Bioactive phenolic compounds from aerial parts of Plinia glomerata

The present work describes the antinociceptive properties and chemical composition of the aerial parts of Plinia glomerata (Myrtaceae). Both of the extracts evaluated, acetonic and methanolic, showed potent antinociceptive action, when analyzed against acetic acid-induced abdominal constrictions in mice, with calculated ID50 (mg/kg, i. p.) values of 24.8 and 3.3, respectively. Through usual chromatographic techniques with an acetonic extract, the following compounds were obtained: 3,4,3'-trimethoxy flavellagic acid (1), 3,4,3'-trimethoxy flavellagic acid 4'-O-glucoside (3) and quercitrin (4), which were identified based on spectroscopic data. Compounds 1 (ID50 = 3.9 mg/kg, i. p., or 10.8 micromol/kg) and 3 (ID50 = 1.3 mg/kg or 2.5 micromol/kg) were notably more active than some well-known analgesic drugs used here for comparison.     

Presenilin-dependent receptor processing is required for axon guidance

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.     

The Binding of κ-Conotoxin PVIIA and Fast C-Type Inactivation of Shaker K+ Channels are Mutually Exclusive

κ-Conotoxin PVIIA (κ-PVIIA), a 27-amino acid peptide identified from the venom of Conus purpurascens, inhibits the Shaker K⁺ channel by blocking its outer pore. The toxin appears as a gating modifier because its binding affinity decreases with relatively fast kinetics upon channel opening, but there is no indication that it interferes with the gating transitions of the wild-type channels (WT), including the structural changes of the outer pore that underlie its slow C-type inactivation. In this report we demonstrate that in two outer pore mutants of Shaker-IR (M448K and T449S), that have high toxin sensitivity and fast C-type inactivation, the latter process is instead antagonized by and incompatible with κ-PVIIA binding. Inactivation is slowed by the necessary preliminary unbinding of κ-PVIIA, whereas toxin rebinding must await recovery from inactivation causing a double-exponential relaxation of the second response to double-pulse stimulations. Compared with the lack of similar effects in WT, these results demonstrate the ability of peptide toxins like κ-PVIIA to reveal possibly subtle differences in structural changes of the outer pore of K⁺ channels; however, they also warn against a naive use of fast inactivating mutants as models for C-type inactivation. Unfolded from the antagonistic effect of inactivation, toxin binding to mutant noninactivated channels shows state- and voltage-dependencies similar to WT: slow and high affinity for closed channels; relatively fast dissociation from open channels at rate increasing with voltage. This supports the idea that these properties depend mainly on interactions with pore-permeation processes that are not affected by the mutations. In mutant channels the state-dependence also greatly enhances the protection of toxin binding against steady-state inactivation at low depolarizations while still allowing large responses to depolarizing pulses that relieve toxin block. Although not obviously applicable to any known combination of natural channel and outer-pore blocker, our biophysical characterization of such highly efficient mechanism of protection from steady-state outer-pore inactivation may be of general interest.     

Karyotypic changes associated to the dispersive potential on Pomacentridae (Pisces, Perciformes)

When comparing karyotypic diversification between freshwater and marine fish, a notable increase in modifications associated to the former environment can be noticed in relation to the latter one. This fact is related to the great number of physical partitions (barriers) that exist in continental waters. Furthermore, in the marine environment, the barriers to the gene flow are complex and not always identifiable, especially when considering the participation of dynamic factors such as currents and winds associated to the dispersive potentialities of a living group. Within Perciformes fish, the Pomacentridae family stands out from the others for its intimate relation with the coral reefs, as well as for its diversity of forms and biological characteristics, providing an adequate model for studying the influence of the pelagic period of the larval stage upon the karyotypic evolution. In the present work, cytogenetic analyses were carried out on three Pomacentridae species, typical representatives of the coral reefs, Abudefduf saxatilis (2n=48, FN=52, 2 m+2sm+44a), Microspathodon chrysurus (2n=48, FN=64, 6 m+10st+32a) and Amphiprion frenatus (2n=48, FN=92, 14 m+22sm+8st+4a) that have different pelagic larval periods. The analyses between subfamilies showed a trend that the lower the dispersive potential, the greater the quantity of fixed pericentric inversions based on the basal karyotype of the Perciformes.     

Tyrosine 3 of poliovirus terminal peptide VPg(3B) has an essential function in RNA replication in the context of its precursor protein, 3AB

Poliovirus (PV) VPg is a genome-linked protein that is essential for the initiation of viral RNA replication. It has been well established that RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine (Y3) in VPg by the viral RNA polymerase 3D(pol), but it is not yet known whether the substrate for uridylylation in vivo is the free peptide itself or one of its precursors. The aim of this study was to use complementation analyses to obtain information about the true in vivo substrate for uridylylation by 3D(pol). Previously, it was shown that a VPg mutant, in which tyrosine 3 and threonine 4 were replaced by phenylalanine and alanine (3F4A), respectively, was nonviable. We have now tested whether wild-type forms of proteins 3B, 3BC, 3BCD, 3AB, 3ABC, and P3 provided either in trans or in cis could rescue the replication defect of the VPg(3F4A) mutations in the PV polyprotein. Our results showed that proteins 3B, 3BC, 3BCD, and P3 were unable to complement the RNA replication defect in dicistronic PV or dicistronic luciferase replicons in vivo. However, cotranslation of the P3 precursor protein allowed rescue of RNA replication of the VPg(3F4A) mutant in an in vitro cell-free translation-RNA replication system, but only poor complementation was observed when 3BC, 3AB, 3BCD, or 3ABC proteins were cotranslated in the same assay. Interestingly, only protein 3AB but not 3B and 3BC, when provided in cis by insertion of a wild-type 3AB coding sequence between the P2 and P3 domains of the polyprotein, supported the replication of the mutated genome in vivo. Elimination of cleavage between 3A and 3B in the complementing 3AB protein, however, led to a complete lack of RNA replication. Our results suggest that (i) VPg has to be delivered to the replication complex in the form of a large protein precursor (P3) to be fully functional in replication; (ii) the replication complex formed during PV replication in vivo is essentially inaccessible to proteins provided in trans, even if the complementing protein is translated from a different cistron of the same RNA genome; (iii) 3AB is the most likely precursor of VPg; and (iv) Y3 of VPg has an essential function in RNA replication in the context of both VPg and 3AB.     

Characterization of the lipopolysaccharide O-antigen of Cronobacter turicensis HPB3287 as a polysaccharide containing a 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic acid (legionaminic acid) residue

Cronobacter turicensis, previously known as Enterobacter sakazakii, is a Gram-negative opportunistic food-borne pathogen that has been reported as a cause of life-threatening neonatal infections. From chemical and physical analyses involving composition analysis, methylation, two-dimensional high-resolution nuclear magnetic resonance, and mass spectrometry methods, the antigenic O-polysaccharide in the smooth-type lipopolysaccharide of C. turicensis (strain HPB 3287) was determined to be a high molecular mass polymer of a repeating pentasaccharide unit composed of d-galactose, d-glucose, 2-acetamido-2-deoxy-d-galactose, and 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic acid (legionaminic acid), in a molar ratio 2:1:1:1, and having the structure:     

Reaction of [Pt{Fe(CO)3(NO)}2(PhCN)2] with diphenyl(2-pyridyl)phosphine selenide. Crystal structure of [(CO)3Fe(μ3-Se){Pt(CO)P(2-C5H4N)Ph2}2] and its theoretical study

The reaction between the linear trinuclear complex [Pt{Fe(CO)₃(NO)}₂(PhCN)₂] and Ph₂(2-C₅H₄N)PSe led to the isolation and characterization of the 46-electron cluster [(CO)₃Fe(μ₃-Se){Pt(CO)P(2-C₅H₄N)Ph₂}₂] (1), whose structure has been determined by X-ray diffraction methods. The cluster typology, which consists of an open triangle Pt---Fe---Pt capped by a μ₃-Se atom, is rather rare. The chemical bonding in 1 and in similar systems has been analyzed through density functional theory (DFT) and qualitative MO approaches. A strict analogy with the well understood L₂M(μ-acetylene)ML₂ systems is invoked by considering 1 as formed by the (CO)₃FeSe tetrahedral unit stabilized by sidewise interactions of the triple bond with two d¹⁰-L₂M fragments. Otherwise, the 18-electron (CO)₃FeSe monomer is unstable as an isolate molecule. This is confirmed by our DFT calculations that indicate how the well characterized dimer (CO)₃Fe(μ-Se₂)Fe(CO)₃ lies as much as, approximately, 58 kcal mol⁻¹ deeper in energy. Finally, by considering an analogy with [L₂M(μ-dichalcogen)ML₂]^{0, +2} redox systems (M=Pd, Pt), reduction of 1 to a dianion has been hypothesized and the structure of the latter has been tentatively explored by DFT calculations.     

Genetic exchange of the S2 and S3 subunits in pertussis toxin

Bordetella pertussis, the causative agent of whooping cough, produces a complex hetero-oligomeric exotoxin, named pertussis toxin (PTX), which is responsible for several of the clinical manifestations associated with whooping cough. The toxin is composed of five dissimilar subunits, named S1 through S5 and arranged in a hexameric structure with a 1S1:1S2:1S3:2S4:1S5 stoichiometry. Although S2 and S3 share 70% amino acid identity, these two subunits were previously thought not to be able to substitute for each other in toxin assembly/secretion and the biological activities of PTX. Here, we show that toxin analogues containing two S3 subunits and lacking S2 (PTXdeltaS2), or containing two S2 subunits and lacking S3 (PTXdeltaS3), can be produced, assembled and secreted by B. pertussis strains, in which the S2-encoding cistron or the S3-coding cistrons have been inactivated by internal in-frame deletions that avoid downstream effects. In fact, PTXdeltaS3 was produced in higher amounts in the bacterial culture supernatants than natural PTX, whereas PTXdeltaS2 was produced in lower amounts than PTX. The action of the toxin analogues on the clustering of Chinese Hamster Ovary cells was also affected differentially by the S2-S3 substitution. These toxin analogues constitute thus interesting probes for the study of cellular functions, in particular immune cell functions, for which natural PTX has already shown its usefulness.     

A micrite-dominated norian carbonate platform from Northern Calabria (Southern Italy)

Summary  This paper is focused on the facies associations of an Early-Middle Norian stratigraphic succession cropping outin the Northern Calabria (Buonvicino, Cosenza). These carbonate deposits, pertaining to the Verbicaro Unit, represent a dolomitized platform characterized by a clear dominance of automicrites over skeletal metazoans. On the basis of the chronostatigraphic data and sedimentary evolution, two stratigraphic units have been distinguished: the Lower Unit (object of the present paper) and the Upper Unit. The Lower Unit consists of an Early-Middle Norian high-relief prograding carbonate platform. The Upper Unit is represented by Middle p.p.-Upper Norian basinal deposits. Three main facies associations, indicative of different depositional settings, have been identified. The inner platform facies association is characterized by automicrite (cauliflower-columnar subordinate planar stromatolites) associated with detrital carbonates (intraclastic breccia, bioclastic grainstone and packstone). Apeculiar facies is represented by megalodontid-bearing beds. Mud-cracked horizons and low-developed teepees occur quite frequently. The margin facies association is dominated by automicrite (planar to low relief stromatolites associated with thrombolitic fenestral boundstone) and detrital carbonates with subaerial exposure features. The slope facies association includes detrital carbonates (breccia/megabreccia) associated with serpulid/sphinctozoan bioconstructions and automicrite (planar stromatolites/thrombolitic boundstone). The whole carbonate body is completely dolomitized; nevertheless the morphology and microarchitecture of carbonate components (cements, grains and automicrite) is still clearly recognizable. The dolomite Mg content ranges from 40 to 48 mole%, sometimes reaching the stoichiometric value. Cements, primary and late, represent a minor component of the rock volume; they occur more frequently on the margin and upper slope setting. Quantitative tacies analyses lead to the following conclusions:

Exercise restores beta-adrenergic vasorelaxation in aged rat carotid arteries

Aging is associated with alterations in beta-adrenergic receptor (beta-AR) signaling and reduction in cardiovascular responses to beta-AR stimulation. Because exercise can attenuate age-related impairment in myocardial beta-AR signaling and function, we tested whether training could also exert favorable effects on vascular beta-AR responses. We evaluated common carotid artery responsiveness in isolated vessel ring preparations from 8 aged male Wistar-Kyoto (WKY) rats trained for 6 wk in a 5 days/wk swimming protocol, 10 untrained age-matched rats, and 10 young WKY rats. Vessels were preconstricted with phenylephrine (10-6 M), and vasodilation was assessed in response to the beta-AR agonist isoproterenol (10-10-3 x 10-8 M), the alpha2-AR agonist UK-14304 (10-9-10-6 M), the muscarinic receptor agonist ACh (10-9-10-6 M), and nitroprusside (10-8-10-5 M). beta-AR density and cytoplasmic beta-AR kinase (beta-ARK) activity were tested on pooled carotid arteries. beta-ARK expression was assessed in two endothelial cell lines from bovine aorta and aorta isolated from a 12-wk WKY rat. beta-AR, alpha2-AR, and muscarinic responses, but not that to nitroprusside, were depressed in untrained aged vs. young animals. Exercise training restored beta-AR and muscarinic responses but did not affect vasodilation induced by UK-14304 and nitroprusside. Aged carotid arteries showed reduced beta-AR number and increased beta-ARK activity. Training counterbalanced these phenomena and restored beta-AR density and beta-ARK activity to levels observed in young rat carotids. Our data indicate that age impairs beta-AR vasorelaxation in rat carotid arteries through beta-AR downregulation and desensitization. Exercise restores this response and reverts age-related modification in beta-ARs and beta-ARK. Our data support an important role for beta-ARK in vascular beta-AR vasorelaxation.