Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity

Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich alpha helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use. We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% alpha-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression pi-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.     

Prenatal Stress Down-Regulates Reelin Expression by Methylation of Its Promoter and Induces Adult Behavioral Impairments in Rats

Prenatal stress causes predisposition to cognitive and emotional disturbances and is a risk factor towards the development of neuropsychiatric conditions like depression, bipolar disorders and schizophrenia. The extracellular protein Reelin, expressed by Cajal-Retzius cells during cortical development, plays critical roles on cortical lamination and synaptic maturation, and its deregulation has been associated with maladaptive conditions. In the present study, we address the effect of prenatal restraint stress (PNS) upon Reelin expression and signaling in pregnant rats during the last 10 days of pregnancy. Animals from one group, including control and PNS exposed fetuses, were sacrificed and analyzed using immunohistochemical, biochemical, cell biology and molecular biology approaches. We scored changes in the expression of Reelin, its signaling pathway and in the methylation of its promoter. A second group included control and PNS exposed animals maintained until young adulthood for behavioral studies. Using the optical dissector, we show decreased numbers of Reelin-positive neurons in cortical layer I of PNS exposed animals. In addition, neurons from PNS exposed animals display decreased Reelin expression that is paralleled by changes in components of the Reelin-signaling cascade, both in vivo and in vitro. Furthermore, PNS induced changes in the DNA methylation levels of the Reelin promoter in culture and in histological samples. PNS adult rats display excessive spontaneous locomotor activity, high anxiety levels and problems of learning and memory consolidation. No significant visuo-spatial memory impairment was detected on the Morris water maze. These results highlight the effects of prenatal stress on the Cajal-Retzius neuronal population, and the persistence of behavioral consequences using this treatment in adults, thereby supporting a relevant role of PNS in the genesis of neuropsychiatric diseases. We also propose an in vitro model that can yield new insights on the molecular mechanisms behind the effects of prenatal stress.     

Paired Siglec receptors generate opposite inflammatory responses to a human-specific pathogen

Paired immune receptors display near-identical extracellular ligand-binding regions but have intracellular sequences with opposing signaling functions. While inhibitory receptors dampen cellular activation by recognizing self-associated molecules, the functions of activating counterparts are less clear. Here, we studied the inhibitory receptor Siglec-11 that shows uniquely human expression in brain microglia and engages endogenous polysialic acid to suppress inflammation. We demonstrated that the human-specific pathogen Escherichia coli K1 uses its polysialic acid capsule as a molecular mimic to engage Siglec-11 and escape killing. In contrast, engagement of the activating counterpart Siglec-16 increases elimination of bacteria. Since mice do not have paired Siglec receptors, we generated a model by replacing the inhibitory domain of mouse Siglec-E with the activating module of Siglec-16. Siglec-E16 enhanced proinflammatory cytokine expression and bacterial killing in macrophages and boosted protection against intravenous bacterial challenge. These data elucidate uniquely human interactions of a pathogen with Siglecs and support the long-standing hypothesis that activating counterparts of paired immune receptors evolved as a response to pathogen molecular mimicry of host ligands for inhibitory receptors.     

Time course study of Bactrocera oleae (Diptera: Tephritidae) pupae predation in soil: the effect of landscape structure and soil condition

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Structure and growth pattern of the bizarre hemispheric prominence on the rostrum of the fossil beaked whale Globicetus hiberus (Mammalia,Cetacea, Ziphiidae)

The rostrum of most ziphiids (beaked whales) displays bizarre swollen regions, accompanied with extreme hypermineralisation and an alteration of the collagenous mesh of the bone. The functional significance of this specialization remains obscure. With the voluminous and dense hemispheric excrescence protruding from the premaxillae, the recently described fossil ziphiid Globicetus hiberus is the most spectacular case. This study describes the histological structure and interprets the growth pattern of this unique feature. Histologically, the prominence in Globicetus is made up of an atypical fibro‐lamellar complex displaying an irregular laminar organization and extreme compactness (osteosclerosis). Its development is suggested to have resulted from a protraction of periosteal accretion over the premaxillae, long after the end of somatic growth. Complex shifts in the geometry of this tissue are likely to have occurred during its accretion and no indication of Haversian remodeling could be found. X‐ray diffraction and Raman spectroscopy indicate that the bone matrix in the premaxillary prominence of Globicetus closely resembles that of the rostrum of the extant beaked whale Mesoplodon densirostris: apatite crystals are of common size and strongly oriented, but the collagenous meshwork within bone matrix seems to be extremely sparse. These morphological and structural data are discussed in the light of functional interpretations proposed for the highly unusual and diverse ziphiid rostrum. J. Morphol. 277:1292–1308, 2016. © 2016 Wiley Periodicals, Inc.     

Down under the southeastern Pacific: marine non-indigenous species in Chile

This article presents the first compilation of marine non-indigenous species (NIS) of algae and macro-invertebrates invading Chilean waters. A total of 32 cosmopolitan and non-cosmopolitan species are reported. Among them there are six species considered as extending their southern range of distribution in connection with El Niño events. The article highlights negative and positive impacts caused by marine NIS invasions. Among the first are Codium fragile var. tomentosoide, considered as a pest in Gracilaria chilensis aquaculture facilities in northern Chile, and Ciona intestinalis, a pest in scallop aquaculture installations. Among the second are bio-engineers species, such as the ascidian Pyura praeputialis and the sea grass Heterozostera tasmanica, which have caused an increase in local biodiversity and enhancement of nursery grounds via the creation of new habitats. Further more, invaders such as the algae Mastocarpus papillosus, Porphyra linearis and P. pseudolinearis represent new exploitable resources, extracted by coastal food gatherers along the coast (M. papillosus) or potential species to develop aquaculture. Additional information is presented on the anemone Anemonia alicemartinae, which appears to be a native species (?), having shown in the past 40–50 years, a geographical southward range extension of approximately 1900 km. The number of NIS reported for Chile is compared with those published for the southwestern Atlantic, South Africa, North America (Atlantic and Pacific coasts) and New Zealand. It is suggested that probably the low number of Chilean NIS is due to the fact that the Chilean coasts are environmentally less stressed than other coasts in the world, due to the scarcity of estuaries, gulfs, enclosed bays, lagoons and low human populations. These kinds of sheltered areas have been suggested as centers for bio-invasions, due to the high rate of human-mediated transfer and increase of pollutants. Furthermore, none of NIS reported from Chile show a fast geographical expansion rate (exception of A. alicemartinae), nor invading strategies such as those described for marine NIS in other latitudes, where notorious ecological unbalances following invasions have been observed. An alternative hypothesis is that the low number of marine NIS invading Chile is underestimated, since the modern list of species generated through specific taxonomically intensive port and harbor surveys is still lacking. Fifteen species (five invertebrate and 10 fish) have been deliberately imported to Chile for aquaculture. The invertebrates appear to be controlled within aquaculture facilities and have not established naturalized populations or caused direct ecological impacts on local communities. On the contrary, several millions of salmoniforms (and rainbow trout) have escaped from farming facilities in southern Chile and established naturalized populations. Studies on ecological impacts are lacking. These escapees are also playing a role in the enhancement of artisanal and sport fishery activities.     

Spatial and temporal variations of hydrological conditions,nutrients and chlorophyll <Emphasis Type="Italic">a</Emphasis> in a Mediterranean coastal lagoon (Mar Menor,Spain)

The Mar Menor is a sheltered and hypersaline lagoon, with salinity ranges between 38 and 51 psu. The lagoon is threatened by several pressures and in the last decades detrimental impact on the natural community structure and dynamics have increased. In the watershed, agricultural practices are rapidly evolving from extensive dry crop farming to intensively irrigated crops, with increasing loads of nutrient and pollutants to the lagoon. Hydrological conditions, nutrients and chlorophyll a concentrations were analysed in 1997 and 2002–2003 in a grid of 20 stations in the lagoon. Different time scales, from daily to inter-annual, were considered. In the considered periods, the dissolved inorganic nitrogen (DIN) increased whilst phosphate decreased significantly. These contrasting patterns depended upon the increased agricultural loading for DIN and were due to the implementation of the wastewater works for phosphates. In 1997 and 2002, the highest nitrate concentrations were usually found on the west coast of the lagoon, close to the mouths of the main watercourses. In parallel, the lowest concentrations were detected at the inner coastline along “La Manga” sandy bar and “El Estacio” channel. Based on weekly data, correlations between chlorophyll a concentrations and environmental variables disagreed with traditional eutrophication models. Relationships between chlorophyll a and nutrients were negative, suggesting that in the short term phytoplankton controlled nutrient concentrations. Moreover, nitrate and phosphorous seemed to alternate as limiting factors. The relationships between chlorophyll a became positive when considering time lags and analysed at longer time scales (monthly or seasonal means), thus suggesting a very rapid response of primary producers to nutrient enrichment. A significant correlation between chlorophyll a concentration and fish larvae density was also found at all time scales analysed, suggesting a top-down control of the trophic web.     

Influence of hydrophobic matching on association of model transmembrane fragments containing a minimised glycophorin A dimerisation motif

The principles that govern the folding and packing of membrane proteins are still not completely understood. In the present work, we have revisited the glycophorin A (GpA) dimerisation motif that mediates transmembrane (TM) helix association, one of the best-suited models of membrane protein oligomerisation. By using artificial polyleucine TM segments we have demonstrated in this study that a pattern of only five amino acids (GVxxGVxxT) promotes specific dimerisation. Further, we have used this minimised GpA motif to assess the influence of hydrophobic matching on the TM helix packing process in detergent micelles and found that this factor modulates helix-helix association and/or dissociation between TM fragments.     

Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection

Previously we improved the neurotensin (NT)-polyplex by the coupling of HA2 fusogenic peptide (FP) and Vp1 SV40 karyophilic peptide (KP). We now report the proportion of [(125)I]-NT, [(3)H]-FP, and poly-l-lysine (PLL) in the NT-polyplex, and some of its biophysical properties. We concluded that the most efficient NT-polyplex comprised 1 NT, 4 FP, and 2 PLL molecules. Electrophoresis revealed that high acidity is detrimental for NT-polyplex stability. Electron microscopy and electrophoresis studies showed that 6 muM KP and 1% serum condensed the plasmid DNA (pDNA) before the appearance of toroid structures. Four plasmids were used to evaluate the transfection efficiency. In vitro, maximum expression was produced at molar ratios (pDNA : [(125)I]-NT-[(3)H]-FP-PLL conjugate) of 1:34 for pEGFP-N1 and 1:27 for pECFP-Nuc. Cotransfection of those plasmids was attained at their optimum molar ratios. In vivo, maximum expression of the pDAT-BDNF-flag in dopamine neurons was produced at a 1:45 molar ratio, whereas that of pDAT-EGFP was at 1:20. The NT-polyplex in the presence of 1 muM SR-48692, an NT-receptor specific antagonist, and untargeted polyplex did not cause transfection in vivo demonstrating the specificity of gene transfer via NT-receptor endocytosis. This information is essential for synthesizing an efficient NT-polyplex that can provide a useful tool for specific gene transfection.