Investigations of peptide structural stability in vacuo

Gas-phase analytical techniques provide very valuable tools for tackling the structural complexity of macromolecular structures such as those encountered in biological systems. Conformational dynamics of polypeptides and polypeptide assemblies underlie most biological functionalities, yet great difficulties arise when investigating such phenomena with the well-established techniques of X-ray crystallography and NMR. In areas such as these ion mobility interfaced with mass spectrometry (IMMS) and molecular modelling can make a significant contribution. During an IMMS experiment analyte ions drift in a chamber filled with an inert gas; measurement of the transport properties of analyte ions under the influence of a weak electric field can lead to determination of the orientationally-averaged collision cross-section of all resolved ionic species. A comparison with cross-sections estimated for model molecular geometries can lead to structural assignments. Thus IMMS can be used effectively to separate gas-phase ions based on their conformation. The drift tube employed in the experiments described herein is thermally regulated, which also enables the determination of collision cross-sections over a range of temperatures, and can provide a view of temperature-dependent conformational dynamics over the experimental (low microsecond) timescale. Studies described herein employ IMMS and a gamut of other MS-based techniques, solution spectroscopy and – importantly – molecular mechanics simulations to assess a) conformational stability of isolated peptide ions, with a focus on small model peptides and proteins, especially the Trp cage miniprotein; and b) structural characteristics of oligomeric aggregates of an amyloidogenic peptide. The results obtained serve to clarify the factors which dominate the intrinsic stability of non-covalent structure in isolated peptides and peptide assemblies. Strong electrostatic interactions are found to play a pivotal role in determining the conformations of isolated proteins. Secondary structures held together by hydrogen bonding, such as helices, are stable in the absence of solvent, however gas-phase protein structures display loss of their hydrophobic cores. The absence of a polar solvent, “self-solvation” is by far the most potent force influencing the gas-phase configuration of these systems. Geometries that are more compact than the folded state observed in solution are routinely detected, indicating the existence of intrinsically stable compact non-native states in globular proteins, illuminating the nature of proteins’ ‘unfolded’ states

Complete mitochondrial genomes of three species of fresh flies of forensic entomology interest from the genus Sarcophaga (Sarcophagidae) from Portugal and Brazil

The Sarcophagidae family of fresh flies bears strong importance in the context of forensic entomology due to their application in the estimation of the Post Mortem Interval (PMI). Sarcophaga is the major genus in the Sarcophagidae family and includes cosmopolitan species, which are distributed worldwide. In this communication, we present the analysis of the complete mitochondrial genome (mtDNA) of two species from Portugal – S. melanura and S. dux – and one from Brazil – S. ruficornis. The mtDNA of these species range from 14,882 bp to 15,190 bp and have 22 tRNA genes, 13 protein-coding genes (PCG), and two rRNAs distributed along both strands. Our data include the first record of complete Sarcophaga mtDNA sequences from species collected in Portugal and in Brazil. These genomes represent an advance in the understanding about this group, expand the database, and can be used for the development of new markers for species identification41237239CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES25/2014; 23038.006839 / 2014-33; 0092/ 17-7This work was funded by Coordenac¸~ao de Aperfeic¸oamento de Pessoal de N ıvel Superior (CAPES) (Edital Ci^ encias Forenses no. 25/2014, Process 23038.006839 / 2014-33), International postdoc fellowship (CAPES 0092/ 17-7) and Conselho Nacional de Desenvolvimento Cient ıfico e Tecnologico (CNPq). Financial support was also provided by CESAM (UID/ AMB/50017 - POCI-01-0145-FEDER-007638), and by FCT/MCTES through national funds (PIDDAC). Co-funding was provided by the FEDER, within the PT2020 Partnership Agreement and Compete 202

Clonagem e expressão do gene bioinseticida TX4(6-1) em sistema heterologo.

bitstream/CNPMS/16145/1/Com_64.pd

Vascular Endothelial Growth Factor Increases During Blood-brain Barrier-enhanced Permeability Caused By Phoneutria Nigriventer Spider Venom.

Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV) causes blood-brain barrier breakdown (BBBb). The PNV-induced excitotoxicity results from disturbances on Na(+), K(+) and Ca(2+) channels and glutamate handling. The vascular endothelial growth factor (VEGF), beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB) and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.201472196

Complete mitochondrial genomes from four species of the genus Oxysarcodexia (Sarcophagidae) with forensic entomology interest

The Sarcophagidae family is one of the most important in forensic entomology. Its richest genus, Oxysarcodexia, is well-distributed in Brazil and Latin America and includes species that can be used in forensic investigations to estimate the Post Mortem Interval (PMI). In this communication, we present the mitochondrial genome (mtDNA) of four Oxysarcodexia species: O. avuncula (one specimen), O. terminalis (one specimen), O. thornax (three specimens), and O. varia (one specimen). These mitochondrial genomes (mtDNA) range from 14,998 to 15,613 bp and have 22 tRNA genes, 13 protein-coding genes (PCG), and 2 rRNAs distributed along both strands. The mitogenomes from Oxysarcodexia showed in the present work represents an important contribution to the knowledge regarding the Sarcophagidae phylogenetic structure and is an important source of information for the development of novel DNA markers for forensic identification4143043

Characterization of a new Autographa californica multiple nucleopolyhedrovirus (AcMNPV) polyhedra mutant

In the very late phase of baculovirus infection, virions are occluded in a crystalline matrix called polyhedra, which is mainly composed of polyhedrin. This protein is highly conserved among baculoviruses and changes in its amino acid sequence may lead to mutant polyhedra. During the purification of an AcMNPV recombinant virus, a mutant virus was isolated. Structural and ultrastrutural analysis by light and transmission electron microscopy (TEM) of insect cells infected with this mutant virus did not show polyhedra formation and differed from the wild-type infection by the presence of a proteinaceous mass dispersed in the cytoplasm and nucleus of the infected cells, which was confirmed by immunogold labelling to be polyhedrin. The polyhedrin gene was amplified by PCR and sequenced. The only change observed was the substitution of a G to a T at the nucleotide +352, which resulted in a Val to Phe change. A recombinant virus was constructed by transferring the mutant gene into a polyhedrin negative virus. The phenotype of this recombinant virus was the same as the mutant one, confirming that this single mutation alone was responsible for the mutant phenotype

Complete mitochondrial genomes from three species of the genus Peckia (Sarcophagidae) with forensic entomology interest

Peckia is one of the most important genera in the Sarcophagidae family of flesh flies. This genus is distributed in Brazil and Latin America, and its species can be used to estimate the Post Mortem Interval (PMI) in forensic investigations. In this communication, we present four mitochondrial genomes (mtDNA) from three Peckia species: P. australis, P. collusor, and P. resona. These mtDNA range from 15,116 bp to 15,234 bp in length and have 22 tRNA genes, 13 protein-coding genes (PCG), and two rRNAs distributed along both the strands. These data expand the knowledge about the Sarcophagidae genomes and present, for the first time, four complete mtDNA sequences of the Peckia genus. We show novel complete mtDNA sequences of flesh fly species of forensic importance. Our data expand the knowledge on the molecular database for the identification of these species, and is an important step towards increasing the databases and can help on the identification of new species, particularly in the forensic context41220221CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESNão tem25/2014; 23038.006839/2014-33; 0092/ 17-7This work was funded by Coordenac¸~ao de Aperfeic¸oamento de Pessoal de N ıvel Superior (CAPES) (Edital Ciências Forenses no. 25/2014, Process 23038.006839/2014-33), International postdoc fellowship (CAPES 0092/ 17-7) and Conselho Nacional de Desenvolvimento Cient ıfico e Tecnologico (CNPq). Financial support was also provided by CESAM (UID/ AMB/50017 - POCI-01-0145-FEDER-007638), and by FCT/MCTES through national funds (PIDDAC). Co-funding provided by the FEDER, within the PT2020 Partnership Agreement and Compete 202

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules

BACKGROUND: Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite. METHODS: To test this hypothesis, adult male rats were submitted to surgical castration followed by estradiol, DHT or 3-beta-diol replacement. Changes in AQP9 expression in the efferent ductules were investigated by using immunohistochemistry and Western blotting assay. RESULTS: Data show that, after castration, AQP9 expression was significantly reduced in the efferent ductules. 3-beta-diol injections restored AQP9 expression, similar to DHT and estradiol. The results were confirmed by Western blotting assay. CONCLUSION: This is the first evidence that 3-beta-diol has biological activity in the male reproductive tract and that this androgen metabolite has estrogen-like activity in the efferent ductules, whose major function is the reabsorption of luminal fluid