Distribution of exoantigens and a 43-kDa glycoprotein (gp43) in the yeast and mycelial forms of Paracoccidioides brasiliensis

Objective. Paracoccidioides brasiliensis antigens (strain 113) were located at ultrastructural level in both yeast and mycelial forms of the fungus. The reactivity of the sera employed was analysed. Materials and methods. Immunofluorescence and ultrastructural protein A-gold immunolabelling techniques were performed using two polyclonal antisera: one against P. brasiliensis exoantigens and the other against a 43-kDa glycoprotein (gp43). Immunoblotting assays were employed to define reactivity of these antisera with somatic and metabolic antigens of both forms of the fungus. Results. The techniques employed revealed in both yeast and mycelial forms of P. brasiliensis a similar antigenic distribution. The antigens deposits were seen within the cytoplasm, and over the cell wall of the fungus. The anti-exoantigen serum recognized several bands in both forms of the fungus. The anti-gp43 serum reacted strongly with the 43-kDa fraction and weakly with few other fractions. Conclusions. Immunocytochemical techniques suggest a protein synthesis within the cytoplasm followed by excretion through the cell wall. Similar results employing both polyclonal antisera were obtained.

Effects of tunicamycin on glycoprotein antigens of Aspergillus fumigatus

Tunicamycin, which inhibits N-glycosylation of proteins, was used as a tool to determine the type of linkage which occurs in glycoprotein antigens of Aspergillus fumigatus. When A. fumigatus extracts were electrophoretically separated and blotted then probed with anti-Aspergillus patients' sera, differences in antigenic profiles were noted when tunicamycin-treated samples were compared with controls. Tunicamycin had no detectable effect on the cellular proteinases of A. fumigatus, most of which are glycosylated. Some enzymatic components were lacking when extracellular proteinases were compared with those of control samples. The major catalase component of A. fumigatus is a concanavalin A (Con A)-binding glycoprotein. In cultures grown in the presence of tunicamycin, partiallydeglycosylated catalase components were obtained which could be distinguished from the native catalase by their altered mobilities in polyacrylamide gels. The effect of deglycosylation on catalase antigens was monitored using an antiserum raised to a ConA-binding fraction of A fumigatus mycelium. These antibodies bound both to the native glycoprotein and the partially deglycosylated material. These latter two were largely unaffected when incubated with an antiserum raised to a non-ConA-binding fraction of A. fumigatus which is essentially carbohydrate free. The ability to produce partially-glycosylated antigens of A. fumigatus offers a model to study the effect of basic structural modifications on both the enzymatic and antigenic activities of these molecules.

Involvement of the major glycoprotein (gp43) of Paracoccidioides brasiliensis in attachment to macrophages

The yeast form of Paracoccidioides brasiliensis, the causative agent of a deep mycosis in humans, is known to be phagocytized by, and to multiply inside, macrophages. In this work we describe the involvement of gp43, a major antigenic protein of P. brasiliensis, in the initial steps of attachment of the fungus to macrophages. Anti-gp43 F(ab) polyclonal fragments were capable of inhibiting phagocytosis in a concentrationdependent manner. Sheep red blood cells sensitized with purified gp43 were more endocytized than SRBC alone, and this process was also inhibited by anti-gp43 F(ab) fragments. Inhibition tests indicated the involvement of fucose and mannose residues in the phagocytosis of the fungus and of SRBC-gp43 by macrophages. Taken together, these results suggest that gp43 may be involved in the adherence and uptake of the fungus by murine peritoneal macrophages, and that this binding may be dependent on monosaccharide residues that are part of the gp43 glycoprotein. © 1998 ISHAM.

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.

Reinforcing and expanding the predictions of the disturbance vicariance hypothesis in Amazonian harlequin frogs: A molecular phylogenetic and climate envelope modelling approach

The disturbance vicariance hypothesis (DV) has been proposed to explain speciation in Amazonia, especially its edge regions, e. g. in eastern Guiana Shield harlequin frogs (Atelopus) which are suggested to have derived from a cool-adapted Andean ancestor. In concordance with DV predictions we studied that (i) these amphibians display a natural distribution gap in central Amazonia; (ii) east of this gap they constitute a monophyletic lineage which is nested within a pre-Andean/western clade; (iii) climate envelopes of Atelopus west and east of the distribution gap show some macroclimatic divergence due to a regional climate envelope shift; (iv) geographic distributions of climate envelopes of western and eastern Atelopus range into central Amazonia but with limited spatial overlap. We tested if presence and apparent absence data points of Atelopus were homogenously distributed with Ripley's K function. A molecular phylogeny (mitochondrial 16S rRNA gene) was reconstructed using Maximum Likelihood and Bayesian Inference to study if Guianan Atelopus constitute a clade nested within a larger genus phylogeny. We focused on climate envelope divergence and geographic distribution by computing climatic envelope models with MaxEnt based on macroscale bioclimatic parameters and testing them by using Schoener's index and modified Hellinger distance. We corroborated existing DV predictions and, for the first time, formulated new DV predictions aiming on species' climate envelope change. Our results suggest that cool-adapted Andean Atelopus ancestors had dispersed into the Amazon basin and further onto the eastern Guiana Shield where, under warm conditions, they were forced to change climate envelopes. © 2010 The Author(s).

EIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer

The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein-protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNAPhe tertiary structure, analogously to the EF-P monomer. © 2012 Springer-Verlag.

Time-frequency analysis combining wavelets and the envelope technique for fault identification in rotary machines

Nowadays the method based on demodulation by envelope finds wide application in industry as a technique for evaluation of bearings and other components in rotating machinery. In recent years the application of Wavelets for fault diagnosis in machinery has also obtained good development. This article demonstrates the effectiveness of the combined application of Wavelets and envelope technique (also known as HFRT High-Frequency Resonance Technique) to remove background noise from signals collected from defect bearings and identification of the characteristic frequencies of defects. A comparison of the results obtained with the isolated application of only one method against the combined technique is performed showing the increased capacity in detection of faults in rolling bearings. © (2013) Trans Tech Publications, Switzerland.

Clonagem e expressão da proteína E2 no vírus da hepatite C Humana: estudo da interação molecular E2-rLDL in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização estrutural e das interações entre a Proteína G do hRSV e potenciais inibidores

Pós-graduação em Microbiologia - IBILCE

Desenvolvimento de um instrumento portátil para detectar falha em rolamento de motor de indução, pela técnica do envelope, usando um DSP de 16 bits e a transformada de Hilbert

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Sínteses e estudos dos diferentes peptídeos de fusão dos flavivirus causadores da dengue

Pós-graduação em Biotecnologia - IQ

Expression of P-glycoprotein, multidrug resistance-associated protein, glutathione-S-transferase pi and p53 in canine transmissible venereal tumor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Impedance-derived electrochemical capacitance spectroscopy for the evaluation of lectin-glycoprotein binding affinity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aeroelastic stability analysis considering a continuous flight envelope

This paper presents a new methodology to analyze aeroelastic stability in a continuous range of flight envelope with varying parameter of velocity and altitude. The focus of the paper is to demonstrate that linear matrix inequalities can be used to evaluate the aeroelastic stability in a region of flight envelope instead of a single point, like classical methods. The proposed methodology can also be used to study if a system remains stable during an arbitrary motion from one point to another in the flight envelope, i.e., when the problem becomes time-variant. The main idea is to represent the system as a polytopic differential inclusion system using rational function approximation to write the model in time domain. The theory is outlined and simulations are carried out on the benchmark AGARD 445.6 wing to demonstrate the method. The classical pk-method is used for comparing results and validating the approach. It is shown that this method is efficient to identify stability regions in the flight envelope. (C) 2014 Elsevier Ltd. All rights reserved.