Influence of Yersinia pseudotuberculosis outer proteins (Yops) on interleukin-12, tumor necrosis factor alpha and nitric oxide production by peritoneal macrophages

An essential key to pathogenicity in Yersinia is the presence of a 70 kb plasmid (pYV) which encodes a type-III secretion system and several virulence outer proteins whose main function is to enable the bacteria to survive in the host. Thus, a specific immune response is needed in which cytokines are engaged. The aim of this study was to assess the influence of Yersinia outer proteins (Yops) released by Yersinia pseudotuberculosis on the production of the proinflammatory cytokines, interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO) by murine peritoneal macrophages. To this end, female Swiss mice were infected intravenously with wild-type Y pseudotuberculosis or with mutant strains unable to secrete specific Yops (YopE, YopH, YopJ, YopM, and YpkA). on the 7th, 14th, 21st, and 28th days after infection, the animals were sacrificed and the cytokines and NO were assayed in the peritoneal macrophages culture supernatants. A fall in NO production was observed during the course of infection with all the strains tested, though during the infection with the strains that did not secrete YopE and YopH, the suppression occurred later. There was, in general, an unchanged or sometimes increased production of TNF-alpha between the 7th and the 21st day after infection, compared to the control group, followed by an abrupt decrease on the last day of infection. The IL-12 production was also suppressed during the infection, with most of the strains tested, except with those that did not secrete YopJ and YopE. The results suggest that Yops may suppress IL-12, TNF-alpha, and NO production and that the most important proteins involved in this suppression are YopE and YopH. (C) 2004 Elsevier B.V. All rights reserved.

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A(2) homologue: An example of function versatility of snake venom proteins

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)S or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA2 homologues and, together with additional strategies, supports the concept of the presence of others bioactive sites distinct from the catalytic site in snake venom myotoxic PLA(2)s. (c) 2005 Elsevier B.V. All rights reserved.

Molecular characterization and phylogenetic analysis of JussuMP-I: A RGD-P-III class hemorrhagic metalloprotease from Bothrops jararacussu snake venom

Snake venom metalloproteases (SVMPs) embody zinc-dependent multidomain enzymes responsible for a relevant pathophysiology in envenomation. including local and systemic hemorrhage. The molecular features responsible for hemorrhagic potency of SVMPs have been associated with their multidomains structures which can target these proteins them to several receptors of different tissues and cellular types. BjussuMP-I. a SVMP isolated from the Bothrops jararacussu venom, has been characterized as a P-III hemorrhagic metalloprotease. The complete cDNA sequence of BjussuMP-I with 1641bp encodes open reading frames of 547 amino acid residues, which conserve the common domains of P-III high molecular weight hemorrhagic metalloproteases: (i) pre-pro-peptide, (ii) metalloprotease, (iii) disintegrin-like and (iv) rich cysteine domain. BjussuMP-I induced lyses in fibrin clots and inhibited collagen- and ADP-induced platelet aggregation. We are reporting, for the first time, the primary structure of an RGD-P-III class snake venom metalloprotease. A phylogenetic analysis of the BjussuMP-1 metalloprotease/catalytic domain was performed to get new insights into the molecular evolution of the metalloproteases. A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive residues were identified as important features of the SVMPs and other metalloproteases. (C) 2006 Elsevier B.V. All rights reserved.

Ultrastructural detection of proteins, lipids and carbohydrates in oocytes of Amblyomma triste (Koch, 1844) (Acari; Ixodidae) during the vitellogenesis process

The ovary of the tick Ainblyomma triste is classified as panoistic, which is characterized by the presence of oogonia without nurse and follicular cells. The present study has demonstrated that the oocytes in all developmental stages (I-IV) are attached to the ovary through a pedicel, a cellular structure that synthesizes and provides carbohydrate, lipids and proteins supplies for the oocytes during the vitellogenesis process. The lipids are deposited during all oocyte stages; they are freely distributed as observed in stages II, III and IV or they form complexes with other elements. The proteins are also deposited in all stages of the oocytes, however, in lower concentration in the stage IV. There is carbohydrate deposition from oocytes in the stage II as well as in stages III and IV. In addition, the present work has demonstrated that the oocyte yolk of A. triste has a glycolipoprotein nature and the elements are deposited in the following sequence: firstly the lipids and proteins, and finally the carbohydrates. (c) 2007 Elsevier Ltd. All rights reserved.

Proteomic profiling of the molecular targets of interactions of the mastoparan peptide Protopolybia MP-III at the level of endosomal membranes from rat mast cells

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

The Venomics of Bothrops alternatus is a Pool of Acidic Proteins with Predominant Hemorrhagic and Coagulopathic Activities

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

Purification, crystallization and preliminary X-ray diffraction analysis of a class P-III metalloproteinase (BmMP-III) from the venom of Bothrops moojeni

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Proteína da matriz do esmalte associada a R.T.G. e vidro bioativo no tratamento de furca grau III em cães

This study investigated, both histologically and histometrically, the efficacy of enamel matrix derived proteins (EMD) associated with bioactive glass (BG) and an absorbable membrane in the treatment of class III furcation defects in mongrel dogs. After surgical defect creation and chronification, the lesions were randomly divided into three groups according to the treatment employed: Test Group 1 - EMD + BG + membrane, Test Group 2 - EMD + membrane and Control Group - BG + membrane. After a 90-day healing period, the dogs were sacrificed. The descriptive analysis and the histometric data showed similar results for the experimental groups in all studied parameters (MANOVA, p > 0.05). The association of Emdogain® with bioglass and GTR, or with GTR only, showed similar results when compared with the ones obtained with bioglass associated with membrane in the treatment of class III furcation defects in dogs. The three modalities of treatment showed partial filling of the furcations, with bone and cementum regeneration limited to the apical portion of the defects.

Effects of treatment of the fat body trophocytes of Melipona quadrifasciata anthidioides nurse workers and virgin queens in culture by juvenile hormone III and ecdysterone (20-HE)

The fat body (FB) consists of two types of cells: throphocytes and oenocytes. Throphocytes are related to intermediary metabolism storing lipids, carbohydrates, and proteins while oenocytes play role in the lipids and lipoproteins production. The vitellogenin is the precursor of egg yolk (vitelline) and is synthesized on FB. The aim of this work was to analyze the effects of hormones acting in bee reproduction, as juvenile hormone (JH) and ecdisteroids (20 HE) on FB cells, where vitellogenin is synthesized. For the study were chose nurse workers that in Melipona quadrifasciata anthidioides present activated ovaries and produce eggs, and virgin queens whose ovaries are not yet activated, presenting only previtellogenic follicles. FB trophocytes from these classes of bees were cultivated in media containing different amounts of JH and 20-HE. The effects on trophocytes cytoplasm reserves of lipids, proteins, and activity of acid phosphatase were compared by observing preparations from cultured FB, treated and control, by transmission electron microscopy (TEM). The results showed that the hormones effects are related to the bee's caste and functional ovary stage. The role of acid phosphatase on mobilization of the trophocyte reserves was also determined. © 2012 Wiley Periodicals, Inc.

Purification, characterization, and specificity determination of a new serine protease secreted by penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl2, KCl, and BaCl, and partially inhibited by CuCl2, CoCl2 and totally inhibited by AlCl3 and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k cat/K m of 10,666 mM-1 s-1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k cat/K m of 7,500 mM -1 s-1. Basic and acidic side chain-containing amino acids performed best at subsite S1. Subsites S2, S3, S′ 2, and S′ 1, S ′ 3 showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k cat/K m were observed for the subsites S2, S3, and S′ 2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level. © 2012 Springer Science+Business Media New York.

Expression of opg, rank, and rankl proteins in tooth repair processes after immediate and delayed tooth

This study evaluated the immunohistochemical expression of OPG, RANK, and RANKL proteins in the repair after immediate and delayed replantation of rat teeth. Fifty-six Wistar rats (Rattus norvegicus albinus) had their maxillary right lateral incisor extracted and then replanted, according to the following conditions: group I (control; n = 8), teeth were not extracted; group II (n = 16), immediate replantation; group III (n = 16), delayed replantation without treatment; and group IV (n = 16), delayed replantation after root surface treatment (periodontal ligament removal and immersion in 2% acidulated-phosphate sodium fluoride) and calcium hydroxide intracanal dressing. Rats in group I were euthanized on the first day of the experiment, while the animals in the other groups were euthanized 10 and 60 days after replantation (n = 8/period). Hematoxylin and eosin-stained sections were obtained for histological analysis. The immunohistochemical analysis revealed expression of OPG and RANKL proteins in all groups and both postreplantation times, except for group II at 60 days. In the experimental groups, RANK expression was observed only at 10 days. In conclusion, there was strong immunostaining for the OPG-RANK-RANKL system at the earlier postreplantation time, suggesting a more effective participation of these proteins at the start of the healing process, as their expression decreased at 60 days. Copyright © 2013 by Mutaz B. Habal, MD.

Proteína derivada da matriz do esmalte associada à regeneração tecidual guiada e vidro bioativo no tratamento de lesões de furca grau III. Estudo histológico em cães

Pós-graduação em Odontologia - FOAR

Purification and structural characterization of snake venom proteins

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

The Identification and Biochemical Properties of the Catalytic Specificity of a Serine Peptidase Secreted by Aspergillus fumigatus Fresenius

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

Bone Morphogenetic Proteins: Structure, biological function and therapeutic applications

Bone Morphogenetic Proteins (BMPs) are multifunctional secreted cytokines, which belong to the TGF-beta superfamily. These glycoproteins act as a disulfide-linked homo- or heterodimers, being potent regulators of bone and cartilage formation and repair, cell proliferation during embryonic development and bone homeostasis in the adult. BMPs are promising molecules for tissue engineering and bone therapy. The present review discusses this family of proteins, their structure and biological function, their therapeutic applications and drawbacks, their effects on mesenchymal stem cells differentiation, and the cell signaling pathways involved in this process. (C) 2014 Published by Elsevier Inc.