Mechanism of Focal Adhesion Kinase Mechanosensing

Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP₂ binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP₂ concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP₂-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments.     

Plasma Ceruloplasmin Ferroxidase Activity Correlates with the Nigral Sonographic Area in Parkinson’s Disease Patients: A Pilot Study

Increased area of the substantia nigra (SN) associated to iron deposition has been proposed as a specific marker for Parkinson’s disease (PD). Echogenicity, assessed by transcranial sonography (TCS), has been used to measure such an iron deposition. On the other hand, ferroxidase activity is known to play a role in brain iron metabolism and thus could be involved in increased SN echogenicity of PD patients. The present study was conducted to search for a possible correlation between both markers: TCS of SN and plasma ferroxidase activity. Twenty-one PD patients and 13 healthy volunteers (HV) were included. Mean SN sonographic areas were 0.31 cm² for PD patients and 0.12 cm² for HV (P < 0.001), while plasma ferroxidase activity was reduced in PD patients (P < 0.001). Interestingly, plasma ferroxidase activity was inversely correlated with the SN size by TCS (R² = 0.31), suggesting a relationship between the two markers.     

Variation in reef fish and invertebrate communities with level of protection from fishing across the Eastern Tropical Pacific seascape

Aim To quantify general differences in reef community structure between well‐enforced and poorly enforced marine protected areas (MPAs) and fished sites across the Eastern Tropical Pacific (ETP) regional seascape Location The Pacific continental margin and oceanic islands of Costa Rica, Panama, Colombia and Ecuador, including World Heritage sites at Galapagos, Coiba, Cocos and Malpelo Methods Densities of reef fishes, mobile and sessile invertebrates, and macroalgae were quantified using underwater visual surveys at 136 ‘no‐take’ and 54 openly fished sites associated with seven large MPAs that encompassed a range of management strategies. Spatial variation in multivariate and univariate community metrics was related to three levels of fishing pressure (high‐protection MPAs, limited‐protection MPAs, fishing zones) for both continental and oceanic reefs. Results High‐protection MPAs possessed a much greater biomass of higher carnivorous fishes, lower densities of asteroids and Eucidaris spp. urchins, and higher coral cover than limited‐protection MPAs and fished zones. These results were generally consistent with the hypothesis that overfishing of predatory fishes within the ETP has led to increased densities of habitat‐modifying macroinvertebrates, which has contributed to regional declines in coral cover. Major differences in ecological patterns were also evident between continental and oceanic biogeographic provinces. Main conclusions Fishing down the food web, with associated trophic cascades, has occurred to a greater extent along the continental coast than off oceanic islands. Poorly enforced MPAs generate food webs more similar to those present in fished areas than in well‐protected MPAs.     

Global human footprint on the linkage between biodiversity and ecosystem functioning in reef fishes

Difficulties in scaling up theoretical and experimental results have raised controversy over the consequences of biodiversity loss for the functioning of natural ecosystems. Using a global survey of reef fish assemblages, we show that in contrast to previous theoretical and experimental studies, ecosystem functioning (as measured by standing biomass) scales in a non-saturating manner with biodiversity (as measured by species and functional richness) in this ecosystem. Our field study also shows a significant and negative interaction between human population density and biodiversity on ecosystem functioning (i.e., for the same human density there were larger reductions in standing biomass at more diverse reefs). Human effects were found to be related to fishing, coastal development, and land use stressors, and currently affect over 75% of the world's coral reefs. Our results indicate that the consequences of biodiversity loss in coral reefs have been considerably underestimated based on existing knowledge and that reef fish assemblages, particularly the most diverse, are greatly vulnerable to the expansion and intensity of anthropogenic stressors in coastal areas.     

Body adiposity and type 2 diabetes: increased risk with a high body fat percentage even having a normal BMI

Obesity is the major risk factor for the development of prediabetes and type 2 diabetes. BMI is widely used as a surrogate measure of obesity, but underestimates the prevalence of obesity, defined as an excess of body fat. We assessed the presence of impaired glucose tolerance or impaired fasting glucose (both considered together as prediabetes) or type 2 diabetes in relation to the criteria used for the diagnosis of obesity using BMI as compared to body fat percentage (BF%). We performed a cross-sectional study including 4,828 (587 lean, 1,320 overweight, and 2,921 obese classified according to BMI) white subjects (66% females), aged 18-80 years. BMI, BF% determined by air-displacement plethysmography (ADP) and conventional blood markers of glucose metabolism and lipid profile were measured. We found a higher than expected number of subjects with prediabetes or type 2 diabetes in the obese category according to BF% when the sample was globally analyzed (P < 0.0001) and in the lean BMI-classified subjects (P < 0.0001), but not in the overweight or obese-classified individuals. Importantly, BF% was significantly higher in lean (by BMI) women with prediabetes or type 2 diabetes as compared to those with normoglycemia (NG) (35.5 ± 7.0 vs. 30.3 ± 7.7%, P < 0.0001), whereas no differences were observed for BMI. Similarly, increased BF% was found in lean BMI-classified men with prediabetes or type 2 diabetes (25.2 ± 9.0 vs. 19.9 ± 8.0%, P = 0.008), exhibiting no differences in BMI or waist circumference. In conclusion, assessing BF% may help to diagnose disturbed glucose tolerance beyond information provided by BMI and waist circumference in particular in male subjects with BMI <25 kg/m(2) and over the age of 40.     

Structural characteristics of immunogenic liver-stage antigens derived from P. falciparum malarial proteins

A fully effective antimalarial vaccine must contain multiple proteins from the different development stages of Plasmodium falciparum parasites involved in host-cell invasion or their biologically active fragments. It must therefore include sporozoite molecules able to induce protective immunity by blocking the parasite’s access to hepatic cells, and/or proteins involved in the development of this stage, amongst which are included the Liver Stage Antigen-1 (LSA-1) and the Sporozoite and Liver Stage Antigen (SALSA).Our studies have focused on the search for an association between the structure of high activity binding peptides (HABPs), including both conserved native and their modified analogues, and their ability to bind to the MHC Class II HLA-DR molecules during formation of the MHCII-peptide-TCR complex leading to inducing the appropriate immune response. These studies are part of a logical and rational strategy for developing multi-stage, multi-component, minimal subunit-based vaccines, mainly against the P. falciparum malaria.     

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K⁺ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an “active” conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca²⁺ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca²⁺ sensor, conferring Ca²⁺ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.M-type channels are generated by the KCNQ (Kv7) family of voltage-gated subtypes (1), and they are found throughout the nervous system where they fulfill dominant roles in the control of excitability and neural discharges (2). Like all Kv channels, the KCNQ α subunits share a common core structure of six transmembrane segments with a voltage sensing domain (S1–S4) and a pore domain (S5 and S6; see Fig. 1) (3). Sequence analysis predicts the presence of four helical regions (A–D) in all family members (4), and helices A and B constitute the binding site for calmodulin (CaM).⁸ CaM is a prototypical Ca²⁺ sensor that confers Ca²⁺ sensitivity to a wide array of proteins, including ion channels (5). CaM is thought to mediate Ca²⁺-dependent inhibition of KCNQ channels (6), and in addition, we have postulated that a direct association with CaM is required for KCNQ2 channels to exit the endoplasmic reticulum (7).Open in a separate windowFIGURE 1.Topological representation of a KCNQ subunit. The consensus IQ residues are shown in bold. Circles and squares correspond to the residues mutated here (the squares indicate the mutations causing BFNC). The boxes indicate the regions with a high probability of adopting an α helix configuration, and the thick lines delineate the region fused to GST.To gain a deeper understanding into the involvement of CaM in channel trafficking, we have studied this interaction in vitro with a set of CaM-binding site-specific mutants. Although we had previously explored the impact of some of these mutants on channel trafficking (7), here we have extended this study to the mutant L339R located in helix A. This mutation, as well as the R353G mutation in helix A, has been linked to benign familial neonatal convulsions (BFNC), a human epileptic syndrome of newborn children (2). Accordingly, we demonstrate that there is a strong correlation between the impact of these mutations on the adoption of an “active” conformation by CaM and channel subunit exit from the ER, lending further support to the concept that CaM is a critical regulator for the exit of the channel from the ER.     

Study of GABA<Subscript>A</Subscript> receptors on the sleep-like behavior in <Emphasis Type="Italic">Coturnix japonica</Emphasis> (Temminck Schlegel, 1849) (Galliformes: Aves)

The present study was carried out to investigate the influence of GABA_A signaling on sleep-like behaviors through systemic administration of bicuculline and picrotoxin (GABA_A antagonists) and thiopental (an allosterical modulator). A thiopental (20 mg/kg) injection increased the eye closure frequency compared to the control group. The birds quickly became sleepy with a low frequency of early behavioral stages, such as rapid oral movement (ROM), feather ruffling and blinking. A bicuculline administration (1 and 4 mg/kg) did not modify the frequency of feather ruffling, ROM, eye closure or blinking responses. A lower dose of picrotoxin (2 mg/kg) stimulated an active awakening status, while an intermediate dose (4 mg/kg) elicited a moderate awakening status, which was associated with an increase in the frequency of ROM, blinking and eye closure. At the higher dose (8 mg/kg), the birds exhibited thermoregulatory-like behaviors and convulsions immediately after the injection. Interestingly, picrotoxin (4 mg/kg) intensified the eye closures when given in combination with thiopental (20 mg/kg). Both barbiturate and picrotoxin-induced sleep-like responses have the same behavioral neuropharmacological properties, conceivably because they are correlated with action at an identical site on the GABA_A receptor.