Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing

Neurofibromatosis type 1 (NF1) is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.     

Effects of supplementation with plant extract product containing carvacrol,cinnamaldehyde and capsaicin on serum metabolites and enzymes during the finishing phase of feedlot-fed bull calves

This study investigated the in vivo effects of a commercial blend of plant extracts (carvacrol, cinnamaldehyde and capsaicin) on serum metabolic parameters closely connected with energy and protein metabolism (glucose; l-lactate; non-esterified fatty acids, NEFA; urea nitrogen, SUN; creatinine; total protein, TSP) and enzymes associated with hepatic function (aspartate-aminotransferase, AST and gamma-glutamyl transferase, GGT) in finishing-stage Belgian Blue bull calves maintained in a commercial feedlot. Monitoring was performed over 86 days in 24 animals randomly allotted to two groups: (1) a control group (CTR, no supplementation; n = 10), and (2) a group receiving dietary supplementation with a commercial blend of plant extracts (PEX, 100 mg/kg DM of concentrate; n = 14). Under the conditions of our study, supplementation with the commercial blend did not give detrimental effects, but the opposite: the decrease in serum l-lactate, NEFA and creatinine levels and the increase in SUN concentrations; suggests an improvement in the energy status and protein turnover of the supplemented animals.     

DFT studies of the phenol adsorption on boron nitride sheets

We perform first principles total energy calculations to investigate the atomic structures of the adsorption of phenol (C₆H₅OH) on hexagonal boron nitride (BN) sheets. Calculations are done within the density functional theory as implemented in the DMOL code. Electron-ion interactions are modeled according to the local-spin-density-approximation (LSDA) method with the Perdew-Wang parametrization. Our studies take into account the hexagonal h-BN sheets and the modified by defects d-BN sheets. The d-BN sheets are composed of one hexagon, three pentagons and three heptagons. Five different atomic structures are investigated: parallel to the sheet, perpendicular to the sheet at the B site, perpendicular to the sheet at the N site, perpendicular to the central hexagon and perpendicular to the B-N bond (bridge site). To determine the structural stability we apply the criteria of minimum energy and vibration frequency. After the structural relaxation phenol molecules adsorb on both h-BN and d-BN sheets. Results of the binding energies indicate that phenol is chemisorbed. The polarity of the system increases as a consequence of the defects presence which induces transformation from an ionic to covalent bonding. The elastic properties on the BN structure present similar behavior to those reported in the literature for graphene.     

Vegetative innervation of the esophagus. II. Intraganglionic laminar endings.

The intraganglionic laminar endings in the esophagus of the cat and the rhesus monkey show absolute equivalence between the results in both species from the morphological standpoint. The different types of apparatus found are described, with their location in the esophagus and their percentage distribution in relation to the different portions of its wall. The osmium tetroxide-zinc iodide technique gives pictures equivalent to those using silver impregnations, with the added advantage that the former brings out the morphological details more clearly, to the point of showing up the peculiar characteristics of the edges with their thorn-like protrusions. The complete independence of these structures within the ganglion is confirmed, and evidence is provided for rejecting the possibility that they might be dendritic prolongations of the neuronal elements composing the intramural ganglia. A possible afferent function is proposed, which, however, must be considered an open question, pending the results of further experimental investigation.     

Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy

The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated. A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic. Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.     

Role of endocannabinoids in brain development.

In addition to those functions that have been extensively addressed in this special issue, such as nociception, motor activity, neuroendocrine regulation, immune function and others, the endogenous cannabinoid system seems to play also a role in neural development. This view is based on a three-fold evidence. A first evidence emerges from neurotoxicological studies that showed that synthetic and plant-derived cannabinoids, when administered to pregnant rats, produced a variety of changes in the maturation of several neurotransmitters and their associated-behaviors in their pups, changes that were evident at different stages of brain development. A second evidence comes from studies that demonstrated the early appearance of elements of the endogenous cannabinoid system (receptors and ligands) during the brain development. The atypical location of these elements during fetal and early postnatal periods favours the notion that this system may play a role in specific molecular events related to neural development. Finally, a third evidence derives from studies using cultures of fetal glial or neuronal cells. Cannabinoid receptors are present in some of these cultured cells and their activation produced a set of cellular effects consistent with a role of this system in the process of neural development. All this likely supports that endocannabinoids, early synthesized in nervous cells, play a role in events related to development, by acting through the activation of second messenger-coupled cannabinoid receptors.     

Metabolic adaptations for isopod specialization in three species of Dysdera spiders from the Canary Islands

The spider genus Dysdera is considered to comprise specialist isopod feeders, although the degree of specialization varies between species, depending on morphological (shape of chelicerae), behavioural (attack tactics) and metabolic (food quality of prey) adaptations. Dysdera has radiated extensively in the Canary Islands (currently 47 endemic species are described) and codistributed species have different cheliceral shapes and body sizes indicating different feeding niches. In the present study, we investigate the existence of metabolic adaptations to feeding on isopods by three endemic species (Dysdera insulana Simon, Dysdera macra Simon and Dysdera verneaui Simon) from Tenerife. We hypothesize that there is enhanced extraction efficiency of fundamental macronutrients from isopods compared with control prey in species with special morphological and behavioural adaptations for this prey type. We measure quantitatively spider growth, dry mass consumption, lipid and nitrogen consumption, and calculate growth efficiency and efficiency of utilization of dry mass, lipid and nitrogen. The results show that all three species are able to utilize both prey types, indicating that none of them are strict isopod specialist. Dysdera insulana shows enhanced growth efficiency and D. macra shows enhanced nitrogen extraction efficiency compared with D. verneaui when feeding on Porcellio rather than on Musca. Both traits indicate likely adaptations for the utilization of isopods. Spider species, sex and prey type all affect lipid and nitrogen extraction efficiencies, indicating that spiders do not simply extract nutrients in the proportions available. The results support the hypothesis that adaptations for enhanced digestion of focal prey evolve in species that already have adaptations for enhanced capture success.