Ecological relevance of sentinels' biomarker responses: A multi-level approach

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Transcriptoma da interação de tangerina satsuma (Citrus unshiu) e laranja doce Hamlin (Citrus sinensis) infectadas com Xanthomonas citri subsp. citri, agente causal do cancro cítrico

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Atividade de fosfomonohidrolases presentes em fígado de girino de rã-touro (Lithobates catesbeianus) durante a metamorfose

Pós-graduação em Biotecnologia - IQ

Matrix metalloproteinase gene polymorphisms and oral cancer

Since oral squamous cell carcinoma (OSCC) is the most prevalent malignant cancer in the oral cavity, several researches have been performed to study the role of important enzymes in this disease. Among them, the matrix metalloproteinases (MMPs) are highlighted, due to the fact that they are proteinases responsible to degrade many extra-cellular matrix components, making possible the invasion of neoplasic cells. Important tools in cancer prognosis have been utilized aiming to correlate high levels of MMPs and OSCC, such as immunohistochemical, zymographic and mRNA detection methods. However, these techniques are usually applied after cancer detection, characterizing a curative but not a preventive medicine. Trying to make interventions before the development of the disease and making possible the identification of people at high risk and, analysis of modifications in MMP genes has been a chance for modern medicine. Recently, polymorphisms in MMP genes have been related to different neoplasias, including OSCC. Despite investigation is beginning, MMP gene polymorphisms seems to have a promising future in oral cancer research and some of the present results have shown that there are MMP polymorphisms related to an increased risk for developing oral cancer. Key words:Oral cancer, polymorphism, matrix metalloproteinase.

Location, morphology and function of nephrocytes in termites

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Thrombocytopenia and platelet hypoaggregation induced by Bothrops asper snake venom Toxins involved and their contribution to metalloproteinase-induced pulmonary hemorrhage

Thrombocytopenia and platelet dysfunction occur in patients bitten by Bothrops sp snakes in Latin America. An experimental model was developed in mice to study the effects of B. asper venom in platelet numbers and function. Intravenous administration of this venom induces rapid and prominent thrombocytopenia and ex vivo platelet hypoaggregation. The drop in platelet numbers was primarily due to aspercetin, a protein of the C-type lectin family which induces von Willebrand factor-mediated platelet aggregation/agglutination. In addition, the effect of class P-III hemorrhagic metalloproteinases on the microvessel wall also contributes to thrombocytopenia since jararhagin, a P-III metalloproteinase, reduced platelet counts. Hypoaggregation was associated with the action of procoagulant and defibrin(ogen)ating proteinases jararacussin-1 (a thrombin-like serine proteinase) and basparin A (a prothrombin activating metalloproteinase). At the doses which induced hypoaggregation, these enzymes caused defibrin(ogen)ation, increments in fibrin(ogen) degradation products and D-dimer and prolongation of the bleeding time. Incubation of B. asper venom with batimastat and α 2-macroglobulin abrogated the hypoaggregating activity, confirming the role of venom proteinases in this effect. Neither aspercetin nor the defibrin(ogen)ating and hypoaggregating components induced hemorrhage upon intravenous injection. However, aspercetin, but not the thrombin-like or the prothrombin-activating proteinases, potentiated the hemorrhagic activity of two hemorrhagic metalloproteinases in the lungs. © 2005 Schattauer GmbH, Stuttgart.

Integrated biomarker responses as environmental status descriptors of a coastal zone (Sao Paulo, Brazil)

Sao Paulo state (Brazil) has one of the most overpopulated coastal zones in South America, where previous studies have already detected sediment and water contamination. However, biological-based monitoring considering signals of xenobiotic exposure and effects are scarce. The present study employed a battery of biomarkers under field conditions to assess the environmental quality of this coastal zone. For this purpose, the activity of CYP 450, antioxidant enzymes, DNA damage, lipid peroxidation and lysosomal membrane were analysed in caged mussels and integrated using Factorial Analysis. A representation of estimated factor scores was performed in order to confirm the factor descriptions characterizing the studied areas. Biomarker responses indicated signals of mussels` impaired health during the monitoring, which pointed to the impact of different sources of contaminants in the water quality and identified critical areas. This integrated approach produced a rapid, sensitive and cost-effective assessment, which could be incorporated as a descriptor of environmental status in future coastal zones biomonitoring. (C) 2011 Elsevier Inc. All rights reserved.

Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 degrees C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. (C) 2011 Elsevier B.V. All rights reserved.

Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from L-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019331, 1245-1262, 2012.

Transcriptional response to GAA deficiency (Pompe disease) in infantile-onset patients

Pompe disease is a genetic disorder resulting from a deficiency of lysosomal acid alpha-glucosidase (GAA) that manifests as a clinical spectrum with regard to symptom severity and rate of progression. In this study, we used microarrays to examine gene expression from the muscle of two cohorts of infantile-onset Pompe patients to identify transcriptional differences that may contribute to the disease phenotype. We found strong similarities among the gene expression profiles generated from biceps and quadriceps, and identified a number of signaling pathways altered in both cohorts. We also found that infantile-onset Pompe patient muscle had a gene expression pattern characteristic of immature or regenerating muscle, and exhibited many transcriptional markers of inflammation, despite having few overt signs of inflammatory infiltrate. Further, we identified genes exhibiting correlation between expression at baseline and response to therapy. This combined dataset can serve as a foundation for biological discovery and biomarker development to improve the treatment of Pompe disease. (C) 2012 Elsevier Inc. All rights reserved.

The first cardiac transplant experience in a patient with mucopolysaccharidosis

Hunter syndrome (MPSII) is a rare X-linked lysosomal storage disorder that can affect multiple systems but primarily affects the heart. We report the case of a previously asymptomatic 23-year-old patient who had an attenuated form of MPSII and presented with refractory heart failure that required a heart transplant. The diagnosis was confirmed by detection of an increase in urinary excretion of glycosaminoglycans, a deficiency in enzymatic activity, and molecular analysis. A myocardial biopsy revealed hypertrophic cardiomyocytes, mild fibrosis, and lysosomal storage in interstitial cells. Molecular analysis identified a novel mutation in the iduronate-2-sulfatase gene. Although the clinical outcome was not favorable, we believe that this approach may be valid in end-stage heart failure. (C) 2012 Elsevier Inc. All rights reserved.

ISOLATION OF MICROSPORUM GYPSEUM IN SOIL SAMPLES FROM DIFFERENT GEOGRAPHICAL REGIONS OF BRAZIL, EVALUATION OF THE EXTRACELLULAR PROTEOLYTIC ENZYMES ACTIVITIES (KERATINASE AND ELASTASE) AND MOLECULAR SEQUENCING OF SELECTED STRAINS

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

A Transcriptomic View of the Proteome Variability of Newborn and Adult Bothrops jararaca Snake Venoms

Background: Snake bite is a neglected public health problem in communities in rural areas of several countries. Bothrops jararaca causes many snake bites in Brazil and previous studies have demonstrated that the pharmacological activities displayed by its venom undergo a significant ontogenetic shift. Similarly, the venom proteome of B. jararaca exhibits a considerable variation upon neonate to adult transition, which is associated with changes in diet from ectothermic prey in early life to endothermic prey in adulthood. Moreover, it has been shown that the Brazilian commercial antibothropic antivenom, which is produced by immunization with adult venom, is less effective in neutralizing newborn venom effects. On the other hand, venom gland transcripts of newborn snakes are poorly known since all transcriptomic studies have been carried out using mRNA from adult specimens. Methods/Principal Findings: Here we analyzed venom gland cDNA libraries of newborn and adult B. jararaca in order to evaluate whether the variability demonstrated for its venom proteome and pharmacological activities was correlated with differences in the structure of toxin transcripts. The analysis revealed that the variability in B. jararaca venom gland transcriptomes is quantitative, as illustrated by the very high content of metalloproteinases in the newborn venom glands. Moreover, the variability is also characterized by the structural diversity of SVMP precursors found in newborn and adult transcriptomes. In the adult transcriptome, however, the content of metalloproteinase precursors considerably diminishes and the number of transcripts of serine proteinases, C-type lectins and bradykinin-potentiating peptides increase. Moreover, the comparison of the content of ESTs encoding toxins in adult male and female venom glands showed some genderrelated differences. Conclusions/Significance: We demonstrate a substantial shift in toxin transcripts upon snake development and a marked decrease in the metalloproteinase P-III/P-I class ratio which are correlated with changes in the venom proteome complexity and pharmacological activities.

Isolation and characterization of moojenin, an acid-active, anticoagulant metalloproteinase from Bothrops moojeni venom

A fibrinogenolytic metalloproteinase from Bothrops moojeni venom, named moojenin, was purified by a combination of ion-exchange chromatography on DEAE-Sephacel and gel filtration on Sephacryl S-300. SDS-PAGE analysis indicated that moojenin consists of a single polypeptide chain and has a molecular mass about 45 kDa. Sequencing of moojenin by Edman degradation revealed the amino acid sequence LGPDIVSPPVCGNELLEV-GEECDCGTPENCQNE, which showed strong identity with many other snake venom metalloproteinases (SVMPs). The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the E beta-chain, and shows no effects on the gamma-chain. Moojenin showed a coagulant activity on bovine plasma about 3.1 fold lower than crude venom. The fibrinogenolytic and coagulant activities of the moojenin were abolished by preincubation with EDTA, 1,10-phenanthroline and beta-mercaptoethanol. Moojenin showed maximum activity at temperatures ranging from 30 to 40 degrees C and its optimal pH was 4.0. Its activity was completely lost at temperatures above 50 degrees C. Moojenin induced necrosis in liver and muscle, evidenced by morphological alterations, but did not cause histological alterations in mouse lungs, kidney or heart. Moojenin rendered the blood uncoagulatable when it was intraperitoneally administered into mice. This metalloproteinase may be of medical interest because of its anticoagulant activity. (C) 2012 Elsevier Ltd. All rights reserved.

Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae)

Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 mu M); CAL2 (17 kDa, pHo 5.5, km 11 mu M Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM L-Leucine-p-nitroanilide), alpha-glucosidase (90 kDa, pHo 5.0, km 5 mM with p-nitrophenyl alpha-D-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans. (C) 2012 Elsevier Ltd. All rights reserved.