Mutant huntingtin impairs the post-Golgi trafficking of brain-derived neurotrophic factor but not its Val66Met polymorphism.

Brain-derived neurotrophic factor (BDNF) polymorphism is associated with the pathophysiology of several neurodegenerative disorders, including Huntington"s disease. In view ofthese data andthe involvement of huntingtin in intracellular trafficking, we examined the intracellular transport and release of Val66Val BDNF (Val-BDNF) and Val66Met BDNF (Met-BDNF) in transfected striatal knock-in cells expressing wild-type or mutant full-length huntingtin. Colocalization studies with specific markers for endoplasmic reticulum showed no differences between the Val-BDNF and Met-BDNF and were not modified by mutant huntingtin. However, post-Golgi trafficking was altered by mutant huntingtin dependent on the BDNF form. Thus, fluorescence recovery after photobleaching (FRAP) and inverse FRAP analysis showed retention of Met-BDNF inthe Golgi apparatus with respectto Val-BDNF in wild-type cells. Strikingly, mutant huntingtin diminished post-Golgi trafficking of Val-BDNF, whereas Met-BDNF was not modified. Accordingly, a reduction in the number of transport vesicles was only observed in mutant huntingtin cells transfected with Val-BDNF but not Met-BDNF. Moreover, mutant huntingtin severely affectedthe KCl-evoked release of Val-BDNF, although it had little effect on Met-BDNF regulated release. The constitutive release of Val-BDNF or Met-BDNF in mutant cells was only slightly reduced. Interestingly, mutant huntingtin only perturbed post-Golgi trafficking of proteins that follow the regulated secretory pathway (epidermal growth factor receptor or atrial natriuretic factor), whereas it did not change those that follow the constitutive pathway (p75 NTR ). We conclude that mutant huntingtin differently affects intracellular transport and release of Val-BDNF and Met-BDNF. In addition, our findings reveal a new role for huntingtin in the regulation of the post-Golgi trafficking of the regulated secretory pathway.

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway

The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER-Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER-Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

Fatores de risco clínicos e ultra-sonográficos relacionados à litíase vesicular assintomática em mulheres

OBJETIVO: Analisar variáveis clínicas e ultra-sonográficas, como presença ou ausência de barro biliar, espessura da parede e medida transversal da vesícula biliar, idade, paridade, presença ou ausência de diabetes mellitus associadas a litíase vesicular assintomática, bem como determinar a sua prevalência em pacientes submetidas ao exame ultra-sonográfico. MATERAIS E MÉTODOS: Foram analisadas, em estudo prospectivo, 265 pacientes do sexo feminino, atendidas na Escola de Ultra-sonografia e Reciclagem Médica de Ribeirão Preto, durante o período de janeiro a setembro de 2001. RESULTADOS: Evidenciou-se diferença estatisticamente significativa relacionada à litíase da vesícula biliar e espessura da parede da vesícula biliar, barro biliar, diâmetro transverso da vesícula biliar, faixa etária, paridade, passando de 4,1% nas nulíparas para 39,1% nas multíparas e diabéticas. A prevalência de litíase na vesícula biliar em pacientes assintomáticas foi de 14,7%. CONCLUSÃO: A litíase vesicular assintomática em mulheres ocorre principalmente com o decorrer da idade e da paridade. Os achados ultra-sonográficos mais freqüentemente encontrados foram presença de barro biliar e de espessamento da parede da vesícula biliar.

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a.

Reggies/flotillins are implicated in trafficking of membrane proteins to their target sites and in the regulation of the Rab11a-dependent targeted recycling of E-cadherin to adherens junctions (AJs). Here we demonstrate a function of reggies in focal adhesion (FA) formation and α5- and β1-integrin recycling to FAs. Downregulation of reggie-1 in HeLa and A431 cells by siRNA and shRNA increased the number of FAs, impaired their distribution and modified FA turnover. This was coupled to enhanced focal adhesion kinase (FAK) and Rac1 signaling and gain in plasma membrane motility. Wild type and constitutively-active (CA) Rab11a rescued the phenotype (normal number of FAs) whereas dominant-negative (DN) Rab11a mimicked the loss-of-reggie phenotype in control cells. That reggie-1 affects integrin trafficking emerged from the faster loss of internalized antibody-labeled β1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and α5- or β1-integrin, trafficking close to the substrate-near membrane and making kiss-and-run contacts with FAs. Thus, reggie-1 in interaction with Rab11a controls Rac1 and FAK activation and coordinates the targeted recycling of α5- and β1-integrins to FAs to regulate FA formation and membrane dynamics.

Trafficking of Estrella lausannensis in human macrophages.

Estrella lausannensis is a new member of the Chlamydiales order. Like other Chlamydia-related bacteria, it is able to replicate in amoebae and in fish cell lines. A preliminary study investigating the pathogenic potential of Chlamydia-related bacteria found a correlation between antibody response to E. lausannensis and pneumonia in children. To further investigate the pathogenic potential of E. lausannensis, we determined its ability to grow in human macrophages and its intracellular trafficking. The replication in macrophages resulted in viable E. lausannensis; however, it caused a significant cytopathic effect. The intracellular trafficking of E. lausannensis was analyzed by determining the interaction of the Estrella-containing inclusions with various endocytic markers as well as host organelles. The E. lausannensis inclusion escaped the endocytic pathway rapidly avoiding maturation into phagolysosomes by preventing both EEA-1 and LAMP-1 accumulation. Compared to Waddlia chondrophila, another Chlamydia-related bacteria, the recruitment of mitochondria and endoplasmic reticulum was minimal for E. lausannensis inclusions. Estrella lausannensis appears to use a distinct source of nutrients and energy compared to other members of the Chlamydiales order. In conclusion, we hypothesize that E. lausannensis has a restricted growth in human macrophages, due to its reduced capacity to control programmed cell death.

VAP-1 in Leukocyte Trafficking

The extravasation of leukocytes from the blood stream into the tissues is a prerequisite for adequate immune surveillance and immune reaction. The leukocyte movement from the bloodstream into the tissues is mediated by molecular bonds. The bonds are formed between adhesion molecules on endothelial cells and their counterparts expressed on leukocytes. Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule mediating leukocyte interactions with endothelium. It is also an enzyme having semicarbazide sensitive amine oxidase (SSAO) activity. The SSAOactivity catalyses deamination of primary amines into corresponding aldehyde and during the enzymatic reaction hydrogen peroxide and ammonia are produced. The aim of this study was to investigate the relationship between the adhesive and enzymatic activities of VAP-1. The role of VAP-1 in leukocyte traffic was studied in vivo under normal and pathological conditions in VAP-1 deficient mice. The results from in vitro flow-based assays indicated that VAP-1 uses both SSAOactivity and its adhesive epitope to bind leukocytes, and both are perquisites for VAP-1 mediated adhesion. Furthermore, in vivo results demonstrated that leukocyte trafficking was impaired in vivo by deleting VAP-1 or inhibiting SSAO-activity. There was impairment in lymphocyte recirculation as well as leukocyte accumulation into the inflamed area. Moreover, the VAP-1 deficient mice did not show generalized defects in antimicrobial responses, whereas significant reduction in tumor progression and neovascularization was observed. These results indicate that VAP-1 could be used as a target in anti-adhesive therapies either by blocking its adhesive epitope with antibodies or by inhibiting its SSAO-activity using inhibitors. Moreover, targeting of VAP-1 may provide a new way of inhibiting neovascularization in tumors.

Intracellular membrane trafficking in Osteoclasts The role of direct Rab protein – Rac 1 interactions in the targeting of intracellular vesicles

Osteoclasts are cells responsible for bone resorption. These cells undergo extensive membrane re-organization during their polarization for bone resorption and form four distinct membrane domains, namely the ruffled border, the basolateral membrane, the sealing zone and the functional secretory domain. The endocytic/biosynthetic pathway and transcytotic route(s) are important for the resorption process, since the endocytic/biosynthetic pathway brings the specific vesicles to the ruffled border whereas the transcytotic flow is believed to transport the degraded bone matrix away from the resorption lacuna to the functional secretory domain. In the present study, we found a new transcytotic route from the functional secretory domain to the ruffled border, which may compensate membrane loss from the ruffled border during the resorption process. We also found that lipid rafts are essential for the ruffled border-targeted late endosomal pathways. A small GTP-binding protein, Rab7, has earlier been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts it is involved in the formation of the ruffled border, which displays several features of late endosomal membranes. Here we discovered a new Rab7-interacting protein, Rac1, which is another small GTP-binding protein and binds to the GTP-form of Rab7 in vitro. We demonstrated further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, this colocalization is mainly perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, we suggest that the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments, thus enabling endosomal vesicles to switch tracks from microtubules to microfilaments before their fusion to the ruffled border. We then studied the role of Rab-Rac1 interaction in the slow recycling pathway. We revealed that Rac1 also binds directly to Rab11 and to some other but not all Rab-proteins, suggesting that Rab-Rac1 interaction could be a general regulatory mechanism to direct the intracellular vesicles from microtubule mediated transport to actin filament mediated transport and vice versa. On the basis of our results we thus propose a new hypothesis for these GTPases in the regulation of intracellular membrane flow.

The Dark Side of International Business? Stakeholder Approach to Human Trafficking in Finnish Construction and Service Sectors

Human trafficking is not a new phenomenon. It has existed in various forms for ages around the world. Some researchers have even compared it to slavery, calling it the modern form of slavery in the 21st century. This study is particularly interested in the role of work-related human trafficking in Finnish business. In order for something to be called work-related human trafficking, the concepts of forced labour and human trafficking have to overlap. From the economic point of view, human trafficking is governed by the laws of supply and demand. In many countries the global pressure on cutting costs has created two trends: the increased supply of migrant workers and the deregulation of labour markets. These competitive pressures can have an adverse impact on the conditions of employment and, in the worst cases, can lead to forced labour and trafficking. In fact, trafficking has become one of the most profitable illicit industries worldwide, generating tremendous profits due to its low costs and huge profits. Therefore, it is important to investigate the phenomenon from the business point of view. This study is a qualitative research conducted by using theme interviews as a research approach. Altogether 13 interviews have been conducted and some secondary data has been used in order to find out what the role of human trafficking is in Finnish business. The special sectors investigated are the Finnish construction and service sectors. The theory framework used in this study follows the stakeholder approach. The relevant stakeholder groups for this study are: ‘institutions and authorities’, ‘law enforcement’, ‘management’ and ‘employees – potential victims’ of trafficking. With the help of these stakeholder groups, a holistic picture of the phenomenon is formed. It can be concluded that the role of human trafficking is complicated but it does exist in Finnish business. It appears in low-cost business sectors where the demand for cheap labour is high. Thus, often the victims are foreigners who do not know the culture or the Finnish conditions of employment. Especially smaller Finnish companies are at risk of getting involved in human trafficking or related exploitation cases since monitoring is much more scarce in these firms than in larger companies. The risk of human trafficking and exploitation is also higher at the bottom of the complicated subcontracting chains or when using foreign recruitment agencies. Thus, the study believes that active and intensive collaboration between the company’s different stakeholder groups is needed in order to prevent work-related human trafficking from flourishing in Finland.

Litíase vesicular assintomática em mulheres: aspectos epidemiológicos e clínicos

OBJETIVO: Analisar variáveis clínicas e ultra-sonográficas como presença ou ausência de barro biliar, espessura da parede e medida transversal da vesícula biliar, idade, paridade, presença ou ausência de diabetes melitus associadas à litíase vesicular assintomática, bem como determinar a sua prevalência em pacientes submetidas ao exame ultra-sonográfico. MÉTODO: Foram analisadas, em estudo prospectivo, 265 pacientes do sexo feminino, atendidas na Escola de Ultra-sonografia e Reciclagem Médica de Ribeirão Preto - EURP, durante o período de janeiro a setembro de 2001. RESULTADOS: Evidenciou-se diferença estatisticamente significativa relacionada à litíase da vesícula biliar e: espessura da parede da vesícula biliar, barro biliar, diâmetro transverso da vesícula biliar, faixa etária, paridade, passando de 4,1% na nulíparas, para 39,1% nas multíparas e diabéticas. A prevalência de litíase na vesícula biliar, em pacientes assintomáticas, foi de 14.7%. CONCLUSÕES: A litíase vesicular assintomática em mulheres ocorre principalmente com o decorrer da idade e da paridade. Os achados ultra-sonográficos mais freqüentemente encontrados foram: presença de barro biliar e de espessamento da parede da vesícula biliar.

Leukocyte trafficking: a special focus on VAP-1 and CLEVER-1

It is crucial that lymphocytes patrol the body against foreign intruders and that leukocytes invade inflamed tissues to ameliorate the infection or injury. The adhesion molecules in leukocytes and endothelial cells play an essential role in the immune response by directing the traffic of leukocytes. However, the same molecules that guide leukocyte traffic under physiological conditions are also involved in pathological situations, when an overly excessive or harmful inflammatory response leads to tissue destruction and organ dysfunction or tumor growth. Vascular adhesion protein-1 (VAP-1) and Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) are endothelial molecules that participate in the adhesion of leukocytes to the endothelia. This study was designed to elucidate, using different inflammation models, the role of VAP-1 and CLEVER-1 in leukocyte migration to the inflamed tissue, and to evaluate the use of antibodies against these molecules as an anti-adhesive therapy. Also, the role of CLEVER-1 during tumorigenesis was studied. Blocking the function of VAP-1 with antibodies significantly decreased the accumulation of leukocytes in the inflamed tissue. Targeting CLEVER-1 prevented cell migration via lymphatic vessels, as well as leukocyte traffic during inflammation. Following the anti-CLEVER-1 antibody treatment the number of immune regulating leukocytes in tumors was reduced, which led to a decrease in tumor growth. However, the normal immune response towards immunization or bacterial infection was not compromised. Thus, VAP-1 and CLEVER-1 are both potential targets for antiinflammatory therapies for preventing the harmful accumulation of leukocytes in inflamed areas. Targeting CLEVER-1 may also inhibit tumor growth by reducing immunosuppressive leukocytes in tumors

PV-1: Leukocyte trafficking molecule and vascular marker

The distinction between lymphatic vessels and blood vessels is a crucial factor in many studies in immunology, vascular biology and cancer biology. They both share several characteristics and perform related, though different functions. They are equally important for the performance of the human immune system with the continuous recirculation of leukocytes from the tissues via lymphatics to the blood vessels and back into the tissue presenting the link between both systems. This study was undertaken to elucidate the differences in the gene expression between primary blood- and lymphatic endothelial cells as well as the two immortalized cell lines HMEC-1 (human microvascular endothelial cell line 1) and TIME (telomerase immortalized microvascular endothelial cell line). Furthermore, we wanted to investigate the mystery surrounding the identity of the antigen recognized by the prototype blood vascular marker PAL-E. In the last step we wanted to study whether the PAL-E antigen would be involved in the process of leukocyte migration from the bloodstream into the surrounding tissue. Our results clearly show that the gene expression in primary blood endothelial cells (BEC), lymphatic endothelial cells (LEC) and the cell lines HMEC-1 and TIME is plastic. Comparison of a large set of BEC- and LEC datasets allowed us to assemble a catalog of new, stable BEC- or LEC specific markers, which we verified in independent experiments. Additionally, several lines of evidence demonstrated that PAL-E recognizes plasmalemma vesicle associated protein 1 (PV-1), which can form complexes with vimentin and neuropilin-1. Finally, numerous in vitro and in vivo experiments identify the first function of the protein PV-1 during leukocyte trafficking, where it acts as regulatory molecule.

Outbreaks of vesicular disease caused by Vaccinia virus in dairy cattle from Goiás State, Brazil (2010-2012)

Cases of vesicular and exanthematic disease by Vaccinia virus (VACV) have been reported in dairy herds of several Brazilian regions, occasionally also affecting humans. The present article describes eight outbreaks of vesicular disease caused by VACV in dairy herds of six counties of Goiás state, Midwestern Brazil (2010-2012), involving a total of 122 cows, 12 calves and 11 people. Dairy cows (3 to 9 years old) were affected in all cases and calves (2 to 9 months old) were affected in five outbreaks, presenting oral lesions. The morbidity ranged between 8 and 100% in cows, and 1.5 to 31% in calves. In the cows, the clinical signs started with vesicles (2-7mm), painful and coalescent papules (3-8 mm), which resulted in ulcers (5-25mm) and scabs in teats, and, occasionally, in the muzzle. The clinical course lasted from 16 to 26 days. The histopathology of bovine skin samples revealed superficial perivascular inflammatory infiltrate of lymphocytes, plasma cells, neutrophils, macrophages and multifocal areas of acanthosis, spongiosis, hipergranulosis and parakeratotic or orthokeratotic hyperkeratosis with adjacent focally extensive ulcers. Eosinophilic inclusion bodies were noted in the cytoplasm of the keratinocytes. PCR to vgf gene of Orthopoxvirus was positive in samples collected from all outbreaks, and in some cases, genomic VACV sequences were identified by nucleotide sequencing of the PCR amplicons. Infectious virus was isolated in cell culture from scabs from one outbreak. Antibodies to Orthopoxvirus were detected in at least 3 or 4 animals in most outbreaks, by ELISA (outbreaks 1, 2, 3, 4, 5 and 7) or virus-neutralization (outbreak 6). Neutralizing titers ranging from 8 to 64 in outbreak 6. In all outbreaks, VACV infection was suspected based on the clinical and pathological findings and it was confirmed by laboratory tests. Upon the etiological confirmation, other agents associated with vesicular disease were discarded. In all outbreaks, at least one milker who handled the affected cows developed malaise, headache, fever, painful vesico-pustular lesions mainly in the hands, but also in the neck and nose. These results confirm the circulation of VACV in the region and call attention for a correct diagnosis and the adoption of prophylactic and control measures.

Descrição morfológica e morfométrica da glândula vesicular de cobaias durante o desenvolvimento pós-natal

Glândulas vesiculares são essenciais para a reprodução, pois suas secreções afetam a função espermática. Cobaias (Cavia porcellus) são um excelente modelo experimental para estudo destas glândulas, contudo não existem dados morfológicos e morfométricos durante seu desenvolvimento. Neste estudo a morfologia (projeções das pregas (PP) e altura das células epiteliais (AE) da túnica mucosa) e a morfometria (massa (MG), volume (VG), comprimento (CG), largura das porções cranial (LCR), média (LM) e caudal (LCA)) das glândulas vesiculares foram determinadas em cobaios (N= 25) com uma (S1), três (S3), cinco (S5), oito (S8) e onze (S11) semanas de idade (N=5/grupo de idade). Em adição massa (MC), comprimento (CC) e altura (AC) corporais e o índice organo-somático (IOS) foram também determinados e o coeficiente de correlação (r) estabelecido entre as variáveis. As glândulas restringiam-se a cavidade pélvica e tinham seu interior repleto de uma massa semi-sólida e, após a S8, ocuparam também a cavidade abdominal e tiveram o conteúdo aumentado significativamente. A túnica mucosa era pregueada e forrada de um epitélio simples colunar. MC, CC e AC aumentaram continuamente com a idade. MG, VG, CG e LCA não se alteraram da S1 a S5 e aumentaram na S8; enquanto que MF, VG e LCA também aumentaram na S11. IOS e PP aumentaram na S8 e S11 e AE na S5, S8 e S11. Houve (r) significativo entre a idade, Bc e MFg,; IOS e MG na S8 e S11; idade e PP; idade e AE e entre PP e AE. Em conclusão, as glândulas vesiculares de cobaios seguiram o padrão morfológico observado em ratos e hamsters, mas diferiram em alguns aspectos de outros histricomorfos, podendo ser utilizadas como modelo experimental e seu desenvolvimento morfológico e morfométrico podem ser divididos em três fases: da S1 a S5, quando são discretos; da S6 a S8 de idade, quando são acentuados de maneira geral e após a S8, quando há incremento intenso da capacidade secretória.

Investigação epidemiológica de Estomatite vesicular por achados clínicos em bovinos e equinos no Estado do Maranhão

Resumo:A Estomatite Vesicular (EV) é uma doença infecciosa que acomete equinos, bovinos, suínos, mamíferos silvestres e humanos. Por apresentar sinais clínicos semelhantes a outras doenças vesiculares, principalmente, febre aftosa, sua presença em determinadas regiões pode interferir no intercâmbio comercial internacional dos animais, seus produtos e subprodutos. Apesar de sua importância, a epidemiologia e a manutenção do vírus no ambiente não estão totalmente esclarecidas dificultando a aplicação de medidas de controle efetivas. A doença já foi diagnosticada em todas as regiões brasileiras. Bovinos com sialorréia, perda do epitélio lingual, lesões abertas com bordas amareladas nas gengivas, lábios, língua e mucosa oral e equinos com sialorréia e lesões abertas na mucosa oral e lábios foram observados e notificados ao Serviço Veterinário Oficial do Estado do Maranhão, Agência Estadual de Defesa Agropecuária do Maranhão (AGRD/MA). Amostras de soro de equinos e bovinos com sintomas de EV foram coletadas para investigação por ELISA e por neutralização viral, além do diagnóstico diferencial para Febre Aftosa (FA). Fragmentos epiteliais de bovinos com lesões na língua foram coletados para identificação molecular do agente. Todos os animais foram negativos para FA. Todos os bovinos e equinos foram reativos para EV nos testes sorológicos. A partir dos fragmentos epiteliais de bovinos enviados ao Instituto Biológico de São Paulo para PCR, foi possível caracterizar o agente como VesiculovirusIndiana III (Alagoas/VSAV).

Efectos citolítico de una fosfolipasa A2, y citopático de una fracción vesicular de Entamoeba histolytica sobre hepatocitos humanos, mediados por leucocitos polimorfonucleares humanos.

Tesis (Maestría en Ciencias con Especialidad en Microbiología) UANL