The heterotrimeric laminin coiled-coil domain exerts anti-adhesive effects and induces a pro-invasive phenotype

Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling.     

Assessing camera traps for surveying the European mink,<Emphasis Type="Italic"> Mustela lutreola</Emphasis> (Linnaeus, 1761), distribution

This study assesses the suitability of camera trapping as a method for detecting the European mink and determining its distribution in a region located in southwestern Europe. Using this technique, 98 river stretches were surveyed, resulting in the detection of 11 species of carnivores. A high photographic rate was obtained for the European mink, and we were able to get a picture of its distribution area in the year 2000. No seasonal differences were found in the efficiency of the method used. Camera trapping is an effective technique that provides quick updates of the distribution of the European mink and may be used in programs monitoring this species.     

Novel Evidence That Attributing Affectively Salient Signal to Random Noise Is Associated with Psychosis

We wished to replicate evidence that an experimental paradigm of speech illusions is associated with psychotic experiences. Fifty-four patients with a first episode of psychosis (FEP) and 150 healthy subjects were examined in an experimental paradigm assessing the presence of speech illusion in neutral white noise. Socio-demographic, cognitive function and family history data were collected. The Positive and Negative Syndrome Scale (PANSS) was administered in the patient group and the Structured Interview for Schizotypy-Revised (SIS-R), and the Community Assessment of Psychic Experiences (CAPE) in the control group. Patients had a much higher rate of speech illusions (33.3% versus 8.7%, OR_adjusted: 5.1, 95% CI: 2.3–11.5), which was only partly explained by differences in IQ (OR_adjusted: 3.4, 95% CI: 1.4–8.3). Differences were particularly marked for signals in random noise that were perceived as affectively salient (OR_adjusted: 9.7, 95% CI: 1.8–53.9). Speech illusion tended to be associated with positive symptoms in patients (OR_adjusted: 3.3, 95% CI: 0.9–11.6), particularly affectively salient illusions (OR_adjusted: 8.3, 95% CI: 0.7–100.3). In controls, speech illusions were not associated with positive schizotypy (OR_adjusted: 1.1, 95% CI: 0.3–3.4) or self-reported psychotic experiences (OR_adjusted: 1.4, 95% CI: 0.4–4.6). Experimental paradigms indexing the tendency to detect affectively salient signals in noise may be used to identify liability to psychosis.     

Cyclic AMP-dependent and Epac-mediated activation of R-Ras by G protein-coupled receptors leads to phospholipase D stimulation

The activation of the Ras-related GTPase R-Ras, which has been implicated in the regulation of various cellular functions, by G protein-coupled receptors (GPCRs) was studied in HEK-293 cells stably expressing the M3 muscarinic acetylcholine receptor (mAChR), which can couple to several types of heterotrimeric G proteins. Activation of the receptor induced a very rapid and transient activation of R-Ras. Studies with inhibitors and activators of various signaling pathways indicated that R-Ras activation by the M3 mAChR is dependent on cyclic AMP formation but is independent of protein kinase A. Similar to the rather promiscuous M3 mAChR, two typical G(s)-coupled receptors also induced R-Ras activation. The receptor actions were mimicked by an Epac-specific cyclic AMP analog and suppressed by depletion of endogenous Epac1 by small interfering RNAs, as well as expression of a cyclic AMP binding-deficient Epac1 mutant, but not by expression of dominant negative Rap GTPases. In vitro studies demonstrated that Epac1 directly interacts with R-Ras and catalyzes GDP/GTP exchange at this GTPase. Finally, it is shown that the cyclic AMP- and Epac-activated R-Ras plays a major role in the M3 mAChR-mediated stimulation of phospholipase D but not phospholipase C. Collectively, our data indicate that GPCRs rapidly activate R-Ras, that R-Ras activation by the GPCRs is apparently directly induced by cyclic AMP-regulated Epac proteins, and that activated R-Ras specifically controls GPCR-mediated phospholipase D stimulation.     

Differences in the abundance and distribution of copepods in two estuaries of the Basque coast (Bay of Biscay) in relation to pollution

The abundance and spatial distribution of copepod species werecompared in the euhaline region of the polluted estuary of Bilbao[mean biological oxygen demand (BOD): 49748.05 kg day^–1in 1996] and the unperturbed estuary of Urdaibai. Sampling wasperformed at fixed salinity sites, where data of temperature,dissolved oxygen saturation, particulate organic matter, Secchidisk depth and chlorophyll were also obtained. Differences betweenthe estuaries and within the estuaries for environmental variablesand copepod abundances were tested statistically. The dominantspecies Acartia clausi and Paracalanus parvus differed significantlyin abundance between estuaries, and most of neritic speciesdiminished more drastically with decreasing salinity in Bilbaoby the effect of pollution. The different responses observedamong species in relation to pollution and water desalinationare discussed. The lack of the brackish-water species in Bilbaoindicated that water quality worsening upward the estuary ofBilbao not only limited the penetration of neritic species butalso prevented the development of autochthonous estuarine species.At the outer euhaline region of Bilbao, however, pollution wasfound to be low enough to enhance the development of tolerantneritic species that are favoured under moderate pollution conditions.Results indicate that comparisons of copepod abundances anddistributions with salinity in estuaries may be a useful toolto evaluate the health of the different estuarine pelagic habitats.     

Effects of estuarine conditions and organic enrichment on the fecundity and hatching success of Acartia clausi in contrasting systems

The fecundity and hatching success of Acartia clausi were analysed at fixed salinity sites (35, 34 and 33 psu) in two nearby estuaries (Bilbao and Urdaibai, Basque coast, Bay of Biscay) from March to June 1997. Field incubations were conducted to estimate egg production rates and hatching success, and the size of eggs and experimental females measured. Water temperature and dissolved oxygen saturation were also determined, as well as seston samples to quantify food abundance and quality. Between-estuary and within-estuary differences were tested statistically, and correlation and regression analyses were used to determine relationships between reproductive and environmental variables. Egg production rates were higher in the organically enriched estuary of Bilbao; this denoting that food supply controls the fertility of A. clausi in these systems. Temporal patterns of egg production differed between estuaries, and were associated with different nutritional factors in each estuary. Within the salinity range analysed, egg production reached higher values at intermediate salinity (≈34 psu) in both estuaries. This was interpreted as the result of the interaction between the positive effect of food increase, and the negative effect of physicochemical conditions with decreasing salinity. Egg size variations mainly occurred temporally in relation to female size, but no clear trade-off between egg size and egg number was observed in any case. A drop in hatching success in Bilbao, mainly in waters of <34 psu, was related to the oxygen depletion caused by organic pollution. This indicates that organic enrichment in Bilbao has opposite effects on the reproductive success, because it enhances egg production but reduces offspring survival.     

Reduced stability and increased dynamics in the human proliferating cell nuclear antigen (PCNA) relative to the yeast homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity.     

Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus

Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265 bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein–DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo.