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Tight Junctions (tight + junction)
Terms modified by Tight Junctions Selected AbstractsAbnormal Endothelial Tight Junctions in Active Lesions and Normal-appearing White Matter in Multiple SclerosisBRAIN PATHOLOGY, Issue 2 2002Jonnie Plumb Blood-brain barrier (BBB) breakdown, demonstrable in vivo by enhanced MRI is characteristic of new and expanding inflammatory lesions in relapsing-remitting and chronic progressive multiple sclerosis (MS). Subtle leakage may also occur in primary progressive MS. However, the anatomical route(s) of BBB leakage have not been demonstrated. We investigated the possible involvement of interendothelial tight junctions (TJ) by examining the expression of TJ proteins (occludin and ZO-1) in blood vessels in active MS lesions from 8 cases of MS and in normal-appearing white (NAWM) matter from 6 cases. Blood vessels (10,50 per frozen section) were scanned using confocal laser scanning microscopy to acquire datasets for analysis. TJ abnormalities manifested as beading, interruption, absence or diffuse cytoplasmic localization of fluorescence, or separation of junctions (putative opening) were frequent (affecting 40% of vessels) in oil-red-O-positive active plaques but less frequent in NAWM (15%), and in normal (<2%) and neurological controls (6%). Putatively "open" junctions were seen in vessels in active lesions and in microscopically inflamed vessels in NAWM. Dual fluorescence revealed abnormal TJs in vessels with pre-mortem serum protein leakage. Abnormal or open TJs, associated with inflammation may contribute to BBB leakage in enhancing MRI lesions and may also be involved in subtle leakage in non-enhancing focal and diffuse lesions in NAWM. BBB disruption due to tight junctional pathology should be regarded as a significant form of tissue injury in MS, alongside demyelination and axonopathy. [source] Mechanisms of transjunctional transport of NaCl and water in proximal tubules of mammalian kidneysACTA PHYSIOLOGICA, Issue 1 2002F. KIILArticle first published online: 30 APR 200 ABSTRACT Tight junctions and the intercellular space of proximal tubules are not accessible to direct measurements of fluid composition and transport rates, but morphological and functional data permit analysis of diffusion and osmosis causing transjunctional NaCl and water transport. In the S2 segment NaCl diffuses through tight junctions along a chloride gradient, but against a sodium gradient. Calculation in terms of modified Nernst,Fick diffusion equation after eliminating electrical terms shows that transport rates (300,500 pmol min,1 mm,1 tubule length) and transepithelial voltage of +2 mV are in agreement with observations. Diffusion coefficients are Dtj=1500 ,m2 s,1 in the S1 segment, and Dtj=90,100 ,m2 s,1 in the S2 segment where apical intercellular NaCl concentration is 132 mM, 1 mM below complete stop (Dtj=0 and Donnan equilibrium). Tight junctions with gap distance 6 Å are impermeable to mannitol (effective molecular radius 4 Å); reflection coefficients are ,=0.92 for NaHCO3 and ,=0.28 for NaCl, because of difference in anion size. The osmotic force is provided by a difference in effective transjunctional osmolality of 10 mOsm kg,1 in the S1 segment and 30 mOsm kg,1 in the S2 segment, where differences in transjunctional concentration contribute with 21 mOsm kg,1 for NaHCO3 and ,4 mOsm kg,1 for NaCl. Transjunctional difference of 30 mOsm kg,1 causes a volume flow of 2 nL min,1 mm,1 tubule length. Luminal mannitol concentration of 30 mM stops all volume flow and diffusive and convective transport of NaCl. In conclusion, transjunctional diffusion and osmosis along gradients generated by transcellular transport of other solutes account for all NaCl transport in proximal tubules. [source] Novel role of nectin: implication in the co-localization of JAM-A and claudin-1 at the same cell,cell adhesion membrane domainGENES TO CELLS, Issue 8 2008Kaori Kuramitsu Tight junctions (TJs) are formed at the apical side of adherens junctions (AJs) in epithelial cells. Major cell adhesion molecules (CAMs) at TJs are JAM and claudin, whereas major CAMs at AJs are nectin and cadherin. We previously showed that nectin initially forms cell,cell adhesion and then recruits cadherin to the nectin-based cell,cell adhesion sites to form AJs, followed by the recruitment of JAM and claudin to the apical side of AJs to form TJs. We investigated the roles of nectin in the formation of TJs by expressing various combinations of CAMs in L fibroblasts with no TJs or AJs. Co-expression of one of the AJ CAMs and one of the TJ CAMs formed two separate cell,cell adhesion membrane domains (CAMDs). Co-expression of nectin-3 and E-cadherin formed the same CAMD, but co-expression of JAM-A and claudin-1 did not form the same CAMD. Co-expression of JAM-A and claudin-1 with nectin-3, but not E-cadherin, made them form the same CAMD, which was separated from the nectin-based CAMD. Nectin-3 required afadin, a nectin- and F-actin-binding protein, for this ability. In conclusion, nectin plays a novel role in the co-localization of JAM and claudin at the same CAMD. [source] Tissue-specific expression of the tight junction proteins claudins and occludin in the rat salivary glandsJOURNAL OF ANATOMY, Issue 4 2004M. Peppi Abstract Tight junctions (TJs) are essential features of endothelial barrier membranes and of fluid-secreting epithelial cells, such as in the salivary glands. Novel integral membrane proteins have been identified as components of TJs, namely claudins and occludin. The aim of the present study was to determine the distribution of occludin and claudins in the large salivary glands of the rat. The parotid, submandibular and sublingual salivary glands were harvested from adult Sprague,Dawley rats and cryostat sections were stained using immunoperoxidase and immunofluorescence methods. Claudin-1 was expressed in endothelial cells of microvessels and in short selected segments of the duct system. Claudin-3 was expressed principally in the acinar cells and intercalated ducts, while claudin-4 was principally expressed by the striated and interlobular ducts. Claudin-5 was specific to endothelial cells of microvessels. Occludin was ubiquitously detected in the duct system. Double labelling and confocal microscopy showed some co-localization of claudin-3 with claudin-4, and minimal co-localization of occludin with claudin-4, in the striated ducts. Claudin 2 was not detected in any of the salivary glands. The results indicate specificity of the chemical composition of tight junctions in the rat salivary glands, and may reflect different physiological roles for TJs in the glandular and duct epithelial cells, and in endothelial cells of salivary gland microvessels. [source] Intercellular Junctions in Rabbit Eye Ora SerrataANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 5 2006L. Nobeschi Summary The aim of this study was to describe and localize the intercellular junctions in the ora serrata region of albino and pigmented rabbit eyes. Eyes of albino and pigmented rabbits were fixed and processed for transmission electron microscopy. Light and electron microscope examination was carried out on semithin and ultrathin sections. The ora serrata region showed adherens, gap and tight junctions in the retinal and ciliary margins of albino and pigmented rabbit eyes. In the retinal margin, zonulae adherens between Müller cells and photoreceptors are associated with tight junctions. In the ciliary margin, epithelial cells are joined by adherens, gap and tight junctions localized between apical and apicolateral cell membranes. Tight junctions appear as zonulae occludens in the non-pigmented apicolateral cell membranes and as tight focal junctions between pigmented and non-pigmented apical cell membranes. Between the ciliary and retinal margins there are adherens and tight focal junctions which attach pigmented apical cell membranes to adjacent cells. There were no differences in the distribution of intercellular junctions between albino and pigmented rabbits. [source] Disruption of tight junction structure in salivary glands from Sjögren's syndrome patients is linked to proinflammatory cytokine exposureARTHRITIS & RHEUMATISM, Issue 5 2010Patricia Ewert Objective Disorganization of acinar cell apical microvilli and the presence of stromal collagen in the acinar lumen suggest that the labial salivary gland (LSG) barrier function is impaired in patients with Sjögren's syndrome. Tight junctions define cell polarity and regulate the paracellular flow of ions and water, crucial functions of acinar cells. This study was undertaken to evaluate the expression and localization of tight junction proteins in LSGs from patients with SS and to determine in vitro the effects of tumor necrosis factor , (TNF,) and interferon-, (IFN,) on tight junction integrity of isolated acini from control subjects. Methods Twenty-two patients and 15 controls were studied. The messenger RNA and protein levels of tight junction components (claudin-1, claudin-3, claudin-4, occludin, and ZO-1) were determined by semiquantitative reverse transcriptase,polymerase chain reaction and Western blotting. Tight junction protein localization was determined by immunohistochemistry. Tight junction ultrastructure was examined by transmission electron microscopy. Isolated acini from control subjects were treated with TNF, and IFN,. Results Significant differences in tight junction protein levels were detected in patients with SS. ZO-1 and occludin were strongly down-regulated, while claudin-1 and claudin-4 were overexpressed. Tight junction proteins localized exclusively to apical domains in acini and ducts of LSGs from controls. In SS patients, the ZO-1 and occludin the apical domain presence of decreased, while claudin-3 and claudin-4 was redistributed to the basolateral plasma membrane. Exposure of isolated control acini to TNF, and IFN, reproduced these alterations in vitro. Ultrastructural analysis associated tight junction disorganization with the presence of endocytic vesicles containing electron-dense material that may represent tight junction components. Conclusion Our findings indicate that local cytokine production in LSGs from SS patients may contribute to the secretory gland dysfunction observed in SS patients by altering tight junction integrity of epithelial cells, thereby decreasing the quality and quantity of saliva. [source] Vascular and Biology 05BRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue S1 2002L. Yea Background: Tight junctions govern the permeability of endothelial and epithelial cells. Changes in tight junction function are thus an early and key event in cancer metastasis and tissue permeability. This study sought to determine the role of oestrogen in the regulation of tight junctions and expression of occludin in endothelial cells. Methods: Human vascular endothelial cell line was incubated with 17-,-oestradiol at different concentrations (from 10,11m to 10,7m) over 1,24 h. Expression of occludin mRNA was determined using RT-PCR, and change of occludin protein using Western blotting. Transendothelial resistance (TER) was measured with an EVOM, and transendothelial cell permeability was determined using fluorescence labelled dextran (FITC-dextran 10) with a multichannel fluorescence reader. Results: 17-,-oestradiol reduced the expression of occludin mRNA in a time and concentration dependent manner with an obvious effect starting from 4 h. Reduced level of occludin protein was similarly seen after treatment with 17-,-oestradiol. Incubation of HECV with 17-,-oestradiol resulted in an increase in paracellular permeability by 9.9 ± 8.9 per cent at 10,10m (P > 0.05 versus control), 42.1 ± 15.2 per cent at 10,9m (P < 0.05 versus control), and 40.1 ± 22.4 per cent at 10,8m (P < 0.05, versus control). A decrease in the transendothelial cell resistance (TER) was seen with oestradiol (a reduction by 18.6 ± 16.6 per cent (P > 0.05 versus control), 31.5 ± 10.6 per cent (P < 0.01), or 44.4 ± 18.4 per cent (P < 0.01), at concentration 10,10, 10,9, 10,8m, respectively. Conclusions: This study shows a perturbation of tight junction functions in endothelial cells by oestrogen, which may have implications in the aetiology of mastalgia and vascular spread of breast cancer. [source] Attaching and effacing pathogen-induced tight junction disruption in vivoCELLULAR MICROBIOLOGY, Issue 4 2006Julian A. Guttman Summary Diarrhoea is a hallmark of infections by the human attaching and effacing (A/E) pathogens, enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). Although the mechanisms underlying diarrhoea induced by these pathogens remain unknown, cell culture results have suggested that these pathogens may target tight junctions. Tight junctions in the colon function as physical intercellular barriers that separate and prevent mixing of the luminal contents with adlumenal regions of the epithelium. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by A/E pathogens could result in a loss of barrier function in the alimentary tract; however, this remains unexamined. Here we demonstrate for the first time that A/E pathogen infection results in the morphological alteration of tight junctions during natural disease. Tight junction alteration, characterized by relocalization of the transmembrane tight junction proteins claudin 1, 3 and 5, is a functional disruption; molecular tracers, which do not normally penetrate uninfected epithelia, pass across pathogen-infected epithelia. Functional junction disruption occurs with a concomitant increase in colon luminal water content. The effects on tissue are dependent upon the bacterial type III effector EspF (E. coli secreted protein F), because bacteria lacking EspF, while able to colonize, are defective for junction disruption and result in decreased proportions of water in the colon compared with wild-type infection. These results suggest that the diarrhoea induced by A/E pathogens occurs as part of functional tight junction disruption. [source] Tight junctions, leaky intestines, and pediatric diseasesACTA PAEDIATRICA, Issue 4 2005Z Liu Abstract Background: Tight junctions (TJs) represent the major barrier within the paracellular pathway between intestinal epithelial cells. Disruption of TJs leads to intestinal hyperpermeability (the so-called "leaky gut") and is implicated in the pathogenesis of several acute and chronic pediatric disease entities that are likely to have their origin during infancy. Aim: This review provides an overview of evidence for the role of TJ breakdown in diseases such as systemic inflammatory response syndrome (SIRS), inflammatory bowel disease, type 1 diabetes, allergies, asthma, and autism. Conclusion: A better basic understanding of this structure might lead to prevention or treatment of these diseases using nutritional or other means. [source] Reprogramming of genetic networks during initiation of the Fetal Alcohol Syndrome,DEVELOPMENTAL DYNAMICS, Issue 2 2007Maia L. Green Abstract Fetal Alcohol Spectrum Disorders (FASD) are birth defects that result from maternal alcohol use. We used a non a priori approach to prioritize candidate pathways during alcohol-induced teratogenicity in early mouse embryos. Two C57BL/6 substrains (B6J, B6N) served as the basis for study. Dosing pregnant dams with alcohol (2× 2.9 g/kg ethanol spaced 4 hr on day 8) induced FASD in B6J at a higher incidence than B6N embryos. Counter-exposure to PK11195 (4 mg/kg) significantly protected B6J embryos but slightly promoted FASD in B6N embryos. Microarray transcript profiling was performed on the embryonic headfold 3 hr after the first maternal alcohol injection (GEO data series accession GSE1074). This analysis revealed metabolic and cellular reprogramming that was substrain-specific and/or PK11195-dependent. Mapping ethanol-responsive KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways revealed down-regulation of ribosomal proteins and proteasome, and up-regulation of glycolysis and pentose phosphate pathway in B6N embryos; and significant up-regulation of tight junction, focal adhesion, adherens junction, and regulation of the actin cytoskeleton (and near-significant up-regulation of Wnt signaling and apoptosis) pathways in both substrains. Expression networks constructed computationally from these altered genes identified entry points for EtOH at several hubs (MAPK1, ALDH3A2, CD14, PFKM, TNFRSF1A, RPS6, IGF1, EGFR, PTEN) and for PK11195 at AKT1. Our findings are consistent with the growing view that developmental exposure to alcohol alters common signaling pathways linking receptor activation to cytoskeletal reorganization. The programmatic shift in cell motility and metabolic capacity further implies cell signals and responses that are integrated by the mitochondrial recognition site for PK11195. Developmental Dynamics 236:613,631, 2007. © 2007 Wiley-Liss, Inc. [source] Disruption effects of monophthalate exposures on inter-Sertoli tight junction in a two-compartment culture modelENVIRONMENTAL TOXICOLOGY, Issue 3 2008Yun-Hui Zhang Abstract Phthalates are suspect environmental endocrine disruptors that may affect male reproduction and development by disturbing androgen synthesis and cell,cell interactions in the seminiferous epithelium. The in vivo metabolites, monophthalates, are thought to be the active agents, and toxicant effects including testicular damage and decreased sperm motility have been described previously. In this study, the aim was to investigate the effect of monophthalates on Sertoli cells using a two-compartment cell culture model, asking whether tight junction protein structures are affected, compromising the blood-testis barrier and contributing to male-mediated toxicity. Sertoli cells were isolated from Sprague Dawley rat testes and seeded onto the filters of two-compartment wells. A Sertoli cell monolayer was allowed to form, whereupon the cultures were treated with 0, 10, 30, 150, and 600 ,mol/L monobutyl phthalate (MBP) or mono-2-ethylhexyl phthalate (MEHP) for 24 h. Effects on the tight junctions between adjacent Sertoli cells were studied by light and transmission electron microscopy, the transepithelial electrical resistance (TEER) assay, and immunofluorescence localization. Results showed that exposures to monophthalates destroyed tight junctional structure in Sertoli cell monolayers in a dose-depended manner, as evidenced by a loss of single-cell layer organization in the cultures, decline of TEER value, and decreased expression of proteins associated with tight junctions such as zonula occludens-1 (ZO-1), F-actin, and Occludin. The changes were observed at doses of 150 and 600 ,mol/L, which is 10,100 times higher relative to estimated human exposures from the environment. These results are consistent with monophthalate-induced damage to tight junctions between adjacent Sertoli cells, suggesting that damage to Sertoli cell tight junctions induced by monophthalates may be an underlying mechanism of their male-mediated reproductive toxicity. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Endothelial barriers: from hypothetical pores to membrane proteins*JOURNAL OF ANATOMY, Issue 6 2002J. A. Firth Abstract The anatomical counterpart of the physiologically defined small pore system of capillary endothelia has proved difficult to establish. In non-brain continuous capillaries, the contributions of caveolar and transmembrane pathways are likely to be small and paracellular clefts are probably the dominant routes. Analogy with epithelial paracellular pathways suggests that tight junctions may be the most restrictive elements. However, structural features of tight junction-based models are incompatible with physiological data; it is more likely that the tight junction acts as a shutter limiting the available cleft area. Proposed molecular sieves elsewhere in the paracellular pathway include the glycocalyx and the cadherin-based complexes of the adherens junctions. The molecular architecture of tight junctions and adherens junctions is moderately well defined in terms of molecular species, and there are differences at both sites between the endothelial and epithelial spectra of protein expression. However, definition of the size-restricting pore remains elusive and may require structural biology approaches to the spatial arrangements and interactions of the membrane molecular complexes surrounding the endothelial paracellular clefts. [source] Blood,brain barrier breakdown in septic encephalopathy and brain tumours*JOURNAL OF ANATOMY, Issue 6 2002D. C. DaviesArticle first published online: 28 JUN 200 Abstract Septic encephalopathy is associated with breakdown of the blood,brain barrier and cerebral oedema. These features are also common properties of brain tumours. Perimicrovessel oedema, disruption of associated astrocyte end feet and neuronal injury occur in a porcine model of acute septic encephalopathy. The adrenergic system has been implicated in the inflammatory response to sepsis and may play a role in controlling blood,brain barrier permeability, since the ,2 -adrenoceptor agonist dopexamine inhibits perimicrovessel oedema formation whereas the ,1 -adrenoceptor agonist methoxamine provokes it. Electron microscopy revealed tight junction opening in high-grade astrocytoma microvessels. Expression of the tight junction protein occludin is reduced in these microvessels and this reduction is inversely correlated with the degree of cerebral oedema. Normal astrocytes secrete factors that induce barrier properties in endothelial cells, whereas high-grade astrocytomas secrete vascular endothelial growth factor, which stimulates angiogenesis, down regulates occludin and increases endothelial cell permeability. The water channel protein aquaporin-4 is normally expressed in astrocyte foot processes around cerebral microvessels. Its expression is massively up-regulated in high-grade astrocytoma and around metastatic adenocarcinoma. There is a significant correlation between aquaporin-4 expression and the degree of cerebral oedema, but it is not clear whether increased aquaporin-4 expression enhances oedema formation or clearance. These results suggest that the pathophysiology of brain oedema is multifactorial, but that there may be common processes operating regardless of the aetiology. [source] Air-liquid interface (ALI) culture of human bronchial epithelial cell monolayers as an in vitro model for airway drug transport studiesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2007Hongxia Lin Abstract Serially passaged normal human bronchial epithelial (NHBE) cell monolayers were established on Transwell® inserts via an air-liquid interface (ALI) culture method. NHBE cells were seeded on polyester Transwell® inserts, followed by an ALI culture from day 3, which resulted in peak TEER value of 766,±,154 ,,×,cm2 on the 8th day. Morphological characteristics were observed by light microscopy and SEM, while the formation of tight junctions was visualized by actin staining, and confirmed successful formation of a tight monolayer. The transepithelial permeability (Papp) of model drugs significantly increased with the increase of lipophilicity and showed a good linear relationship, which indicated that lipophilicity is an important factor in determining the Papp value. The expression of P-gp transporter in NHBE cell monolayers was confirmed by the significantly higher basolateral to apical permeability of rhodamine123 than that of reverse direction and RT-PCR of MDR1 mRNA. However, the symmetric transport of fexofenadine,·,HCl in this NHBE cell monolayers study seems to be due to the low expression of P-gp transporter and/or to its saturation with high concentration of fexofenadine,·,HCl. Thus, the development of tight junction and the expression of P-gp in the NHBE cell monolayers in this study imply that they could be a suitable in vitro model for evaluation of systemic drug absorption via airway delivery, and that they reflect in vivo condition better than P-gp over-expressed cell line models. ©2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:341,350, 2007 [source] Tumour necrosis factor-, affects blood,brain barrier permeability and tight junction-associated occludin in acute liver failureLIVER INTERNATIONAL, Issue 8 2010Sa Lv Abstract Background: Cerebral oedema leading to cerebral herniation is a major cause of death during acute liver failure (ALF), but the underlying mechanism is not clear. Aims: We investigated the role of tumour necrosis factor (TNF)-, in changing the permeability of the blood,brain barrier (BBB) during ALF. Methods: ALF animal models were generated by administering d -galactosamine (GalN) and lipopolysaccharide, or GalN and TNF-,. ALF induction was blocked by first administering anti-TNF-,,IgG or anti-TNF-,-R1. We investigated the BBB permeability with Evans blue staining, and the structure with electron microscopy. Results: BBB permeability increased in ALF mice and correlated with elevated serum TNF-, levels. No vascular endothelial cell (EC) apoptosis was detected, but electron microscopy of cells from human and mouse ALF tissues revealed tight junction (TJ) disruptions and EC shrinkage, as well as increased vesicles and vacuoles. In addition, the expression of the TJ-associated protein occludin was significantly decreased in both ALF mice and patients, although the expression of occludin mRNA did not change. Changes in BBB permeability, brain tissue ultrastructure and occludin expression in ALF-induced mice could be prevented by prophylaxis treatment with either antibody to TNF-,,IgG or antibody to TNF-,-R1. Conclusions: Our results suggest that TNF-, plays a critical role in the development of brain oedema in ALF, and that both vasogenic and cytotoxic mechanisms may be involved. Increased BBB permeability may be because of the disruption of TJs, and loss of the TJ-associated protein occludin. [source] Tumor necrosis factor-, and interferon-, directly impair epithelial barrier function in cultured mouse cholangiocytesLIVER INTERNATIONAL, Issue 1 2003Hanada Shinichiro Abstract:Background/Aims: In primary biliary cirrhosis (PBC), cytokines from CD4 + T lymphocytes were suggested to contribute to the intralobular bile duct damage together with cellular immunity by CD8 + T lymphocytes. Recently, we reported that immunolocalization of 7H6 , a tight junction (TJ)-associated protein , was significantly diminished in cholangiocytes in the PBC liver. In this study, we examined the direct effects of several cytokines , tumor necrosis factor-, (TNF-,), interferon-, (IFN-,), interleukin-2 and 4 (IL-2 and 4) , on TJ in immortalized mouse cholangiocytes. Moreover, we examined the inhibitory effect of ursodeoxycholic acid (UDCA)on cytokine-induced changes in paracellular permeability. Methods: Barrier function of TJ was evaluated by measuring transepithelial electrical resistance (TER) and 3H-inulin flux. We also performed immunostaining and immunoblotting for TJ-associated proteins , claudin-1 and -3, occludin, zonula occluden-1 (ZO-1) and 7H6. Results: TNF-, and IFN-,, but neither IL-2 nor IL-4, significantly decreased TER (P < 0.005). 3H-inulin flux studies confirmed IFN-,-induced increases in paracellular permeability of cholangiocytes (P < 0.001). In immunostaining and immunoblotting studies, TJ-associated proteins were well preserved in TNF-,- or IFN-,-treated cells. Ursodeoxycholic acidhas been found to have no inhibitory effect on increased paracellular permeability induced by TNF-, or IFN-,. Conclusion: These findings show that TNF-, and IFN-, disrupt barrier function of TJ in cholangiocytes without major structural changes to TJ and suggest that disruption of TJ function and subsequent leakage of the bile constituents may influence the aggravation of cholestasis in PBC. [source] Epidermal growth factor receptor and claudin-2 participate in A549 permeability and remodeling: Implications for non-small cell lung cancer tumor colonizationMOLECULAR CARCINOGENESIS, Issue 6 2009Yakov Peter Abstract Tumor colonization involves changes in cell permeability and remodeling. Paracellular permeability is regulated by claudins, integrated tight junction (TJ) proteins, located on the apicolateral portion of epithelial cells. Epidermal growth factor (EGF) was reported to modify cellular claudin levels and induce remodeling. To investigate a role for EGF receptor (EGFR) activation in tumor colonization we studied the effect of EGF and claudin-2 overexpression on permeability and cell reorganization in the human A549 non-small cell lung cancer (NSCLC) cell line. Our data demonstrated that A549 cells possess functional TJs and that EGF treatment increased levels of claudin-2 expression by 46%. Furthermore, EGFR signaling reduced monolayer permeability to choline and triggered cellular remodeling. The mitogen-activated protein kinase inhibitor PD98059 blocked the effect on A549 permeability and remodeling. EGF stimulation also exacerbated a fourfold increase in cell colonization elicited by claudin-2 upregulation. Our findings are consistent with the hypothesis that EGFR signaling plays an important role in A549 cell physiology and acts synergistically with claudin-2 to accelerate tumor colonization. Understanding the influence of EGF on A549 cell permeability and reorganization will help shed light on NSCLC tumor colonization and contribute to the development of novel anti-cancer treatments. © 2008 Wiley-Liss, Inc. [source] Molecular dynamics of the blood,testis barrier components during murine spermatogenesisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2010Masataka Chihara The blood,testis barrier (BTB) separates the seminiferous epithelium into the adluminal and basal compartments. During murine spermatogenesis, preleptotene/leptotene spermatocytes migrate from the basal to the adluminal compartment through the BTB during stages VIII,IX. In the present study, we focused on the tight junction (TJ) molecules and analyzed their spatiotemporal expression during the murine seminiferous epithelial cycle. Structural analysis revealed that the principal components of the BTB, for example, claudin-3, claudin-11, occludin, and zonula occludens-1 (ZO-1), were localized at the basal and luminal sides of the preleptotene/leptotene spermatocytes during the migration stages (VIII,IX). Although we detected claudin-11, occludin, and ZO-1 throughout spermatogenesis, claudin-3 was only detected during stages VI,IX. Quantitative PCR using dissected seminiferous tubules from three stages (Early: II,VI, Middle: VII,VIII, Late: IX,I) clarified that the mRNA levels of TJ molecules were not correlated with the histoplanimetrical protein levels during spermatogenesis. Additionally, tubulobulbar complexes, considered to be involved in the internalization of TJ, were observed at the BTB site. Furthermore, a significant reduction in the mRNA levels of genes for the degradation of occludin (Itch) and endocytic recycling (Rab13) were observed during the Late and Middle stages, respectively. Therefore, we hypothesized that the lag between mRNA and protein expression of TJ molecules may be due to posttranslational modulation, for example, tubulobulbar complexes and endocytic recycling processes. In conclusion, these findings indicate that the integrity of the BTB is maintained throughout spermatogenesis, and the stage-specific localization of claudin-3 protein plays an important role in regulating BTB permeability. Mol. Reprod. Dev. 77: 630,639, 2010. © 2010 Wiley-Liss, Inc. [source] Isolation, propagation and characterization of primary tubule cell culture from human kidney (Methods in Renal Research)NEPHROLOGY, Issue 2 2007WEIER QI SUMMARY: Proximal tubule cells (PTC) are the major cell type in the cortical tubulointerstitium. Because PTC play a central role in tubulointerstitial pathophysiology, it is essential to prepare pure PTC from kidney tissue to explore the mechanisms of tubulointerstitial pathology. The authors have successfully refined and characterized primary cultures of human PTC using Percoll density gradient centrifugation as a key PTC enrichment step. The cells obtained by this method retain morphological and functional properties of PTC and are minimally contaminated by other renal cells. In particular, the primary isolates have characteristics of epithelial cells with uniform polarized morphology, tight junction and well-formed apical microvilli. Cytokeratin is uniformly and strongly expressed in the isolates. Brush border enzyme activities and PTC transport properties are retained in the isolates. This method therefore provides an excellent in vitro model for the physiologic study of the human proximal tubule. [source] Claudin-5 is Restricted to the Tight Junction Region of Uterine Epithelial Cells in the Uterus of Pregnant/Gravid Squamate ReptilesTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 5 2008Joanna M. Biazik Abstract Claudin-5, a tight junctional protein associated with ion and size selectivity, has been found in the uterus of skinks. This study has generated critical information about the molecular assembly of the tight junction at various stages of the reproductive cycle in the skink uterus. Recent studies looking at tight junctional proteins found occludin expression in the tight junction region of uterine epithelial cells in the skink uterus; however, occludin did not disclose any further information about the ions and size of ions permeating across the paracellular pathway. A ,22-kDa claudin-5 band was detected in the uterus of the skinks present in this study and immunohistochemistry revealed that claudin-5 redistributes to the tight junction region of the lateral plasma membrane of uterine epithelial cells in late stage pregnancy/gravidity. This finding indicates that the tight junction becomes more assembled to precisely regulate ion and solute permeation in late stage pregnancy/gravidity. Claudin-5 with its functional role as a molecular sieve due to the formation of ion and size selective pores suggests that permeation of ions smaller than 0.8 kDa are restricted when claudin-5 is redistributed to the tight junction region of the later plasma membrane. This report is the first description of the molecular mechanisms that may be involved in regulating nutrient provision in the reptilian uterus. Anat Rec, 291:547,556, 2008. © 2008 Wiley-Liss, Inc. [source] Rhinovirus infection-induced alteration of tight junction and adherens junction components in human nasal epithelial cellsTHE LARYNGOSCOPE, Issue 2 2010Nam-Kyung Yeo MD Abstract Objectives/Hypothesis: Manifestations of rhinovirus (RV) infections include mucus overproduction, increased vascular permeability, and secondary bacterial infection. These effects may reflect disrupted epithelial barrier functions, which are mainly regulated by intercellular junctions, referred to as tight junctions (TJs) and adherens junctions (AJs). The objective of this study was to investigate changes in the components of TJs (ZO-1, occluding, and claudin-1) and AJs (E-cadherin) after RV infection in cultured nasal epithelial cells. Methods: Primary human nasal epithelial cells grown at an air-liquid interface were infected apically with RV. RV-induced changes in the expression of epithelial TJ and AJ proteins were determined using real-time reverse transcriptase-polymerase chain reaction, confocal microscopy, and Western blot analyses. Functional changes in the integrity of junctional proteins were assessed by measuring transepithelial resistance (TER) using a voltmeter. Results: RV infection decreased mRNA levels of ZO-1, occludin, claudin-1, and E-cadherin to 64.2%, 51.8%, 56.2%, and 56.3%, respectively, of those in controls (P < .05). Decreases in ZO-1, occludin, claudin-1, and E-cadherin protein levels in RV-infected cells were evident in immunofluorescent confocal microscopic images. Expression levels of these proteins were also lower in the RV-infected group in Western blot analyses. RV infection reduced the mean TER from 143.1 ,/cm2 (controls) to 122.6 ,/cm2. Conclusions: RV infection decreased the expression of TJ and AJ components and reduced TER in primary cultured human nasal epithelial cells, indicating that RV infection may exert a harmful effect on nasal epithelial barrier function. Laryngoscope, 2010 [source] Effect of lectins on the transport of food ingredients in Caco-2 cell culturesBIOFACTORS, Issue 1-4 2004Y. Ohno Abstract We investigated the effect of several lectins, such as soy bean lectin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA), on the transport of some food ingredients (isoflavones, quercetin glycosides, carnosine/anserine) across Caco-2 cell monolayers. After incubation of food ingredients (0.03,2 mmol/L) in the presence or absence of lectins (1,180 ,/ml) on the apical side, aliquots were taken from the apical and basolateral solution, and were subjected to HPLC analysis. We also examined the effect of lectins on the permeability of the tight junction by measuring the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayer. Isoflavones, which was not transported to the basolateral solution without lectins, could be transported in the presence of lectins, whereas their aglycones were detected at the same levels with or without the lectin treatment. The transport of quercetin glycosides also increased in the presence of lectins, however, that of peptides was not affected by the lectins. Con A and WGA, but SBA, decreased the TER value, indicating that Con A and WGA increased the transport via paracellular pathway, whereas SBA did via a different pathway. [source] Disrupting specific PDZ domain-mediated interactions for therapeutic benefitBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2009Miles D Houslay The past two decades have seen an immense increase in our appreciation of the vast range of signalling processes and supporting machinery that occur in cells. Pivotal to this is the notion of signal compartmentalization (compartmentation). Targeting by protein domains is critical in allowing signalling complexes to be assembled at defined intracellular locales so as to confer correct function. This issue of the BJP contains two intriguing articles that address functional protein,protein interactions involving PDZ domains [Post-synaptic density protein-95 (PSD95), Drosophila disc large tumour suppressor (DlgA) and Zonula occludens-1 protein (zo-1)] and their implications for signalling. One involves targeting of neuronal nitric oxide synthase to the N-methyl D-aspartic acid (NMDA) receptor via the PDZ-containing signal scaffold, PSD95. The other involves controlling multiple receptor inputs into regulation of epithelial Na+K+ -ATPase through the PDZ-containing signal scaffold Pals-associated tight junction. Highlighted is not only the use of dominant-negative strategies to identify the importance of targeting at specific types of PDZ domains but also the exciting notion that small molecule disruptors of interaction at specific PDZ domains can be generated for potential therapeutic application. [source] Reticulocyte binding protein homologues are key adhesins during erythrocyte invasion by Plasmodium falciparumCELLULAR MICROBIOLOGY, Issue 11 2009Tony Triglia Summary The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invades human erythrocytes through multiple ligand,receptor interactions. The P. falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1 and PfRh4, members of this protein family, bind to red blood cells and function in merozoite invasion during which they undergo a series of proteolytic cleavage events before and during entry into the host cell. The ectodomain of PfRh1 and PfRh4 are processed to produce fragments consistent with cleavage in the transmembrane domain and released into the supernatant, at about the time of invasion, in a manner consistent with rhomboid protease cleavage. Processing of both PfRh1 and PfRh4, and by extrapolation all membrane-bound members of this protein family, is important for function and release of these proteins on the merozoite surface and they along with EBA-175 are important components of the tight junction, the transient structure that links the erythrocyte via receptor,ligand interactions to the actin,myosin motor in the invading merozoite. [source] Mechanisms of transjunctional transport of NaCl and water in proximal tubules of mammalian kidneysACTA PHYSIOLOGICA, Issue 1 2002F. KIILArticle first published online: 30 APR 200 ABSTRACT Tight junctions and the intercellular space of proximal tubules are not accessible to direct measurements of fluid composition and transport rates, but morphological and functional data permit analysis of diffusion and osmosis causing transjunctional NaCl and water transport. In the S2 segment NaCl diffuses through tight junctions along a chloride gradient, but against a sodium gradient. Calculation in terms of modified Nernst,Fick diffusion equation after eliminating electrical terms shows that transport rates (300,500 pmol min,1 mm,1 tubule length) and transepithelial voltage of +2 mV are in agreement with observations. Diffusion coefficients are Dtj=1500 ,m2 s,1 in the S1 segment, and Dtj=90,100 ,m2 s,1 in the S2 segment where apical intercellular NaCl concentration is 132 mM, 1 mM below complete stop (Dtj=0 and Donnan equilibrium). Tight junctions with gap distance 6 Å are impermeable to mannitol (effective molecular radius 4 Å); reflection coefficients are ,=0.92 for NaHCO3 and ,=0.28 for NaCl, because of difference in anion size. The osmotic force is provided by a difference in effective transjunctional osmolality of 10 mOsm kg,1 in the S1 segment and 30 mOsm kg,1 in the S2 segment, where differences in transjunctional concentration contribute with 21 mOsm kg,1 for NaHCO3 and ,4 mOsm kg,1 for NaCl. Transjunctional difference of 30 mOsm kg,1 causes a volume flow of 2 nL min,1 mm,1 tubule length. Luminal mannitol concentration of 30 mM stops all volume flow and diffusive and convective transport of NaCl. In conclusion, transjunctional diffusion and osmosis along gradients generated by transcellular transport of other solutes account for all NaCl transport in proximal tubules. [source] Disruption effects of monophthalate exposures on inter-Sertoli tight junction in a two-compartment culture modelENVIRONMENTAL TOXICOLOGY, Issue 3 2008Yun-Hui Zhang Abstract Phthalates are suspect environmental endocrine disruptors that may affect male reproduction and development by disturbing androgen synthesis and cell,cell interactions in the seminiferous epithelium. The in vivo metabolites, monophthalates, are thought to be the active agents, and toxicant effects including testicular damage and decreased sperm motility have been described previously. In this study, the aim was to investigate the effect of monophthalates on Sertoli cells using a two-compartment cell culture model, asking whether tight junction protein structures are affected, compromising the blood-testis barrier and contributing to male-mediated toxicity. Sertoli cells were isolated from Sprague Dawley rat testes and seeded onto the filters of two-compartment wells. A Sertoli cell monolayer was allowed to form, whereupon the cultures were treated with 0, 10, 30, 150, and 600 ,mol/L monobutyl phthalate (MBP) or mono-2-ethylhexyl phthalate (MEHP) for 24 h. Effects on the tight junctions between adjacent Sertoli cells were studied by light and transmission electron microscopy, the transepithelial electrical resistance (TEER) assay, and immunofluorescence localization. Results showed that exposures to monophthalates destroyed tight junctional structure in Sertoli cell monolayers in a dose-depended manner, as evidenced by a loss of single-cell layer organization in the cultures, decline of TEER value, and decreased expression of proteins associated with tight junctions such as zonula occludens-1 (ZO-1), F-actin, and Occludin. The changes were observed at doses of 150 and 600 ,mol/L, which is 10,100 times higher relative to estimated human exposures from the environment. These results are consistent with monophthalate-induced damage to tight junctions between adjacent Sertoli cells, suggesting that damage to Sertoli cell tight junctions induced by monophthalates may be an underlying mechanism of their male-mediated reproductive toxicity. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Development of an in vitro blood,brain barrier model to study the effects of endosulfan on the permeability of tight junctions and a comparative study of the cytotoxic effects of endosulfan on rat and human glial and neuronal cell culturesENVIRONMENTAL TOXICOLOGY, Issue 3 2006Melissa P. L. Chan Abstract Endosulfan, an organochlorine (OC) insecticide that belongs to the cyclodiene group, is one of the most commonly used pesticides to control pests in vegetables, cotton, and fruits. Porcine brain microvascular endothelial cells were used to develop a model to study the effects of endosulfan on the permeability of tight junctions in the blood,brain barrier (BBB). BBB permeability, measured as transendothelial electrical resistance, decreased in a dose- and time-dependent manner when treated with ,-endosulfan, ,-endosulfan, or endosulfan sulfate. Cytotoxicity testing revealed that the three endosulfans did not cause cell death at concentrations of 10 ,M and below. The ratio of the average permeability of the filter-grown endothelial cell monolayer to 14C-endosulfan (Pe) going from the outer to the inner compartments with that going from the inner to the outer compartments was approximately 1:1.2,2.1 after exposure to concentrations of 0.01,10 ,M. ,-Endosulfan, ,-endosulfan, and endosulfan sulfate had cytotoxic effects on rat glial (C6) and neuronal (PC12) cell cultures as well as on human glial (CCF-STTG1) and neuronal (NT2) cell cultures. The effects of ,-endosulfan were highly selective, with a wide range of LC50 values found in the different cultures, ranging from 11.2 ,M for CCF-STTG1 cells to 48.0 ,M for PC12 cells. In contrast, selective neurotoxicity was not so manifest in glial and neuronal cell cultures after exposure to endosulfan sulfate, as LC50 values were in the range of 10.4,21.6 ,M. CCF-STTG1 cells were more sensitive to ,-endosulfan and endosulfan sulfate, whereas NT2 cells were more sensitive to ,-endosulfan. © 2006 Wiley Periodicals, Inc. Environ Toxicol 21: 223,235, 2006. [source] Erythropoietin protects the in vitro blood,brain barrier against VEGF-induced permeabilityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2003Ofelia María Martínez-Estrada Abstract The blood,brain barrier (BBB) ensures the homeostasis of the brain microenvironment, mostly through complex tight junctions between brain endothelial cells that prevent the passage of hydrophilic molecules from blood to brain and vice versa. A recent study has shown in vivo that systemic administration of erythropoietin (Epo) protects against brain injury. Using an in vitro model of the bovine BBB, we observed that the expression of the Epo receptor is modulated by its ligand and hypoxic stimuli such as vascular endothelial growth factor (VEGF) treatment. In addition, Epo protects against the VEGF-induced permeability of the BBB, decreases the levels of endothelial nitric oxide synthase and restores junction proteins. The kinetic transport experiments revealed the capacity of Epo to cross the in vitro BBB in a saturable and specific way. Our results suggest a new mechanism for Epo-induced neuroprotection, in which circulating Epo controls and maintains the BBB through an Epo receptor signalling pathway and the re-establishment of cell junctions. [source] Involvement of integrin-induced activation of protein kinase C in the formation of adherens junctionsGENES TO CELLS, Issue 5 2007Misa Ozaki In epithelial cells, tight junctions (TJs) and adherens junctions (AJs) form junctional complexes. At AJs, cadherins and nectins are the major cell-cell adhesion molecules. Nectins first form cell,cell adhesions and then recruit cadherins to the nectin-based cell,cell adhesion sites to form AJs in coordination with the activation of integrin ,v,3, followed by the formation of TJs. We previously demonstrated that when MDCK cells precultured at a low Ca2+ concentration were treated with the protein kinase C (PKC) activator 12- O -tetradecanoyl-phorbol-13-acetate (TPA), incomplete AJs and a TJ-like structure were achieved. However, it remains unknown how PKC is activated and how it regulates the formation of cell,cell junctions. When MDCK cells precultured at a low Ca2+ concentration were treated with TPA, incomplete AJs were formed without the activation of integrin ,v,3. Treatment of cells with TPA also enhanced the phosphorylation of FAK, which transmits the outside-in signal of integrin and plays a role in the nectin-induced formation of AJs. In addition, inhibition of PKC suppressed the formation of AJs. These results indicate that the activation of PKC functions downstream of integrin ,v,3 and upstream of FAK, and is important for the nectin-induced formation of AJs. [source] Expression and distribution of ZO-3, a tight junction MAGUK protein, in mouse tissuesGENES TO CELLS, Issue 11 2003Akihito Inoko Background:, Three related MAGUK proteins, ZO-1, ZO-2 and ZO-3, are concentrated at the cytoplasmic surface of tight junctions. However, in contrast to ZO-1/ZO-2, our knowledge of the expression and distribution of ZO-3 is still fragmentary, partly due to a lack of antibodies that specifically distinguish ZO-3 from ZO-1 and ZO-2. Results:, We generated one pAb and one mAb that specifically recognized ZO-3 on Western blotting. The immunofluorescence signals obtained with these antibodies completely disappeared from ZO-1/ZO-2-positive tight junctions in the liver of ZO-3-deficient mice, indicating that the antibodies can be used to localize ZO-3 in various tissues by immunofluorescence microscopy. Immunofluorescence microscopy with these antibodies revealed that ZO-3 was concentrated at tight junctions in various types of epithelium, but not in endothelium or at cadherin-based cell-cell adhesion sites (spot adherens junctions of fibroblasts and intercalated discs of cardiac muscle cells), where ZO-1 and ZO-2 are concentrated. Conclusions:, We conclude that ZO-3 is expressed in a more epithelium-specific manner than ZO-1 and ZO-2. These observations provide for a better understanding of the functions of tight junction-associated MAGUKs. [source] | |||||||||||||||||||||||||||||||||||||