Genetic Diversity Within and among Disjunct Populations of the Mediterranean Island Endemic Delphinium pictum and D. requienii (Ranunculaceae)

Allozyme diversity was assessed in 13 populations of Delphinium pictum and D. requienii, two short-lived and closely related insular Mediterranean endemics. While D. pictum has scattered distribution in Corsica, Sardinia and Majorca, D. requienii is found in a few small populations restricted to the Hyères Archipelago. Eleven enzyme systems were assayed, and 15 loci were resolved. Both species harboured moderate levels of genetic diversity, comparable to the values expected for endemic plants. All genetic parameters suggested higher diversity in D. pictum (A?=?1.93, P?=?40, H _e?=?0.106) than in D. requienii (A?=?1.30, P?=?26.7, H _e?=?0.096); F _IS values revealed higher inbreeding in D. requienii. Although the two species did not harbour species-specific alelles in the surveyed loci, the UPGMA dendrogram based on Nei’s genetic similarity index supported divergence between them. These results, together with significant morphological similarity and evidence of successful hybridization between the species, support the hypothesis of a recent speciation event. The moderate levels of both genetic variability and population divergence observed for D. pictum are likely attributable to its ability to establish seed banks. Given that the allozyme variation detected for D. pictum was significantly partitioned among islands, we suggest the implementation of conservation programs throughout its distribution range, and not only in Corsica as it occurs nowadays. Management strategies are also desirable for D. requienii to maintain population size and control inbreeding.     

Cannabinoids prevent the opposite regulation of astroglial connexin43 hemichannels and gap junction channels induced by pro‐inflammatory treatments

Brain injuries as well as neurodegenerative diseases, are associated with neuro‐inflammation characterized by astroglial and microglial activation and/or proliferation. Recently, we reported that lipopolysaccharide (LPS)‐activation of microglia inhibits junctional channels and promotes hemichannels, two connexin43 functions in astrocytes. This opposite regulation is mediated by two pro‐inflammatory cytokines, interleukin‐1 beta and tumor necrosis factor‐alpha, released from activated microglia. Because cannabinoids (CBs) have anti‐inflammatory properties and their receptors are expressed by glial cells, we investigated on primary cortical cultures the effects of CB agonists, methanandamide and synthetic CBs on (i) cytokines released from LPS‐activated microglia and (ii) connexin43 functions in astrocytes subjected to pro‐inflammatory treatments. We observed that CBs inhibited the LPS‐induced release of interleukin‐1 beta and tumor necrosis factor‐alpha from microglia. Moreover, the connexin43 dual regulation evoked by the pro‐inflammatory treatments, was prevented by CB treatments. Pharmacological characterizations of CB actions on astrocytic connexin43 channels revealed that these effects were mainly mediated through CB1 receptors activation, although non‐CB1/CB2 receptors seemed to mediate the action of the methanandamide. Altogether these data demonstrate that in inflammatory situations CBs exert, through the activation of different sub‐types of glial CB receptors, a regulation on two functions of connexin43 channels in astrocytes known to be involved in neuron survival.     

Cellular levels of heme affect the activity of dimeric glutamyl-tRNA reductase

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.     

Phospholipid packing and hydration in pulmonary surfactant membranes and films as sensed by LAURDAN

The efficiency of pulmonary surfactant to stabilize the respiratory surface depends critically on the ability of surfactant to form highly packed films at the air-liquid interface. In the present study we have compared the packing and hydration properties of lipids in native pulmonary surfactant and in several surfactant models by analyzing the pressure and temperature dependence of the fluorescence emission of the LAURDAN (1-[6-(dimethylamino)-2-naphthyl]dodecan-1-one) probe incorporated into surfactant interfacial films or free-standing membranes. In interfacial films, compression-driven changes in the fluorescence of LAURDAN, evaluated from the generalized polarization function (GPF), correlated with changes in packing monitored by surface pressure. Compression isotherms and GPF profiles of films formed by native surfactant or its organic extract were compared at 25 or 37 °C to those of films made of dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), DPPC/phosphatidylglycerol (PG) (7:3, w/w), or the mixture DPPC/POPC/palmitoyloleoylphosphatidylglycerol (POPG)/cholesterol (Chol) (50:25:15.10), which simulates the lipid composition of surfactant. In general terms, compression of surfactant films at 25 °C leads to LAURDAN GPF values close to those obtained from pure DPPC monolayers, suggesting that compressed surfactant films reach a dehydrated state of the lipid surface, which is similar to that achieved in DPPC monolayers. However, at 37 °C, the highest GPF values were achieved in films made of full surfactant organic extract or the mixture DPPC/POPC/POPG/Chol, suggesting a potentially important role of cholesterol to ensure maximal packing/dehydration under physiological constraints. Native surfactant films reached high pressures at 37 °C while maintaining relatively low GPF, suggesting that the complex three-dimensional structures formed by whole surfactant might withstand the highest pressures without necessarily achieving full dehydration of the lipid environments sensed by LAURDAN. Finally, comparison of the thermotropic profiles of LAURDAN GPF in surfactant model bilayers and monolayers of analogous composition shows that the fluorophore probes an environment that is in average intrinsically more hydrated at the interface than inserted into free-standing bilayers, particularly at 37 °C. This effect suggests that the dependence of membrane and surfactant events on the balance of polar/non-polar interactions could differ in bilayer and monolayer models, and might be affected differently by the access of water molecules to confined or free-standing lipid structures.     

Immuno-enzymatic evaluation of the recombinant TSSA-II protein of Trypanosoma cruzi in dogs and human sera: a tool for epidemiological studies

The rTSSA-II (recombinant Trypomastigote Small Surface II) antigen was evaluated by ELISA to detect anti-Trypanosoma cruzi antibodies in sera from naturally infected dogs and humans. For this evaluation ELISA-rTSSA-II was standardized and groups were classified according to the results obtained through xenodiagnosis, ELISA and PCR. Sensitivity (Se), Specificity (Sp), Kappa index (KI) and area under curve (AUC) were determined. The Se was determined by using 14 sera from dogs infected with T. cruzi VI (TcVI) whereas Sp was determined by using 95 non-chagasic sera by xenodiagnosis, ELISA-Homogenate and PCR. The performance of ELISA-rTSSA-II in dog sera was high (AUC=0·93 and KI=0·91). The Se was 92·85% (1 false negative) and Sp was 100%. Two sera from dogs infected with TcI and 1 with TcIII were negative. For patients infected with T. cruzi, reactivity was 87·8% (36/41), there was only 1 indeterminate, and Sp was 100%. Fifty-four sera from non-chagasic and 68 sera from patients with cutaneous leishmaniasis did not react with rTSS-II. ELISA-rTSSA-II showed a high performance when studying sera from naturally infected dogs and it also presented 100% Sp. This assay could be an important tool to carry out sero-epidemiological surveys on the prevalence of T. cruzi circulating lineages in the region.     

Toxin-Antitoxin Systems in the Mobile Genome of Acidithiobacillus ferrooxidans

Toxin-antitoxin (TA) systems are genetic modules composed of a pair of genes encoding a stable toxin and an unstable antitoxin that inhibits toxin activity. They are widespread among plasmids and chromosomes of bacteria and archaea. TA systems are known to be involved in the stabilization of plasmids but there is no consensus about the function of chromosomal TA systems. To shed light on the role of chromosomally encoded TA systems we analyzed the distribution and functionality of type II TA systems in the chromosome of two strains from Acidithiobacillus ferrooxidans (ATCC 23270 and 53993), a Gram-negative, acidophilic, environmental bacterium that participates in the bioleaching of minerals. As in other environmental microorganisms, A. ferrooxidans has a high content of TA systems (28-29) and in twenty of them the toxin is a putative ribonuclease. According to the genetic context, some of these systems are encoded near or within mobile genetic elements. Although most TA systems are shared by both strains, four of them, which are encoded in the active mobile element ICEAfe1, are exclusive to the type strain ATCC 23270. We demostrated that two TA systems from ICEAfe1 are functional in E. coli cells, since the toxins inhibit growth and the antitoxins counteract the effect of their cognate toxins. All the toxins from ICEAfe1, including a novel toxin, are RNases with different ion requirements. The data indicate that some of the chromosomally encoded TA systems are actually part of the A. ferrooxidans mobile genome and we propose that could be involved in the maintenance of these integrated mobile genetic elements.     

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.     

Cloning and functional expression of a rodent brain cDNA encoding a novel protein with agmatinase activity, but not belonging to the arginase family

A rat brain cDNA encoding for a novel protein with agmatinase activity was cloned and functionally expressed. The protein was expressed as a histidine-tagged fusion product with a molecular weight of about 63 kDa. Agmatine hydrolysis was strictly dependent on Mn(2+); K(m) and k(cat) values were 2.5+/-0.2 mM and 0.8+/-0.2 s(-1), respectively. The product putrescine was a linear competitive inhibitor (K(i)=5+/-0.5 mM). The substrate specificity, metal ion requirement and pH optimum (9.5) coincide with those reported for Escherichia coli agmatinase, the best characterized of the agmatinases. However, as indicated by the k(cat)/K(m) (320 M(-1)s(-1)), the recombinant protein was about 290-fold less efficient than the bacterial enzyme. The deduced amino sequence revealed great differences with all known agmatinases, thus excluding the protein from the arginase family. It was, however, highly identical (>85%) to the predicted sequences for fragments of hypothetical or unnamed LIM domain-containing proteins. As a suggestion, the agmatinase activity is adscribed to a protein with an active site that promiscuously catalyze a reaction other than the one it evolved to catalyze.