Molecular Characterisation and Phylogenetic Analysis of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Zc-hepc1), from the Coral Fish Moorish idol, Zanclus cornutus (Linnaeus,1758)

Hepcidin is a family of short cysteine-rich antimicrobial peptides (AMPs) participating in various physiological functions with inevitable role in host immune responses. Present study deals with identification and characterisation of a novel hepcidin isoform from coral fish Zanclus cornutus. The 81 amino acid (aa) preprohepcidin obtained from Z. cornutus consists of a hydrophobic aa rich 22 mer signal peptide, a highly variable proregion of 35 aa and a bioactive mature peptide with 8 conserved cysteine residues which contribute to the disulphide back bone. The mature hepcidin, Zc-hepc1 has a theoretical isoelectric point of 7.46, a predicted molecular weight of 2.43 kDa and a net positive charge of ?1. Phylogenetic analysis grouped Z. cornutus hepcidin with HAMP2 group hepcidins confirming the divergent evolution of hepcidin-like peptide in fishes. Zc-hepc1 can attain a b-hairpin-like structure with two antiparallel b-sheets. This is the first report of an AMP from the coral fish Z. cornutus.

The function of SIFamide, PDF, and further neuropeptides in the circadian system of Rhyparobia maderae

Der täglich Wechsel von Hell- und Dunkelphasen führte während der Evolution zur Entwicklung innerer Uhren in nahezu allen Organismen. In der Schabe Rhyparobia maderae lokalisierten Läsions- und Transplantationsexperimente die innere Uhr in der akzessorischen Medulla (AME). Dieses kleine birnenförmige Neuropil am ventromedianen Rand der Medulla ist mit etwa 240 Neuronen assoziiert, die eine hohe Anzahl an zum Teil kolokalisierten Neuropeptiden und Neurotransmittern exprimieren. Diese Signalstoffe scheinen essentiell zu sein für die Synchronisation der inneren Uhr mit der Umwelt, der Kopplung der beiden bilateralen AME, der Aufrechterhaltung des circadianen Rhythmus sowie der zeitlichen Steuerung bestimmter Verhaltensweisen. Während die Funktion einiger dieser neuronalen Botenstoffe bereits gut untersucht ist, fehlt sie für andere. Zudem ist noch ungeklärt, wann einzelne Botenstoffe im circadianen Netzwerk agieren. Im Fokus dieser Studie lag daher die Erforschung der Funktion von SIFamide und Corazonin im circadianen Netzwerk sowie die weitere Untersuchung der Funktionen der Neuropeptide MIP und PDF. Es konnte gezeigt werden, dass SIFamide auch in R. maderae in vier großen neurosekretorischen Zellen in der pars intercerebralis exprimiert wird. Varikosenreiche SIFamide-immureaktive (-ir) Fasern innervieren eine Vielzahl an Neuropilen und finden sich auch in der Hüllregion der AME. Injektionsexperimente resultierten in einer monophasischen Phasen-Antwort-Kurve (PRC) mit einer Verzögerung zur frühen subjektiven Nacht. SIFamide ist also ein Eingangssignal für das circadiane Netzwerk und könnte in der Kontrolle der Schalf/Wach-Homöostase involviert sein. Auch Corazonin fungiert als Eingangssignal. Da die Injektionsexperimente in einer monophasischen PRC mit einem Phasenvorschub zur späten subjektiven Nacht resultierten, ist davon auszugehen, dass die Corazonin-ir AME-Zelle Bestandteil des Morning-Oszillator-Netzwerkes in R. maderae ist. Darüber hinaus zeigten Backfill-Experimente, dass MIP an der Kopplung beider AMAE beteiligt ist. ELISA-Quantifizierungen der PDF-Level im Tagesverlauf ergaben Schwankungen in der Konzentration, die auf eine Ausschüttung des Peptids während des Tages hindeuten – ähnlich wie es in Drosophila melanogaster der Fall ist. Dies spiegelt sich in der vervollständigten bimodalen PDF-PRC wieder. Hier führen Injektionen zu einem Phasenvorschub, bevor maximale Peptidlevel erreicht werden, sowie zu einer Phasenverzögerung, sobald die Peptidlevel wieder zu sinken beginnen. Die PRCs erlauben somit Rückschlüsse auf den Zeitpunkt der maximalen Peptidfreisetzung. PDF-ir Neuriten findet sich zudem in sämtlichen Ganglien des ventralen Strickleiternervensystems, was eine Funktion in der Kontrolle der Prozesse impliziert, die durch die Mustergeneratoren in Thorakal- und Abdominalganglien gesteuert werden.

Statistical evidences of cyclic changes in volcanic gas chemistry composition by inverse modelling

The identification of compositional changes in fumarolic gases of active and quiescent volcanoes is one of the most important targets in monitoring programs. From a general point of view, many systematic (often cyclic) and random processes control the chemistry of gas discharges, making difficult to produce a convincing mathematical-statistical modelling. Changes in the chemical composition of volcanic gases sampled at Vulcano Island (Aeolian Arc, Sicily, Italy) from eight different fumaroles located in the northern sector of the summit crater (La Fossa) have been analysed by considering their dependence from time in the period 2000-2007. Each intermediate chemical composition has been considered as potentially derived from the contribution of the two temporal extremes represented by the 2000 and 2007 samples, respectively, by using inverse modelling methodologies for compositional data. Data pertaining to fumaroles F5 and F27, located on the rim and in the inner part of La Fossa crater, respectively, have been used to achieve the proposed aim. The statistical approach has allowed us to highlight the presence of random and not random fluctuations, features useful to understand how the volcanic system works, opening new perspectives in sampling strategies and in the evaluation of the natural risk related to a quiescent volcano

3D Analysis of the TCR/pMHCII Complex Formation in Monkeys Vaccinated with the First Peptide Inducing Sterilizing Immunity against Human Malaria

T-cell receptor gene rearrangements were studied in Aotus monkeys developing high antibody titers and sterilizing immunity against the Plasmodium falciparum malaria parasite upon vaccination with the modified synthetic peptide 24112, which was identified in the Merozoite Surface Protein 2 (MSP-2) and is known to bind to HLA-DR beta 1*0403 molecules with high capacity. Spectratyping analysis showed a preferential usage of V beta 12 and V beta 6 TCR gene families in 67% of HLA-DR beta 1*0403-like genotyped monkeys. Docking of peptide 24112 into the HLA-DR beta 1*0401-HA peptide-HA1.7TCR complex containing the VDJ rearrangements identified in fully protected monkeys showed a different structural signature compared to nonprotected monkeys. These striking results show the exquisite specificity of the TCR/pMHCII complex formation needed for inducing sterilizing immunity and provide important hints for a logical and rational methodology to develop multiepitopic, minimal subunit-based synthetic vaccines against infectious diseases, among them malaria.

Enfermedad tiroidea autoinmune en pacientes colombianos con lupus eritematoso sistémico

Objetivos: Determinar la prevalencia y los factores asociados con el desarrollo de hipotiroidismo autoinmune (HA) en una cohorte de pacientes con lupus eritematoso sistémico (LES), y analizar la información actual en cuanto a la prevalencia e impacto de la enfermedad tiroidea autoinmune y la autoinmunidad tiroidea en pacientes con LES. Métodos: Este fue un estudio realizado en dos pasos. Primero, un total de 376 pacientes con LES fueron evaluados sistemáticamente por la presencia de: 1) HA confirmado, 2) positividad para anticuerpos tiroperoxidasa/tiroglobulina (TPOAb/TgAb) sin hipotiroidismo, 3) hipotiroidismo no autoinmune, y 4) pacientes con LES sin hipotiroidismo ni positividad para TPOAb/TgAb. Se construyeron modelos multivariados y árboles de regresión y clasificación para analizar los datos. Segundo, la información actual fue evaluada a través de una revisión sistemática de la literatura (RLS). Se siguieron las guías PRISMA para la búsqueda en las bases de datos PubMed, Scopus, SciELO y Librería Virtual en Salud. Resultados: En nuestra cohorte, la prevalencia de HA confirmado fue de 12% (Grupo 1). Sin embargo, la frecuencia de positividad para TPOAb y TgAb fue de 21% y 10%, respectivamente (Grupo 2). Los pacientes con LES sin HA, hipotiroidismo no autoinmune ni positividad para TPOAb/TgAb constituyeron el 40% de la corhorte. Los pacientes con HA confirmada fueron estadísticamente significativo de mayor edad y tuvieron un inicio tardío de la enfermedad. El tabaquismo (ORA 6.93, IC 95% 1.98-28.54, p= 0.004), la presencia de Síndrome de Sjögren (SS) (ORA 23.2, IC 95% 1.89-359.53, p= 0.015) y la positividad para anticuerpos anti-péptido cíclico citrulinado (anti-CCP) (ORA 10.35, IC 95% 1.04-121.26, p= 0.047) se asociaron con la coexistencia de LES-HA, ajustado por género y duración de la enfermedad. El tabaquismo y el SS fueron confirmados como factores predictivos para LES-HA (AUC del modelo CART = 0.72). En la RSL, la prevalencia de ETA en LES varío entre 1% al 60%. Los factores asociados con esta poliautoinmunidad fueron el género femenino, edad avanzada, tabaquismo, positividad para algunos anticuerpos, SS y el compromiso articular y cutáneo. Conclusiones: La ETA es frecuente en pacientes con LES, y no afecta la severidad del LES. Los factores de riesgo identificados ayudarán a los clínicos en la búsqueda de ETA. Nuestros resultados deben estimular políticas para la suspensión del tabaquismo en pacientes con LES.

Synthesis of antimicrobial peptides derived from BP100 and BPC194

In the present PhD thesis we studied the solid-phase peptide synthesis of antimicrobial peptides derived from the lead peptides BP100 and BPC194. First, peptides derived from BP100 containing D-amino acids at different positions of the sequences were prepared. Moreover, peptidotriazoles derived from BP100 were also synthesized containing the triazole ring at the side-chain of different amino acids. Then, we proceeded to perform studies for the synthesis of multivalent peptides derived from BPC194. To achieve this objective, the synthesis of cyclic peptides containig a triazole ring at amino acids side-chain with different elongations was carried out. Finally, we prepared various carbopeptides containing 2 and 4 units of BP100 and/or its derivatives. The evaluation of the biological activity allowed the identification of active sequences against the economically important phytopathogenic bacteria and fungi and not toxic against eukaryotic cells.

Enantioselective Intramolecular Michael Addition of Nitronates onto Conjugated Esters: Access to Cyclic γ-Amino Acids with up to Three Stereocenters

A highly stereoselective synthesis of conformationally constrained cyclic γ-amino acids has been devised. The key step involves an intramolecular cyclization of a nitronate onto a conjugated ester, promoted by a bifunctional thiourea catalyst. This methodology has been successfully applied to generate a variety of γ-amino acids, including some containing three contiguous stereocenters, with very high diastereoselectivity and excellent enantioselectivity. It is postulated that an interaction that is key to the success of the process is the simultaneous coordination of the thiourea functionality to both the conjugated ester and the nitronate. Finally, the synthetic utility of these compounds is demonstrated in the synthesis of two dipeptides derived from the C- and N-termini.

Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein

CSRP3 or muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with alpha-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerase I and II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative nuclear localization signal (NLS) of MLP, nuclear import of the protein in cultured rat neonatal myocytes was inhibited. The NLS of MLP also localizes to the nucleolus. Inhibition of nuclear translocation prevented the increased protein accumulation in response to phenylephrine. Furthermore, cyclic strain of myocytes after prior NLS treatment to remove nuclear MLP resulted in disarrayed sarcomeres. Increased protein synthesis and brain natriuretic peptide expression were also prevented suggesting that MLP is required for remodeling of the myo filaments and gene expression. These findings suggest that nucleocytoplasmic shuttling MLP plays an important role in the regulation of the myocyte remodeling and hypertrophy and is required for adaptation to hypertrophic stimuli. (C) 2009 Elsevier Inc. All rights reserved.

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL), for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins constituting the vast majority of species in any proteome, as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis. Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification / detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL capture is at least twice that of control, untreated sample.

Cell-penetrating peptide-morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo

The cellular uptake of PMOs (phosphorodiamidate morpholino oligomers) can be enhanced by their conjugation to arginine-rich CPPs (cell-penetrating peptides). Here, we discuss our recent findings regarding (R-Ahx-R)(4)AhxB (Ahx is 6-aminohexanoic acid and B is beta-alanine) CPP-PMO conjugates in DMD (Duchenne muscular dystrophy) and murine coronavirus research. An (R-Ahx-R)(4)AhxB-PMO conjugate was the most effective compound in inducing the correction of mutant dystrophin transcripts in myoblasts derived from a canine model of DMD. Similarly, normal levels of dystrophin expression were restored in the diaphragms of mdx mice, with treatment starting at the neonatal stage, and protein was still detecTable 22 weeks after the last dose of an (R-Ahx-R)(4)AhxB-PMO conjugate. Effects of length, linkage and carbohydrate modification of this CPP on the delivery of a PMO were investigated in a coronavirus mouse model. An (R-Ahx-R)(4)AhxB-PMO conjugate effectively inhibited viral replication, in comparison with other peptides conjugated to the same PMO. Shortening the CPP length, modifying it with a mannosylated serine moiety or replacing it with the R(9)F(2) CPP significantly decreased the efficacy of the resulting PPMO (CPP-PMO conjugate). We attribute the success of this CPP to its stability in serum and its capacity to transport PMO to RNA targets in a manner superior to that of poly-arginine CPPs.

Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains

The stable signal peptide (SSP) of the lymphocytic choriomeningitis virus surface glycoprotein precursor has several unique characteristics. The SSP is unusually long, at 58 amino acids, and contains two hydrophobic domains, and its sequence is highly conserved among both Old and New World arenaviruses. To better understand the functions of the SSP, a panel of point and deletion mutants was created by in vitro mutagenesis to target the highly conserved elements within the SSP. We were also able to confirm critical residues required for separate SSP functions by trans-complementation. Using these approaches, it was possible to resolve functional domains of the SSP. In characterizing our SSP mutants, we discovered that the SSP is involved in several distinct functions within the viral life cycle, beyond translocation of the viral surface glycoprotein precursor into the endoplasmic reticulum lumen. The SSP is required for efficient glycoprotein expression, posttranslational maturation cleavage of GP1 and GP2 by SKI-1/S1P protease, glycoprotein transport to the cell surface plasma membrane, formation of infectious virus particles, and acid pH-dependent glycoprotein-mediated cell fusion.