conclusion, we report that the oxidative aggression generates modifications in the redox status of the active site of the TR and induces an alteration of the Trx/TR system, concomitant with those of the other antioxidant systems that could explain the causes of oxidative stress related to RA disease.     

Intracellular cAMP: the "switch" that triggers on "spontaneous transient outward currents" generation in freshly isolated myocytes from thoracic aorta

Spontaneous transient outward currents (STOCs) have been reported in resistance and small arteries but have not yet been found in thoracic aorta. Do thoracic aorta myocytes possess cellular machinery that generates STOCs? It was found that the majority of aortic myocytes do not generate STOCs. STOCs were generated in 8.7% of freshly isolated aortic myocytes. Myocytes that did not generate STOCs we have called "silent" myocytes and myocytes with STOCs have been called "active." STOCs recorded in active myocytes were voltage dependent and were inhibited by ryanodine, caffeine, and charybdotoxin. Forskolin was reported to increase STOCs frequency in myocytes isolated from resistance arteries. Forskolin (10 microM) triggered STOCs generation in 35.1% of silent aortic myocytes. In 36.8% percent of silent myocytes, forskolin did not trigger STOCs but increased the amplitude of charybdotoxin-sensitive outward net current to 136.1 +/- 8.5% at 0 mV. Membrane-permeable 8BrcAMP triggered STOCs generation in 38.7% of silent myocytes. Forskolin- or 8BrcAMP-triggered STOCs were inhibited by charybdotoxin. 8BrcAMP also increased open probability of BK(Ca) channels in BAPTA-AM-pretreated cells. Our data demonstrate that, in contrast to resistance arteries, STOCs are present just in the minority of myocytes in the thoracic aorta. However, cellular machinery that generates STOCs can be "switched" on by cAMP. Such an inactive cellular mechanism could modulate the contractility of the thoracic aorta in response to physiological demand.     

Coxiella burnetii infection in C57BL/6 mice aged 1 or 14 months

The objective of this study was to investigate the effects of age on infection with Coxiella burnetii, the agent of Q fever. Bacterial burden and granuloma number were increased in the spleens of 14-month-old as compared with 1-month-old mice. This increase was not the result of an anti-inflammatory macrophage response, because inflammatory and anti-inflammatory cytokines were induced in macrophages from young mice but were repressed in mature mice. In addition, macrophage microbicidal competence was similar in mature and young mice. These results suggest the importance of individual host factors in the pathophysiology of an infectious disease such as Q fever.