PaCATB, a secreted catalase protecting Podospora anserina against exogenous oxidative stress

A differential mass spectrometry analysis of secreted proteins from juvenile and senescent Podospora anserina cultures revealed age-related differences in protein profiles. Among other proteins with decreased abundance in the secretome of senescent cultures a catalase, termed PaCATB, was identified. Genetic modulation of the abundance of PaCATB identified differential effects on the phenotype of the corresponding strains. Deletion of PaCatB resulted in decreased resistance, over-expression in increased resistance against hydrogen peroxide. While the lifespan of the genetically modified strains was found to be unaffected under standard growth conditions, increased exogenous hydrogen peroxide stress in the growth medium markedly reduced the lifespan of the PaCatB deletion strain but extended the lifespan of PaCatB over-expressors. Overall our data identify a component of the secretome of P. anserina as a new effective factor to cope with environmental stress, stress that under natural conditions is constantly applied on organisms and influences aging processes

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi

Comparison of the Proteomes of Three Yeast Wild Type Strains: CEN.PK2, FY1679 and W303

Yeast deletion strains created during gene function analysis projects very often show drastic phenotypic differences depending on the genetic background used. These results indicate the existence of important molecular differences between the CEN.PK2, FY1679 and W303 wild type strains. To characterise these differences we have compared the protein expression levels between CEN.PK2, FY1679 and W303 strains using twodimensional gel electrophoresis and identified selected proteins by mass spectrometric analysis. We have found that FY1679 and W303 strains are more similar to each other than to the CEN.PK2 strain. This study identifies 62 proteins that are differentially expressed between the strains and provides a valuable source of data for the interpretation of yeast mutant phenotypes observed in CEN.PK2, FY1679 and W303 strains

Molecular adaptations to advanced fungus farming in leaf-cutting ant symbiosis

This paper addresses several aspects of legal regulation concerning cosmetics, homeopathy products and medical devices. The Portuguese legal framework, based mainly upon European directives, is analyzed concerning the administrative legal environment of the production, distribution and marketing of these products. It is stressed that legislation aims to achieve a balance between the values of free trade and enterprise, on one side, and the protection of public health protection, on the other side.1. Cosméticos – Regime jurídico dos cosméticos (Decreto-Lei n.º 296/98, de 25 de Setembro; Decreto-Lei n.º 100/2001, de 28 de Março; Decreto-Lei n.º 206/99, de 9 de Junho). 1.1. Noção funcional de produtos cosméticos e de higiene corporal, ilustrada mediante uma lista (indicativa) de exemplos por categorias de produtos cosméticos e de higiene corporal. 1.2. Desnecessidade de obtenção de autorização administrativa prévia, mas dever de notificação ao INFARMED. 1.3. Requisitos de qualidade e regras de composição e de experimentação (Decreto-Lei n.º 100/2001, de 28 de Março, alterado pelo Decreto-Lei n.º 151/2003, de 11 de Julho). 1.4. Obrigação de assistência por um técnico responsável. 1.5. Rotulagem. 1.6. Requisitos da actividade industrial. 1.7. A protecção da confidencialidade (Decreto-Lei n.º 206/99, de 9 de Junho). 1.8. Sanções. 1.8.1. Poderes de controlo e fiscalização do INFARMED. 1.8.2. Suspensão da comercialização dos produtos por razões de saúde pública. 1.8. 3. As contra-ordenações 2. Produtos Homeopáticos. 2.1. Linhas gerais do Regime jurídico dos produtos homeopáticos (Decreto-Lei n.º 94/95, de 9 de Maio). 2.1.1. Garantia da qualidade e da segurança de utilização dos produtos homeopáticos como salvaguarda da saúde pública.2.1.2. Garantia aos seus utilizadores do fornecimento de informações claras sobre o seu carácter homeopático e a sua inocuidade). 2.2. Noção e modalidades de produtos homeopáticos. 2.2.1. Medicamentos homeopáticos. 2.2.2. Produtos farmacêuticos homeopáticos. 2.3. Delimitação do âmbito de aplicação da lei dos produtos homeopáticos aos produtos farmacêuticos homeopáticos e aplicação do regime jurídico dos medicamentos para uso humano (Decreto-Lei n.º 72/91, 8.2) aos medicamentos homeopáticos. 2.3.1. Comercialização de medicamentos homeopáticos entre fabricantes, grossistas, laboratórios e farmácias. 2.3.2. Venda de medicamentos homeopáticos ao público. 2.4. Regime de registo simplificado da introdução no mercado dos produtos farmacêuticos homeopáticos. 2.4.1. O pedido de registo. 2.4.2. Necessidade de autorização para o fabrico de produtos farmacêuticos homeopáticos. 2.4.3. Exigência de direcção técnica. 2.4.4. Requisitos relativos à rotulagem e ao folheto informativo. 2.5. Fiscalização e contra-ordenações. 3. Dispositivos Médicos – Regime jurídico dos dispositivos médicos (Decreto-Lei n.º 273/95, de 23 de Outubro, alterado pelo Decreto-Lei n.º 30/2003, de 14 de Fevereiro). 3.1. Noção e modalidades de dispositivos médicos. 3.2. Delimitação positiva e negativa do âmbito de aplicação do regime geral dos dispositivos médicos; regimes especiais, como o dos dispositivos médicos para diagnóstico in vitro (Decreto-Lei n.º 189/2000, de 12 de Agosto - que transpõe a Directiva 98/79/CE do Parlamento Europeu e do Conselho, de 27 de Outubro). 3.3. Requisitos de colocação no mercado. 3.3.1 As normas técnicas e os procedimentos de avaliação da conformidade. 3.3.2. Cláusula de salvaguarda – os poderes especiais do presidente do Conselho de Administração do INFARMED. 3.4. O sistema de vigilância (vide Portaria n.º 196/2004, de 1 de Março: aprova o Regulamento do Sistema Nacional de Vigilância de Dispositivos Médicos). 3.5. Fiscalização e contraordenaçõe

Chlorination and oxidation of the extracellular matrix protein laminin and basement membrane extracts by hypochlorous acid and myeloperoxidase

Basement membranes are specialized extracellular matrices that underlie arterial wall endothelial cells, with laminin being a key structural and biologically-active component. Hypochlorous acid (HOCl), a potent oxidizing and chlorinating agent, is formed in vivo at sites of inflammation via the enzymatic action of myeloperoxidase (MPO), released by activated leukocytes. Considerable data supports a role for MPO-derived oxidants in cardiovascular disease and particularly atherosclerosis. These effects may be mediated via extracellular matrix damage to which MPO binds. Herein we detect and quantify sites of oxidation and chlorination on isolated laminin-111, and laminin in basement membrane extracts (BME), by use of mass spectrometry. Increased modification was detected with increasing oxidant exposure. Mass mapping indicated selectivity in the sites and extent of damage; Met residues were most heavily modified. Fewer modifications were detected with BME, possibly due to the shielding effects. HOCl oxidised 30 (of 56 total) Met and 7 (of 24) Trp residues, and chlorinated 33 (of 99) Tyr residues; 3 Tyr were dichlorinated. An additional 8 Met and 10 Trp oxidations, 14 chlorinations, and 18 dichlorinations were detected with the MPO/H2O2/Cl- system when compared to reagent HOCl. Interestingly, chlorination was detected at Tyr2415 in the integrin-binding region; this may decrease cellular adhesion. Co-localization of MPO-damaged epitopes and laminin was detected in human atherosclerotic lesions. These data indicate that laminin is extensively modified by MPO-derived oxidants, with structural and functional changes. These modifications, and compromised cell-matrix interactions, may promote endothelial cell dysfunction, weaken the structure of atherosclerotic lesions, and enhance lesion rupture. Keywords: Extracellular matrix, Hypochlorous acid, Laminin, Protein oxidation, 3-chlorotyrosine, Myeloperoxidas

The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments

INTRODUCTION: Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. RESULTS: Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell--along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. SUMMARY: We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance