tight junction proteins Dotaz Zobrazit nápovědu
The skin is known to be the largest organ in human organism creating interface with outer environment. The skin provides protective barrier against pathogens, physical and chemical insults, and against uncontrolled loss of water. The barrier function was primarily attributed to the stratum corneum (SC) but recent studies confirmed that epidermal tight junctions (TJs) also play important role in maintaining barrier properties of the skin. Independent observations indicate that barrier function and its recovery is impaired in aged skin. However, trans-epidermal water loss (TEWL) values remains rather unchanged in elderly population. UV radiation as major factor of photoageing impairs TJ proteins, but TJs have great self-regenerative potential. Since it may be possible that TJs can compensate TEWL in elderly due to its regenerative and compensatory capabilities, important question remains to be answered: how are TJs regulated during skin ageing? This review provides an insight into TJs functioning as epidermal barrier and summarizes current knowledge about the impact of ageing on the barrier function of the skin and epidermal TJs.
- MeSH
- epidermis metabolismus patologie účinky záření MeSH
- kožní absorpce MeSH
- lidé MeSH
- permeabilita MeSH
- perspiratio insensibilis MeSH
- stárnutí kůže * účinky záření MeSH
- stárnutí metabolismus patologie MeSH
- těsný spoj metabolismus patologie účinky záření MeSH
- ultrafialové záření škodlivé účinky MeSH
- věkové faktory MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc.
- MeSH
- alosterická regulace MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- klonování DNA MeSH
- lidé MeSH
- ligandy MeSH
- peptidy chemie genetika metabolismus MeSH
- protein zonula occludens 1 chemie genetika metabolismus MeSH
- proteinové domény MeSH
- proteinové inženýrství MeSH
- rekombinantní fúzní proteiny chemie genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky * MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Many respiratory pathogens compromise epithelial barrier function during lung infection by disrupting intercellular junctions, such as adherens junctions and tight junctions, that maintain intercellular integrity. This includes Streptococcus pneumoniae, a leading cause of pneumonia, which can successfully breach the epithelial barrier and cause severe infections such as septicemia and meningitis. Fluorescence microscopy analysis on intercellular junction protein manipulation by respiratory pathogens has yielded major advances in our understanding of their pathogenesis. Unfortunately, a lack of automated image analysis tools that can tolerate variability in sample-sample staining has limited the accuracy in evaluating intercellular junction organization quantitatively. We have created an open source, automated Python computer script called "Intercellular Junction Organization Quantification" or IJOQ that can handle a high degree of sample-sample staining variability and robustly measure intercellular junction integrity. In silico validation of IJOQ was successful in analyzing computer generated images containing varying degrees of simulated intercellular junction disruption. Accurate IJOQ analysis was further confirmed using images generated from in vitro and in vivo bacterial infection models. When compared in parallel to a previously published, semi-automated script used to measure intercellular junction organization, IJOQ demonstrated superior analysis for all in vitro and in vivo experiments described herein. These data indicate that IJOQ is an unbiased, easy-to-use tool for fluorescence microscopy analysis and will serve as a valuable, automated resource to rapidly quantify intercellular junction disruption under diverse experimental conditions.
We aimed to investigate the effects of brain-derived neurotrophic factor (BDNF) on apoptosis of intestinal epithelial cells (IECs) and alterations of intestinal barrier integrity using BDNF knock-out mice model. Colonic tissues from BDNF(+/+) mice and BDNF(+/-) mice were prepared for this study. The integrity of colonic mucosa was evaluated by measuring trans-mucosa electrical resistance and tissue conductance in Ussing chamber. The colonic epithelial structure was analyzed by transmission electron microscopy. Apoptosis involvement was determined with TUNEL staining, active caspase-3 immunostaining and Western blotting for the protein expression of active caspase-3, Bax and Bcl-2. The expression levels and distribution of tight junction proteins were evaluated by immunohistochemistry or Western blots. Compared with BDNF(+/+) mice, BDNF(+/-) mice displayed impaired integrity and ultrastructure alterations in their colonic mucosa, which was characterized by diminished microvilli, mitochondrial swelling and epithelial cells apoptosis. Altered intestinal barrier function was linked to excessive apoptosis of IECs demonstrated by the higher proportion of TUNEL-positive apoptotic cells and enhanced caspase activities in BDNF(+/-) mice. Increased expression of Bax and claudin-2 proteins and reduced Bcl-2 and tight junction proteins (occludin, ZO-1 and claudin-1) expression were also detected in the colonic mucosa of BDNF(+/-) mice. BDNF may play a role in the maintenance of intestinal barrier integrity via its anti-apoptotic properties.
- MeSH
- apoptóza * MeSH
- epitelové buňky fyziologie MeSH
- kaspasa 3 metabolismus MeSH
- mozkový neurotrofický faktor fyziologie MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- proteiny těsného spoje metabolismus MeSH
- střevní sliznice metabolismus ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Gap junctional intercellular communication (GJIC) is an important mechanism that is involved and affected in many diseases and injuries. So far, the effect of nanosecond pulsed electric fields (nsPEFs) on the communication between cells was not investigated. An in vitro approach is presented with rat liver epithelial WB-F344 cells grown and exposed in a monolayer. In order to observe sub-lethal effects, cells were exposed to pulsed electric fields with a duration of 100ns and amplitudes between 10 and 20kV/cm. GJIC strongly decreased within 15min after treatment but recovered within 24h. Gene expression of Cx43 was significantly decreased and associated with a reduced total amount of Cx43 protein. In addition, MAP kinases p38 and Erk1/2, involved in Cx43 phosphorylation, were activated and Cx43 became hyperphosphorylated. Immunofluorescent staining of Cx43 displayed the disassembly of gap junctions. Further, a reorganization of the actin cytoskeleton was observed whereas tight junction protein ZO-1 was not significantly affected. All effects were field- and time-dependent and most pronounced within 30 to 60min after treatment. A better understanding of a possible manipulation of GJIC by nsPEFs might eventually offer a possibility to develop and improve treatments.
- MeSH
- aktiny metabolismus MeSH
- aktivace enzymů MeSH
- buněčné linie MeSH
- časové faktory MeSH
- elektřina * MeSH
- fosforylace MeSH
- konexin 43 genetika metabolismus MeSH
- krysa rodu rattus MeSH
- mezerový spoj metabolismus MeSH
- mezibuněčná komunikace * MeSH
- mitogenem aktivovaná proteinkinasa 1 metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 3 metabolismus MeSH
- mitogenem aktivované proteinkinasy p38 metabolismus MeSH
- protein zonula occludens 1 metabolismus MeSH
- regulace genové exprese MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Non-melanoma skin cancer (NMSC) is the most frequent human cancer with continuously rising incidences worldwide. Herein, we investigated the molecular basis for the impaired skin barrier function of organotypic NMSC models. We unraveled disturbed epidermal differentiation by reflectance confocal microscopy and histopathological evaluation. While the presence of claudin-4 and occludin were distinctly reduced, zonula occludens protein-1 was more wide-spread, and claudin-1 was heterogeneously distributed within the NMSC models compared with normal reconstructed human skin. Moreover, the cancer altered stratum corneum lipid packing and profile with decreased cholesterol content, increased phospholipid amount, and altered ceramide subclasses. These alterations contributed to increased surface pH and to 1.5 to 2.6-fold enhanced caffeine permeability of the NMSC models. Three topical applications of ingenol mebutate gel (0.015%) caused abundant epidermal cell necrosis, decreased Ki-67 indices, and increased lactate dehydrogenase activity. Taken together, our study provides new biological insights into the microenvironment of organotypic NMSC models, improves the understanding of the disease model by revealing causes for impaired skin barrier function in NMSC models at the molecular level, and fosters human cell-based approaches in preclinical drug evaluation.
- MeSH
- diterpeny farmakologie MeSH
- esterasy metabolismus MeSH
- fibroblasty metabolismus MeSH
- keratinocyty metabolismus MeSH
- kofein farmakologie MeSH
- kokultivační techniky MeSH
- kůže účinky léků metabolismus MeSH
- lidé MeSH
- metabolismus lipidů MeSH
- nádorové buněčné linie MeSH
- nádory kůže metabolismus MeSH
- proteiny těsného spoje metabolismus MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A balanced microbiota of the gastrointestinal tract (GIT) is a prerequisite for a healthy host. The GIT microbiota in preterm infants is determined by the method of delivery and nutrition. Probiotics can improve the GIT microbiota balance and suitable animal models are required to verify their harmlessness. Preterm gnotobiotic piglets were colonized with Lactobacillus rhamnosus GG (LGG) to evaluate its safety and possible protective action against infection with an enteric pathogen, Salmonella Typhimurium (ST). Clinical signs (anorexia, somnolence, fever and diarrhea), bacterial interference and translocation, intestinal histopathology, transcriptions of claudin-1, occludin and interferon (IFN)-γ, intestinal and systemic protein levels of interleukin (IL)-8, IL-12/23 p40 and IFN-γ were compared among (i) germ-free, (ii) LGG-colonized, (iii) ST-infected and (iv) LGG-colonized and subsequently ST-infected piglets for 24 h. Both LGG and ST-colonized the GIT; LGG translocated in some cases into mesenteric lymph nodes and the spleen but did not cause bacteremia and clinical changes. ST caused clinical signs of gastroenteritis, translocated into mesenteric lymph nodes, the spleen, liver and blood, increased claudin-1 and IFN-γ transcriptions, but decreased occludin transcription and increased local and systemic levels of IL-8 and IL-12/23 p40. Previous colonization with LGG reduced ST colonization in the jejunum and translocation into the liver, spleen and blood. It partially ameliorated histopathological changes in the intestine, reduced IL-8 levels in the jejunum and plasma and IL-12/23 p40 in the jejunum. The preterm gnotobiotic piglet model of the vulnerable preterm immunocompromised infant is useful to verify the safety of probiotics and evaluate their protective effect.
- MeSH
- bakteriální translokace MeSH
- cytokiny analýza MeSH
- gnotobiologické modely MeSH
- Lacticaseibacillus rhamnosus * MeSH
- prasata MeSH
- předčasný porod mikrobiologie MeSH
- probiotika farmakologie MeSH
- proteiny těsného spoje genetika MeSH
- Salmonella typhimurium růst a vývoj MeSH
- střeva mikrobiologie patologie MeSH
- střevní mikroflóra MeSH
- těhotenství MeSH
- zvířata MeSH
- Check Tag
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whether i.v. butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins, and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins (ZO-1, claudin-7, E-cadherin), antimicrobial peptides (Defa 8, Rd5, RegIIIγ), and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10, and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/pathogenic genera, which might contribute to its overall beneficial effect.
- MeSH
- biodiverzita MeSH
- butyráty farmakologie MeSH
- fenotyp MeSH
- fylogeneze MeSH
- ileum účinky léků patologie MeSH
- kolon účinky léků patologie MeSH
- lymfatické uzliny účinky léků metabolismus MeSH
- lymfocyty účinky léků metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- modely u zvířat MeSH
- muciny biosyntéza MeSH
- Panethovy buňky účinky léků metabolismus MeSH
- parenterální výživa * MeSH
- peptidy genetika metabolismus MeSH
- permeabilita MeSH
- potkani Wistar MeSH
- potravní doplňky * MeSH
- proteiny těsného spoje metabolismus MeSH
- regulace genové exprese účinky léků MeSH
- střeva patologie MeSH
- střevní mikroflóra * účinky léků MeSH
- tenké střevo účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Intestinal milieu disorders are strongly related to the occurrence of inflammatory bowel diseases (IBDs), which results from mucosa destruction, epithelium disruption, and tight junction (TJ) proteins loss. Excess of H2 S in the intestinal milieu produced by the sulfate-reducing bacteria metabolism contributes to development of IBDs via epithelial barrier breakdown. Conventional interventions, such as surgery and anti-inflammatory medications, are considered not completely effective because of frequent recurrence and other complications. Herein, a novel oral delivery system, a hydroxypropyl methylcellulose acetate succinate (HPMCAS)-based polymer-coated Zr-based metal-organic framework (UiO-66) with a Cux -rhodamine B (CR) probe (hereinafter referred to as HUR), is produced via a co-flow microfluidic approach with the ability to reduce H2 S levels, thus restoring the intestinal lumen milieu. HPMCAS serves as an enteric coating that exposes UiO-66@CR at the pH of the intestine but not the acidic pH of the stomach. The synthesized HUR exhibits notable therapeutic efficacy, including mucosa recovery, epithelium integrity restoration, and TJ proteins upregulation via H2 S scavenging to protect against intestinal barrier damage and microbiome dysbiosis. Thus, HUR is verified to be a promising theranostic platform able to decrease the H2 S content for intestinal milieu disorder treatment. The presented study therefore opens the door for further exploitation for IBDs therapy.
Puwainaphycins (PUW) and minutissamides (MIN) are cyanobacterial lipopeptides found in various cyanobacterial species. The first possible target of human exposure to them is intestinal epithelium but effect of PUW/MIN on enterocytes is not known at all. Using differentiated Caco-2 cells, PUW F was found to be cytotoxic from 5 µM concentration based on lactate dehydrogenase release assay and total protein concentration. However, it is also able to induce production of interleukin 8 in non-cytotoxic concentrations 1 and 2.5 µM detected by ELISA. Effects of MIN A and C were similar but less pronounced compared to PUW F. On the other hand, MIN D was the least toxic compound with no significant pro-inflammatory effects. Surprisingly, pro-inflammatory activation of the cells by PUW F and MIN C resulted in an increase in tight junction (TJ) protein claudin 4 expression determined by western blot analysis and confirmed by confocal microscopy. Furthermore, decrease in expression of zonula occludens 3, another TJ protein, was observed after the exposure to PUW F. Taken together, these cytotoxic lipopeptides, especially PUW F, are to be studied more deeply due to their capability to activate and/or deregulate human enterocytes in low concentrations.
- MeSH
- Caco-2 buňky MeSH
- lidé MeSH
- lipopeptidy * MeSH
- sinice * MeSH
- střevní sliznice MeSH
- těsný spoj MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
