Function and conformation analyses of an aspartate substitution of the invariant glycine in the integrin βI domain α1-α1′ helix

We showed that the αLβ2 integrin with the non-functional mutation G150D cannot be induced with Mg/EGTA to express the mAb KIM127 epitope, which reports the leg-extended conformation. We extended the study to the αIIbβ3, an integrin without an αI domain. The equivalent mutation, i.e. G161D, also resulted in an expressible, but non-adhesive αIIbβ3 integrin. An NMR study of synthetic peptides spanning the α1-α1′ helix of the β3 I domain shows that both wild-type and mutant peptides are α-helical. However, whereas in the wild-type peptide this helix is continuous, the mutant presents a discontinuity, or kink, precisely at the site of mutation G161D. Our results suggest that the mutation may lock integrin heterodimers in a bent conformation that prevents integrin activation via conformational extension.     

Taphonomic signature of golden eagles (Aquila chrysaetos) on bone prey remains

Abstract

The golden eagle (Aquila chrysaetos) is one of the most important birds of prey in the Northern Hemisphere. This raptor is used to building large nests in high cliffs to which they return for several breeding years accumulating important amounts of their prey skeletal remains. This makes the golden eagle one of the major predators able to accumulate faunal remains in archaeological sites. Despite this fact, the taphonomic signature of golden eagles has not been properly characterized. Here we present the analysis of ingested and non-ingested faunal remains predated and accumulated by this raptor in two different nesting areas from the Iberian Peninsula. Results show how the faunal taxonomic record may vary depending on the ecological zone. Leporids and terrestrial carnivores are the best represented. The observed anatomical representation, breakage and bone surface modification patterns are discussed for different taxa. The taphonomic pattern varies depending on the type of prey and the origin of skeletal materials (non-ingested vs. pellets). Finally, after comparing our results with marks left by other predators, several characteristic features are noted to recognise golden eagles as agents of animal bones accumulations in the fossil record.     

Distribution, ecology, mode of reproduction and karyology of freshwater planarians (Platyhelminthes; Turbellaria; Tricladida) in the springs of the central Pyrenees

The distribution, reproductive modality and karyology of populations of freshwater planarians were studied together with physico-chemical variables m 149 springs in the central Pyrenees Diversity was limited to four species (Dugesia (Dugesia) subtentaculata, Polycelis felina. Crenobia alpina and Phagocata vitta), already known from streams and lakes Planarians were found rarely (only a third of springs contain them), m low-moderate abundance, and any single spring did not harbour more than one species The distribution of potential predators and preys among the springs sampled was fairly uniform So. we assume that whereas predation is unlikely to influence triclad distribution, the oligotrophic character of these habitats may explain, through food shortage and ensuing interspecific competition, the presence of a single species in each spring Besides interspecific competition, the distribution of each species can also be influenced by particular abiotic parameters Temperature and current velocity appeared to be the dominant factors, the first separating C alpina from the rest of species, and the second favouring the presence of P felina in a large set of springs These factors also seem to determine planarian distribution in streams and rivers Other environmental factors such as calcium bicarbonated water (for D subtentaculata) and preference for karst systems (for Phagocata vitta) are of importance Some peculiarities of distribution, like the scarcity of C alpina in reophile habitats and the thermal tolerance of Phagocata vitta, may be due both to competition from P felina and to historical factors in the former and to morphological (presence of pigment) and ecological (epigean habits) features m the latter, respectively Karyological analyses demonstrated triploidy in all populations of D subtentaculata. a polyploid origin for C alpina and the finding in Phagocata vitta of a new polyploid series leading to the highest chromosome number in turbellarians (14n = 238) Furthermore, the basic chromosome number of C alpina (n = 21) and Phagocata vitta (n = 17) differs from those found in northern European populations (n = 7 in both, respectively) This suggests that under the specific names C alpina and Phagocata vitta more than one species actually occurs, this being a problem for further studies Whereas polyploidy has often been related to living in cold, harsh habitats, asexual reproduction has been linked to environments where biotic stress is low The preeminence of asexual reproduction (by fissiparity) and polyploid forms in freshwater planarians inhabiting the unproductive, biotically unsaturated springs of the central Pyrenees adds new evidence to strengthen such links     

The Brazilian Amaryllidaceae as a source of acetylcholinesterase inhibitory alkaloids

Nine Brazilian Amaryllidaceae species were studied for their alkaloid composition and acetylcholinesterase (AChE) inhibitory activity via GC–MS and a modified Ellman assay, respectively. A total of thirty-six alkaloids were identified in these plants, of which Hippeastrum papilio and H. glaucescens exhibited the highest galanthamine content and the best IC₅₀ values against AChE. Furthermore, Hippeastrum vittatum and Rhodophiala bifida also showed notable AChE inhibitory effects. X-ray crystallographic data for four galanthamine-type compounds revealed significant differences in the orientation of the N-methyl group, which are shown to be related to AChE inhibition.     

Global DNA Methylation of Ischemic Stroke Subtypes

Ischemic stroke (IS), a heterogeneous multifactorial disorder, is among the leading causes of mortality and long-term disability in the western world. Epidemiological data provides evidence for a genetic component to the disease, but its epigenetic involvement is still largely unknown. Epigenetic mechanisms, such as DNA methylation, change over time and may be associated with aging processes and with modulation of the risk of various pathologies, such as cardiovascular disease and stroke. We analyzed 2 independent cohorts of IS patients. Global DNA methylation was measured by luminometric methylation assay (LUMA) of DNA blood samples. Univariate and multivariate regression analyses were used to assess the methylation differences between the 3 most common IS subtypes, large-artery atherosclerosis (LAA), small-artery disease (SAD), and cardio-aortic embolism (CE). A total of 485 IS patients from 2 independent hospital cohorts (n = 281 and n = 204) were included, distributed across 3 IS subtypes: LAA (78/281, 59/204), SAD (97/281, 53/204), and CE (106/281, 89/204). In univariate analyses, no statistical differences in LUMA levels were observed between the 3 etiologies in either cohort. Multivariate analysis, adjusted by age, sex, hyperlipidemia, and smoking habit, confirmed the lack of differences in methylation levels between the analyzed IS subtypes in both cohorts. Despite differences in pathogenesis, our results showed no global methylation differences between LAA, SAD, and CE subtypes of IS. Further work is required to establish whether the epigenetic mechanism of methylation might play a role in this complex disease.     

Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum

Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen’s endogenous concentration, by combining the kinetic exclusion assay and Biacore’s calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.     

The nervous system in planarians: Peripheral and gastrodermal plexuses,pharynx innervation,and the relationship between central nervous system structure and the acoelomate organization

The Champy-Maillet osmium tetroxide-zinc iodide technique and a new method using azur B-sodium thioglycolate were used to study the general nervous tissue structure in planarians. A subepidermal and a submuscular nerve plexus, partially reported by earlier authors, are described, and a gastrodermal plexus is reported for the first time in triclads. The possible functions for each one of these plexuses are discussed. By the Champy-Maillet method, the innervation within the parenchyma appears as an array of numerous single nerve fibers that course between the parenchyma cells making apparent synaptic contacts. The pharynx has outer and inner nerve nets similar in structure to the submuscular nerve plexus. Both nerve nets are connected to each other by radial nerves. The central nervous system has a sponge-like structure with many lacunae filled with cell bodies, dorso-ventral muscle fibers, parenchymal cell processes and excretory ducts. The existence of this sponge-like nervous tissue structure is discussed in relation to the still incomplete centralization of the nervous tissue in these organisms, to the lack of a true vascular system and to the acoelomate level of organization. A comparison with the nervous tissue structure of more advanced groups like polyclads and nemertines is suggested.