Cytoplasmic Extensions Research Articles

A Potential Role of Progestin-Induced Laminin-5/α6-Integrin Signaling in the Formation of Side Branches in the Mammary Gland

Mammary organoids from adult mice produce tubules, analogous to mammary ducts in vivo, in response to hepatocyte growth factor (HGF) when cultured in collagen gels. The combination of HGF plus progestin (R5020) causes reduced tubule number and length. We hypothesized that the inhibitory effect on tubulogenesis was due to progestin-mediated alteration of HGF/c-Met signaling. Using molecular inhibitors and short hairpin RNA, it was determined that HGF activation of Ras-related C3 botulinum toxin substrate (Rac1) was required for the formation of cytoplasmic extensions, the first step of tubulogenesis, and that Rac1 activity was Src kinase (Src) and focal adhesion kinase (FAK) dependent. The highly novel finding was that R5020 reduced tubulogenesis by up-regulating and increasing extracellular laminin and α6-integrin ligation to reduce activation of the Src, focal adhesion kinase, and Rac1 pathway. Receptor activator of nuclear factor-κB ligand, another progesterone-induced paracrine factor, did not replicate this effect of R5020. The inhibitory effect of R5020 on tubulogenesis was likely mediated through progesterone receptor (PR) isoform A (PRA), because PRA is the predominant PR isoform expressed in the organoids, and the progestin-induced effect was prevented by the PR antagonist RU486. These results provide a plausible mechanism that explains progestin/PRA-mediated blunting of HGF-induced tubulogenesis in vitro and is proposed to be relevant to progesterone/PRA-induced side-branching in vivo during pregnancy.

The ATPase Activity of the P-glycoprotein Drug Pump Is Highly Activated When the N-terminal and Central Regions of the Nucleotide-binding Domains Are Linked Closely Together

The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 Å) or long (22 Å) cross-linkers to identify domain-domain interactions that activate ATPase activity. It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.

Qualitative study of young, adult, and aged wistar rats temporomandibular synovial membrane employing light, scanning, and transmission electron microscopy

The aim of this study was to analyze the rat temporomandibular joint (TMJ) synovial membrane at different ages using light, scanning, and transmission electron microscopy. Under light microscopic analysis, the TMJ structures were observed such as condyle, capsule, disk, the synovial membrane collagen type, and cells distribution. In the scanning electron microscopy, the synovial membrane surface exhibited a smooth aspect in young animals and there was an increase with ageing in the number of folds. The transmission electron microscopic analysis showed more synoviocytes in the synovial layer in the young group and still a great number of vesicles and cisterns dilation of rough endoplasmic reticulum in the aged group. In the three groups, a dense layer of collagen fibers in the synovial layer and cytoplasmic extensions were clearly seen. It was possible to conclude that synovial membrane structures in aged group showed alterations contributing to the decrease in joint lubrication and in the sliding between disk and joint surfaces. These characteristic will reflect in biomechanics of chewing, and may cause the TMJ disorders, currently observed in clinical processes.

Multiscale grooved titanium processed with femtosecond laser influences mesenchymal stem cell morphology, adhesion, and matrix organization

The femtosecond laser processing enabled the structuring of six types of surfaces on titanium-6aluminium-4vanadium (Ti-6Al-4V) plates. The obtained hierarchical features consisted of a combination of microgrooves and oriented nanostructures. By adjusting beam properties such as laser polarization, the width of the microgrooves (20 or 60 μm) and the orientation of the nanostructures (parallel or perpendicular to the microgrooves) can be precisely controlled. Mesenchymal stem cells (MSCs) grown on these structured surfaces produced cytoplasmic extensions with focal contacts, while on the smooth titanium, the cells were found to be well spread and without any focal contact 12 h postseeding. The 600-nm wide nanostructures on their own were sufficient to orient the MSCs. For the multiscale structured areas, when the orientation of the nanostructures was orthogonal in relation to the microgrooves, there was an important decrease in or even a loss of cell alignment signifying that cells were sensitive to the directional nanostructures in the microgrooves. At 7 days, cell proliferation was not affected but the direction of nanostructures controlled the matrix organization. The ultrafast laser, as a new method for producing micro-nanohybrid surfaces, is a promising approach to promote desired tissue organization for tissue engineering.

Tunneling Nanotubes

Tunneling nanotubes are actin-based cytoplasmic extensions that function as intercellular channels in a wide variety of cell types.There is a renewed and keen interest in the examination of modes of intercellular communication in cells of all types, especially in the field of cancer biology. Tunneling nanotubes –which in the literature have also been referred to as “membrane nanotubes,” “’intercellular’ or ‘epithelial’ bridges,” or “cytoplasmic extensions” – are under active investigation for their role in facilitating direct intercellular communication. These structures have not, until recently, been scrutinized as a unique and previously unrecognized form of direct cell-to-cell transmission of cellular cargo in the context of human cancer. Our recent study of tunneling nanotubes in human malignant pleural mesothelioma and lung adenocarcinomas demonstrated efficient transfer of cellular contents, including proteins, Golgi vesicles, and mitochondria, between cells derived from several well-established cancer cell lines. Further, we provided effective demonstration that such nanotubes can form between primary malignant cells from human patients. For the first time, we also demonstrated the in vivo relevance of these structures in humans, having effectively imaged nanotubes in intact solid tumors from patients. Here we provide further analysis and discussion on our findings, and offer a prospective ‘road map’ for studying tunneling nanotubes in the context of human cancer. We hope that further understanding of the mechanisms, methods of transfer, and particularly the role of nanotubes in tumor-stromal cross-talk will lead to identification of new selective targets for cancer therapeutics.

Human invariant chain isoform p35 restores thymic selection and antigen presentation in CD74‐deficient mice

The invariant chain (Ii) has pleiotropic functions and is a key factor in antigen presentation. Ii associates with major histocompatibility complex class II molecules in the endoplasmic reticulum (ER) and targets the complex in the endocytic pathway to allow antigenic peptide loading. The human Iip35 isoform includes a cytoplasmic extension containing a di-arginine motif causing ER retention. This minor isoform does not exist in mice and its function in humans has not been thoroughly investigated. We have recently generated transgenic mice expressing Iip35 and these were crossed with Ii-deficient mice to generate animals (Tgp35/mIiKO) expressing exclusively the human isoform. In these mice, we show that Iip35 is expressed in antigen presenting cells and is inducible by interferon gamma (IFN-γ). Despite the low constitutive expression of the protein and some minor differences in the Vβ repertoire of Tgp35/mIiKO mice, Iip35 restored thymic selection of CD4(+) T cells and of invariant natural killer T cells. In vitro functional assays using purified primary macrophages treated with IFN-γ showed that Iip35 allows presentation of an Ii-dependent ovalbumin T-cell epitope. Altogether, our results suggest that Iip35 is functional and does not require co-expression of other isoforms for antigen presentation.

Molecular anatomy and physiology of exocytosis in sensory hair cells

Hair cells mediate our senses of hearing and balance by synaptic release of glutamate from somatic active zones (AZs). They share conserved mechanisms of exocytosis with neurons and other secretory cells of diverse form and function. Concurrently, AZs of these neuro-epithelial hair cells employ several processes that differ remarkably from those of neuronal synaptic terminals of the brain. Their unique molecular anatomy enables them to better respond to small, graded changes in membrane potential and to produce unsurpassed rates of exocytosis. Here, we focus on the AZs of cochlear inner hair cells (IHCs). As in other hair cells, these AZs are occupied by a cytoplasmic extension of the presynaptic density, called the synaptic ribbon: a specialized protein complex required for normal physiological function. Some proteins found at IHC synapses are uniquely expressed or enriched there, where their disruption can beget deafness in humans and in animal models. Other proteins, essential for regulation of conventional neuronal Ca2+-triggered fusion, are apparently absent from IHCs. Certain common synaptic proteins appear to have extra significance at ribbon-type AZs because of their interactions with unique molecules, their unusual concentrations, or their atypical localization and regulation. We summarize the molecular–anatomical specializations that underlie the unique synaptic physiology of hair cells.

Role of the Juxtamembrane Region of Cytoplasmic Loop 3 in the Gating and Conductance of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

Opening and closing of the cystic fibrosis transmembrane conductance regulator chloride channel are controlled by interactions of ATP with its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected to the transmembrane pore via four cytoplasmic loops. These loops have been suggested to play roles both in channel gating and in forming a cytoplasmic extension of the channel pore. To investigate the structure and function of one of these cytoplasmic loops, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced into loop 3. We find that methanethiosulfonate (MTS) reagents modify cysteines introduced at 14 of 16 sites studied in the juxtamembrane region of loop 3, in all cases leading to inhibition of channel function. In most cases, both the functional effects of modification and the rate of modification were similar for negatively and positively charged MTS reagents. Single-channel recordings indicated that, at all sites, inhibition was the result of an MTS reagent-induced decrease in channel open probability; in no case was the Cl– conductance of open channels altered by modification. These results indicate that loop 3 is readily accessible to the cytoplasm and support the involvement of this region in the control of channel gating. However, our results do not support the hypothesis that this region is close enough to the Cl– permeation pathway to exert any influence on permeating Cl– ions. We propose that either the cytoplasmic pore is very wide or cytoplasmic Cl– ions use other routes to access the transmembrane pore.

Effect of direct co-culture of mesenchymal stem cells and osteoblasts on the osteoblast phenotype expression

Effect of direct co-culture of mesenchymal stem cells and osteoblasts on the osteoblast phenotype expression

Development of the osteogenic phenotype in vitro on titanium surface microtopography functionalized with a type I collagen-derived synthetic peptide

Development of the osteogenic phenotype in vitro on titanium surface microtopography functionalized with a type I collagen-derived synthetic peptide

Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino

Os primeiros estudos demonstrando o potencial de trandiferenciação neural das células-tronco mesenquimais (CTMs) provenientes da medula óssea (MO) foram conduzidos em camundogos e humanos no início da década de 2000. Após esse período, o número de pesquisas e publicações com o mesmo propósito tem aumentado, mas com raros ou escassos estudos na espécie equina. Nesse sentindo, o objetivo desse trabalho foi avaliar o potencial in vitro da transdiferenciação neural das CTMs provenientes da MO de equinos utilizando-se dois protocolos: P1 (forksolin e ácido retinóico) e P2 (2-βmecarptoetanol). Após a confirmação das linhagens mesenquimais, pela positividade para o marcador CD90 (X=97,94%), negatividade para o marcador CD34 e resposta positiva a diferenciação osteogênica, as CTMs foram submetidas a transdiferenciação neural (P1 e P2) para avaliação morfológica e expressão dos marcadores neurais GFAP e β3 tubulina por citometria de fluxo. Os resultados revelaram mudanças morfológicas em graus variados entre os protocolos testados. No protocolo 1, vinte quatro horas após a incubação com o meio de diferenciação neural, grande proporção de células (>80%) apresentaram morfologia semelhante a células neurais, caracterizadas por retração do corpo celular e grande número de projeções protoplasmáticas (filopodia). Por outro lado, de forma comparativa, já nos primeiros 30 minutos após a exposição ao antioxidante β-mercaptoetanol (P2) as CTMs apresentaram rápida mudança morfológica caracterizada principalmente por retração do corpo celular e menor número de projeções protoplasmáticas. Também ficou evidenciado com o uso deste protocolo, menor aderência das células após tempo de exposição ao meio de diferenciação, quando comparado ao P1. Com relação a análise imunofenotípica foi observado uma maior (P<0,001) expressão dos marcadores GFAP e β3 tubulina ao término do P2 quando comparado ao P1. A habilidade das CTMs em gerar tipos celulares relacionados a linhagem neural é complexa e multifatorial, dependendo não só dos agentes indutores, mas também do ambiente no qual estas células são cultivadas. Desta forma um maior número de estudos é necessário para o melhor entendimento do processo de transdiferenciação neural a partir de CTMs de equinos.

Development of the osteogenic phenotype in vitro on titanium surface nanotopographies functionalized with GDF-5

Development of the osteogenic phenotype in vitro on titanium surface nanotopographies functionalized with GDF-5

Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma.

Tunneling nanotubes are long, non-adherent F-actin-based cytoplasmic extensions which connect proximal or distant cells and facilitate intercellular transfer. The identification of nanotubes has been limited to cell lines, and their role in cancer remains unclear. We detected tunneling nanotubes in mesothelioma cell lines and primary human mesothelioma cells. Using a low serum, hyperglycemic, acidic growth medium, we stimulated nanotube formation and bidirectional transfer of vesicles, proteins, and mitochondria between cells. Notably, nanotubes developed between malignant cells or between normal mesothelial cells, but not between malignant and normal cells. Immunofluorescent staining revealed their actin-based assembly and structure. Metformin and an mTor inhibitor, Everolimus, effectively suppressed nanotube formation. Confocal microscopy with 3-dimensional reconstructions of sectioned surgical specimens demonstrated for the first time the presence of nanotubes in human mesothelioma and lung adenocarcinoma tumor specimens. We provide the first evidence of tunneling nanotubes in human primary tumors and cancer cells and propose that these structures play an important role in cancer cell pathogenesis and invasion.

Characterizing Phospholamban to Sarco(endo)plasmic Reticulum Ca2+-ATPase 2a (SERCA2a) Protein Binding Interactions in Human Cardiac Sarcoplasmic Reticulum Vesicles Using Chemical Cross-linking

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys(27) of PLB was cross-linked to the Ca(2+) pump at the cytoplasmic extension of M4 (at or near Lys(328)) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca(2+) or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca(2+) affinity conformation of the Ca(2+)-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca(2+)-ATPase activity and Ca(2+) transport. The results suggest that the inhibitory effect of PLB on Ca(2+)-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca(2+) to the Ca(2+) pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca(2+) pump are largely unchanged in failing myocardium.

Panobinostat (LBH589)-induced acetylation of tubulin impairs megakaryocyte maturation and platelet formation

Drug-induced thrombocytopenia often results from dysregulation of normal megakaryocytopoiesis. In this study, we investigated the mechanisms responsible for thrombocytopenia associated with the use of Panobinostat (LBH589), a histone deacetylase inhibitor with promising anti-cancer activities. The effects of LBH589 were tested on the cellular and molecular aspects of megakaryocytopoiesis by utilizing an ex vivo system in which mature megakaryocytes (MK) and platelets were generated from human primary CD34(+) cells. We demonstrated that LBH589 did not affect MK proliferation or lineage commitment but inhibited MK maturation and platelet formation. Although LBH589 treatment of primary MK resulted in hyperacetylation of histones, it did not interfere with the expression of genes that play important roles during megakaryocytopoiesis. Instead, we found that LBH589 induced post-translational modifications of tubulin, a nonhistone protein that is the major component of the microtubule cytoskeleton. We then demonstrated that LBH589 treatment induced hyperacetylation of tubulin and alteration of microtubule dynamics and organization required for proper MK maturation and platelet formation. This study provides new insights into the mechanisms underlying LBH589-induced thrombocytopenia and provides a rationale for using tubulin as a target for selective histone deacetylase inhibitor therapies to treat thrombocytosis in patients with myeloproliferative neoplasms.

Phospholamban does not Interact with E2 Intermediates of the Cardiac Calcium Pump Stabilized by Metal Fluorides

The phospholamban (PLB) monomer binds in the groove between M2, M4, and M9 of the cardiac calcium pump (SERCA2a) and inhibits enzyme activity. We previously demonstrated that cross-linking of PLB with engineered Cys-residues to this region is prevented by micromolar Ca, but is stimulated by the nucleotides ATP or ADP. We proposed that PLB inhibits the catalytic activity of the enzyme by stabilizing a unique, nucleotide bound, Ca-free intermediate of SERCA2a (E2•ATP). Stabilization of E2•ATP prevents Ca binding at the high affinity Ca binding sites and formation of E1, thus blocking the kinetic cycle. To further test our model, we have now checked if PLB interacts with other E2 conformations, notably, SERCA2a with the bound metal fluorides (MgF42-, AlF4-, or BeF3-), which mimic the E2•Pi, E2P, and E2-P states, respectively. Interactions between PLB and SERCA2a were examined by cross-linking of N30C-PLB to K328 of WT-SERCA2a at the cytoplasmic extension of M4 and of V49C-PLB to V89C-SERCA2a at the C-terminal end of M2. Insect cell microsomes co-expressing N30C-PLB/SERCA2a or V49C-PLB/V89C-SERCA2a were first incubated with the metal fluorides in Ca-free buffer, and then subjected to chemical cross-linking. All these metal fluorides prevented cross-linking of PLB to SERCA2a at both the cytoplasmic and transmembrane regions, suggesting that PLB does not interact with E2•Pi, E2P, and E2-P formed in the absence of nucleotide. Remarkably, however, the cross-linking of PLB to SERCA2a was completely restored upon addition of 3mM ATP, allowing formation of E2•ATP. These results strongly support our model in which E2•ATP is the single conformation binding PLB, and further suggest that the binding interaction between E2•ATP and PLB is very strong, sufficient to prevent formation of E2•Pi, E2P, or E2-P, even when stabilized by metal fluorides.

Alimentary Canal of the Adult Blow Fly,Chrysomya megacephala(F.) (Diptera: Calliphoridae)—Part I: Ultrastructure of Salivary Glands

The salivary gland ultrastructure of the adult male blow fly, Chrysomya megacephala (F.) (Diptera: Calliphoridae), was investigated at the ultrastructural level using light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The salivary glands are paired structures composed of a single median deferent duct bifurcated into two long, narrow efferent ducts connected to the coiled tubular glands. The SEM image of the gland surface revealed that the basal lamina is relatively smooth in general, but the whole surface appeared as a trace of rough swollen insertion by intense tracheal ramification. Ultrastructurally, the salivary gland is enclosed within the basal lamina, and interdigitation cytoplasmic extensions were apparent between the adjacent gland cells. The basement membrane appeared infoldings that is similar to the complex of the labyrinth channel. The cytoplasm characteristic of the gland revealed high activity, based on the abundance of noticeable secretory granules, either singly or in an aggregated reservoir. In addition, mitochondria were found to intersperse among rich parallel of arrays rough endoplasmic reticulum. Thick cuticle, which was well-delineated and electron dense, apically lined the gland compartments, with discontinuity of the double-layer cuticle revealing a trace of secretion discharged into the lumen. Gross anatomy of the adult salivary gland was markedly different from that of the third instar of the same species, and structural dissimilarity is discussed briefly.

Impact of compound alpha treatment in vivo on egg production by the liver fluke, Fasciola hepatica

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were treated with compound alpha at a dosage of 15mg/kg at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24h, 48h and 72h post-treatment (pt). They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system: specifically, the uterus, Mehlis’ gland, ovary and vitellaria. As judged by the appearance of the uterus, normal egg production ceased within 24h of treatment; this phenomenon preceded significant changes to the other reproductive organs.Over the 3-day pt period, there was a progressive decline in the number of oogonia in the ovary, together with an increase in the number of eosinophilic and apoptotic oocytes and vacuolation and shrinkage of the ovarian tubules. There was a shift in the cell population within the vitelline follicles at 48h pt, with relatively greater numbers of mature vitelline cells and fewer immature cells. The follicles were vacuolated and the shell globule clusters in the mature cells were disorganised. Greater disruption was seen at 72h pt, with a reduction in the size of the follicles and rupture of cells, releasing their content into the lumen of the follicles. These histological observations were confirmed and extended at the TEM level. Thus, examination of electron micrographs showed that disruption of the shell globule clusters was evident at 48h pt, which coincided with the start of the breakdown of the mature cells and of the nurse cell network. These degenerative changes were more conspicuous at 72h pt. In the Mehlis’ gland, shrinkage and vacuolation of the cells and their cytoplasmic extensions became progressively greater from 48h to 72h pt, and secretory activity declined. The changes in the reproductive organs and inhibition of egg production are put in context of the overall time-course of drug action.

Spermatological characters in the diphyllobothriidean Schistocephalus solidus (Cestoda)

AbstractLevron, C., Yoneva, A. and Kalbe, M. 2011. Spermatological characters in the diphyllobothriidean Schistocephalus solidus (Cestoda). —Acta Zoologica (Stockholm) 00: 1–8.The spermiogenesis and the mature spermatozoon of Schistocephalus solidus (Cestoda: Diphyllobothriidea) are described using transmission electron microscopy. Spermiogenesis in S. solidus begins with the formation in the spermatid of a differentiation zone surrounded by cortical microtubules and delimited by arching membranes. This conical area presents two centrioles associated with striated rootlets and a median cytoplasmic extension between them. The centrioles are separated by an intercentriolar body composed of three electron‐dense plates dividing four electron‐lucent plates. The centrioles give rise to two flagella that undergo a rotation and later fuse proximodistally with the median cytoplasmic expansion. The presence of an electron‐dense material in the distal part of the differentiation zone is observed in the early stage of spermiogenesis. This pattern corresponds to Type I spermiogenesis according to the classification proposed by Bâ and Marchand (Mémoires du Muséum National d’Histoire Naturelle 1995; 166: 87). The mature spermatozoon of S. solidus presents the Type I pattern defined by Levron et al. (Biological Reviews 2010; 85: 523). It consists of five regions that exhibit two axonemes, parallel cortical microtubules, nucleus and electron‐dense zones. The anterior tip of the spermatozoon possesses only a few singlets. The axonemes are of a 9 + ’1’ trepaxonematan pattern and do not reach the posterior extremity of the mature spermatozoon.

Differential expression of occludin in patients with ulcerative colitis and healthy controls

To the Editor: The etiology of inflammatory bowel disease (IBD) remains unknown. However, it has been postulated that IBD patients have a disruption of the intestinal barrier integrity that facilitates the trigger of an intestinal pathological process after exposure to several exogenous antigens. Barrier function is highly regulated, allowing the epithelium to control transmucosal permeability to solutes, water, and electrolytes. Occludin (OCLN) has a large cytoplasmic extension that may coordinate with the tight junctional complex, comprising a large number of membrane-associated proteins. OCLN is the first integral tight junction protein and the most ubiquitously expressed at the apical-most membranes. In order to confirm the contribution of OCLN gene in the pathogenesis of ulcerative colitis (UC), this is the first study to our best knowledge that explores the OCLN gene expression in colonic mucosa from patients with UC. We measured the OCLN gene expression from colonic biopsies of UC patients from September 2010 to July 2011. We studied a total of 54 individuals divided into three groups: 1) active UC (n 1⁄4 20); 2) remission UC (n 1⁄4 16); and 3) a healthy control group (n 1⁄4 18). Expression of mRNA OCLN was measured with the realtime polymerase chain reaction (RTPCR) method. The following primers were used: OCLN forward ggatgagc aatgccctttag and reverse aggaaccgaga gccaggt; and GADPH: forward gcccaa tacgaccaaatcc and reverse agccacatcgct cagaca for normalization. The OCLN mRNA expression was significantly decreased from colonic mucosa in UC remission patients as compared with the healthy control group (P 1⁄4 0.0001). Differential levels of expression were found between active and remission UC groups in OCLN gene expression, where active UC had significantly increased levels of OCLN compared to UC in remission (P 1⁄4 0.026), as shown in Figure 1. Our findings confirmed the potential role of OCLN in the pathogenesis of UC. This differential expression of OCLN between active and remission UC patients could be explained by the maintenance of epithelial cell polarity of the tight junction during the inflammatory process. Other proteins involved in the tight junction, like claudin-1 and claudin-2, were increased in patients with active IBD. In conclusion, expression of OCLN was elevated in colonic mucosa from the active UC compared to UC in remission and healthy control groups. Jesus K. Yamamoto-Furusho, MD, PhD, MSc Edgar J. Mendivil-Rangel, BS Gabriela Fonseca-Camarillo, PhD Inflammatory Bowel Disease Clinic Department of Gastroenterology Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico