IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment.

Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/ß-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a "balloon-like" morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with "cilia rigidity" morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation.

Branched actin networks push against each other at adherens junctions to maintain cell-cell adhesion.

Adherens junctions (AJs) are mechanosensitive cadherin-based intercellular adhesions that interact with the actin cytoskeleton and carry most of the mechanical load at cell-cell junctions. Both Arp2/3 complex-dependent actin polymerization generating pushing force and nonmuscle myosin II (NMII)-dependent contraction producing pulling force are necessary for AJ morphogenesis. Which actin system directly interacts with AJs is unknown. Using platinum replica electron microscopy of endothelial cells, we show that vascular endothelial (VE)-cadherin colocalizes with Arp2/3 complex-positive actin networks at different AJ types and is positioned at the interface between two oppositely oriented branched networks from adjacent cells. In contrast, actin-NMII bundles are located more distally from the VE-cadherin-rich zone. After Arp2/3 complex inhibition, linear AJs split, leaving gaps between cells with detergent-insoluble VE-cadherin transiently associated with the gap edges. After NMII inhibition, VE-cadherin is lost from gap edges. We propose that the actin cytoskeleton at AJs acts as a dynamic push-pull system, wherein pushing forces maintain extracellular VE-cadherin transinteraction and pulling forces stabilize intracellular adhesion complexes.

Internacionalização, ciência e saúde: a medicina regenerativa global e os mercados paralelos / Internationalization, science and health: global regenerative medicine and the parallel markets

A medicina regenerativa implica em uma mudança de paradigma, a regeneração do organismo ao nível celular ou tecidual – um assunto contemporâneo controverso e de difícil estandardização. O artigo apresenta um resumo das tendências científicas, econômicas, sociais e de regulamentação global nessa área, analisadas em relação a dilemas teóricos relevantes em antropologia médica e sociologia da ciência e da saúde. Em especial, aqueles que tratam da construção de um ‘aparato coletivo de sentido’ para as novas entidades biológicas e ontológicas, a formação da cidadania biológica e a governança pela incerteza. Apresentam-se, também, evidências empíricas sobre um fenômeno chave para a governança e a regulamentação, qual seja a instalação de uma nova demanda transnacional em pesquisa e saúde através de mercados paralelos de óvulos e de terapias celulares em experimentação. Utilizam-se dados qualitativos coletados para uma pesquisa mais abrangente, resenhas jornalísticas e entrevistas com lideranças internacionais. Conclui-se com uma reflexão sobre a importância da governança internacional em ensaios clínicos e dos caminhos a serem explorados, visando uma harmonização da diversidade de práticas normativas.

Regenerative medicine involves a paradigm change due to organism regeneration at cellular and tissue level – a controversial contemporary issue and difficult to regulate. This article presents a summary of the main scientific, economic, social and regulatory global trends, analyzed according to relevant theoretical dilemmas in medical anthropology and in the sociology of science and health. This is especially true of the construction of a ‘collective frame of reference’ on the new biological and ontological entities, the shaping of biological citizenship, and governance through uncertainty. Empirical evidence is also presented on a key aspect in regulation and governance, namely the emergence of a new transnational demand in health research through the establishment of parallel markets for ova and experimental cellular therapies. Qualitative data collected for a broader research paper is analyzed, as well as journal reviews and information gathered during interviews with international leaders. The paper concludes with a discussion on the importance on international governance of clinical trials and on further exploration, towards a multilevel harmonization of a diversity of normative practices.

Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells.

Giardia duodenalis is a parasitic protozoan that causes diarrhea and other symptoms which together constitute a disease known as giardiasis. Although the disease has been well defined, the mechanisms involving the establishment of the infection have not yet been fully elucidated. In this study, we show that after 24h of interaction between parasites and intestinal Caco-2 cells, there was an alteration of the paracellular permeability, as observed by an approximate 42% of reduction in the transepithelial electrical resistance and permeation to ruthenium red, which was concomitant with ultrastructural changes. Nevertheless, epithelium viability was not affected. We also demonstrate that there was no change in expression of junctional proteins (tight and adherens) but that the distribution of these proteins in Caco-2 cells after parasite adhesion was significantly altered, as observed via laser scanning confocal microscopy 3D reconstruction. The present work shows that adhesion of Giardia duodenalis trophozoites to intestinal cells in vitro induces disturbances of the tight, adherens and desmosomal junctions.

Prostaglandin E2-EP1 and EP2 receptor signaling promotes apical junctional complex disassembly of Caco-2 human colorectal cancer cells.

BACKGROUND: The apical junctional complex (AJC) is a dynamic structure responsible to maintain epithelial cell-cell adhesions and it plays important functions such as, polarity, mechanical integrity, and cell signaling. Alteration of this complex during pathological events leads to an impaired epithelial barrier by perturbation of the cell-cell adhesion system. Although clinical and experimental data indicate that prostaglandin E(2) (PGE2) plays a critical function in promoting cell motility and cancer progression, little is known concerning its role in AJC disassembly, an event that takes place at the beginning of colorectal tumorigenesis. Using Caco-2 cells, a cell line derived from human colorectal cancer, we investigated the effects of prostaglandin E(2) (PGE(2)) treatment on AJC assembly and function. RESULTS: Exposition of Caco-2 cells to PGE(2) promoted differential alteration of AJC protein distribution, as evidenced by immunofluorescence and immunoblotting analysis and impairs the barrier function, as seen by a decrease in the transepithelial electric resistance and an increase in the permeability to ruthenium red marker. We demonstrated the involvement of EP1 and EP2 prostaglandin E(2) receptor subtypes in the modulation of the AJC disassembly caused by prostanoid. Furthermore, pharmacological inhibition of protein kinase-C, but not PKA and p38MAPK significantly prevented the PGE(2) effects on the AJC disassembly. CONCLUSION: Our findings strongly suggest a central role of Prostaglandin E2-EP1 and EP2 receptor signaling to mediate AJC disassembly through a mechanism that involves PKC and claudin-1 as important target for the TJ-related effects in human colorectal cancer cells (Caco-2).

Changes in the adherens junctions of human endothelial cells infected with spotted fever group rickettsiae.

Rickettsiae of the spotted fever group are obligately intracellular bacteria that primarily infect the vascular endothelium, invade adjacent cells propelled by actin polymerization, and cause severe systemic diseases. Endothelial dysfunction and vascular leakage develop as a consequence; this effect is the pathophysiological mechanism that explains most clinical manifestations. Here we report that rickettsial infection of cultured primary human endothelial cells is associated with the formation of gaps in the interendothelial adherens junctions, occurring late during the course of in vitro infections but not early, even when rickettsial loads are significant.

Cell junctions in the germinal epithelium may play an important role in spermatogenesis of the catfish P. fasciatum (Pisces, Siluriformes).

We identified adhesive junctions and gap junctions between Sertoli cells, between Sertoli and germ cells and between germ cells in the testis of P. fasciatum, a catfish of commercial relevance. To investigate the role of these junctions in spermatogenesis, as well as the molecular composition of the junctions, we performed an immunohistochemistry light microscopy as well as an immunogold labelling electron microscopy study with antibodies to adhesive and gap junctions proteins. Testes that were at different stages of spermatogenesis were used. Based on our morphological studies we speculate that Sertoli-germ and germ-germ cell adhesive junctions are important for maintaining the three-dimensional structure of the germinal cysts and an organized arrangement of the germ cells inside the cysts. Connexin 32 was identified in the germ cells and in the cysts walls. Our observations also suggest that Sertoli-germ and germ-germ cells gap junctions may be involved in the mechanism of synchronous development of germ cells.

Evidence that increased tyrosine phosphorylation causes disassembly of adherens junctions but does not perturb paracellular permeability in Caco-2 cells.

In this study, we report on the apparent effect of increased tyrosine phosphorylation events on the assembly and integrity of adherens junctions (AJs) and on paracellular permeability in Caco-2 cells. Cell monolayers were incubated with the phosphotyrosine phosphatase inhibitor vanadate/H2O2. Addition of this compound to monolayer resulted in disruption of the AJs, as revealed by electron microscopy and by a loss of membrane association of the AJ-associated protein uvomorulin/E-cadherin (U/E-c). However, tight junctions (TJs) were unaltered, as determined by measuring the transepithelial resistance (Rt), by ruthenium red labeling, as seen by transmission electron microscopy, and the distribution of TJ strands as seen in freeze-fracture replicas and by hyperphosphorylation of triton-insoluble occludin. Also examination of vanadate/H2O2 treated cells indicated a specific increase in AJ-associated phosphotyrosine residues as evaluated by immunofluorescence microscopy, but no modification of F-actin distribution, as revealed by confocal laser scanning microscopy analysis. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we tested a range of distinct protein kinase inhibitors. Of the three inhibitors tested (tyrphostin 25, genistein and staurosporine), tyrphostin 25 completely blocked the effects of vanadate/ H2O2 on assembly and integrity of AJs, redistribution of U/E-c and phosphotyrosine labeling. Our results indicate that, after addition of vanadate/H2O2 to Caco-2 monolayers, specific tyrosine phosphorylation of proteins cause disruption of AJs, but no modifications of the TJs' structure and functionality. These observations suggest that, in contrast to what happens with epithelial cells, TJs and AJs of Caco-2 cells are regulated by independent mechanisms.