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

bitstream/CNPMS/16145/1/Com_64.pd

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

Epicrates crassus SNAKE: COMPLETE MITOCHONDRIAL GENOME SEQUENCE

Epicrates crassus Cope, 1862 is a snake distributed in South America and a target species for illegal animal trafficking. In this work, we sequenced and described this species complete mitochondrial genome sequence. The mtDNA of E. crassus has 17,379 bp, with 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes, and two non-coding control regions. Phylogenetic reconstruction, based on the complete mitogenome sequence of E. crassus and 18 other mitogenomes, confirmed the best grouping of this species with other members of the Boidae family. These new data can contribute to correctly identifying E. crassus, which would represent one more tool in the fight against the illegal trafficking of this species

Peptide Fragments of a β-Defensin Derivative with Potent Bactericidal Activity

β-Defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine β-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this β-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide

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

Comparison of five methods of extraction of staphylococcus aureus dna for molecular detection by pcr

Introduction: Molecular techniques for the detection of pathogens have been shown to be effective diagnostic tools with high sensitivity and short turnaround times. Methods: This study compared five Staphylococcus aureus DNA extraction methods for detection by the polymerase chain reaction. Results: The concentration and purity of the extracted DNA showed that the methods did not yield DNA of significant quality. However, most protocols yielded 100% positivity, even with low DNA concentrations. Conclusions: Although one protocol seemed more efficient than the others, PCR was sensitive enough to allow for detection of S. aureus with all the protocol

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