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.

Graduate entry to medical studies: thoughts from 'down under' (editorial)

This invited editorial, reflecting on expectations of changing to graduate entry, eg enhanced maturity in the student cohort with greater self-sufficiency and taking of responsibility for learning in the context of adoption of a problem-based learning model, examines experiences of early post-change years and raises questions for contemplation by medical schools considering graduate entry.

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.

χ-conopeptide MrIA partially overlaps desipramine and cocaine binding sites on the human norepinephrine transporter

The interactions of chi-conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [H-3] norepinephrine (K-i 1.89 muM) than [H-3] dopamine (K-i 4.33 muM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA ( to 7 muM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [H-3] norepinephrine uptake for three mutations ( in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrIA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine.

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.

Phylogeny of the Sporobolus indicus complex, based on internal transcribed spacer (ITS) sequences

The entire internal transcribed spacer ( ITS) region, including the 5.8S subunit of the nuclear ribosomal DNA ( rDNA), was sequenced by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified fragments. The study included 40 Sporobolus ( Family Poaceae, subfamily Chloridoideae) seed collections from 14 putative species ( all 11 species from the S. indicus complex and three Australian native species). These sequences, along with those from two out-group species [ Pennisetum alopecuroides ( L.) Spreng. and Heteropogon contortus ( L.) P. Beauv. ex Roemer & Schultes, Poaceae, subfamily Panicoideae], were analysed by the parsimony method (PAUP; version 4.0b4a) to infer phylogenetic relationships among these species. The length of the ITS1, 5.8S subunit and ITS2 region were 222, 164 and 218 base pairs ( bp), respectively, in all species of the S. indicus complex, except for the ITS2 region of S. diandrus P. Beauv. individuals, which was 217 bp long. Of the 624 characters included in the analysis, 245 ( 39.3%) of the 330 variable sites contained potential phylogenetic information. Differences in sequences among the members of the S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur & Schinz and S. jacquemontii Kunth. collections were 0%, while differences ranged from 0 to 2% between these and other species of the complex. Similarly, differences in sequences among collections of S. laxus B. K. Simon, S. sessilis B. K. Simon, S. elongatus R. Br. and S. creber De Nardi were 0%, compared with differences of 1-2% between these four species and the rest of the complex. When comparing S. fertilis ( Steud.) Clayton and S. africanus (Poir.) Robyns & Tourney, differences between collections ranged from 0 to 1%. Parsimony analysis grouped all 11 species of the S. indicus complex together, indicating a monophyletic origin. For the entire data set, pair-wise distances among members of the S. indicus complex varied from 0.00 to 1.58%, compared with a range of 20.08-21.44% among species in the complex and the Australian native species studied. A strict consensus phylogenetic tree separated 11 species of the S. indicus complex into five major clades. The phylogeny, based on ITS sequences, was found to be congruent with an earlier study on the taxonomic relationship of the weedy Sporobolus grasses revealed from random amplified polymorphic DNA ( RAPD). However, this cladistic analysis of the complex was not in agreement with that created on past morphological analyses and therefore gives a new insight into the phylogeny of the S. indicus complex.

Editorial: Celebrating 30 years - 1973-2003

In 2003 The Australian Journal of Indigenous Education celebrates its 30th anniversary. Beginning in 1973 as The Aboriginal Child at School, the journal was a practical response to a recommendation made at the National Workshop on Aboriginal Education held in May 1971 “that a periodical publication be commenced to provide a medium for the exchange of ideas and developments in the teaching of Aborigines, for the examination of practical implications of research findings and for the recording of Aboriginal achievements” (Watts, 1973, p. 2). Funded by the Department of Aboriginal Affairs in Canberra and housed in the Department of Education and later the Aboriginal and Torres Strait Islander Studies Unit at the University of Queensland, The Aboriginal Child at School was published at two monthly intervals. It aimed to provide a medium whereby teachers in the field of Aboriginal and Torres Strait Islander education could share their thinking and their strategies for successful teaching and thereby enter into a meaningful and productive dialogue with one another (Watts, 1973, p. 2). An overarching concern of the journal was to improve and optimise children’s development and the types of pedagogies employed to provide challenging and rewarding learning experiences for Aboriginal and Torres Strait Islander children.The journal was enthusiastically embraced by a broad range of professionals and proved to be a useful resource for both experienced and beginning teachers, particularly in the primary sector.

Relat??rio de gest??o 2003

Apresenta a necessidade de vincular a capacita????o de servidores p??blicos aos objetivos estrat??gicos do governo e das institui????es, bem como formar quadros para carreiras do servi??o p??blico, a ENAP estabeleceu como diretrizes ser escola com padr??o de qualidade, espa??o de reflex??o e debate e centro de refer??ncia em gest??o das pol??ticas p??blicas.Desta forma, s??o apresentadas as atividades realizadas pela ENAP no ano de 2003, observando a Norma de Execu????o no 002/DG/SFC/CGU-PR, de 23 de dezembro de 2003

Formar dirigentes, capacitar gestores, desenvolver gerentes: do que estamos falando? A experi??ncia da ENAP-Brasil no per??odo 2003-2007

Este texto busca em sua primeira parte analisar as contribui????es ofertadas pela Carta Iberoamericana da Fun????o P??blica na constru????o de uma linguagem comum entre nossos governos e organiza????es formadoras e depois se dedica a apresentar alguns elementos contextualizadores da atua????o da Escola Nacional de Administra????o P??blica (ENAP ??? Brasil) no que diz respeito ?? forma????o e capacita????o de dirigentes p??blicos

Balan??o de gest??o 2003-2010 Escola Nacional de Administra????o P??blica

O balan??o destes oito anos de trabalho da ENAP durante os dois mandatos do Presidente Luiz In??cio Lula da Silva busca descrever a a????o desta Escola para o fortalecimento da fun????o capacita????o de servidores na administra????o p??blica federal

Relat??rio de atividades 2003

A ENAP, Escola Nacional de Administra????o P??blica, ?? uma funda????o p??blica vinculada ao Minist??rio do Planejamento, Or??amento e Gest??o, cuja miss??o ?? desenvolver compet??ncias de servidores p??blicos para aumentar a capacidade de governo na gest??o das pol??ticas p??blicas. A ENAP realizou suas a????es no ano de 2003 seguindo as de governo: difus??o de conhecimento e tecnologia gerencial para servidores p??blicos; desenvolvimento de gerentes e servidores respons??veis pela execu????o dos Planos Plurianuais; desenvolvimento de compet??ncias de gest??o de pessoas, envolvidas em pol??ticas governamentais de car??ter transversal, e difus??o de metodologias de planejamento estrat??gico para apoiar as institui????es na elabora????o do Plano Plurianual 2004-2007.

Contradi????es do processo de implementa????o de pol??ticas p??blicas: uma an??lise do Programa Bolsa Fam??lia 2003 ??? 2006

O Programa Bolsa Fam??lia, programa de transfer??ncia condicionada de renda, vem se firmando no cen??rio mundial como uma das mais expressivas iniciativas dessa natureza para o enfrentamento da pobreza e a redu????o da desigualdade. Presente em todos os 5.564 munic??pios brasileiros, caracteriza- se por ser um programa federal que n??o prescinde dos estados e, em especial, dos munic??pios para a sua execu????o. No sentido de estabelecer uma gest??o compartilhada entre todas as esferas governamentais, o governo federal tem buscado implantar mecanismos flex??veis de parceria, com defini????o clara de pap??is e compartilhamento de responsabilidades, bem como auxiliar os governos subnacionais, por meio da transfer??ncia de recursos para a gest??o, na cria????o das condi????es institucionais e de infra-estrutura necess??rias ?? operacionaliza????o de um programa dessa magnitude. Este texto analisa as rela????es intergovernamentais existentes no ??mbito do Programa Bolsa Fam??lia, identificando os avan??os e os desafios na busca da coordena????o federativa. A primeira parte, sobre o federalismo brasileiro, ap??ia-se basicamente em estudos do pesquisador Luiz Fernando Abrucio. J?? os dados e informa????es sobre o Programa foram fornecidos pelo Minist??rio do Desenvolvimento Social e Combate ?? Fome.

Educação escolar Tupinikim e Guarani : experiências de interculturalidade em aldeias de Aracruz, no Estado do Espírito Santo

A pesquisa analisa as relações entre interculturalidade, práxis e educação escolar indígena Tupinikim e Guarani do município de Aracruz, Espírito Santo, Brasil. Investiga a práxis da educação intercultural no espaço da educação escolar indígena como meio de revitalização das culturas Tupinikim e Guarani. Objetiva problematizar a formação inicial e continuada dos professores indígenas; discutir a práxis da interculturalidade no contexto da educação escolar indígena; e, identificar outros espaços educativos da cultura e educação indígena. Analisa aspectos teóricos e práticos sobre cultura (WILLIAMS, 2008; BRANDÃO, 1989; FORQUIN, 1993; CANDAU, 2011; GEERTZ, 1989), interculturalidade (D‘AMBROSIO,1996; FLEURI, 2002; 2003; SCANDIUZZI, 2009;), identidade e alteridade (MELIÁ, 2000; FREIRE, 1981; 1987; LITAIFF, 2004) e práxis (FREIRE, 1989; VÁSQUEZ, 2011; SEMERARO, 2006) e educação (escolar) indígena de acordo com a legislação vigente. Realiza pesquisa interpretativa (GEERTZ, 1989) na educação escolar indígena junto aos professores indígenas Guarani das Aldeias de Boa Esperança e Três Palmeiras (2009-2010) e professores indígenas Tupinikim da Aldeia de Comboios (2011-2013) na perspectiva de um diálogo intercultural. Contribuem nos processos investigativos para produção, sistematização e análise de dados a realização de observações, entrevistas semiestruturadas, registros no caderno de campo, fotografias, gravações em áudio e em vídeo e análise documental sobre a educação escolar indígena de Aracruz. (ANDRÉ, 2007; GIL, 1999; 2004). Os resultados deste trabalho levantam questões relativas a duas realidades de educação escolar nas comunidades indígenas pesquisadas que se constituem em aspectos de sobrevivência e desencadeia formas para interagir e reagir em defesa de sua identidade e dignidade. Nesse sentido, a escola é um local de vivências e de encontro, vista e sentida pelas lideranças e pela comunidade como uma possibilidade real para desenvolver um elo entre as formas tradicionais de vida e as formas contemporâneas. O desafio de garantir uma escola nestes termos significa concretizar a proposta de um projeto de educação escolar para os povos indígenas, constituído por especificidades de como trabalhar a terra, pelo reconhecimento de suas tradições, das línguas e da memória coletiva. Distante de apresentar respostas conclusivas propõe uma educação escolar, coletiva e participativa, que critica e dialoga com todos os envolvidos no processo educativo.

Análise histopatológica de produtos de adenotonsilectomia de janeiro de 2001 a maio de 2003

As tonsilas palatinas e faríngeas são agregados encapsulados incompletos de nódulos linfóides em contato direto com o epitélio de revestimento do trato aerodigestivo. TIPO DE ESTUDO: Estudo retrospectivo baseado na revisão de prontuários dos pacientes submetidos a adenotonsilectomias no Hospital de Clínicas da Faculdade de Medicina de Marília durante o período de janeiro de 2001 a maio de 2003. OBJETIVO: Relatar o perfil dos pacientes e as principais alterações histopatológicas em 250 pacientes com hipertrofia de tonsilas palatinas e faríngea, infecções de repetição ou ambos. MATERIAL E MÉTODOS: Análise histopatológica de 250 pacientes submetidos a adenotonsilectomia ou tonsilectomia entre adultos e crianças. RESULTADOS: Dos 250 pacientes, 117 (46,8%) são do sexo feminino e 133 (53,2%) masculino. A idade média foi de 7,3 anos e variou de 2 a 34 anos. A principal indicação cirúrgica foi a concomitância de infecções de repetição e hipertrofia de tonsilas palatinas e faríngea, sendo que em 160 (64%) foram classificadas em 3+/4+. Em 205 (82%) pacientes foi encontrada hiperplasia linfóide ou linfóide folicular. 45 (18%) também apresentaram inflamação aguda supurativa focal. Dentre estes, 2 pacientes tiveram cistos de inclusão epidermóide, outros 2 com colônias de Actinomyces sp e 1 paciente com lesão compatível com Doença da Arranhadura de Gato. DISCUSSÃO: Os dados apresentados neste trabalho nos mostram uma possível relação das tonsilites de repetição com as hipertrofias tonsilas palatinas e faríngea. CONCLUSÃO: O exame anatomopatológico de rotina das tonsilectomias apresenta uma relação custo/benefício negativa, no entanto, por problemas de ordem legal, ético, fica o médico sujeito à solicitação desse exame.