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Find video protocols related to scientific articles indexed in Pubmed.
Tight junction, selective permeability, and related diseases.
Semin. Cell Dev. Biol.
PUBLISHED: 08-15-2014
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The tight junction forms a barrier against unlimited paracellular passage but some of the tight junction proteins just do the opposite, they form extracellular channels zigzagging between lateral membranes of neighboring cells. All of these channel-forming proteins and even some of the barrier formers exhibit selectivity, which means that they prefer certain substances over others. All channel formers exhibit at least one of the three types of selectivity: for cations (claudin-2, -10b, -15), for anions (claudin-10a, -17) or for water (claudin-2). Also some, but not all, barrier-forming claudins are charge-selective (claudin-4, -8, -14). Moreover, occludin and tricellulin turned out to be relevant for barrier formation against macromolecule passage. Tight junction proteins are dysregulated or can be genetically defective in numerous diseases, which may lead to three effects: (i) impaired paracellular transport e.g. causing magnesium loss in the kidney, (ii) increased paracellular transport of solutes and water e.g. causing leak-flux diarrhea in the intestine, and (iii) increased permeability to large molecules e.g. unwanted intestinal pathogen uptake fueling inflammatory processes. This review gives an overview on the properties of tight junction proteins featuring selective permeability, and in this context explains how these proteins induce or aggravate diseases.
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Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins.
Mol. Biol. Cell
PUBLISHED: 07-16-2014
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Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon ? and tumor necrosis factor ? increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.
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Physiological and transcriptional memory in guard cells during repetitive dehydration stress.
New Phytol.
PUBLISHED: 06-30-2014
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Arabidopsis plants subjected to a daily dehydration stress and watered recovery cycle display physiological and transcriptional stress memory. Previously stressed plants have stomatal apertures that remain partially closed during a watered recovery period, facilitating reduced transpiration during a subsequent dehydration stress. Guard cells (GCs) display transcriptional memory that is similar to that in leaf tissues for some genes, but display GC-specific transcriptional memory for other genes. The rate-limiting abscisic acid (ABA) biosynthetic genes NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) and ALDEHYDE OXIDASE 3 (AAO3) are expressed at much higher levels in GCs, particularly during the watered recovery interval, relative to their low levels in leaves. A genetic analysis using mutants in the ABA signaling pathway indicated that GC stomatal memory is ABA-dependent, and that ABA-dependent SNF1-RELATED PROTEIN KINASE 2.2 (SnRK2.2), SnRK2.3 and SnRK2.6 have distinguishable roles in the process. SnRK2.6 is more important for overall stomatal control, while SnRK2.2 and SnRK2.3 are more important for implementing GC stress memory in the subsequent dehydration response. Collectively, our results support a model of altered ABA production in GCs that maintains a partially closed stomatal aperture during an overnight watered recovery period.
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Different gene-specific mechanisms determine the 'revised-response' memory transcription patterns of a subset of A. thaliana dehydration stress responding genes.
Nucleic Acids Res.
PUBLISHED: 04-17-2014
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Plants that have experienced several exposures to dehydration stress show increased resistance to future exposures by producing faster and/or stronger reactions, while many dehydration stress responding genes in Arabidopsis thaliana super-induce their transcription as a 'memory' from the previous encounter. A previously unknown, rather unusual, memory response pattern is displayed by a subset of the dehydration stress response genes. Despite robustly responding to a first stress, these genes return to their initial, pre-stressed, transcript levels during the watered recovery; surprisingly, they do not respond further to subsequent stresses of similar magnitude and duration. This transcriptional behavior defines the 'revised-response' memory genes. Here, we investigate the molecular mechanisms regulating this transcription memory behavior. Potential roles of abscisic acid (ABA), of transcription factors (TFs) from the ABA signaling pathways (ABF2/3/4 and MYC2), and of histone modifications (H3K4me3 and H3K27me3) as factors in the revised-response transcription memory patterns are elucidated. We identify the TF MYC2 as the critical component for the memory behavior of a specific subset of MYC2-dependent genes.
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ATX1/AtCOMPASS and the H3K4me3 marks: how do they activate Arabidopsis genes?
Curr. Opin. Plant Biol.
PUBLISHED: 03-31-2014
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Despite the proven correlation between gene transcriptional activity and the levels of tri-methyl marks on histone 3 lysine4 (H3K4me3) of their nucleosomes, whether H3K4me3 contributes to, or 'registers', activated transcription is still controversial. Other questions of broad relevance are whether histone-modifying proteins are involved in the recruitment of Pol II and the general transcription machinery and whether they have roles other than their enzyme activities. We address these questions as well as the roles of the ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1), of the COMPASS-related (AtCOMPASS) protein complex, and of their product, H3K4me3, at ATX1-dependent genes. We suggest that the ambiguity about the role of H3K4me3 as an activating mark is due to the unknown duality of the ATX1/AtCOMPASS to facilitate PIC assembly and to generate H3K4me3, which is essential for activating transcriptional elongation.
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ABA signaling is necessary but not sufficient for RD29B transcriptional memory during successive dehydration stresses in Arabidopsis thaliana.
Plant J.
PUBLISHED: 03-07-2014
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Plants subjected to a prior dehydration stress were seen to have altered transcriptional responses during a subsequent dehydration stress for up to 5 days after the initial stress. The abscisic acid (ABA) inducible RD29B gene of Arabidopsis thaliana was strongly induced after the first stress and displayed transcriptional memory with transcript levels nine-fold higher during the second dehydration stress. These increased transcript levels were due to an increased rate of transcription and are associated with an altered chromatin template during the recovery interval between the dehydration stresses. Here we use a combination of promoter deletion/substitutions, mutants in the trans-acting transcription factors and their upstream protein kinases, and treatments with exogenous ABA or dehydration stress to advance our understanding of the features required for transcriptional memory of RD29B. ABA Response Elements (ABREs) are sufficient to confer transcriptional memory on a minimal promoter, although there is a context effect from flanking sequences. Different mutations in Snf1 Related Protein Kinase 2 (SnRK2) genes positively and negatively affected the response, suggesting that this effect is important for transcriptional memory. Although exogenous ABA treatments could prime transcriptional memory, a second ABA treatment was not sufficient to activate transcriptional memory. Therefore, we concluded that transcriptional memory requires ABA and an ABA-independent factor that is induced or activated by a subsequent dehydration stress and directly or indirectly results in a more active RD29B chromatin template. These results advance our knowledge of the cis- and trans-acting factors that are required for transcriptional memory of RD29B.
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Dehydration stress memory genes of Zea mays; comparison with Arabidopsis thaliana.
BMC Plant Biol.
PUBLISHED: 02-27-2014
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Pre-exposing plants to diverse abiotic stresses may alter their physiological and transcriptional responses to a subsequent stress, suggesting a form of "stress memory". Arabidopsis thaliana plants that have experienced multiple exposures to dehydration stress display transcriptional behavior suggesting "memory" from an earlier stress. Genes that respond to a first stress by up-regulating or down-regulating their transcription but in a subsequent stress provide a significantly different response define the 'memory genes' category. Genes responding similarly to each stress form the 'non-memory' category. It is unknown whether such memory responses exists in other Angiosperm lineages and whether memory is an evolutionarily conserved response to repeated dehydration stresses.
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Laurate permeates the paracellular pathway for small molecules in the intestinal epithelial cell model HT-29/B6 via opening the tight junctions by reversible relocation of claudin-5.
Pharm. Res.
PUBLISHED: 02-24-2014
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To mechanistically analyze effects of the medium-chain fatty acid laurate on transepithelial permeability in confluent monolayers of the intestinal epithelial cell line HT-29/B6, in context with an application as an absorption enhancer improving transepithelial drug permeation.
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?-Haemolysin of Escherichia coli in IBD: a potentiator of inflammatory activity in the colon.
Gut
PUBLISHED: 02-17-2014
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?-Haemolysin (HlyA) influences host cell ionic homeostasis and causes concentration-dependent cell lysis. As a consequence, HlyA-producing Escherichia coli is capable of inducing 'focal leaks' in colon epithelia, through which bacteria and antigens translocate. This study addressed the role of HlyA as a virulence factor in the pathogenesis of colitis according to the 'leaky gut' concept.
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H3K27me3 and H3K4me3 chromatin environment at super-induced dehydration stress memory genes of Arabidopsis thaliana.
Mol Plant
PUBLISHED: 01-30-2014
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Pre-exposure to a stress may alter the plant's cellular, biochemical, and/or transcriptional responses during future encounters as a 'memory' from the previous stress. Genes increasing transcription in response to a first dehydration stress, but producing much higher transcript levels in a subsequent stress, represent the super-induced 'transcription memory' genes in Arabidopsis thaliana. The chromatin environment (histone H3 tri-methylations of Lys 4 and Lys 27, H3K4me3, and H3K27me3) studied at five dehydration stress memory genes revealed existence of distinct memory-response subclasses that responded differently to CLF deficiency and displayed different transcriptional activities during the watered recovery periods. Among the most important findings is the novel aspect of the H3K27me3 function observed at specific dehydration stress memory genes. In contrast to its well-known role as a chromatin repressive mechanism at developmentally regulated genes, H3K27me3 did not prevent transcription from the dehydration stress-responding genes. The high H3K27me3 levels present during transcriptionally inactive states did not interfere with the transition to active transcription and with H3K4me3 accumulation. H3K4me3 and H3K27me3 marks function independently and are not mutually exclusive at the dehydration stress-responding memory genes.
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Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins.
Pflugers Arch.
PUBLISHED: 01-22-2014
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Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.
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Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice.
Nucleic Acids Res.
PUBLISHED: 09-02-2013
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The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5 coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.
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Four distinct types of dehydration stress memory genes in Arabidopsis thaliana.
BMC Plant Biol.
PUBLISHED: 08-11-2013
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How plants respond to dehydration stress has been extensively researched. However, how plants respond to multiple consecutive stresses is virtually unknown. Pre-exposure to various abiotic stresses (including dehydration) may alter plants subsequent responses by improving resistance to future exposures. These observations have led to the concept of stress memory implying that during subsequent exposures plants provide responses that are different from those during their first encounter with the stress. Genes that provide altered responses in a subsequent stress define the memory genes category; genes responding similarly to each stress form the non-memory category.
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CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity.
Cell Commun. Signal
PUBLISHED: 06-04-2013
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Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation. In targeting adherens and tight junctions (TJs), CK2 modulates the strength and dynamics of epithelial cell-cell contacts. Occludin previously was identified as a substrate of CK2, however the functional consequences of CK2-dependent occludin phosphorylation on TJ function were unknown.
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Contribution of tight junction proteins to ion, macromolecule, and water barrier in keratinocytes.
J. Invest. Dermatol.
PUBLISHED: 02-14-2013
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Tight junctions (TJs) form a selective barrier for ions, water, and macromolecules in simple epithelia. In keratinocytes and epidermis, TJs were shown to be involved in individual barrier functions. The absence of the TJ protein claudin-1 (Cldn1) in mice results in a skin-barrier defect characterized by lethal water loss. However, detailed molecular analyses of the various TJ barriers in keratinocytes and the contribution of distinct TJ proteins are missing. Herein, we discriminate TJ-dependent paracellular resistance from transcellular resistance in cultured keratinocytes using the two-path impedance spectroscopy. We demonstrate that keratinocyte TJs form a barrier for Na(+), Cl(-), and Ca(2+), and contribute to barrier function for water and larger molecules of different size. In addition, knockdown of Cldn1, Cldn4, occludin, and zonula occludens-1 increased paracellular permeabilities for ions and larger molecules, demonstrating that all of these TJ proteins contribute to barrier formation. Remarkably, Cldn1 and Cldn4 are not critical for TJ barrier function for water in submerged keratinocyte cultures. However, Cldn1 influences stratum corneum (SC) proteins important for SC water barrier function, and is crucial for TJ barrier formation for allergen-sized macromolecules.
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Comment on "Large volcanic aerosol load in the stratosphere linked to Asian monsoon transport".
Science
PUBLISHED: 02-09-2013
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Bourassa et al. (Reports, 6 July 2012, p. 78) report on the 13 June 2011 eruption of the Nabro volcano and satellite observations of stratospheric aerosol that they attribute to troposphere to stratosphere ascent via the Asian monsoon. They claim (citing another source) that the 13 June top injection height was well below the tropopause. We will show that the 13 June Nabro eruption plume was clearly stratospheric and contained both volcanic gases and aerosols. Moreover, we will show height-resolved stratospheric sulfur dioxide and volcanic aerosol enhancements 1 to 3 days old, unaffected by the Asian monsoon, precisely connected to the volcano. The observed stratospheric aerosols and gases are fully explained by the 13 June eruption and do not require a monsoon vehicle.
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Improved Cell Line IPEC-J2, Characterized as a Model for Porcine Jejunal Epithelium.
PLoS ONE
PUBLISHED: 01-01-2013
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Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER) and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ) proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS) or species-specific (porcine serum, PS) conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS), compared to conventional FBS culture (IPEC-J2/FBS), the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell lines initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function.
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Aerolysin from Aeromonas hydrophila perturbs tight junction integrity and cell lesion repair in intestinal epithelial HT-29/B6 cells.
J. Infect. Dis.
PUBLISHED: 09-16-2011
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Aeromonads cause a variety of infections, including gastroenteritis, sepsis, and wound necrosis. Pathogenesis of Aeromonas hydrophila and its hemolysin has been characterized, but the mechanism of the epithelial barrier dysfunction is currently poorly understood.
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Cell polarity-determining proteins Par-3 and PP-1 are involved in epithelial tight junction defects in coeliac disease.
Gut
PUBLISHED: 08-24-2011
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Epithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage.
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Sheep rumen and omasum primary cultures and source epithelia: barrier function aligns with expression of tight junction proteins.
J. Exp. Biol.
PUBLISHED: 08-12-2011
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The forestomachs of cows and sheep have historically served as important models for the study of epithelial transport. Thus, the ruminal epithelium was among the first tissues in which absorption of chloride against an electrochemical gradient was observed, requiring a tight paracellular barrier to prevent back-leakage. However, little is known about ruminal barrier function, despite the considerable implications for ruminant health. The tight junction proteins of the omasum have never been investigated, and no cell culture model exists. We present a new method for the isolation of cells from forestomach epithelia. Protein expression of cells and source tissues of sheep were studied using western blot, PCR and confocal laser scanning microscopy. Cultured cells were characterized by transepithelial resistance (TER) measurements and patch clamping. Cells developed TER values of 729±134 ? cm(2) (rumen) and 1522±126 ? cm(2) (omasum). Both primary cells and source epithelia of rumen and omasum expressed cytokeratin, occludin and claudins 1, 4 and 7 (but not claudins 2, 3, 5, 8 and 10), consistent with the observed paracellular sealing properties. Staining for claudin-1 reached the stratum basale. The full mRNA coding sequence of claudins 1, 4 and 7 (sheep) was obtained. Patch-clamp analyses of isolated cells proved expression of an anion conductance with a permeability sequence of gluconate
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Oral and fecal Campylobacter concisus strains perturb barrier function by apoptosis induction in HT-29/B6 intestinal epithelial cells.
PLoS ONE
PUBLISHED: 05-19-2011
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Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t)) and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t) either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05), by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001), suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6) cm/s in control, P<0.05) but showed no difference in permeability for 4 kDa FITC-dextran (FD-4). The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and fecal C. concisus strains could mainly be assigned to apoptotic leaks together with moderate TJ changes, demonstrating a leak-flux mechanism that parallels the clinical manifestation of diarrhea.
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Endothelin antagonism as an active principle for glaucoma therapy.
Br. J. Pharmacol.
PUBLISHED: 05-12-2011
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Endothelin, the most potent vasoactive peptide known to date, has been suggested to play a potential role in the pathogenesis of open-angle glaucoma. Open-angle glaucoma is the most common optic nerve head neuropathy and is associated with a loss of retinal ganglion cells and visual field damage. Although an increased intraocular pressure is a major risk factor for glaucomatous optic neuropathy, other factors such as a reduced ocular blood flow play an important role for appearance of the disease. Thus, treatment of glaucoma is focused on lowering of intraocular pressure and preventing the occurrence or progression of glaucomatous optic neuropathy. Endothelin participates in the regulation of intraocular pressure by an effect on trabecular outflow, the main route for aqueous humour outflow from the eye. Trabecular outflow is modulated by trabecular meshwork contractility which is affected by endothelin. In addition to the effects of endothelin in the anterior part of the eye, the vasoconstrictor causes a decrease in ocular blood flow followed by pathological changes in the retina and the optic nerve head which is assumed to contribute to the degeneration of retinal ganglion cells. In sum, inhibition of endothelin signalling leads to lowering of intraocular pressure and exerts neuroprotective effects. Thus, endothelin antagonism in the eye represents a promising approach for pharmacological treatment of glaucoma.
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Epithelial barriers in intestinal inflammation.
Antioxid. Redox Signal.
PUBLISHED: 05-11-2011
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The gastrointestinal epithelium transports solutes and water between lumen and blood and at the same time forms a barrier between these compartments. This highly selective and regulated barrier permits ions, water, and nutrients to be absorbed, but normally restricts the passage of harmful molecules, bacteria, viruses and other pathogens. During inflammation, the intestinal barrier can be disrupted, indicated by a decrease in transcellular electrical resistance and an increase in paracellular permeability for tracers of different size. Such inflammatory processes are accompanied by increased oxidative stress, which in turn can impair the epithelial barrier. In this review, we discuss the role of inflammatory oxidative stress on barrier function with special attention on the epithelial tight junctions. Diseases discussed causing barrier changes include the inflammatory bowel diseases Crohns disease, ulcerative colitis, and microscopic colitis, the autoimmune disorder celiac disease, and gastrointestinal infections. In addition, the main cytokines responsible for these effects and their role during oxidative stress and intestinal inflammation will be discussed, as well as therapeutic approaches and their mode of action.
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The Arabidopsis trithorax-like factor ATX1 functions in dehydration stress responses via ABA-dependent and ABA-independent pathways.
Plant J.
PUBLISHED: 04-01-2011
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Emerging evidence suggests that the molecular mechanisms driving the responses of plants to environmental stresses are associated with specific chromatin modifications. Here, we demonstrate that the Arabidopsis trithorax-like factor ATX1, which trimethylates histone H3 at lysine?4 (H3K4me3), is involved in dehydration stress signaling in both abscisic acid (ABA)-dependent and ABA-independent pathways. The loss of function of ATX1 results in decreased germination rates, larger stomatal apertures, more rapid transpiration and decreased tolerance to dehydration stress in atx1 plants. This deficiency is caused in part by reduced ABA biosynthesis in atx1 plants resulting from decreased transcript levels from NCED3, which encodes a key enzyme controlling ABA production. Dehydration stress increased ATX1 binding to NCED3, and ATX1 was required for the increased levels of NCED3 transcripts and nucleosomal H3K4me3 that occurred during dehydration stress. Mechanistically, ATX1 affected the quantity of RNA polymerase?II bound to NCED3. By upregulating NCED3 transcription and ABA production, ATX1 influenced ABA-regulated pathways and genes. ATX1 also affected the expression of ABA-independent genes, implicating ATX1 in diverse dehydration stress-response mechanisms in Arabidopsis.
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Transforming growth factor-?, a whey protein component, strengthens the intestinal barrier by upregulating claudin-4 in HT-29/B6 cells.
J. Nutr.
PUBLISHED: 03-23-2011
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TGF? (isoforms 1-3) has barrier-protective effects in the intestine. The mechanisms involved in regulating tight junction protein expression are poorly understood. The aim of this study was to elucidate TGF?-dependent protective effects with special attention to promoter regulation of tight junction proteins using the HT-29/B6 cell model. In addition, the effects of whey protein concentrate 1 (WPC1), a natural source of TGF? in human nutrition, were examined. For this purpose, the claudin-4 promoter was cloned and tested for its activity. It exhibited transactivation in response to TGF?1, which was intensified when Smad-4 was cotransfected, indicating a Smad-4-dependent regulatory component. Shortening and mutation of the promoter altered and attenuated this effect. WPC1 induced an increase in the claudin-4 protein level and resistance of HT-29/B6 cell monolayers. Anti-TGF?(1-3) antibodies blocked these whey protein effects, suggesting that a main part of this function was mediated through TGF?. This effect was observed on intact monolayers as well as when barrier function was impaired by preexposure to IFN?. In conclusion, TGF?1 affects claudin-4 gene expression via Smad-4-dependent and -independent transcriptional regulation, resulting in barrier protection, a cytokine effect that is also found in whey protein concentrates used in enteral nutrition.
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Two distinct roles of ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) at promoters and within transcribed regions of ATX1-regulated genes.
Plant Cell
PUBLISHED: 01-25-2011
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The Arabidopsis thaliana trithorax-like protein, ATX1, shares common structural domains, has similar histone methyltransferase (HMT) activity, and belongs in the same phylogenetic subgroup as its animal counterparts. Most of our knowledge of the role of HMTs in trimethylating lysine 4 of histone H3 (H3K4me3) in transcriptional regulation comes from studies of yeast and mammalian homologs. Little is known about the mechanism by which ATX1, or any other HMT of plant origin, affects transcription. Here, we provide insights into how ATX1 influences transcription at regulated genes, playing two distinct roles. At promoters, ATX1 is required for TATA binding protein (TBP) and RNA Polymerase II (Pol II) recruitment. In a subsequent event, ATX1 is recruited by a phosphorylated form of Pol II to the +300-bp region of transcribed sequences, where it trimethylates nucleosomes. In support of this model, inhibition of phosphorylation of the C-terminal domain of Pol II reduced the amounts of H3K4me3 and ATX1 bound at the +300-nucleotide region. Importantly, these changes did not reduce the occupancy of ATX1, TBP, or Pol II at promoters. Our results indicate that ATX1 affects transcription at target genes by a mechanism distinct from its ability to trimethylate H3K4 within genes.
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Glucocorticoid receptor is indispensable for physiological responses to aldosterone in epithelial Na+ channel induction via the mineralocorticoid receptor in a human colonic cell line.
Eur. J. Cell Biol.
PUBLISHED: 01-05-2011
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The epithelial Na+ channel (ENaC) plays a crucial role in electrogenic Na(+) absorption in the distal colon. ENaC induction via the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) is differentially regulated by modulatory components. As most existing epithelial cell lines including colonic epithelial cell lines miss the co-expression of functional GR and MR, signaling on ENaC is only poorly characterized regarding the interplay of glucocorticoids and mineralocorticoids. In the present study, we show that GR expression and activity are indispensable for MR-dependent induction of ENaC-mediated Na(+) transport. Cooperativity of the two receptors has been studied in the highly differentiated, epithelial colonic cell line HT-29/B6-GR/MR which is equipped with the complete receptor repertoire of both GR and MR due to stable transfection. In contrast to HT-29/B6 cells solely expressing the MR, this cell line displays a physiological response to aldosterone regarding ENaC induction. To achieve this, a pre-incubation step with the GR agonist dexamethasone was required to allow for the subsequent stimulation of ENaC by aldosterone. As a result of cooperative effects between the activated GR and the MR, MR protein levels were elevated and MR-dependent transcription of ENaC subunits ? and ? was increased. As an additional mechanism involved, transcription of SGK-1 (serum- and glucocorticoid-induced kinase 1) and GILZ (glucocorticoid-induced leucin zipper)--both essential for the insertion of ENaC into the apical enterocyte membrane--were also augmented by the activated MR.
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TNFalpha-induced and berberine-antagonized tight junction barrier impairment via tyrosine kinase, Akt and NFkappaB signaling.
J. Cell. Sci.
PUBLISHED: 11-09-2010
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TNF?-mediated tight junction defects contribute to diarrhea in inflammatory bowel diseases (IBDs). In our study, the signaling pathways of the TNF? effect on barrier- or pore-forming claudins were analyzed in HT-29/B6 human colon monolayers. Berberine, a herbal therapeutic agent that has been recently established as a therapy for diabetes and hypercholesterinemia, was able to completely antagonize the TNF?-mediated barrier defects in the cell model and in rat colon. Ussing chamber experiments and two-path impedance spectroscopy revealed a decrease of paracellular resistance after TNF? to 11±4%, whereas transcellular resistance was unchanged. The permeability of the paracellular marker fluorescein was increased fourfold. Berberine alone had no effect while it fully prevented the TNF?-induced barrier defects. This effect on resistance was confirmed in rat colon. TNF? removed claudin-1 from the tight junction and increased claudin-2 expression. Berberine prevented TNF?-induced claudin-1 disassembly and upregulation of claudin-2. The effects of berberine were mimicked by genistein plus BAY11-7082, indicating that they are mediated via tyrosine kinase, pAkt and NF?B pathways. In conclusion, the anti-diarrheal effect of berberine is explained by a novel mechanism, suggesting a therapeutic approach against barrier breakdown in intestinal inflammation.
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Yersinia enterocolitica induces epithelial barrier dysfunction through regional tight junction changes in colonic HT-29/B6 cell monolayers.
Lab. Invest.
PUBLISHED: 10-18-2010
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Yersinia enterocolitica is a common cause of acute gastroenteritis. This study aimed to clarify the mechanisms leading to barrier dysfunction and diarrhea. Exposure of human colonic HT-29/B6 cells to Y. enterocolitica resulted in a decrease in transepithelial resistance from 404±23 to 163±21 ? cm² (P<0.001) in parallel with an increase in mannitol (182?Da) and fluorescein (332?Da) permeability, whereas short circuit current did not change. This effect was time dependent, required the presence of living bacteria, could not be triggered by bacterial supernatants and was not due to Yersinia outer proteins. Concomitantly, Y. enterocolitica induced necrosis as indicated by an increase in lactate dehydrogenase-release, whereas epithelial apoptosis was not upregulated. Local changes in conductivity were detected by conductance scanning, indicating leaky regions within the epithelium that were visualized by biotinylation and confocal microscopy. In these regions, claudin-3 and -4 and, especially claudin-8, were redistributed off the tight junction (TJ) into the cytoplasm. In addition, the expression of claudin-2, -3, -8, -10 and ZO-1 was diminished as quantified by immunoblotting. Moreover, we found claudin-8 to be regulated by the c-Jun N-terminal kinase, the inhibition of which attenuated the Y. enterocolitica-induced decrease in transepithelial resistance and restored claudin-8 protein level. In conclusion, barrier dysfunction in Y. enterocolitica infection is due to circumscribed epithelial TJ protein changes and necrotic cell loss, as a consequence of which leak flux diarrhea and antigen-uptake provoking extraintestinal arthritis may be triggered.
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Tight junction proteins contribute to barrier properties in human pleura.
Respir Physiol Neurobiol
PUBLISHED: 09-07-2010
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The permeability of pleural mesothelium helps to control the volume and composition of the liquid lubricating pleural surfaces. Information on pleural barrier function in health and disease, however, is scarce. Tissue specimens of human pleura were mounted in Ussing chambers for measurement of transmesothelial resistance. Expression of tight junction (TJ) proteins was studied by Western blots and immune fluorescence confocal microscopy. Both visceral and parietal pleura showed barrier properties represented by transmesothelial resistance. Occludin, claudin-1, -3, -5, and -7, were detected in visceral pleura. In parietal pleura, the same TJ proteins were detected, except claudin-7. In tissues from patients with pleural inflammation these tightening claudins were decreased and in visceral pleura claudin-2, a paracellular channel former, became apparent. We report that barrier function in human pleura coincides with expression of claudins known to be key determinants of epithelial barrier properties. In inflamed tissue, claudin expression indicates a reduced barrier function.
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Dynamic changes in genome-wide histone H3 lysine 4 methylation patterns in response to dehydration stress in Arabidopsis thaliana.
BMC Plant Biol.
PUBLISHED: 06-29-2010
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The molecular mechanisms of genome reprogramming during transcriptional responses to stress are associated with specific chromatin modifications. Available data, however, describe histone modifications only at individual plant genes induced by stress. We have no knowledge of chromatin modifications taking place at genes whose transcription has been down-regulated or on the genome-wide chromatin modification patterns that occur during the plants response to dehydration stress.
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Acute HIV infection induces mucosal infiltration with CD4+ and CD8+ T cells, epithelial apoptosis, and a mucosal barrier defect.
Gastroenterology
PUBLISHED: 06-10-2010
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A barrier defect of the intestinal mucosa is thought to affect the progression of human immunodeficiency virus (HIV) infection. It is not clear whether the mucosal barrier impairment already is present in acute infection and what mechanisms cause this defect. We analyzed T-cell subsets, epithelial apoptosis, and barrier function of the duodenal mucosa in patients with acute HIV infection.
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Activated AMPK and prostaglandins are involved in the response to conjugated linoleic acid and are sufficient to cause lipid reductions in adipocytes.
J. Nutr. Biochem.
PUBLISHED: 05-20-2010
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trans-10, cis-12 Conjugated linoleic acid (t10c12 CLA) reduces triglyceride levels in adipocytes. AMP-activated protein kinase (AMPK) and inflammation were recently demonstrated to be involved in the emerging pathways regulating this response. This study further investigated the role of AMPK and inflammation by testing the following hypotheses: (1) a moderate activation of AMPK and an inflammatory response are sufficient to reduce triglycerides, and (2) strong activation of AMPK is also sufficient. Experiments were performed by adding compounds that affect these pathways and by measuring their effects in 3T3-L1 adipocytes. A comparison of four AMPK activators (metformin, phenformin, TNF-? and t10c12 CLA) found a correlation between AMPK activity and triglyceride reduction. This correlation appeared to be modulated by the level of cyclo-oxygenase (COX)-2 mRNA produced. Inhibitors of the prostaglandin (PG) biosynthetic pathway interfered with t10c12 CLAs ability to reduce triglycerides. A combination of metformin and PGH2, or phenformin alone, efficiently reduced triglyceride levels in adipocytes. Microarray analysis indicated that the transcriptional responses to phenformin or t10c12 CLA were very similar, suggesting similar pathways were activated. 3T3-L1 fibroblasts were found to weakly induce the integrated stress response (ISR) in response to phenformin or t10c12 CLA and to respond robustly as they differentiated into adipocytes. This indicated that both chemicals required adipocytes at the same stage of differentiation to be competent for this response. These results support the above hypotheses and suggest compounds that moderately activate AMPK and increase PG levels or robustly activate AMPK in adipocytes may be beneficial for reducing adiposity.
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Tight junctions form a barrier in human epidermis.
Eur. J. Cell Biol.
PUBLISHED: 05-19-2010
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Tight junctions (TJ) are cell-cell junctions that have proved to form a paracellular barrier for solutes and water between cells of epithelia, including the stratum granulosum of the stratified epithelium of the epidermis of newborn mice. In mice lacking claudin-1, a major barrier-forming TJ component, this barrier was abolished. However, the role of TJ in human skin is controversially discussed as unambiguous data were missing so far. Here, we investigated TJ barrier function in healthy human skin as well as in skin samples from psoriatic lesions which are characterized by an altered localization of TJ proteins. We demonstrate for human skin that occludin- and claudin-1-positive sites in the stratum granulosum form a barrier for extracellular biotin-SH (557Da) and that in psoriatic skin the localization of the barrier and the TJ proteins are altered in parallel.
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Claudin-2, a component of the tight junction, forms a paracellular water channel.
J. Cell. Sci.
PUBLISHED: 05-11-2010
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Whether or not significant amounts of water pass the tight junction (TJ) of leaky epithelia is still unresolved, because it is difficult to separate transcellular water flux from TJ-controlled paracellular water flux. Using an approach without differentiating technically between the transcellular and paracellular route, we measured transepithelial water flux with and without selective molecular perturbation of the TJ to unequivocally attribute changes to the paracellular pathway. To this end, MDCK C7 cells were stably transfected with either claudin-2 or claudin-10b, two paracellular cation-channel-forming TJ proteins that are not endogenously expressed in this cell line. Claudin-2 is typical of leaky, water-transporting epithelia, such as the kidney proximal tubule, whereas claudin-10b is present in numerous epithelia, including water-impermeable segments of the loop of Henle. Neither transfection altered the expression of endogenous claudins or aquaporins. Water flux was induced by an osmotic gradient, a Na(+) gradient or both. Under all conditions, water flux in claudin-2-transfected cells was elevated compared with vector controls, indicating claudin-2-mediated paracellular water permeability. Na(+)-driven water transport in the absence of an osmotic gradient indicates a single-file mechanism. By contrast, claudin-10b transfection did not alter water flux. We conclude that claudin-2, but not claudin-10b, forms a paracellular water channel and thus mediates paracellular water transport in leaky epithelia.
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Femtosecond lentotomy: generating gliding planes inside the crystalline lens to regain accommodation ability.
J Biophotonics
PUBLISHED: 05-04-2010
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Based on Helmholtz Theory for accommodation the increasing sclerosis of lens nucleus and cortex is the main cause for the developments of presbyopia. Existing therapies, however, do not reverse the stiffness of the crystalline lens and thus do not regain real accommodation ability. A new approach to restore the flexibility of the lens could be realized by photodisruption using ultrafast laser pulses. This process, known as fs-lentotomy, was used to create micro-incisions which act as gliding planes inside the crystalline lens without opening the eye globe.
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Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes.
Biochim. Biophys. Acta
PUBLISHED: 03-11-2010
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The paracellular barrier of epithelia and endothelia is established by several tight junction proteins including claudin-3. Although claudin-3 is present in many epithelia including skin, lung, kidney, and intestine and in endothelia, its function is unresolved as yet. We therefore characterized claudin-3 by stable transfection of MDCK II kidney tubule cells with human claudin-3 cDNA. Two clone systems were analyzed, exhibiting high or low claudin-2 expression, respectively. Expression of other claudins was unchanged. Ultrastructurally, tight junction strands were changed toward uninterrupted and rounded meshwork loops. Functionally, the paracellular resistance of claudin-3-transfected monolayers was strongly elevated, causing an increase in transepithelial resistance compared to vector controls. Permeabilities for mono- and divalent cations and for anions were decreased. In the high-claudin-2 system, claudin-3 reduced claudin-2-induced cation selectivity, while in the low-claudin-2 system no charge preference was observed, the latter thus reflecting the "intrinsic" action of claudin-3. Furthermore, the passage of the paracellular tracers fluorescein (332Da) and FD-4 (4kDa) was decreased, whereas the permeability to water was not affected. We demonstrate that claudin-3 alters the tight junction meshwork and seals the paracellular pathway against the passage of small ions of either charge and uncharged solutes. Thus, in a kidney model epithelium, claudin-3 acts as a general barrier-forming protein.
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Tricellulin forms homomeric and heteromeric tight junctional complexes.
Cell. Mol. Life Sci.
PUBLISHED: 02-11-2010
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Sealing of the paracellular cleft by tight junctions is of central importance for epithelia and endothelia to function as efficient barriers between the extracellular space and the inner milieu. Occludin and claudins represent the major tight junction components involved in establishing this barrier function. A special situation emerges at sites where three cells join together. Tricellulin, a recently identified tetraspan protein concentrated at tricellular contacts, was reported to organize tricellular as well as bicellular tight junctions. Here we show that in MDCK cells, the tricellulin C-terminus is important for the basolateral translocation of tricellulin, whereas the N-terminal domain appears to be involved in directing tricellulin to tricellular contacts. In this respect, identification of homomeric tricellulin-tricellulin and of heteromeric tricellulin-occludin complexes extends a previously published model and suggests that tricellulin and occludin are transported together to the edges of elongating bicellular junctions and get separated when tricellular contacts are formed.
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Disruption of the K+ channel beta-subunit KCNE3 reveals an important role in intestinal and tracheal Cl- transport.
J. Biol. Chem.
PUBLISHED: 01-05-2010
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The KCNE3 beta-subunit constitutively opens outwardly rectifying KCNQ1 (Kv7.1) K(+) channels by abolishing their voltage-dependent gating. The resulting KCNQ1/KCNE3 heteromers display enhanced sensitivity to K(+) channel inhibitors like chromanol 293B. KCNE3 was also suggested to modify biophysical properties of several other K(+) channels, and a mutation in KCNE3 was proposed to underlie forms of human periodic paralysis. To investigate physiological roles of KCNE3, we now disrupted its gene in mice. kcne3(-/-) mice were viable and fertile and displayed neither periodic paralysis nor other obvious skeletal muscle abnormalities. KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they might facilitate transepithelial Cl(-) secretion through basolateral recycling of K(+) ions and by increasing the electrochemical driving force for apical Cl(-) exit. Indeed, cAMP-stimulated electrogenic Cl(-) secretion across tracheal and intestinal epithelia was drastically reduced in kcne3(-/-) mice. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3(-/-) mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. Further, these results suggest KCNE3 as a potential modifier gene in cystic fibrosis.
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Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine.
J. Comp. Physiol. B, Biochem. Syst. Environ. Physiol.
PUBLISHED: 01-05-2010
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In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (R (epi)). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest R (epi), followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (R (trans) or TER) overestimated true R (epi) by approximately 60%. In colon, strongest expression of "tightening" claudins 1, 3, 4, 5, and 8 was detected. In accordance with R (epi) the most proximal of the small intestinal segments, duodenum exhibited highest expression of "tightening" claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content.
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Klaus Hierholzer (1929-2007) and his impact on our understanding of the renal effects of steroid hormones.
J. Nephrol.
PUBLISHED: 12-17-2009
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Klaus Hierholzer (1929-2007) dissected various functions influenced by steroids in the distal tubule and showed that aldosterone in low doses reversed the sodium and potassium transport defect in adrenalectomized rats, through a rapid activation of Na+,K+-ATPase. Subsequent studies addressed the role of 11-beta-hydroxysteroid oxidoreductase (11-HSD) and showed that the undisturbed functioning of 11-HSD is a prerequisite for selective mineralocorticosteroid regulation of epithelial transport. Another set of original experiments showed that 11-HSD was equally important in the distal colon, thus establishing that the large intestine acts in parallel with the distal nephron. Hierholzer, born in Konstanz on June 8, 1929, was laureated in medicine on May 25, 1954. Subsequently he worked at the Department of Pharmacology of the University of Freiburg, Cornell University with J. F. Pitts, the Department of Medicine of the University of Frankfurt-am-Main, the University of Copenhagen with H. H. Ussing, and the Institute of Physiology of the Freie Universitaet in Berlin where he became full professor and head of the Institute of Clinical Physiology in 1968. He held that position until 1998. He died in Allensbach in the family house on February 27, 2007. Hierholzer was a member of the Naturforscher Leopoldina Academy and of many other scientific societies, including the Academy of Science and Technology in Berlin, and received various awards including an honorary professorship at the University of Naples, the Bezold Medal, the Volhard Medal, the Schoeller/Junkman Award, and the Malpighi Medal (in memoriam). He published nearly 300 papers including various seminal books. Noteworthy also are his papers on the history of physiology of the kidney and acid-base balance. A total of 26 scientists who trained in his laboratory became professors.
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The Arabidopsis chromatin modifier ATX1, the myotubularin-like AtMTM and the response to drought.
Plant Signal Behav
PUBLISHED: 11-15-2009
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Plants respond to environmental stresses by altering transcription of genes involved in the response. The chromatin modifier ATX1 regulates expression of a large number of genes; consequently, factors that affect ATX1 activity would also influence expression from ATX1-regulated genes. Here, we demonstrate that dehydration is such a factor implicating ATX1 in the plants response to drought. In addition, we report that a hitherto unknown Arabidopsis gene, At3g10550, encodes a phosphoinositide 3-phosphatase related to the animal myotubularins (AtMTM1). Myotubularin activities in plants have not been described and herein, we identify an overlapping set of genes co-regulated by ATX1 and AtMTM under drought conditions. We propose that these shared genes represent the ultimate targets of partially overlapping branches of the pathways of the nuclear ATX1 and the cytoplasmic AtMTM1. Our analyses offer first genome-wide insights into the relationship of an epigenetic factor and a lipid phosphatase from the other end of a shared drought responding pathway in Arabidopsis.
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Conjugated linoleic acid activates AMP-activated protein kinase and reduces adiposity more effectively when used with metformin in mice.
J. Nutr.
PUBLISHED: 10-14-2009
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Trans-10, cis-12 (t10c12) conjugated linoleic acid (CLA) reduces lipid levels in adipocytes, but the mechanisms involved are still emerging. The hypotheses of this study were that t10c12 CLA treatment activated AMP-activated protein kinase (AMPK) and that the effectiveness of a low dose of t10c12 CLA would be increased when combined with an AMPK activator. We demonstrated t10c12 CLA, directly or indirectly, activated AMPK and increased the amount of phosphorylated acetyl-CoA carboxylase (ACC) in 3T3-L1 adipocytes. Compound C, a potent inhibitor of AMPK, attenuated the phosphorylation of ACC, integrated stress response (ISR), inflammatory response, reduction in key lipogenic transcription factors, and triglyceride (TG) reduction that otherwise occurred in t10c12 CLA-treated adipocytes. Treatment of adipocytes or mice with a low dose of t10c12 CLA in conjunction with the AMPK activator metformin resulted in more TG loss than treatment with the individual chemicals. Additionally, although an inflammatory response was required for robust TG reduction, the combination of t10c12 CLA with AMPK activators had a similar TG loss with a reduced inflammatory response. A microarray analysis of the transcriptional response to either t10c12 CLA, metformin, or the combination, indicated the responses were very similar, with a correlation coefficient of 0.91 or better for genes in the ISR or lipid-related pathways. Altogether, these results support our hypotheses that t10c12 CLA activates AMPK, directly or indirectly, and that metformin increases the effectiveness of t10c12 CLA in reducing TG amounts in adipocytes.
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Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro: a novel model for studying the pathomechanisms of inflammatory bowel disease cytokines.
Scand. J. Gastroenterol.
PUBLISHED: 08-07-2009
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In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms.
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Arcobacter butzleri induces barrier dysfunction in intestinal HT-29/B6 cells.
J. Infect. Dis.
PUBLISHED: 07-17-2009
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Arcobacter butzleri causes watery diarrhea and bacteremia. Although, recently, more cases of diarrhea have been caused by Arcobacter species, very little is known about its pathogenesis, the identification of which is the aim of this study.
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Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key role in the regulation of the epithelial sodium channel.
FASEB J.
PUBLISHED: 06-30-2009
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Aldosterone is the principal hormonal regulator of sodium homeostasis in vertebrates. It exerts its actions through the mineralocorticoid receptor (MR) that regulates the transcription of specific target genes. In recent years, a number of MR target genes have been identified that are involved in the regulation of the epithelial sodium channel (ENaC), a key modulator of renal sodium absorption. Here we report the identification of cnksr3 as a direct MR target gene that is up-regulated in response to physiological concentrations of aldosterone. The cnksr3 promoter exhibits two functional aldosterone-responsive regions, which were bound by the MR as assessed by chromatin immunoprecipitation (ChIP). In vivo, CNKSR3 was highly expressed in the renal cortical collecting duct (CCD), the prime target segment of aldosterone-regulated sodium retention in the kidney. CCD cell lines stably overexpressing or silencing CNKSR3 were electrophysiologically analyzed and show that CNKSR3 expression correlated with and is required for ENaC-mediated transepithelial sodium transport. In parallel, CNKSR3 expression led to decreased MEK phosphorylation. We conclude that CNKSR3, a homologue of scaffold proteins involved in MAPK pathway regulation, is a direct target of MR and is required for the maintenance of transepithelial sodium transport in the kidney.
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Epithelial tight junctions in intestinal inflammation.
Ann. N. Y. Acad. Sci.
PUBLISHED: 06-23-2009
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The epithelium in inflamed intestinal segments of patients with Crohns disease is characterized by a reduction of tight junction strands, strand breaks, and alterations of tight junction protein content and composition. In ulcerative colitis, epithelial leaks appear early due to micro-erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin-2. Th1-cytokine effects by interferon-gamma in combination with TNFalpha are important for epithelial damage in Crohns disease, while interleukin-13 (IL-13) is the key effector cytokine in ulcerative colitis stimulating apoptosis and upregulation of claudin-2 expression. Focal lesions caused by apoptotic epithelial cells contribute to barrier disturbance in IBD by their own conductivity and by confluence toward apoptotic foci or erosions. Another type of intestinal barrier defect can arise from alpha-hemolysin harboring E. coli strains among the physiological flora, which can gain pathologic relevance in combination with proinflammatory cytokines under inflammatory conditions. On the other hand, intestinal barrier impairment can also result from transcellular antigen translocation via an initial endocytotic uptake into early endosomes, and this is intensified by proinflammatory cytokines as interferon-gamma and may thus play a relevant role in the onset of IBD. Taken together, barrier defects contribute to diarrhea by a leak flux mechanism (e.g., in IBD) and can cause mucosal inflammation by luminal antigen uptake. Immune regulation of epithelial functions by cytokines may cause barrier dysfunction not only by tight junction impairments but also by apoptotic leaks, transcytotic mechanisms, and mucosal gross lesions.
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Barrier effects of nutritional factors.
Ann. N. Y. Acad. Sci.
PUBLISHED: 06-23-2009
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High dietary intake of fruits and vegetables is associated with a reduced disease risk. Therefore, clinical interest is growing in therapies based on dietary supplements and effects of food components. Immune-modulatory and barrier-protective effects have been described for the amino acid glutamine and the trace element zinc. In Caco-2-cells, zinc is necessary to maintain the expression of proteins like ZO-1 and occludin, and experimental evidence exists that glutamine has enterocyte-protective effects and modulates intestinal barrier function in stressed animals and humans. Polyunsaturated fatty acids (PUFA) improve paracellular permeability after IL-4 incubation. Enhancement of barrier properties by long-chain PUFA is discussed controversially, but a beneficial role preventing the redistribution of occludin and ZO-1 and reduction of epithelial resistance by IFN-gamma and TNF-alpha exists. In addition, a group of secondary plant compounds, the polyphenols, are supposed to be important in this respect. The flavonoid quercetin and its metabolite DHBA increased epithelial resistance of Caco-2-cells to 157 +/- 4% of control values, and DHBA up to 119 +/- 4% of control values, respectively. This is due to a 2.3 +/- 0.1-fold expression rate of the tight junction protein claudin-4.
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Tight junction proteins as channel formers and barrier builders.
Ann. N. Y. Acad. Sci.
PUBLISHED: 06-23-2009
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Tight junctions form the paracellular barrier for ions and uncharged solutes not only in "tight" but also in "leaky" epithelia. In the premolecular era of tight junction research, this was believed to be achieved in a perfect or less perfect way, depending mainly on the amount of horizontally oriented tight junction strands. During the past decade it emerged that tight junction molecules, such as claudin-1 and many others, strengthen the barrier, while a few claudins, such as claudin-2 or -10, weaken it. This report focuses on three claudins: one channel former and two barrier builders. Claudin-2 represents the prototype of a paracellular, channel-forming, tight junction protein responsible for specific transfer of solutes across the epithelium without entering the cells. This channel is selective for small cations but nearly impermeable to anions and uncharged solutes of any size. In contrast, claudin-5, a tight junction protein typical for all endothelia but also found in some epithelia, was characterized as a potent barrier builder. Claudin-8, another barrier builder, was demonstrated to be regulated by Na(+) uptake in surface epithelial cells of human colon. Here, aldosterone enhanced Na(+) absorption by dual action: transcellularly by inducing the epithelial sodium channel and paracellularly by preventing back leakage of absorbed Na(+) by upregulating claudin-8.
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High-resolution analysis of barrier function.
Ann. N. Y. Acad. Sci.
PUBLISHED: 06-23-2009
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High-resolution analysis of epithelial barrier function adds substantial information to that provided by conventional transepithelial electrical resistance (TER) measurements. This chapter describes three high-resolution techniques. First, two variants of impedance spectroscopy are delineated. One-path impedance spectroscopy discriminates vertically between serial pathways, namely resistances of the epithelial cell layer and of subepithelial tissues. As a typical application, measurements on human sigmoid colon biopsies from patients suffering from Crohns disease are reported. Two-path impedance spectroscopy allows to discriminate between trans- and paracellular resistance, and the general principle of this technique is outlined. Second, the conductance scanning technique is presented, which discriminates horizontally between optically distinct parallel pathways over a wide range of spatial resolutions. Using this technique, it was shown that occludin--in contrast to the then prevailing opinion--is not irreplaceable to barrier function. Third, three-dimensional confocal fluorescence imaging for depicting transepithelial transport processes is introduced. Using this method the transepithelial translocation of bacteria which generate focal leaks was discovered.
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Tight junctions: molecular structure meets function.
Ann. N. Y. Acad. Sci.
PUBLISHED: 06-23-2009
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Tight junctions of epithelial and endothelial cells form selective barriers that regulate paracellular transport of solutes, immune cells, and drugs. Tight junctions consist of proteins that physically "seal" the tight junction but also form channels that allow for permeation between the cells, resulting in epithelial surfaces of different tightness. The tight junction proteins occludin, tricellulin, and at least 24 members of the claudin family are characterized by four transmembranal domains and two extracellular loops that, like teeth of a zipper, contact the appropriate loops from opposing cell membranes. Tight junctions are regulated in their molecular composition, ultrastructure, and function by intracellular scaffolding proteins and the cytoskeleton; such regulation serves normal, physiologic adaptation but also occurs in numerous diseases.
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Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability.
Mol. Biol. Cell
PUBLISHED: 06-17-2009
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Tricellulin is a tight junction protein localized in tricellular tight junctions (tTJs), the meeting points of three cells, but also in bicellular tight junctions (bTJs). To investigate its specific barrier functions in bTJs and tTJs, TRIC-a was expressed in low-level tricellulin-expressing cells, and MDCK II, either in all TJs or only in tTJs. When expressed in all TJs, tricellulin increased paracellular electrical resistance and decreased permeability to ions and larger solutes, which are associated with enhanced ultrastructural integrity of bTJs toward enhanced strand linearity. In tTJs in contrast, ultrastructure was unchanged and tricellulin minimized permeability to macromolecules but not to ions. This paradox is explained by properties of the tTJ central tube which is wide enough for passage of macromolecules, but too rare to contribute significantly to ion permeability. In conclusion, at low tricellulin expression the tTJ central tube forms a pathway for macromolecules. At higher expression, tricellulin forms a barrier in tTJs effective only for macromolecules and in bTJs for solutes of all sizes.
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Claudin-16 affects transcellular Cl- secretion in MDCK cells.
J. Physiol. (Lond.)
PUBLISHED: 06-15-2009
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Claudin-16 (paracellin-1) is a tight junction protein localized mainly in the thick ascending limb of Henles loop and also in the distal nephron. Its defect causes familial hypomagnesaemia with hypercalciuria and nephrocalcinosis. This had been taken as an indication that claudin-16 conveys paracellular Mg(2+) and Ca(2+) transport; however, evidence is still conflicting. We studied paracellular ion permeabilities as well as effects of claudin-16 on the driving forces for passive ion movement. MDCK-C7 cells were stably transfected with wild-type (wt) and mutant (R146T, T233R) claudin-16. Results indicated that paracellular permeability to Mg(2+) but not to Ca(2+) is increased in cells transfected with wt compared to mutant claudin-16 and control cells. Increased basolateral Mg(2+) concentration activated a transcellular Cl(-) current which was greatly enhanced in cells transfected with wt and T233R claudin-16, as compared to R146T claudin-16-transfected or control cells. This current was triggered by the basolateral calcium-sensing receptor causing Ca(2+) release from internal stores, thus activating apical Ca(2+)-sensitive Cl(-) channels and basolateral Ca(2+)-sensitive K(+) channels. Immunohistochemical data suggest that the Cl(-) channel involved is bestrophin. We conclude that claudin-16 itself possesses only moderate paracellular Mg(2+) permeability but governs transcellular Cl(-) currents by interaction with apical Ca(2+)-activated Cl(-) channels, presumably bestrophin. As the transepithelial voltage generated by such a current alters the driving force for all ions, this may be the major mechanism to regulate Mg(2+) and Ca(2+) absorption in the kidney.
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Two-path impedance spectroscopy for measuring paracellular and transcellular epithelial resistance.
Biophys. J.
PUBLISHED: 05-22-2009
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Solutes are transported across epithelial cell layers via transcellular and paracellular pathways. The transcellular pathway leads across the apical and basolateral cell membrane, whereas the paracellular pathway is directed through the tight junction. Tight junction proteins (claudins, occludin, tricellulin) can not only form barriers but also paracellular channels that are--in concert with membrane channels and transporters--regulated in a wide range in health and disease states. Thus, it is desirable to determine para- and transcellular resistance (R(para), R(trans)) separately. This cannot be achieved by conventional transepithelial resistance (TER) measurements. We present an impedance spectroscopic approach that is optimized for differentiation between these two pathways. The method is based on a transepithelial impedance measurement in specialized Ussing chambers, combined with a Ca(2+)-dependent modulation of R(para) through EGTA and flux measurements of a nonradioactive paracellular marker, fluorescein. The prerequisites are a paracellular marker that varies in parallel to 1/R(para), an experimental regime that alters R(para) without affecting R(trans), and exact knowledge of the resistance of subepithelial components. The underlying prerequisites and the applicability as a routine method were verified on cell lines of different tightness including HT-29/B6 colon cells and Madin-Darby canine kidney tubule cells C7 and C11.
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A rice kinase-protein interaction map.
Plant Physiol.
PUBLISHED: 05-15-2009
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Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.
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Inflamed pouch mucosa possesses altered tight junctions indicating recurrence of inflammatory bowel disease.
Int J Colorectal Dis
PUBLISHED: 05-13-2009
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The etiology of pouchitis after coloproctomucosectomy with ileal pouch-anal anastomosis in patients with ulcerative colitis is still unknown. Beside changes in luminal antigens, the immunological predisposition is assumed to be responsible. In previous electrophysiological studies, we showed that mucosal barrier and transport function in pouchitis is markedly reduced. Thus, the aim of the present study was to analyze barrier function on the molecular level.
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Femtosecond laser induced flexibility change of human donor lenses.
Vision Res.
PUBLISHED: 04-23-2009
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According to the Helmholtz theory of accommodation the loss of accommodation amplitude is caused by the growing sclerosis of the crystalline lens, whereas the ciliary muscle and the lens capsule are mainly uneffected by age. A permanent treatment method for presbyopia which offers a dynamic accommodation ability is a recent field of study. The concept followed in this paper uses femtosecond laser pulses to potentially overcome the loss of deformation ability of the crystalline lens by creating gliding planes inside the lens tissue to improve its flexibility.
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Claudin-10 exists in six alternatively spliced isoforms that exhibit distinct localization and function.
J. Cell. Sci.
PUBLISHED: 04-21-2009
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The tight junction protein claudin-10 is known to exist in two isoforms, resulting from two alternative exons, 1a and 1b (Cldn10a, Cldn10b). Here, we identified and characterized another four claudin-10 splice variants in mouse and human. One (Cldn10a_v1) results from an alternative splice donor site, causing a deletion of the last 57 nucleotides of exon 1a. For each of these three variants one further splice variant was identified (Cldn10a_v2, Cldn10a_v3, Cldn10b_v1), lacking exon 4. When transfected into MDCK cells, Cldn10a, Cldn10a_v1 and Cldn10b were inserted into the tight junction, whereas isoforms of splice variants lacking exon 4 were retained in the endoplasmic reticulum. Cldn10a transfection into MDCK cells confirmed the previously described increase in paracellular anion permeability. Cldn10a_v1 transfection had no direct effect, but modulated Cldn10a-induced organic anion permeability. At variance with previous reports in MDCK-II cells, transfection of high-resistance MDCK-C7 cells with Cldn10b dramatically decreased transepithelial resistance, increased cation permeability, and changed monovalent cation selectivity from Eisenman sequence IV to X, indicating the presence of a high field-strength binding site that almost completely removes the hydration shell of the permeating cations. The extent of all these effects strongly depended on the endogenous claudins of the transfected cells.
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Listeriolysin O affects barrier function and induces chloride secretion in HT-29/B6 colon epithelial cells.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 04-16-2009
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Listeria monocytogenes is a food-borne pathogen, which is able to induce diarrhea when residing in the intestine. We studied the effect of listeriolysin O (LLO), an extracellular virulence factor of L. monocytogenes, on intestinal transport and barrier function in monolayers of HT-29/B6 human colon cells using the Ussing technique to understand the pathomechanisms involved. Mucosal addition of LLO, but not a LLO mutant, induced a dose- and pH-dependent increase in short-circuit current (I(SC)). Sodium and chloride tracer flux and DIDS sensitivity studies revealed that I(SC) was mainly due to electrogenic chloride secretion. Barrier function was impaired by LLO, as assessed by transepithelial resistance (R(t)) and mannitol flux measurements. Intracellular signal transduction occurred through Ca(2+) release from intracellular stores and PKC activation. In conclusion, listeriolysin induces chloride secretion and perturbs epithelial barrier function, thus potentially contributing to Listeria-induced diarrhea.
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Visualization of femtosecond laser pulse-induced microincisions inside crystalline lens tissue.
J Cataract Refract Surg
PUBLISHED: 03-16-2009
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To evaluate a new method for visualizing femtosecond laser pulse-induced microincisions inside crystalline lens tissue.
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Na+ absorption defends from paracellular back-leakage by claudin-8 upregulation.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-28-2009
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In distal colon, the limiting factor for Na(+) absorption is represented by the epithelial sodium channel (ENaC). During absorption, high transepithelial Na(+) gradients are observed. In human colon and in HT-29/B6-GR cells, we investigated whether Na(+) back-leakage is prevented by paracellular sealing. Tissues and cells were incubated with corticosteroids. Barrier properties were analyzed in electrophysiological experiments. Subsequently, analysis of ENaC and tight junction protein expression, localization, and regulation was performed. In colon, nanomolar aldosterone induced sodium absorption via ENaC. Concomitantly, paracellular (22)Na(+) permeability was reduced by half and claudin-8 within the tight junction complex was nearly doubled. Real-time PCR validated an increase of claudin-8 transcripts. Two-path impedance spectroscopy following ENaC induction in HT-29/B6-GR revealed a specific increase of paracellular resistance. These results represent an important physiological implication: Na(+) absorption is paralleled by claudin-8-mediated sealing of the paracellular barrier to prevent Na(+) back-leakage, supporting steep Na(+) gradients in distal colon.
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A colonic mineralocorticoid receptor cell model expressing epithelial Na+ channels.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-25-2009
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In the distal colon, the epithelial sodium channel (ENaC) is rate limiting for sodium absorption. Progress in the molecular characterization of ENaC expression and trafficking in response to the mineralocorticoid aldosterone has been hampered, since no epithelial colonic cell line existed expressing functional ENaC stimulated by nanomolar aldosterone via mineralocorticoid receptor (MR). Here, we present a human colonic epithelial cell line inducibly expressing the MR (HT-29/B6-Tet-On-MR) which exhibits aldosterone-dependent ENaC-mediated sodium transport in the presence of the short-chain fatty acid butyrate. Butyrate was necessary for high-level expression of MR which allowed for aldosterone-dependent upregulation of beta- and gamma-ENaC expression. As butyrate alone was not capable of promoting ENaC-mediated sodium transport, aldosterone-induced GILZ (glucocorticoid-induced leucine zipper protein) was identified as a candidate factor increasing apical ENaC levels.
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Molecular basis for cation selectivity in claudin-2-based paracellular pores: identification of an electrostatic interaction site.
J. Gen. Physiol.
PUBLISHED: 02-20-2009
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Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.
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Glucocorticoids and tumor necrosis factor-alpha synergize to induce absorption by the epithelial sodium channel in the colon.
Gastroenterology
PUBLISHED: 02-03-2009
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The epithelial sodium channel (ENaC) mediates electrogenic sodium absorption in distal colon. In patients with inflammatory bowel disease (IBD), ENaC induction is impaired, mainly through transcriptional suppression by proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha. Glucocorticoid therapy rapidly increases sodium absorption; we investigated the molecular mechanisms underlying the interaction among TNF-alpha, glucocorticoids, and ENaC induction.
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Protein-protein interactions of tandem affinity purified protein kinases from rice.
PLoS ONE
PUBLISHED: 02-01-2009
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Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.
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Claudins and other tight junction proteins.
Compr Physiol
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Epithelial transport relies on the proper function and regulation of the tight junction (TJ), other-wise uncontrolled paracellular leakage of solutes and water would occur. They also act as a fence against mixing of membrane proteins of the apical and basolateral side. The proteins determining paracellular transport consist of four transmembrane regions, intracellular N and C terminals, one intracellular and two extracellular loops (ECLs). The ECLs interact laterally and with counterparts of the neighboring cell and by this achieve a general sealing function. Two TJ protein families can be distinguished, claudins, comprising 27 members in mammals, and TJ-associated MARVEL proteins (TAMP), comprising occludin, tricellulin, and MarvelD3. They are linked to a multitude of TJ-associated regulatory and scaffolding proteins. The major TJ proteins are classified according to the physiological role they play in enabling or preventing paracellular transport. Many TJ proteins have sealing functions (claudins 1, 3, 5, 11, 14, 19, and tricellulin). In contrast, a significant number of claudins form channels across TJs which feature selectivity for cations (claudins 2, 10b, and 15), anions (claudin-10a and -17), or are permeable to water (claudin-2). For several TJ proteins, function is yet unclear as their effects on epithelial barriers are inconsistent (claudins 4, 7, 8, 16, and occludin). TJs undergo physiological and pathophysiological regulation by altering protein composition or abundance. Major pathophysiological conditions which involve changes in TJ protein composition are (1) effects of pathogens binding to TJ proteins, (2) altered TJ protein composition during inflammation and infection, and (3) altered TJ protein expression in cancers.
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ATX1-generated H3K4me3 is required for efficient elongation of transcription, not initiation, at ATX1-regulated genes.
PLoS Genet.
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Tri-methylated H3 lysine 4 (H3K4me3) is associated with transcriptionally active genes, but its function in the transcription process is still unclear. Point mutations in the catalytic domain of ATX1 (ARABIDOPSIS TRITHORAX1), a H3K4 methyltransferase, and RNAi knockdowns of subunits of the AtCOMPASS-like (Arabidopsis Complex Proteins Associated with Set) were used to address this question. We demonstrate that both ATX1 and AtCOMPASS-like are required for high level accumulation of TBP (TATA-binding protein) and Pol II at promoters and that this requirement is independent of the catalytic histone modifying activity. However, the catalytic function is critically required for transcription as H3K4me3 levels determine the efficiency of transcription elongation. The roles of H3K4me3, ATX1, and AtCOMPASS-like may be of a general relevance for transcription of Trithorax-activated eukaryotic genes.
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Cross regulation of sirtuin 1, AMPK, and PPAR? in conjugated linoleic acid treated adipocytes.
PLoS ONE
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Trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) reduces triglyceride (TG) levels in adipocytes through multiple pathways, with AMP-activated protein kinase (AMPK) generally facilitating, and peroxisome proliferator-activated receptor ? (PPAR?) generally opposing these reductions. Sirtuin 1 (SIRT1), a histone/protein deacetylase that affects energy homeostasis, often functions coordinately with AMPK, and is capable of binding to PPAR?, thereby inhibiting its activity. This study investigated the role of SIRT1 in the response of 3T3-L1 adipocytes to t10c12 CLA by testing the following hypotheses: 1) SIRT1 is functionally required for robust TG reduction; and 2) SIRT1, AMPK, and PPAR? cross regulate each other. These experiments were performed by using activators, inhibitors, or siRNA knockdowns that affected these pathways in t10c12 CLA-treated 3T3-L1 adipocytes. Inhibition of SIRT1 amounts or activity using siRNA, sirtinol, nicotinamide, or etomoxir attenuated the amount of TG loss, while SIRT1 activator SRT1720 increased the TG loss. SRT1720 increased AMPK activity while sirtuin-specific inhibitors decreased AMPK activity. Reciprocally, an AMPK inhibitor reduced SIRT1 activity. Treatment with t10c12 CLA increased PPAR? phosphorylation in an AMPK-dependent manner and increased the amount of PPAR? bound to SIRT1. Reciprocally, a PPAR? agonist attenuated AMPK and SIRT1 activity levels. These results indicated SIRT1 increased TG loss and that cross regulation between SIRT1, AMPK, and PPAR? occurred in 3T3-L1 adipocytes treated with t10c12 CLA.
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The plant-derived glucocorticoid receptor agonist Endiandrin A acts as co-stimulator of colonic epithelial sodium channels (ENaC) via SGK-1 and MAPKs.
PLoS ONE
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In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR), the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR). GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC) was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc)) in Ussing chambers. Endiandrin A (End A) has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF)-?. This was in part due to up-regulation of ?- and ?-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK) activation induced by dexamethasone and/or TNF-? and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk)-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of ?- and ?-ENaC-subunits along with induction of sgk-1.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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