Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: Involvement of an acidic targeting motif

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen I protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of the trans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process.

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.

Early pregnancy factor suppresses the infiltration of lymphocytes and macrophages in the spinal cord of rats during experimental autoimmune encephalomyelitis but has no effect on apoptosis

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties that has been shown to suppress acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP) in Lewis rats. EAE is associated with infiltration of the central nervous system (CNS) with inflammatory cells. Spontaneous recovery involves the loss of T lymphocytes from the CNS and the selective apoptosis of Vbeta8.2(+) cells. In the present study, T cell, macrophage (CD11b/c(+)) and B cell (CD45RA(+)) populations in spinal cord and popliteal lymph nodes (LN) of Lewis rats with EAE were quantitated and apoptosis was studied. Rats were treated with EPF or vehicle. Following treatment on day 14 after inoculation with MBP, neither 1 x 100 mug nor 2 x 100 mug doses of EPF affected the total number of cells infiltrating the spinal cord on day 15, although the higher dose caused a decrease in the number of CD5(+) and CD11b/c(+) cells. Treatment with 2 x 100 mug/day from days 10 to 14 decreased the total number of infiltrating cells, and the numbers of CD5(+), CD11b/c(+) and CD45RA(+) cells. Apoptosis was unaffected. No alteration on the number or type of inflammatory cells in the popliteal LN was observed after treatment on days 10-14. However, treatment with EPF from days 0 to 11 increased the total number of T and B cells and CD5(+) T cells found on day 12 in the LN. Similarly, there was an increase in the frequency of MBP-reactive cells in the LN as determined by limiting dilution analysis. These results suggest that EPF treatment reduces the numbers of lymphocytes and macrophages in the CNS, possibly through an effect on cell trafficking. (C) 2003 Elsevier B.V. All rights reserved.

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.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response [1, 2]. TNFalpha plays a key role in inflammatory disease [3]; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types [4, 5] was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

Identification of cholinoceptive glycinergic neurons in the mammalian retina

The light-evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain beta2alpha3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of beta2alpha3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine-immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and gamma-aminobutyric acid (GABA)(A) receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of beta2alpha3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of beta2alpha3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic beta2alpha3 nAChRs. (C) 2002 Wiley-Liss, Inc.

A política antidrogas brasileira : velhos dilemas

O debate atual sobre drogas tem sido organizado em torno de discursos científicos que tendem a configurar a questão ora como problema de segurança pública (relacionado ao tráfico e à repressão), ora como problema de saúde pública (relacionado à repressão da demanda por um lado e à redução de danos por outro). O presente texto traz uma reflexão que busca configurar como a política de enfrentamento às drogas no Brasil enseja em suas proposições uma luta entre as lógicas de segurança pública e de saúde pública expressas no embate entre as duas políticas instituídas pelo governo brasileiro no enfrentamento à questão – a política nacional antidrogas regulamentada em 2003 pela Secretaria Nacional Antidrogas (estrutura criada no governo Fernando Henrique Cardoso – FHC - por meio da medida provisória nº 1669, de 1998, e modificada no governo Lula para "Política Pública Sobre Drogas") e a Política de Atenção Integral ao Usuário de Álcool e Drogas do Ministério da Saúde (também formulada no governo FHC)

Vendas de bebidas alcóolicas : questões (im)pertinentes

O objetivo deste estudo é analisar a disponibilidade e o acesso à bebida alcoólica num bairro da cidade de Vitória/ES. Os dados foram obtidos através de pesquisa de campo na região selecionada utilizando a observação simples e a entrevista através da aplicação de questionários numa amostra de 10% dos estabelecimentos encontrados. Os pontos de venda funcionam 7 dias por semana; 68,8% vendem a credito e a um preço médio de R$ 0,41 (a dose de cachaça). 93,8% dos entrevistados não solicitam documento de identidade ao cliente antes de lhe vender bebidas. A relação entre número de moradias e número de pontos de venda foi de 3:1. A alta concentração de estabelecimentos que vendem bebidas alcoólicas no bairro aponta para a necessidade de pensar o entorno (regiões vizinhas) da mesma. Estas regiões envolvem áreas marginalizadas onde ocorre tráfico de drogas, fazendo da região estudada uma área importante para o comércio, pela facilidade de acesso aos outros bairros adjacentes da cidade.

As políticas da insegurança : da Scuderie Detetive Le Cocq às masmorras do novo Espírito Santo

A presente dissertação tem como objetivo a análise das políticas de segurança pública e justiça criminal no Espírito Santo entre 1989 e 2013, utilizando metodologia historiográfica e observando a distância entre os objetivos oficiais e as consequências práticas. No primeiro capítulo, me concentro na contextualização histórica das políticas criminais, analisando a formação organizacional do sistema punitivo brasileiro. Coloco ênfase, de um lado, no processo de militarização, isto é, a adoção de hierarquia, disciplina e formação militares nas agências de segurança pública, e de outro lado, e nas sucessivas legislações penais aprovadas pelo Congresso Nacional. Tais processos nacionais se refletem no Espírito Santo, onde se difundiram “grupos de extermínio” como a Scuderie Le Cocq, mas não havia política de segurança pública. A primeira surge em meio a grave crise política, entre 1999 e 2002. Mas os seus propósitos são mais avançados com o processo de reforma administrativa após 2003, quando o governo se esforça por impôr modelos de gestão empresariais e parcerias público privadas à administração estadual, incluindo a segurança pública e sistema penitenciário. Com isto, ocorre uma rápida expansão do encarceramento seletivo em condições extremas de superlotação e violência, desenvolvendo uma indústria carcerária. No segundo capítulo, realizo uma análise na qual relaciono informações criminais, penitenciárias, econômicas e demográficas, tanto no contexto do Brasil quanto do Espírito Santo. Constato que a repressão estatal tem “preferência” por homens, negros, jovens e de baixa escolaridade; por crimes de drogas e contra o patrimônio, com a utilização cada vez maior da prisão provisória. No Espírito Santo o encarceramento seletivo cresce em maior velocidade que na média nacional, o que se reflete no perfil da população carcerária, sendo esta ainda mais negra, jovem, de baixa escolaridade e presa por tráfico e drogas e em regime provisório, com frequentes denúncias fundamentadas de torturas, mortes e desaparecimentos forçados entre as populações criminalizadas.

Benzedeiras de Maruípe: uma prática de cuidado humano em extinção

Estudo realizado com benzedeiras de uma área de saúde do município de Vitória - ES, objetivando identificá-las, conhecer suas histórias de vida e o interesse das mesmas em articularem-se com os profissionais das unidades básicas de saúde locais. Por se tratar de uma região marcada pela violência advinda do tráfico de drogas, tornou-se impossível identificar o universo dessas mulheres, face à impossibilidade de acesso a alguns desses bairros; assim posto, nossa amostra ficou limitada a cinco benzedeiras. A coleta de material do estudo se deu através de entrevistas e observações registradas em um diário de campo. O material transcrito e os apontamentos do diário de campo possibilitaram a narrativa de inspiração cartográfica deste estudo. Essas benzedeiras são mulheres entre 64 a 88 anos de idade, residem em locais inóspitos e em moradias humildes. Algumas benzem apenas crianças, outras todos aqueles que as procuram, inclusive para benzimento de seus animais. Nenhuma delas cobra e tão pouco aceita agradecimento pela atenção prestada, pois segundo elas, o agradecimento deve ser dirigido a Deus. São mulheres humildes, todas moradoras antigas da área, ora reconhecidas como importantes pelo dom que têm, ora rechaçadas como demoníacas por grupos religiosos. No tocante a uma aproximação com as equipes locais de saúde, todas as benzedeiras se mostraram avessas à ideia, no entendimento de que tal aproximação significaria uma demanda de benzimentos aumentada e obrigatória, o que contraria a lógica da atenção prestada pelas mesmas, que só benzem de acordo com a conveniência: sentindo-se bem, praticam o benzimento; estando desvitalizadas, evitam benzer. Por se tratar de mulheres idosas, as benzedeiras encontram-se ameaçadas de extinção, visto que aprender o oficio não tem sido objeto de interesse das novas gerações.

New insights into functional regulation in MS-based drug profiling

We present a novel data analysis strategy which combined with subcellular fractionation and liquid chromatography-mass spectrometry (LC-MS) based proteomics provides a simple and effective workflow for global drug profiling. Five subcellular fractions were obtained by differential centrifugation followed by high resolution LC-MS and complete functional regulation analysis. The methodology combines functional regulation and enrichment analysis into a single visual summary. The workflow enables improved insight into perturbations caused by drugs. We provide a statistical argument to demonstrate that even crude subcellular fractions leads to improved functional characterization. We demonstrate this data analysis strategy on data obtained in a MS-based global drug profiling study. However, this strategy can also be performed on other types of large scale biological data.

Chitosan-alginate microparticulate delivery system for an alternative route of administration of BCG vaccine

Immunisation against M. tuberculosis with current available BCG vaccine lacks efficacy in preventing adult pulmonary tuberculosis. Targeting nasal mucosa is an attractive option for a more effective immunization. The delivery of BCG via the intranasal route involves overcoming barriers such as crossing the physical barrier imposed by the mucus layer and ciliar remotion, cellular uptake and intracellular trafficking by antigen presenting cells. Due to its biodegradable, immunogenic and mucoadhesive properties, chitosan particulate delivery systems can act both as vaccine carrier and adjuvant, improving the elicited immune response. In this study, different combinations of Chitosan/Alginate/TPP microparticles with BCG were produced as vaccine systems. The developed microparticle system successfully modulates BCG surface physicochemical properties and promotes effective intracellular uptake by human macrophage cell lines Preliminary immune responses were evaluated after s.c. and intranasal immunisation of BALB/c mice. BCG vaccination successfully stimulated the segregation of IgG2a and IgG1, where intranasal immunisation with chitosan/alginate particulate system efficiently elicited a more equilibrated cellular/humoral immune response.

CCTα

In the cell, the correct folding of many proteins depends on the function of preexisting ones known as Molecular Chaperones (for a review see Hartl and Hayer-Hartl 2009). These, were defined as proteins that bind to and stabilize an otherwise unstable conformation of another protein, and by controlling binding and release, facilitate its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation. Molecular chaperones do not convey steric information specifying correct folding: instead, they prevent incorrect interactions within and between nonnative peptides, thus typically increasing the yield but not the rate of folding reactions. Molecular chaperones are ubiquitous and comprise several protein families that are structurally unrelated (Hartl and Hayer-Hartl 2009). The Hsp70s and the Chaperonin families have been extensively studied.