Karyotype characterization reveals an up and down of 45S and 5S rDNA sites in Crotalaria (Leguminosae-Papilionoideae) species of the section Hedriocarpae subsection Macrostachyae

The genus Crotalaria is one of the largest within the family Leguminosae-Papilionoideae, with more than 600 species. However, few karyotypes have been described. In the present paper, five species belonging to the section Hedriocarpae were studied (subsection Machrostachyae), in order to better understand chromosomal evolution in Crotalaria. The results reveals that all species presented 2n = 2x = 16 with symmetrical karyotypes, and slight differences in the chromosome morphology. A secondary constriction was identified at short arm of the pair 1. The 45S rDNA was mapped in the secondary constriction and adjacent heterochromatin (NOR-heterochromatin) and a minor site was identified in C. ochroleuca. The 5S rDNA was mapped linked to 45S rDNA at chromosome 1 short arm in all species. Additional sites for 5S rDNA were identified in C. pallida, C. striata and C. mucronata. Heterochromatin blocks around the centromeres are not CMA(+) neither DAPI(+). The karyotypes of the subsection Macrostachyae are characterized by an inversion at chromosome pair one in relation to previous specialized floral species analyzed. Additional sites of 45S and 5S rDNA were assumed to be a result of transposition events by different ways. The results suggest heterochromatin differentiation and the position of ribosomal genes indicates chromosomal rearrangements during evolution. Karyotype characteristics corroborate the morphological infrageneric classification.

Characterization of selectivity and pharmacophores of type 1 sea anemone toxins by screening seven Na-v sodium channel isoforms

During their evolution, animals have developed a set of cysteine-rich peptides capable of binding various extracellular sites of voltage-gated sodium channels (VGSC). Sea anemone toxins that target VGSCs delay their inactivation process, but little is known about their selectivities. Here we report the investigation of three native type 1 toxins (CGTX-II, delta-AITX-Bcg1a and delta-AITX-Bcg1b) purified from the venom of Bunodosoma cangicum. Both delta-AITX-Bcg1a and delta-AITX-Bcg1b toxins were fully sequenced. The three peptides were evaluated by patch-clamp technique among Nav1.1-1.7 isoforms expressed in mammalian cell lines, and their preferential targets are Na(v)1.5 > 1.6 > 1.1. We also evaluated the role of some supposedly critical residues in the toxins which would interact with the channels, and observed that some substitutions are not critical as expected. In addition, CGTX-II and delta-AITX-Bcg1a evoke different shifts in activation/inactivation Boltzmann curves in Nav1.1 and 1.6. Moreover, our results suggest that the interaction region between toxins and VGSCs is not restricted to the supposed site 3 (S3-54 linker of domain IV), and this may be a consequence of distinct surface of contact of each peptide vs. targeted channel. Our data suggest that the contact surfaces of each peptide may be related to their surface charges, as CGTX-II is more positive than delta-AITX-Bcg1a and delta-AITX-Bcg1b. (C) 2011 Elsevier Inc. All rights reserved.

Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom

The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K-m of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 mu g of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC50: 22.7 mu g/mL) and on MCF-7 cell line (breast adenocarcinoma, IC50:1.41 mu g/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC50: 2.22 mu g/nnL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions. (C) 2012 Elsevier Ltd. All rights reserved.

Molecular characterization of a mariner-like element in the Atta sexdens rubropilosa genome

Mobile elements are widely present in eukaryotic genomes. They are repeated DNA segments that are able to move from one locus to another within the genome. They are divided into two main categories, depending on their mechanism of transposition, involving RNA (class I) or DNA (class II) molecules. The mariner-like elements are class II transposons. They encode their own transposase, which is necessary and sufficient for transposition in the absence of host factors. They are flanked by a short inverted terminal repeat and a TA dinucleotide target site, which is duplicated upon insertion. The transposase consists of two domains, an N-terminal inverted terminal repeat binding domain and a C-terminal catalytic domain. We identified a transposable element with molecular characteristics of a mariner-like element in Atta sexdens rubropilosa genome. Identification started from a PCR with degenerate primers and queen genomic DNA templates, with which it was possible to amplify a fragment with mariner transposable-element homology. Phylogenetic analysis demonstrated that this element belongs to the mauritiana subfamily of mariner-like elements and it was named Asmar1. We found that Asmar1 is homologous to a transposon described from another ant, Messor bouvieri. The predicted transposase sequence demonstrated that Asmar1 has a truncated transposase ORF. This study is part of a molecular characterization of mobile elements in the Atta spp genome. Our finding of mariner-like elements in all castes of this ant could be useful to help understand the dynamics of mariner-like element distribution in the Hymenoptera.

In Vitro Characterization of Chitosan Gels for Buccal Delivery of Celecoxib: Influence of a Penetration Enhancer

Celecoxib (Cx) shows high efficacy in the treatment of osteoarthritis and rheumatoid arthritis as a result of its high specificity for COX-2, without gastrolesivity or interference with platelet function at therapeutic concentrations. Besides of anti-inflammatory effects, Cx also has a potential role for oral cancer chemoprevention. For these conditions, oral administration in long-term treatment is a concern due to its systemic side effects. However, local application at the site of injury (e.g., buccal inflammation conditions or chemoprevention of oral cancer) is a promising way to reduce its toxicity. In this study, the in vitro characterization of mucoadhesive chitosan (CHT) gels associated to AzoneA (R) was assessed to explore the potential buccal mucosal administration of Cx in this tissue. Rheological properties of gels were analyzed by a rheometer with cone-plate geometry. In vitro Cx release and permeability studies used artificial membranes and pig cheek mucosa, respectively. Mucoadhesion were measured with a universal test machine. CHT gels (3.0%) containing 2.0% or 3.0% Az showed more appropriate characteristics compared to the others: pH values, rheology, higher amount of Cx retained in the mucosa, and minimal permeation through mucosa, besides the highest mucoadhesion values, ideal for buccal application. Moreover, the flux (J) and amounts of drug released decreased with increased CHT and Az concentrations. CHT gels (3.0%) associated with 2.0% or 3.0% Az may be considered potential delivery systems for buccal administration of Cx.

STRUCTURAL CHARACTERIZATION OF CANOPIES OF Eucalyptus spp. USING RADIOMETRIC DATA FROM TM/Landsat 5

Empirical approaches and, more recently, physical approaches, have grounded the establishment of logical connections between radiometric variables derived from remote data and biophysical variables derived from vegetation cover. This study was aimed at evaluating correlations of dendrometric and density data from canopies of Eucalyptus spp., as collected in Capao Bonito forest unit, with radiometric data from imagery acquired by the TM/Landsat-5 sensor on two orbital passages over the study site (dates close to field data collection). Results indicate that stronger correlations were identified between crown dimensions and canopy height with near-infrared spectral band data (rho(s)4), irrespective of the satellite passage date. Estimates of spatial distribution of dendrometric data and canopy density (D) using spectral characterization were consistent with the spatial distribution of tree ages during the study period. Statistical tests were applied to evaluate performance disparities of empirical models depending on which date data were acquired. Results indicated a significant difference between models based on distinct data acquisition dates.

Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Early Amyloid-like Oligomers and Their Implication in alpha-Synuclein Aggregation

Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of alpha-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally, it is demonstrated that in vitro the early oligomers present in the glyceraldehyde-3-phosphate dehydrogenase fibrillation pathway promote alpha-synuclein aggregation. Taking into account the toxicity of alpha-synuclein prefibrillar species, the heparin-induced glyceraldehyde-3-phosphate dehydrogenase early oligomers might come in useful as a novel therapeutic strategy in Parkinson disease and other synucleinopathies.

Synthesis and characterization of [Ru(eta(6)-C10H14)(dppf)X][PF6] (X = Cl, Br, I, SnF3) compounds: The X-ray structure of [Ru(eta(6)-C10H14)(dppf)Cl][SnCl3]center dot 0.45CH(2)Cl(2)

The arene-ruthenium complex [Ru(eta(6)-C10H14)(dppf)Cl]PF6 (1) was used as a precursor for the syntheses of the [Ru(eta(6)-C10H14)(dppf)Br]PF6 (2), [Ru(eta(6)-C10H14)(dppf)I]PF6 (3). [Ru(eta(6)-C10H14)(dppf)SnF3]PF6 (4) and [Ru(eta(6)-C10H14)(dppf)Cl][SnCl3]center dot 0.45CH(2)Cl(2) (5) complexes by its reactions with KBr, Kl, SnF2 and SnCl2. respectively. All of the compounds were characterized by NMR, IR, Fe-57 and Sn-119-Mossbauer spectroscopy, and cyclic voltammetry. The single-crystal X-ray structure analysis of the [Ru(eta(6)-C10H14)(dppf)Cl] [SnCl3]center dot 0.45CH(2)Cl(2) complex revealed the expected piano-stool geometry. Cyclic voltammograms of the complexes showed only one quasi-reversible electrochemical process, involving the oxidation of Fe(II) and Ru(II) at the same potential, which was confirmed by exhaustive electrolysis experiments. Fe-57-Mossbauer parameters obtained for the complexes (1-5) were fitted with one doublet corresponding to a site of one iron(II). The Sn-119-Mossbauer parameters of the complex (4) indicate that tin is tetra covalent. (c) 2012 Elsevier Ltd. All rights reserved.

Protomers: formation, separation and characterization via travelling wave ion mobility mass spectrometry

Travelling wave ion mobility mass spectrometry (TWIM-MS) with post-TWIM and pre-TWIM collision-induced dissociation (CID) experiments were used to form, separate and characterize protomers sampled directly from solutions or generated in the gas phase via CID. When in solution equilibria, these species were transferred to the gas phase via electrospray ionization, and then separated by TWIM-MS. CID performed after TWIM separation (post-TWIM) allowed the characterization of both protomers via structurally diagnostic fragments. Protonated aniline (1) sampled from solution was found to be constituted of a ca. 5:1 mixture of two gaseous protomers, that is, the N-protonated (1a) and ring protonated (1b) molecules, respectively. When dissociated, 1a nearly exclusively loses NH3, whereas 1b displays a much diverse set of fragments. When formed via CID, varying populations of 1a and 1b were detected. Two co-existing protomers of two isomeric porphyrins were also separated and characterized via post-TWIM CID. A deprotonated porphyrin sampled from a basic methanolic solution was found to be constituted predominantly of the protomer arising from deprotonation at the carboxyl group, which dissociates promptly by CO2 loss, but a CID-resistant protomer arising from deprotonation at a porphyrinic ring NH was also detected and characterized. The doubly deprotonated porphyrin was found to be constituted predominantly of a single protomer arising from deprotonation of two carboxyl groups. Copyright (C) 2012 John Wiley & Sons, Ltd.

Retinal projections and neurochemical characterization of the pregeniculate nucleus of the common marmoset (Callithrix jacchus)

In mammals, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) are the main components of the circadian timing system. The SCN is the site of the endogenous biological clock that generates rhythms and synchronizes them to environmental cues. The IGL is a key structure that modulates SCN activity and is responsible for the transmission of non-photic information to the SCN, thus participating in the integration between photic and non-photic stimuli. Both the SCN and IGL receive projections of retinal ganglion cells and the IGL is connected to the SCN through the geniculohypothalamic tract. Little is known about these structures in the primate brain and the pregeniculate nucleus (PGN) has been suggested to be the primate equivalent of the rodent IGL. The aim of this study was to characterize the PGN of a primate, the common marmoset (Callithrix jacchus), and to analyze its retinal afferents. Here, the marmoset PGN was found to be organized into three subsectors based on neuronal size, pattern of retinal projections, and the distribution of neuropeptide Y-, GAD-, serotonin-, enkephalin- and substance P-labeled terminals. This pattern indicates that the marmoset PGN is equivalent to the IGL. This detailed description contributes to the understanding of the circadian timing system in this primate species considering the importance of the IGL within the context of circadian regulation. (C) 2012 Elsevier B.V. All rights reserved.

Structural and functional characterization of Group B Streptococcus class C sortases

In Group B Streptococcus (GBS) three structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies and in vivo mutagenesis we performed a broad characterization of GBS sortase C. The high resolution X-ray structure of the enzymes revealed that the active site, located into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the “lid”. We show that the catalytic triad and the predicted N- and C-terminal trans-membrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild type protein.

The role of the NG2 proteoglycan in migration and characterization of the interaction with the intracellular binding partner, Syntenin

NG2 is a transmembrane proteoglycan with two N-terminal LNS domains and a C-terminal PDZ-binding motif. It is expressed in the developing and adult CNS by oligodendroglial precursor cells and subpopulations of perisynaptic glia and elsewhere by many immature cell types. In order to elucidate the functions of the protein and the heterogenous cell population which expresses it, we undertook to identify and characterise interaction partners of the molecule. The presence of the C-terminal PDZ recognition site in NG2 suggested PDZ-domain proteins as intracellular binding partners. In this work, interaction between the PDZ protein Syntenin and NG2 has been characterised. Syntenin is known to be involved in plasma membrane dynamics, metastasis and adhesion. Syntenin may thus link NG2 to the cytoskeleton, mediating migration of developing oligodendrocytes to axonal tracts prior to myelination, as well as process movement of NG2+ perisynaptic glia. NG2 is involved in cell spreading and polyclonal antibodies against NG2 inhibit the migration of immature glia and cell lines expressing the molecule. In this work we have characterised the segments of the extracellular portion of NG2 that are involved in migration. We found that the extracellular region immediately preceding the transmembrane segment is most important for cell motility. As part of this thesis, biochemical approaches to identify a trans-binding ligand interacting with the extracellular part of NG2 was also explored.

Synthesis and characterization of polyelectrolyte brushes

On the pathway to synthesizing synthetic model systems for human cartilage, macroinitiators for the ATRP of styrene sulfonate esters with different chain lengths and initiation site densities from 10 % to 100 % were synthesized. Polymer brushes from styrene sulfonate ethyl ester and styrene sulfonate dodecyl ester with varying grafting density, backbone length and side chain length were synthesized and characterized by 1H-NMR, AUC, AFM, TEM, and in the case of the ethyl esters, GPC-MALLS. Polyelectrolyte brushes from styrene sulfonate were synthesized from the corresponding esters. These brushes were characterized in solution (GPC-MALLS, static and dynamic light scattering, SANS, 1H-NMR) and on solid interfaces (AFM and TEM). It was shown that these brushes may form extended aggregates in solution. The aggregation behavior and the size and shape of the aggregates depend on the side chain length and the degree of saponification. For samples with identical backbone and side chain length, but varying degrees of ester hydrolysis, marked differences in the aggregation behavior were observed. A functionalized ATRP macroinitiator with a positively charged head group was synthesized and employed for the synthesis of a functionalized polyelectrolyte brush. These brushes were found to form complexes with negatively charged latex particles and are thus suitable as proteoglycan models in the proteoglycan-hyaluronic acid complex.

Synthesis and characterization of structurally well-defined polymer-layered silicate nanocomposites

Zusammenfassung Nanokomposite aus Polymeren und Schichtsilikaten werden zumeist auf der Basis natürlicher Tone wie Montmorillonit hergestellt. Für NMR- und EPR-Untersuchungen der Tensidschicht, die das Silikat mit dem Polymer kompatibilisiert, ist der Eisengehalt natürlicher Tone jedoch abträglich, weil er zu einer Verkürzung der Relaxationszeiten und zu einer Linienverbreiterung in den Spektren führt. Dieses Problem konnte überwunden werden, indem als Silikatkomponente eisenfreies, strukturell wohldefiniertes Magadiit hydrothermal synthetisiert und für die Kompositbildung eingesetzt wurde. Die Morphologie des Magadiits wurde durch Rasterelektronenmikroskopie charakterisiert und der Interkalationsgrad von schmelzinterkalierten Polymer-Nanokompositen wurde durch Weitwinkelröntgenstreuung bestimmt. Polymere mit Carbonylgruppen scheinen leichter zu interkalieren als solche ohne Carbonylgruppen. Polycaprolacton interkalierte sowohl in Oragnomagadiite auf der Basis von Ammoniumtensiden als auch in solche auf der Basis von Phosphoniumtensiden. Die Dynamik auf einer Nanosekundenzeitskala und die Struktur der Tensidschicht wurden mittels ortsspezifisch spinmarkierter Tensidsonden unter Nutzung von Dauerstrich- (CW) und Puls-Methoden der elektronenparamagnetischen Resonanzspektroskopie (EPR) untersucht. Zusätzlich wurde die statische 2H-Kernmagnetresonanz (NMR) an spezifisch deuterierten Tensiden angewendet, um die Tensiddynamik auf einer komplementären Zeitskala zwischen Mikrosekunden und Millisekunden zu erfassen. Sowohl die CW-EPR- als auch die 2H-NMR-Ergebnisse zeigen eine Beschleunigung der Tensiddynamik durch Interkalation von Polycaprolacton auf, während sich in den nichtinterkalierten Mikrokompositen mit Polystyrol die Tensiddynamik verlangsamt. Die Rotationskorrelationszeiten und Aktivierungsenergien offenbaren verschiedene Regime der Tensiddynamik. In Polystyrol-Mikrokompositen entspricht die Übergangstemperatur zwischen den Regimen der Glasübergangstemperatur von Polystyrol, während sie in Polycaprolacton-Nanokompositen bei der Schmelztemperatur von Polycaprolacton liegt. Durch die erhebliche Verlängerung der Elektronenspin-Relaxationszeiten bei Verwendung von eisenfreiem Magadiit können Messdaten hoher Qualität mit Puls-EPR-Experimenten erhalten werden. Insebsondere wurden die Vier-Puls-Elektron-Elektron-Doppelresonanz (DEER), die Elektronenspinechoenveloppenmodulation (ESEEM) und die Elektronen-Kern-Doppelresonanz (ENDOR) an spinmarkierten sowie spezifisch deuterierten Tensiden angewandt. Die ENDOR-Ergebnisse legen ein Model der Tensidschicht nahe, in dem zusätzlich zu den Oberflächenlagen auf dem Silikat eine wohldefinierte mittlere Lage existiert. Dieses Modell erklärt auch Verdünnungseffekte durch das Polymer in Kompositen mit Polycaprolacton und Polystyrol. Die umfangreiche Information aus den Magnetresonanztechniken ergänzt die Information aus konventionellen Charakterisierungstechniken wie Röntgendiffraktion und Transmissionselektronenmikroskopie und führt so zu einem detaillierteren Bild der Struktur und Dynamik der Tensidschicht in Nanokompositen aus Polymeren und Schichtsilikaten.

Identification and functional characterization of human cytomegalovirus Fc binding proteins

ABSTRACT Human cytomegalovirus (HCMV) employs many different mechanisms to escape and subvert the host immune system surveillance. Among these different mechanisms the role of human IgG Fc receptors (FcγR) in HCMV pathogenesis is still unclear. In mammalians, FcγRs are expressed on the surface of all haematopoietic cells and have a multifaceted role in regulating the activity of antibodies to generate a well-balanced immune response. Viral proteins with Fcγ binding ability are highly diffuse among herpesviruses. They interfere with the host receptors functions in order to counteract immune system recognition. So far, two human HCMV Fcγ binding proteins have been described: UL119 and RL11. This work was aimed to the identification and characterization of HCMV Fcγ binding proteins. The study is divided in two parts: first the characterization of UL119 and RL11; second the identification and characterization of novel HCMV Fcγ binding proteins. Regarding the first part, we demonstrated that both UL119 and RL11 internalize Fcγ fragments from transfected cells surface through a clathrin dependent pathway. In infected cells both proteins were found in the viral assembly complex and on virions surface as envelope associated glycoproteins. Moreover, internalized Fcγ in infected cells do not undergo lysosomal degradation but rather traffic in early endosomes up to the viral assembly complex. Regarding the second part, we were able to identify two novels Fcγ binding protein coded by CMV: RL12 and RL13. The latter was also further characterized as recombinant protein in terms of cellular localization, Fc binding site and IgG internalization ability. Finally binding specificity of both RL12 and RL13 seems to be confined to human IgG1 and IgG2. Taken together, these data show that HCMV codes for up to 4 FcγR and that they could have a double role both on virus and on infected cells.