Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

Lipopeptides Produced by a Soil Bacillus Megaterium Strain

A soil microorganism identified as Bacillum megaterium was found to produce several antibiotics substances after growth for 20 h at 37A degrees C in a mineral culture medium. Analysis both by electron spray ionization (ESI) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) identified these substances as lipopeptides. Predominant peaks at m/z 1,041 and m/z 1,065 revealed ions which are compatible with surfactins and lichenysins, respectively. Two other ions m/z 1,057 and m/z 1,464 were further studied by collision-induced dissociation (CID) unveiling an iturin A at the first and fengycins A and B at the second m/z peaks. The CID spectrum of the m/z 1,464 ion also suggests the existence of fengycins A and B variants in which Ile was changed to Val in the position 10 of the peptide moiety. Raw mixtures of all these compounds were also assayed for antibiotic features. The data enlighten the unusual diversity of the lipopeptide mixture produced by a sole Bacillus species.

The Potential of p38 MAPK Inhibitors to Modulate Periodontal Infections

Periodontal disease initiation and progression occurs as a consequence of the host immune inflammatory response to oral pathogens. The innate and acquired immune systems are critical for the proper immune response. LPS, an outer membrane constituent of periodontal pathogenic bacteria, stimulates the production of inflammatory cytokines IL-1 beta TNF alpha IL-6 and RANKL either directly or indirectly. In LPS-stimulated cells, the induction of cytokine expression requires activation of several signaling pathways including the p38 MAPK pathway. This review will discuss the significance of the p38 MAPK pathway in periodontal disease progression and the potential therapeutic consequences of pharmacological antagonism of this pathway in the treatment of periodontal diseases.

Coat protein and RNAs of Cole latent virus are not present within chloroplasts of Chenopodium quinoa-infected cells

O vírus latente da couve (Cole latent virus, CoLV), gênero Carlavirus, foi estudado, por microscopia eletrônica de transmissão e técnicas bioquímicas, em relação à ultra-estrutura das células infetadas de Chenopodium quinoa, e de sua associação com os cloroplastos. O CoLV foi observado como partículas dispersas pelo citoplasma entremeadas com vesículas membranosas e ribossomos e/ou como densas massas de partículas. Estes partículas reagiram por imunomarcação com anti-soro policlonal para o CoLV. Morfologicamente, cloroplastos, mitocôndrias e núcleos mostraram-se inalterados e partículas virais não foram encontradas dentro dessas organelas. Entretanto, agregados de partículas virais foram freqüentemente vistos em associação com a membrana externa dos cloroplastos e ocasionalmente com peroxissomos. Cloroplastos foram purificados em gradiente de Percoll e as proteínas e os RNA foram extraídos e analisados, respectivamente, por Western blot e Northern blot. Proteína capsidial e RNA associados ao CoLV não foram detectados nessa organela. Os resultados aqui obtidos indicam que a associação CoLV/cloroplastos, observada nos estudos de microscopia eletrônica, é possivelmente um evento casual dentro da célula hospedeira e que o vírus não se multiplica dentro dessa organela.

Utilização experimental de hidroxiapatita sintética em alvéolos dentários de gatos domésticos (Felis catus): estudo clínico, radiográfico e histomorfométrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide 0 side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Use of lipopolysaccharides to characterize Bradyrhizobium spp.

Three methods of extraction of lipopolysaccharide (LPS) were compared-the conventional hot phenol-water method with 40% phenol, a modified form of this method using 10% phenol, and the hot saline method. Good recovery of LPS was achieved by each of the three methods, with the LPS found in the aqueous phase with the two phenol-based procedures. The application of SDS-PAGE to the LPS extracts, followed by silver staining, showed similar banding with all three methods of extraction. When the hot saline extraction LPS fraction from eight strains of Bradyrhizobium spp. and eight strains of Bradyrhizobium japonicum was compared with SDS-PAGE, characteristic profiles were achieved. Serological analysis of eight strains of Bradyrhizobium spp., using antisera prepared against whole cells in agglutination reactions, showed extensive sharing of antigens. When antisera was prepared using outer membrane LPS, extracted by the hot saline method, the amount of cross-reaction was reduced greatly. The results indicated that LPS provide an efficient means of obtaining monospecific antisera to be used for serological identification of strains of Bradyrhizobium spp. and that the hot saline extraction method is recommended for a fast, simple and efficient way to obtain LPS and characterize this bacterium.

Peptídeos inibidores de topoisomerases bacterianas: síntese e estudos de transporte através da membrana celular empregando moléculas peptídicas carreadoras

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Secretoma da bactéria fitopatogênica Xanthomonas citri subsp. citri

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Método de utilização de membrana de celulose bacteriana intra articular sobre superfície lesada de patela

The present invention discloses the use of the bacterial cellulose membrane in ligament, tendon and synovial capsule reconstructions according to the methods described in the technical description of the invention. Said material could be used in the reconstruction of ligaments and tendons (the knee cruciate ligaments, patellar ligament, Achilles tendon, quadriceps tendon, etc.) and the synovial capsule.

Green tea extract and its major constituent epigallocatechin-3-gallate inhibit growth and halitosis-related properties of Solobacterium moorei

Background: Solobacterium moorei is a volatile sulfide compound (VSC)-producing Gram-positive anaerobic bacterium that has been associated with halitosis. The aim of this study was to investigate the effects of green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) on growth and severalhalitosis-related properties of S. moorei.Methods: A microplate dilution assay was used to determine the antibacterial activity of green tea extract and EGCG against S. moorei. Their effects on bacterial cell membrane integrity were investigated by transmission electron microscopy and a fluorescence-based permeability assay. Biofilm formation was quantified by crystal violet staining. Adhesion of FITC-labeled S. moorei to oral epithelial cells was monitored by fluorometry. The modulation of beta-galactosidase gene expression in S. moorei was evaluated by quantitative RT-PCR.Results: The green tea extract as well as EGCG inhibited the growth of S. moorei, with MIC values of 500 and 250 mu g/ml, respectively. Transmission electron microscopy analysis and a permeabilization assay brought evidence that the bacterial cell membrane was the target of green tea polyphenols. Regarding the effects of green tea polyphenols on the S. moorei colonization properties, it was found that biofilm formation on EGCG-treated surfaces was significantly affected, and that green tea extract and EGCG can cause the eradication of pre-formed S. moorei biofilms. Moreover, both the green tea extract and EGCG were found to reduce the adherence of S. moorei to oral epithelial cells. The beta-galactosidase activity of S. moorei, which plays a key role in VSC production, was dose-dependently inhibited by green tea polyphenols. In addition, EGCG at 1/2 MIC significantly decreased the beta-galactosidase gene expression.Conclusion: Our study brought evidence to support that green tea polyphenols possess a number of properties that may contribute to reduce S. moorei-related halitosis. Therefore, these natural compounds may be of interest to be used to supplement oral healthcare products.

Scaffold nanoestruturado utilizando-se celulose bacteriana/fosfatos de cálcio para regeneração óssea

Pós-graduação em Biotecnologia - IQ

Organization of Sporulated Oocysts of Eimeria funduli in the Gulf Killifish, Fundulus grandis

Oocysts of Eimeria funduli were studied by transmission electron microscopy in naturally-infected livers of the Gulf killifish, Fundulus grandis. Tissues were cryo-processed because membranous structures in the oocyst appear to hinder routine fixation and embedment. The oocyst wall (about 25 nm thick) was adjacent to the host cell and consisted of an outer membrane that limited the host cell cytoplasm and an inner membrane separated from the outer membrane by a narrow space. In some specimens, dense material was applied to the inner face of the inner membrane. Individual sporocysts were surrounded by a membranous "veil" (about 25 nm thick) that consisted of two unit membranes. Sporopodia, projections of the sporocyst wall, supported the veil. The sporocyst wall (130-150 nm thick) consisted of two layers, a thin electron-lucent outer layer (about 10 nm thick) and a thick electron dense inner layer (about 130 nm thick). Depending on the plane of section, the inner layer had transverse striations with periods of 3 to 4 nm or 12 to 15 nm. A narrow fissure, broadest at the anterior pole of the sporocyst, extended about one-third the length of the sporocyst wall. The posterior pole of the sporocyst was characterized by a bulbous swelling. Although this swelling resembled a Stieda body in light microscopic preparations, ultrastructurally, the swelling was a knoblike thickening in the sporocyst wall and did not plug a gap in this wall

Ultrastructure of the Interface Between Stages of Eimeria funduli/i> (Apicomplexa) and Hepatocytes of the Longnose Killifish, Fundulus similis

The interface between stages of Eimeria funduli and hepatocytes of the experimentally infected killifish Fundulus similis was studied ultrastructurally. Parasitophorous vacuoles (PV's) in which meronts, macrogamonts, and microgamonts developed were lined by an inner, smooth membrane and an outer, ribosome-studded membrane. The outer membrane bordered on the cytoplasm of the host cell, whereas the inner one limited the PV. The origins of these membranes have not been determined with certainty, but images were observed in which both membranes appeared to be continuous with the outer nuclear membrane of the host cell. Furthermore, the outer PV membrane was continuous with membranes of rough endoplasmic reticulum in the host cell. For stages which were rapidly growing or differentiating, the inner membrane blebbed into the PV. Blebbing ceased and ribosomes detached from the outer membrane after maturation of the meront or fertilization of the macrogamont. Blebbing appears to be a mechanism by which nutrients transfer from the host to the parasite. During sporogony, the inner PV membrane acquired a thin layer of electron dense material, but otherwise membranes lining the PV remained intact. The two PV membranes, probably together with dense material of parasitic origin lining the inner membrane, appear to serve as the oocyst wall enclosing the sporocysts until they are released in the intermediate host.

Immune receptors and adhesion molecules in human pulmonary leptospirosis

Pulmonary involvement in leptospirosis has been increasingly reported in the last 20 years, being related to the severity and mortality of the disease. The pathogenesis of pulmonary hemorrhage in leptospirosis is not understood. Lung endothelial cells have been proposed as targets in the pathogenesis of lung involvement in leptospirosis through the activation of Toll-like receptor 2 or the complement system, which stimulates the release of cytokines that lead to the activation of adhesion molecules. The aim of this study was to investigate the involvement of immune pathways and of the intercellular and vascular cell adhesion molecules (intercellular adhesion molecule and vascular cell adhesion molecule, respectively) in the lungs of patients with pulmonary involvement of leptospirosis. We studied the lungs of 18 patients who died of leptospirosis and compared them with 2 groups of controls: normal and noninfectious hemorrhagic lungs. Using immunohistochemistry and image analysis, we quantified the expression of the C3a anaphylatoxin receptor, intercellular adhesion molecule, vascular cell adhesion molecule, and Toll-like receptor 2 in small pulmonary vessels and in the alveolar septa. There was an increased expression of intercellular adhesion molecule (P <.03) and C3a anaphylatoxin receptor (P <.008) in alveolar septa in the leptospirosis group compared with the normal and hemorrhagic controls. In the vessels of the leptospirosis group, there was an increased expression of intercellular adhesion molecule (P=.004), vascular cell adhesion molecule (P=.030), and Toll-like receptor 2 (P=.042) compared with the normal group. Vascular cell adhesion molecule expression in vessels was higher in the leptospirosis group compared with the hemorrhagic group (P=.015). Our results indicate that immune receptors and adhesion molecules participate in the phenomena leading to pulmonary hemorrhage in leptospirosis. (C) 2012 Elsevier Inc. All rights reserved.