Avaliação de dois métodos de extração de DNA de material parafinado para amplificação em PCR

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

PAR(2) and Temporomandibular Joint Inflammation in the Rat

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

Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

The 20S proteasome alpha(5) subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the Lettuce mosaic potyvirus HcPro protein

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

Background: Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.Results: The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), beta-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions.Conclusion: Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/ organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.

The venomous secrets of the web droplets from the viscid spiral of the orb-weaver spider Nephila clavipes (Araneae, Tetragnatidae)

The capture web of N. clavipes presents viscous droplets, which play important roles in web mechanics and prey capture. By using scanning and transmission electron microscopy, it was demonstrated that the web droplets are constituted of different chemical environments, provided by the existence both of an aqueous and a lipid layer, which, in turn, present a suspension of tenths of vesicles containing polypeptides and/or tipids. GC/EI-MS Analysis of the contents of these vesicles led to the identification of some saturated fatty acids, such as decanoic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, octadecanoic acid, and icosanoic acid, while other components were unsaturated fatty acids, such as (Z)-tetradec-9-enoic acid, (Z)-octadec-9-enoic acid, and (Z)-icosa-11-enoic acid; and polyunsaturated fatty acids like (9Z,12Z)-octadeca-9,12-dienoic acid, (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid, and (11Z,14Z)-icosa-11,14-dienoic acid. Toxic proteins such as calcium-activated proteinase and metalloproteinase jararhagin-like precursor were also identified by using a proteomic approach, indicating the possible involvement of these enzymes in the pre-digestion of spiders' preys web-captured. Apparently, the mixture of fatty acids are relatively toxic to insects by topical application (LD50 64.3 +/- 7.6 ng mg(-1) honeybee), while the proteins alone present no topical effect; however, when injected into the prey-insects, these proteins presented a moderate toxicity (LD50 40.3 +/- 4.8 ng mg(-1) honeybee); the mixture of fatty acids and proteins is very toxic to the preys captured by the web droplets of the viscid spiral of Nephila clavipes when topically applied on them (LD50 14.3 +/- 1.8ng mg(-1) honeybee).

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

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

Purification of papain from fresh latex of Carica papaya

No presente trabalho apresenta-se um método de cristalização da papaína oriunda do látex fresco de mamão, o qual apresenta uma alta produtividade em relação aos métodos previamente descritos. A metodologia aqui descrita não envolve o uso de reagentes sulfidrílicos, a papaína foi obtida de forma praticamente pura, apresentando uma simples banda quando submetida a eletroforese, e com propriedades idênticas àquelas obtidas por outros métodos. A atividade específica foi determinada utilizando Z-gly-pNP e BAEE como substrato. A papaína obtida por essa metodologia, sem uso de substâncias tais como cisteína e ditiotreitol, apresenta-se na forma de um complexo com inibidores naturais, os quais podem ser removidos por diálise.

Thrombomodulin-independent activation of protein C and specificity of hemostatically active snake venom serine proteinases - Crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 angstrom resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl1- instead of SO42-.

Crystallization and preliminary X-ray crystallographic studies of Protac (R), a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 angstrom resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b = 63.3 and c = 48.2 angstrom, alpha = gamma = 90.0 degrees and beta = 90.8 degrees. (C) 2005 Elsevier B.V. All rights reserved.

Pseudechis australis Venomics: Adaptation for a Defense against Microbial Pathogens and Recruitment of Body Transferrin

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

Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis

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

Oral colonization by yeasts in hiv-positive patients in Brazil

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

Enzymatic Activity, Sensitivity to Antifungal Drugs and Baccharis dracunculifolia Essential Oil by Candida Strains Isolated from the Oral Cavities of Breastfeeding Infants and in Their Mothers' Mouths and Nipples

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

Presence of Staphylococcus spp. and Candida spp. in the human oral cavity

A presença de leveduras do gênero Candida e Staphylococcus na cavidade bucal humana é de extrema importância, pois podem atuar como microbiota suplementar e em determinadas situações causar doença bucal ou sistêmica. O objetivo do presente trabalho foi estudar a prevalência de Candida spp. e Staphylococcus spp. na cavidade bucal humana. Enxagüe bucal foi coletado de 68 indivíduos segundo a técnica proposta por Samaranayake e MacFarlane e a seguir semeados em ágar Sabouraud dextrose com cloranfenicol e ágar Baird-Parker. Após crescimento, os microrganismos foram isolados e identificados através de provas bioquímicas. Os dados foram analisados através de análise de variância (ANOVA). Leveduras do gênero Candida foram encontradas em 61,76% dos indivíduos examinados, sendo C. albicans a mais frequentemente isolada. Staphylococcus spp. foram isolados em 95,60% das cavidades bucais, sendo 41 cepas (63%) coagulase-negativas. Das cepas coagulase-positivas, nove eram S. aureus, 11 S. hyicus, e quatro S. schleiferi subespécie coagulans. Não foi observada correlação entre as contagens (UFC) de Candida spp. e Staphylococcus spp. encontradas nos enxagües bucais dos indivíduos examinados.