Annexin II regulates multivesicular endosome biogenesis in the degradation pathway of animal cells

Proteins of the annexin family are believed to be involved in membrane-related processes, but their precise functions remain unclear. Here, we have made use of several experimental approaches, including pathological conditions, RNA interference and in vitro transport assays, to study the function of annexin II in the endocytic pathway. We find that annexin II is required for the biogenesis of multivesicular transport intermediates destined for late endosomes, by regulating budding from early endosomes-but not the membrane invagination process. Hence, the protein appears to be a necessary component of the machinery controlling endosomal membrane dynamics and multivesicular endosome biogenesis. We also find that annexin II interacts with cholesterol and that its subcellular distribution is modulated by the subcellular distribution of cholesterol, including in cells from patients with the cholesterol-storage disorder Niemann-Pick C. We conclude that annexin II forms cholesterol-containing platforms on early endosomal membranes, and that these platforms regulate the onset of the degradation pathway in animal cells.

Measurement of muscle contraction with ultrasound imaging

To investigate the ability of ultrasonography to estimate musactivity, we measured architectural parameters (pennation angles, fascicle lengths, and muscle thickness) of several human muscles (tibialis anterior, biceps brachii, brachialis, transversus abdominis, obliquus internus abdominis, and obliquus externus abdominis) during isometric contractions of from 0 to 100% maximal voluntary contraction (MVC). Concurrently, electromyographic (EMG) activity was measured with surface (tibialis anterior only) or fine-wire electrodes. Most architectural parameters changed markedly with contractions up to 30% MVC but changed little at higher levels of contraction. Thus, ultrasound imaging can be used to detect low levels of muscle activity but cannot discriminate between moderate and strong contractions. Ultrasound measures could reliably detect changes in EMG of as little as 4% MVC (biceps muscle thickness), 5% MVC (brachialis muscle thickness), or 9% MVC (tibialis anterior pennation angle). They were generally less sensitive to changes in abdominal muscle activity, but it was possible to reliably detect contractions of 12% MVC in transversus abdominis (muscle length) and 22% MVC in obliquus internus (muscle thickness). Obliquus externus abdominis thickness did not change consistently with muscle contraction, so ultrasound measures of thickness cannot be used to detect activity of this muscle. Ultrasound imaging can thus provide a non-invasive method of detecting isometric muscle contractions of certain individual muscles.

GRIP domain-mediated targeting of two new coiled-coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the traps-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of Hela cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the traps-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.

Contextual binding of p120ctn to E-cadherin at the basolateral plasma membrane in polarized epithelia

E-cadherin-catenin complexes mediate cell-cell adhesion on the basolateral membrane of epithelial cells. The cytoplasmic tail of E-cadherin supports multiple protein interactions, including binding of beta-catenin at the C terminus and of p120(ctn) to the juxtamembrane domain. The temporal assembly and polarized trafficking of the complex or its individual components to the basolateral membrane are not fully understood. In Madin-Darby canine kidney cells at steady state and after treatment with cycloheximide or temperature blocks, E-cadherin and beta-catenin localized to the Golgi complex, but p120ctn was found only at the basolateral plasma membrane. We previously identified a dileucine sorting motif (Leu(586)-Leu(587), termed S1) in the juxtamembrane domain of E-cadherin and now show that it is required to target full-length E-cadherin to the basolateral membrane. Removal of S1 resulted in missorting of E-cadherin mutants (EcadDeltaS1) to the apical membrane; beta-catenin was simultaneously missorted and appeared at the apical membrane. p120(ctn) was not mistargeted with EcadDeltaS1, but could be recruited to the E-cadherin-catenin complex only at the basolateral membrane. These findings help define the temporal assembly and sorting of the E-cadherin-catenin complex and show that membrane recruitment of p120(ctn) in polarized cells is contextual and confined to the basolateral membrane.

Characterization of E-cadherin endocytosis in isolated MCF-7 and Chinese hamster ovary cells - The initial fate of unbound E-cadherin

The endocytosis of E-cadherin has recently emerged as an important determinant of cadherin function with the potential to participate in remodeling adhesive contacts. In this study we focused on the initial fate of E-cadherin when it predominantly exists free on the cell surface prior to adhesive binding or incorporation into junctions. Surface-labeling techniques were used to define the endocytic itinerary of E-cadherin in MCF-7 cells and in Chinese hamster ovary cells stably expressing human E-cadherin. We found that in this experimental system E-cadherin entered a transferrin-negative compartment before transport to the early endosomal compartment, where it merged with classical clathrin-mediated uptake pathways. E-cadherin endocytosis was inhibited by mutant dynamin, but not by an Eps15 mutant that effectively blocked transferrin internalization. Furthermore, sustained signaling by the ARF6 GTPase appeared to trap endocytosed E-cadherin in large peripheral structures. We conclude that in isolated cells unbound E-cadherin on the cell surface is predominantly endocytosed by a clathrin-independent pathway resembling macropinocytotic internalization, which then fuses with the early endosomal system. Taken with earlier reports, this suggests the possibility that multiple pathways exist for E-cadherin entry into cells that are likely to reflect cell context and regulation.

alpha-conotoxins PnIA and [A10L] PnIA stabilize different states of the alpha 7-L247T nicotinic acetylcholine receptor

The effects of the native alpha-conotoxin PnIA, its synthetic derivative [ A10L] PnIA and alanine scan derivatives of [ A10L] PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L] PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nM, respectively. Rates of onset of inhibition were similar for PnIA and [ A10L] PnIA; however, the rate of recovery was slower for [ A10L] PnIA, indicating that the increased potency of [ A10L] PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [ A10L] PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [ A10L] PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nM. In contrast, [ A10L] PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [ A10L] PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [ A10L] PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.

Structure of the HERG K+ channel S5P extracellular linker - Role of an amphipathic alpha-helix in C-type inactivation

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp(585)-Ile(593) and Gly(604)-Tyr(611) of the channel. The Trp(585)-Ile(593) helix has distinct hydrophilic and hydrophobic surfaces. The Gly(604)-Tyr(611) helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.

Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

The formative platform of the Congress of Panama (1810-1826): the Pan-American conjecture revisited

This article examines the formative platform of the Congress of Panama of 1826. It seeks to support the hypothesis that the nature and scope of the first test of integration in the Western Hemisphere depended critically on the platform created by Simón Bolívar and other Latin American Independence heroes from the Declaration of Independence of Venezuela in 1810 until the last bilateral agreement of 1826. In that respect, it corroborates the Latin American Identity of the initiative.

The undergraduate level (1st study cycle) as a fundamental platform/standard for the development of Urban and Regional Planning in higher education programs

LUDA is a research project of Key Action 4 "City of Tomorrow & Cultural Heritage" of the programme "Energy, Environment and Sustainable Development" within the Fifth Framework Programme of the European Commission

O antropólogo Herbert Baldus

Herbert Baldus foi um antropólogo teuto-brasileiro que exerceu importante papel na constituição da pesquisa e dos conhecimentos antropológicos no Brasil. Seu trabalho científico se desenvolveu intimamente ligado ao curso de sua própria vida, que transcorreu, em sua maior parte, neste país, dedicada ao ensino, à pesquisa, à divulgação científica e à tentativa de instituir uma política indigenista comprometida com a preservação das etnias indígenas. A contribuição de seu pensamento teórico, tendo iniciado com explanações sobre as culturas materiais e não materiais, passou por abordagens funcionalistas e estruturalistas, lançando as bases dos estudos das sociedades indígenas em situação de contato e de mudança cultural.

Encenação e interpretação de Ella de Herbert Achternbusch

Foi Jean-Pierre Sarrazac quem me deu o texto, aí por volta de 1985, depois de eu ter encenado o seu Lázaro também ele sonhava com o Eldorado, nas instalações dos Modestos, encenação que ele viera ver aquando da sua primeira viagem ao Porto. Disse-me que era um texto para mim. Na primeira leitura, o desejo de fazer a peça colou-se-me ao corpo. Não que pudesse antever uma experiência que me marcaria profundamente de tanto procurar dar corpo a esse ser galináceo que de ora em diante me habitaria. Ler Ella foi, na altura, a descoberta das possibilidades de um teatro que fazia do que era pobre, da infra-língua, dos neurónios desaparafusados e do corpo deficiente, a matéria de uma teatralidade insuspeitada, de um teatro ainda por fazer. Na realidade a primeira abordagem foi difícil, o francês estropiado da tradução não era de leitura imediata e a percepção que tive da relevância da peça foi mais intuitiva, mais sensação do que compreensão. O texto tornou-se mais claro pouco tempo depois ao ler Théâtres Intimes do Sarrazac, a sua tese sobre a simbiose entre o íntimo e o político como futuro do teatro e ainda as suas considerações sobre o récit de vie, “relato de uma vida” à falta de melhor tradução, no teatro de Beckett e de Achternbush. A vontade de fazer a peça foi ficando, mas a oportunidade não surgia. Tinha saído de Évora em confronto com o teatro que lá se fazia e procurava justamente essa dimensão subjectiva que me parecia necessária a um teatro da história que continuava a defender e querer praticar – Ella era para mim a revelação desse teatro, uma palavra dita na primeira pessoa, mas dita como negação do sujeito, palavra tomada de empréstimo desde logo pelo autor. Herbert Achternbusch fala de um familiar próximo: «Ella é minha tia, eu sou o seu tutor», de uma realidade em que quotidiano e história se reencontram num contínuo fluxo e refluxo de causas e consequências. Ele escutou a palavra de Ella e transpô-la para cena, fazendo do filho, Joseph, o seu fiel depositário. Duplo empréstimo, portanto, que sinaliza o mutismo e a inacção do verdadeiro sujeito do relato de vida. Se é o nome da mãe, Ella, que dá título à peça, a sua história de vida só nos chega regurgitada pelo filho que lhe está para sempre umbilicalmente ligado. A oportunidade surgiu quando a companhia de Coimbra Escola da Noite, que pretendia fazer Susn do mesmo autor, me possibilitou realizar a encenação com produção sua. Foi nesse contexto e já em 1992 que decidi fazer o espectáculo com a equipa do Teatro da Rainha, que entretanto começava a refazer-se autonomamente. A essa equipa juntava-se agora a Amélia Varejão, nome histórico do teatro português, vinda do longínquo TEP de António Pedro, mestra de costura e figura excepcional em cena que fizera connosco muitos guarda-roupas e com quem tinha uma relação de grande proximidade, amizade e respeito profissional. Pela minha parte, decidi encenar Ella e também interpretar Joseph, tarefa que teria sido impossível sem a orientação, assumida como direcção de ensaios, da Isabel Lopes, que vinha de Évora no intervalo das suas tarefas de actriz no CENDREV. O que foi esta experiência de encenação feita no corpo do Joseph? Foi fundamentalmente descobrir duas coisas: uma, o modo feminino do comportamento gestual de uma criatura que toda a vida fez trabalhos forçados, violentos, outra, a descoberta das etapas sincopadas, desfazendo as brancas mentais de uma cabeça fundida e incapaz de lógica, de raciocínio, através da memória imediata do que é gestual e físico, realizando a única acção concebida por Achternbusch para a execução da peça, fazer um café. Essa acção única, partida em mil e um fragmentos e dispersões, foi realmente construída no trabalho de ensaios como pura descoberta de jogo apoiada pela definição do espaço e pela manipulação dos objectos. Poderei dizer que encenar Ella foi descobrir a teatralidade de um corpo bloqueado por uma cabeça limitada pela deficiência desde o nascimento, deficiência essa acrescentada pela experiência de vida e pelas circunstâncias históricas. Pela sua rebeldia, a sua não conformação às soluções de aniquilamento do eu, Ella foi submetida durante toda a vida a uma tortura constante e se há uma descoberta que tenha feito com esta peça é a de que a vitalidade de uma sobrevivente não morre diante da maior repressão e a de que a rebeldia salutar pode expressar alegria vital na condição mais inumana. Nenhum ódio, nenhuma raiva e uma capacidade de surpresa perante o mais acessível e irrelevante face à biografia trágica, um moinho de café estimado e tratado como um objecto de altar, o pouco que se tem como um céu alcançado, o café, extraordinária nova possibilidade e prazer – a peça desenrola-se já a partir da sociedade de consumo e a sua retrospectiva elabora-se a partir desse presente. Um outro aspecto decisivo foi descobrir a comicidade como uma via paradoxal do trágico contemporâneo, de uma infra-tragédia que oscila entre a incontinência verbal e a afasia. Em Ella, a comicidade da palavra e do gesto não são uma via menor em termos dramáticos, pelo contrário, amplificam as possibilidades autenticamente populares da expressão linguística. E é importante referir nesta introdução a extraordinária tradução da Profª Idalina Aguiar e Melo cujo trabalho de procura dos equivalentes linguísticos do bávaro alemão de Achternbusch e da palavra deficiente, estropiada, foi notável revelando um profundo conhecimento de falares e expressões regionais e uma capacidade inventiva extraordinária da palavra agramatical e falha de lógica vinda de uma cabeça muito particular.

Development and validation of a novel method for the analysis of chlorinated pesticides in soils using microwave-assisted extraction–headspace solid phase microextraction and gas chromatography–tandem mass spectrometry

A new procedure for determining eleven organochlorine pesticides in soils using microwave-assisted extraction (MAE) and headspace solid phase microextraction (HS-SPME) is described. The studied pesticides consisted of mirex, α- and γ-chlordane, p,p’-DDT, heptachlor, heptachlor epoxide isomer A, γ-hexachlorocyclohexane, dieldrin, endrin, aldrine and hexachlorobenzene. The HS-SPME was optimized for the most important parameters such as extraction time, sample volume and temperature. The present analytical procedure requires a reduced volume of organic solvents and avoids the need for extract clean-up steps. For optimized conditions the limits of detection for the method ranged from 0.02 to 3.6 ng/g, intermediate precision ranged from 14 to 36% (as CV%), and the recovery from 8 up to 51%. The proposed methodology can be used in the rapid screening of soil for the presence of the selected pesticides, and was applied to landfill soil samples.

Analysis of PCBs in soils and sediments by microwave-assisted extraction, headspace-SPME and high resolution gas chromatography with ion-trap tandem mass spectrometry

A procedure for the determination of seven indicator PCBs in soils and sediments using microwave-assisted extraction (MAE) and headspace solid-phase microextraction (HS-SPME) prior to GC-MS/MS is described. Optimization of the HS-SPME was carried out for the most important parameters such as extraction time, sample volume and temperature. The adopted methodology has reduced consumption of organic solvents and analysis runtime. Under the optimized conditions, the method detection limit ranged from 0.6 to 1 ng/g when 5 g of sample was extracted, the precision on real samples ranged from 4 to 21% and the recovery from 69 to 104%. The proposed method, which included the analysis of a certified reference material in its validation procedure, can be extended to several other PCBs and used in the monitoring of soil or sediments for the presence of PCBs.

Screening of grapes and wine for azoxystrobin, kresoxim-methyl and trifloxystrobin fungicides by HPLC with diode array detection

The Quinone outside Inhibitors (QoI) are one of the most important and recent fungicide groups used in viticulture and also allowed by Integrated Pest Management. Azoxystrobin, kresoxim-methyl and trifloxystrobin are the main active ingredients for treating downy and powdery mildews that can be present in grapes and wines. In this paper, a method is reported for the analysis of these three QoI-fungicides in grapes and wine. After liquid–liquid extraction and a clean-up on commercial silica cartridges, analysis was by isocratic HPLC with diode array detection (DAD) with a run time of 13 min. Confirmation was by solid-phase micro-extraction (SPME), followed by GC/MS determination. The main validation parameters for the three compounds in grapes and wine were a limit of detection up to 0.073mg kg-1, a precision not exceeding 10.0% and an average recovery of 93% ±38.