PH-sauvagine from the skin secretion of Phyllomedusa hypochondrialis: a novel CRF-like peptide with smooth muscle contraction activity

Amphibian skin, and particularly that of south/Central American phyllomedusine frogs, is supposed to be "a huge factory and store house of a variety of active peptides". The 40 amino acid amphibian CRF-like peptide, sauvagine, is a prototype member of a unique family of these Phyllomedusa skin peptides. In this study, we describe for the first time the structure of a mature novel peptide from the skin secretion of the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, which belongs to the amphibian CRF/sauvagine family. Partial amino acid sequence from the N-terminal was obtained by automated Edman degradation with the following structure: pGlu-GPPISIDLNMELLRNMIEI-. The biosynthetic precursor of this novel sauvagine peptide, consisted of 85 amino acid residues and was deduced from cDNA library constructed from the same skin secretion. Compared with the standard sauvagine from the frog, Phyllomedusa sauvagei, this novel peptide was found to exert similar contraction effects on isolated guinea-pig colon and rat urinary bladder smooth muscle preparations.

Strategies for detection and quantification of cysteine cathepsins-evolution from bench to bedside

The cysteine cathepsins are a family of closely related thiol proteases, normally found in the endosomal and lysosomal compartments of cells. A growing body of evidence has clearly linked the dysregulated activity of these proteases with many diseases and pathological conditions, offering therapeutic, prognostic and diagnostic potential. However, these proteases are synthesised as inactive precursors and once activated, are controlled by factors such as pH and presence of endogenous inhibitors, meaning that overall protein and activity levels do not necessarily correlate. In order to fully appreciate the role and potential of these proteases, tools are required that can detect and quantify overall cathepsin activity. Two main strategies have evolved; synthetic substrates and protease-labelling with affinity-binding probes (or activity-based probes). This review examines recent innovations in these approaches as the field moves towards developing tools that could ultimately be used in patients for diagnostic or prognostic applications.

Molecular and physiological roles of Pellino E3 ubiquitin ligases in immunity

The sensing of foreign agents by the innate and adaptive immune system triggers complex signal transduction cascades that culminate in expression of gene patterns that facilitate host protection from the invading agent. Post-translational modification of intracellular signaling proteins in these pathways is a key regulatory mechanism with ubiquitination being one of the important processes that controls levels and activities of signaling molecules. E3 ubiquitin ligases are the determining enzymes in dictating the ubiquitination status of individual proteins. Among these hundred E3 ubiquitin ligases are a family of Pellino proteins that are emerging to be important players in immunity and beyond. Herein, we review the roles of the Pellino E3 ubiquitin ligases in innate and adaptive immunity. We discuss their early discovery and characterization and how this has been aided by the highly conserved nature of innate immune signaling across evolution. We describe the molecular roles of Pellino proteins in immune signaling with particular emphasis on their involvement in pathogen recognition receptor (PRR) signaling. The growing appreciation of the importance of Pellino proteins in a wide range of immune-mediated diseases are also evaluated.

The roles of Pellino E3 ubiquitin ligases in immunity

Pellino proteins were initially characterized as a family of E3 ubiquitin ligases that can catalyse the ubiquitylation of interleukin-1 receptor-associated kinase 1 (IRAK1) and regulate innate immune signalling pathways. More recently, physiological and molecular roles for members of the Pellino family have been described in the regulation of innate and adaptive immune responses by ubiquitylation. This Review describes the emerging roles of Pellino proteins in innate and adaptive immunity and discusses the mechanistic basis of these functions.

The white dwarf's carbon fraction as a secondary parameter of Type Ia supernovae

Context. Binary stellar evolution calculations predict thatChandrasekhar-mass carbon/oxygen white dwarfs (WDs) show a radiallyvarying profile for the composition with a carbon depleted core. Manyrecent multi-dimensional simulations of Type Ia supernovae (SNe Ia),however, assume the progenitor WD has a homogeneous chemicalcomposition.
Aims: In this work, we explore the impact ofdifferent initial carbon profiles of the progenitor WD on the explosionphase and on synthetic observables in the Chandrasekhar-mass delayeddetonation model. Spectra and light curves are compared to observationsto judge the validity of the model.
Methods: The explosion phaseis simulated using the finite volume supernova code Leafs, which isextended to treat different compositions of the progenitor WD. Thesynthetic observables are computed with the Monte Carlo radiativetransfer code Artis. Results: Differences in binding energies ofcarbon and oxygen lead to a lower nuclear energy release for carbondepleted material; thus, the burning fronts that develop are weaker andthe total nuclear energy release is smaller. For otherwise identicalconditions, carbon depleted models produce less 56Ni.Comparing different models with similar 56Ni yields showslower kinetic energies in the ejecta for carbon depleted models, butonly small differences in velocity distributions and line velocities inspectra. The light curve width-luminosity relation (WLR) obtained formodels with differing carbon depletion is roughly perpendicular to theobserved WLR, hence the carbon mass fraction is probably only asecondary parameter in the family of SNe Ia.
Tables 3 and 4 are available in electronic form at http://www.aanda.org

Phylloseptin-PBa—A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities.

An in vitro model of nociceptors in human trigeminal nerves

Background: The oro-facial region is densely innervated by the trigeminal nerve, which when stimulated can induce noxious pain sensation and contribute to neurogenic inflammation in local tissues. Recent research on the expression of specialised ion channels on the trigeminal nerve has highlighted the need to undertake more extensive studies on ion channel expression/functionality with the aim of elucidating their role in pain sensations. A major family of such ion channels is the transient receptor potential (TRP) channels which are activated by a wide variety of thermal, mechanical or chemical stimuli and merit investigation as possible druggable targets for future analgesics.
Objective: Study of TRP channel expression and regulation in oro-facial tissues is hindered by the fact that the cell bodies of neurons innervating these tissues are located in the trigeminal ganglion. Using dental pulp stem cells differentiated towards peripheral neuronal equivalents (PNEs), we sought to determine TRP channel expression, functionality and potential modulation by cytokines in this novel model.
Method: Dental pulp stem cells (DPSCs) were grown on substrate-coated tissue culture plates and differentiated towards a neuronal phenotype using neuronal induction media. Quantitative polymerase chain reaction (qPCR) was performed on PNEs +/-cytokine treatment. Ion channel functionality was investigated using whole cell patch clamping.
Result: qPCR analysis showed that PNEs expressed the TRP channels TRPA1, TRPV1, TRPV4 and TRPM8. TRPA1 was the most abundantly expressed TRP channel studied whereas TRPM8 was lowly expressed. TRP channel expression was shown to be regulated by treatment with inflammatory cytokines. Patch clamp studies using specific agonists and antagonists for TRPA1 and TRPV1 showed these channels were functional.
Conclusion: PNEs differentiated from DPSCs provide a suitable model for TRP channel expression, regulation, and sensistisation in oro-facial tissues. This human neuronal model has potential for use in pre-clinical studies of novel analgesics.

Immunocytochemical Localization of Substance P Receptors (NK-1 Receptors) in Human Gingival Tissue

Substance P (SP) is a member of the structurally related family of neuropeptides known as the tachykinins. In addition to neurotransmitter roles, the tachykinins are also known to modulate local inflammation which depends on signalling between the neuropeptide molecules and target cells and tissues. SP mediates its effects through a specific receptor, known as the substance P receptor or the neurokinin 1 (NK-1) receptor. The NK-1 receptor is a G-protein associated integral membrane protein and although it has been studied in a wide range of tissues, to date there has been no published data on the localisation of the NK-1 receptor in human gingival tissue. Objective: The aim of this study was to examine the distribution of the NK-1 receptor in human gingival tissue using immunocytochemistry. Method: Gingival tissue was obtained from patients undergoing periodontal surgery. Tissue was fixed in paraformaldehyde and embedded in wax for sectioning. Sections were dewaxed in xylene and then rehydrated in alcohols and phosphate buffered saline. Rehydrated sections were probed with rabbit polyclonal antibody to human NK-1 receptor which was subsequently detected using anti-rabbit horseradish peroxidase conjugate and diaminobenzidine as substrate. Results: Immunocytochemistry revealed that the NK-1 receptor was distributed along nerve fibres and blood vessel endothelial cells, suggesting these areas are main targets for the actions of SP via the NK-1 receptor. Conclusion: This is the first immunocytochemical report of NK-1 receptors in human gingival tissue and provides evidence for possible NK-1 mediated biological effects of SP in human gingival tissue from periodontitis patients.

A class of efficient short-chain fluorinated catanionic surfactants

There have been many attempts to find suitable replacements for fluorinated surfactants due to problems related to their bioaccumulation and resistance to biodegradation. Meeting the exceptional performances of these compounds, however, remains largely an unaccomplished challenge. Currently, a solution might be found through the synthesis of new classes of fluorinated compounds that possess reduced environmental impact by following the recommended strategies for their greener and safer design. In this article we report a novel approach by designing a family of catanionic fluorinated surfactants that shows potential for improved degradation, good water solubility, and low Krafft points. Furthermore, these surfactants exhibit excellent fluorine efficiency and effectiveness of surface tension reduction.

A generalization of the Hoffman-Lovász upper bound on the independence number of a regular graph

A family of quadratic programming problems whose optimal values are upper bounds on the independence number of a graph is introduced. Among this family, the quadratic programming problem which gives the best upper bound is identified. Also the proof that the upper bound introduced by Hoffman and Lovász for regular graphs is a particular case of this family is given. In addition, some new results characterizing the class of graphs for which the independence number attains the optimal value of the above best upper bound are given. Finally a polynomial-time algorithm for approximating the size of the maximum independent set of an arbitrary graph is described and the computational experiments carried out on 36 DIMACS clique benchmark instances are reported.

Studies of epithelial response to streptozotocin-induced hyperglycemia

A hiperglicemia é a principal característica da diabetes mellitus (DM), uma das causas de morte que mais cresce em Portugal, e cujas complicações a longo prazo são mais debilitantes e mais sobrecarregam os sistemas de saúde. No entanto, os mecanismos subjacentes à resposta fisiológica de alguns tecidos à hiperglicemia não estão completamente esclarecidos. Este estudo teve como objetivo avaliar de que forma o tempo de exposição à hiperglicemia afeta dois tecidos epiteliais, o endotélio e as glândulas salivares, que são frequentemente associados a complicações da DM, utilizando um modelo animal. Adicionalmente, procurou-se encontrar novos biomarcadores para avaliar a suscetibilidade a complicações orais em diabéticos, analisando as modificações pós-traducionais (PTMs) da família de proteínas mais representativa na saliva humana: proteínas ricas em prolina (PRPs). A disfunção vascular está na origem de várias complicações da diabetes. Neste sentido, observou-se uma progressiva disfunção endotelial com o aumento do tempo de exposição à hiperglicemia, que resulta do aumento de danos no endotélio e da diminuição da capacidade de mobilização de células progenitoras. Simultaneamente, o aumento observado na atividade da via de ativação do sistema de complemento mediada por lectinas (MBL), sugere um envolvimento do sistema de imunidade inata na patogénese da disfunção vascular. Outra complicação comum da DM é o desenvolvimento de doenças orais, nomeadamente as relacionadas com a redução da secreção salivar. Na análise às glândulas submandibulares, observou-se uma resposta inicial à hiperglicemia com fortes variações na expressão de proteínas, mas a longo prazo, estas variações foram atenuadas, sugerindo um mecanismo de adaptação à hiperglicemia crónica. Adicionalmente, as proteínas relacionadas com a secreção, como as anexinas, apresentaram-se sobre-expressas, enquanto as calicreinas e proteínas metabólicas estavam sub-expressas. Estas variações sugerem que, apesar de uma diminuição da capacidade de regeneração, as células tentam superar a perda de tecido por meio do aumento da secreção, embora sem êxito. O comprometimento funcional das glândulas salivares tem consequências na composição e funções da saliva. Analisando as PTMs das PRPs salivares humanas, observou-se um aumento da frequência de péptidos com ciclização de resíduos N-terminais de glutamina a piroglutamato, o que confere uma resistência à atividade proteolítica que, por sua vez, se encontra aumentada em diabéticos. Assim, a presença de péptidos com N-piroglutamato poderá ser um potencial biomarcador da suscetibilidade a complicações orais em diabéticos.

Evolution of matrix and bone gamma-carboxyglutamic acid proteins in vertebrates

The evolution of calcified tissues is a defining feature in vertebrate evolution. Investigating the evolution of proteins involved in tissue calcification should help elucidate how calcified tissues have evolved. The purpose of this study was to collect and compare sequences of matrix and bone γ-carboxyglutamic acid proteins (MGP and BGP, respectively) to identify common features and determine the evolutionary relationship between MGP and BGP. Thirteen cDNAs and genes were cloned using standard methods or reconstructed through the use of comparative genomics and data mining. These sequences were compared with available annotated sequences (a total of 48 complete or nearly complete sequences, 28 BGPs and 20 MGPs) have been identified across 32 different species (representing most classes of vertebrates), and evolutionarily conserved features in both MGP and BGP were analyzed using bioinformatic tools and the Tree-Puzzle software. We propose that: 1) MGP and BGP genes originated from two genome duplications that occurred around 500 and 400 million years ago before jawless and jawed fish evolved, respectively; 2) MGP appeared first concomitantly with the emergence of cartilaginous structures, and BGP appeared thereafter along with bony structures; and 3) BGP derives from MGP. We also propose a highly specific pattern definition for the Gla domain of BGP and MGP. Previous Section Next Section BGP1 (bone Gla protein or osteocalcin) and MGP (matrix Gla protein) belong to the growing family of vitamin K-dependent (VKD) proteins, the members of which are involved in a broad range of biological functions such as skeletogenesis and bone maintenance (BGP and MGP), hemostasis (prothrombin, clotting factors VII, IX, and X, and proteins C, S, and Z), growth control (gas6), and potentially signal transduction (proline-rich Gla proteins 1 and 2). VKD proteins are characterized by the presence of several Gla residues resulting from the post-translational vitamin K-dependent γ-carboxylation of specific glutamates, through which they can bind to calcium-containing mineral such as hydroxyapatite. To date, VKD proteins have only been clearly identified in vertebrates (1) although the presence of a γ-glutamyl carboxylase has been reported in the fruit fly Drosophila melanogaster (2) and in marine snails belonging to the genus Conus (3). Gla residues have also been found in neuropeptides from Conus venoms (4), suggesting a wider prevalence of γ-carboxylation.

Molecular structure and functional analysis of runx3 in zebrafish

Tese de doutoramento, Ciências Biomédicas, Universidade do Algarve, Departamento de Ciências Biomédicas e Medicina, 2014

Role of plasmodium translationally repressed gene products in malaria transmission

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2015

Application of continuous wavelet transform to the analysis of the modulus of the fractional Fourier transform bands for resolving two component mixture

In this paper, the fractional Fourier transform (FrFT) is applied to the spectral bands of two component mixture containing oxfendazole and oxyclozanide to provide the multicomponent quantitative prediction of the related substances. With this aim in mind, the modulus of FrFT spectral bands are processed by the continuous Mexican Hat family of wavelets, being denoted by MEXH-CWT-MOFrFT. Four modulus sets are obtained for the parameter a of the FrFT going from 0.6 up to 0.9 in order to compare their effects upon the spectral and quantitative resolutions. Four linear regression plots for each substance were obtained by measuring the MEXH-CWT-MOFrFT amplitudes in the application of the MEXH family to the modulus of the FrFT. This new combined powerful tool is validated by analyzing the artificial samples of the related drugs, and it is applied to the quality control of the commercial veterinary samples.