Junctional Barrier Research Articles

Critical pH Level of Lye (NaOH) for Esophageal Injury

Lye (NaOH) ingestion in humans often results in alkaline damage to the esophagus, but knowledge about this process is limited. Here, we explore the effects of lye on esophageal epithelial structure and function using rabbit esophageal epithelium as a model of lye ingestion. Rabbit esophageal epithelium was mounted in Ussing chambers so that the electrical potential difference (PD), short-circuit current (I (sc)), and transepithelial resistance (R (T)) could be monitored before, during, and after mucosal exposure to lye (NaOH) at pHs ranging from 7.4 to 12.1. Histopathology and dextran fluxes were also performed and correlated with the electrical data. Mucosal exposure to lye at pHs <11.5 had no damaging effects on the esophagus. However, at pHs >or=11.5, damage was both time- and pH-dependent, as noted by increases in PD and I (sc), and declines in R (T). Further, the electrical changes were paralleled morphologically by epithelial liquefaction necrosis and increases in dextran flux. Also, by pretreating tissues with ouabain, the early lye-induced rise in PD and I (sc) was shown to result from a combination of increased active (sodium) transport and passive (sodium) diffusion which indicates that, even early on, the damaging effects of lye include changes in both apical cell membranes and tight junctions of this epithelium. Lye (NaOH) injury to the esophageal epithelium is both pH- and time-dependent, but requires a minimum pH of 11.5. At pHs >or=11.5, lye produces liquefaction necrosis, an injury that involves both cellular and junctional barriers, and which markedly increases epithelial permeability to ions and uncharged molecules. Based on these results, non-industrial cleaning products in the home are likely to be safer if they have a concentration of lye below pH 11.5.

Dynamics of neutrophil extravasation and vascular permeability are uncoupled during aseptic cutaneous wounding

Transport of macromolecules and transmigration of leukocytes across vascular endothelium are regulated by a tight molecular junction, but the mechanisms by which these two inflammatory events are differentially controlled in time and magnitude during aseptic cutaneous wounding remain elusive. A real-time fluorescence imaging technique was developed to simultaneously track influx of Alexa 680-labeled albumin and genetically tagged enhanced green fluorescent protein-neutrophils [polymorphonuclear neutrophils (PMN)] within the wound bed. Vascular permeability increased approximately threefold more rapidly than the rate of PMN influx, reaching a maximum at 12 h, on the order of approximately 0.15% per minute versus approximately 0.05% per minute for PMN influx, which peaked at 18 h. Systemic depletion of PMN with antibody blocked their extravasation to the wound but did not alter the increase in vascular permeability. In contrast, pretreatment with antiplatelet GPIb decreased permeability by 25% and PMN influx by 50%. Hyperpermeability stimulated by the endothelium-specific agonists VEGF or thrombin at 24 h postwounding was completely inhibited by blocking Rho-kinase-dependent signaling, whereas less inhibition was observed at 1 h and neutrophil influx was not perturbed. These data suggest that in aseptic wounds, the endothelium maintains a tight junctional barrier to protein leakage that is independent of neutrophil transmigration, partially dependent on circulating platelets, and associated with Rho-kinase-dependent signaling.

Critical role of Cdc42 in mediating endothelial barrier protection in vivo

Activation of the Rho GTPase Cdc42 has been shown in endothelial cell monolayers to prevent disassembly of interendothelial junctions and the increase in endothelial permeability. Here, we addressed the in vivo role of Cdc42 activity in mediating endothelial barrier protection in lungs by generating mice expressing the dominant active mutant V12Cdc42 protein in vascular endothelial cells targeted via the VE-cadherin promoter. These mice developed normally and exhibited constitutively active GTP-bound Cdc42. The increase in lung vascular permeability and gain in tissue water content in response to intraperitoneal lipopolysaccharide challenge (7 mg/kg) were markedly attenuated in the transgenic mice. To address the basis of the protective effect, we observed that expression of V12Cdc42 mutant in endothelial monolayers reduced the decrease in transendothelial electrical resistance, a measure of opening of interendothelial junctions, thus indicating that Cdc42 activity preserved junctional integrity. RhoA activity in V12Cdc42-expressing endothelial monolayers was reduced compared with untransfected cells, suggesting that activated Cdc42 functions by counteracting the canonical RhoA-mediated mechanism of endothelial hyperpermeability. Therefore, Cdc42 activity of microvessel endothelial cells is a critical determinant of junctional barrier restrictiveness and may represent a means of therapeutically modulating increased lung vascular permeability and edema formation.

Effect of Focused Ultrasound Applied With an Ultrasound Contrast Agent on the Tight Junctional Integrity of the Brain Microvascular Endothelium

Previous studies have investigated a potential method for targeted drug delivery in the central nervous system that uses focused ultrasound bursts combined with an ultrasound contrast agent to temporarily disrupt the blood-brain barrier (BBB). The purpose of this work was to investigate the integrity of the tight junctions (TJs) in rat brain microvessels after this BBB disruption. Ultrasound bursts (1.5-MHz) in combination with a gas contrast agent (Optison) was applied at two locations in the brain in 25 rats to induce BBB disruption. Using immunoelectron microscopy, the distributions of the TJ-specific transmembrane proteins occludin, claudin-1, claudin-5, and of submembranous ZO-1 were examined at 1, 2, 4, 6 and 24 h after sonication. A quantitative evaluation of the protein expression was made by counting the number of immunosignals per micrometer in the junctional clefts. BBB disruption at the sonicated locations was confirmed by the leakage of i.v. administered horseradish peroxidase (HRP, m.w. 40,000 Da) and lanthanum chloride (La 3+, m.w. ∼ 139 Da). Leakage of these agents was observed at 1 and 2 h and, in a few vessels, at 4 h after ultrasound application. These changes were paralleled by the apparent disintegration of the TJ complexes, as evidenced by the redistribution and loss of the immunosignals for occludin, claudin-5 and ZO-1. Claudin-1 seemed less involved. At 6 and 24 h after sonication, no HRP or lanthanum leakage was observed and the barrier function of the TJs, as indicated by the localization and density of immunosignals, appeared to be completely restored. This study provides the first direct evidence that ultrasound bursts combined with a gas contrast agent cause disassembling of the TJ molecular structure, leading to loss of the junctional barrier functions in brain microvessels. The BBB disruption appears to last up to 4 h after sonication and permits the paracellular passage of agents with molecular weights up to at least 40 kDa. These promising features can be exploited in the future development of this method that could enable the delivery of drugs, antibodies or genes to targeted locations in the brain. (E-mail: njm@bwh.harvard.edu)

Septate junctions are required for ommatidial integrity and blood–eye barrier function in Drosophila

The anatomical organization of the Drosophila ommatidia is achieved by specification and contextual placement of photoreceptors, cone and pigment cells. The photoreceptors must be sealed from high ionic concentrations of the hemolymph by a barrier to allow phototransduction. In vertebrates, a blood–retinal barrier (BRB) is established by tight junctions (TJs) present in the retinal pigment epithelium and endothelial membrane of the retinal vessels. In Drosophila ommatidia, the junctional organization and barrier formation is poorly understood. Here we report that septate junctions (SJs), the vertebrate analogs of TJs, are present in the adult ommatidia and are formed between and among the cone and pigment cells. We show that the localization of Neurexin IV (Nrx IV), a SJ-specific protein, coincides with the location of SJs in the cone and pigment cells. Somatic mosaic analysis of nrx IV null mutants shows that loss of Nrx IV leads to defects in ommatidial morphology and integrity. nrx IV hypomorphic allelic combinations generated viable adults with defective SJs and displayed a compromised blood–eye barrier (BEB) function. These findings establish that SJs are essential for ommatidial integrity and in creating a BEB around the ion and light sensitive photoreceptors. Our studies may provide clues towards understanding the vertebrate BEB formation and function.

Lymphocyte transmigration in the brain: a new way of thinking

Lymphocytes depend on endothelial adhesion molecules such as ICAM-1 and VCAM-1, upregulated with inflammation, to facilitate transmigration across junctional barriers. New data show that ALCAM replaces VCAM-1 in the CNS during the development of neuroinflammatory diseases such as multiple sclerosis.

Effects of puromycin aminonucleoside on the cytoskeletal changes of glomerular epithelial cells

Purpose : This study was designed to clarify the mechanism of proteinuria in nephrotic syndrome patients by using puromycin aminonucleoside (PAN) nephrosis model. Methods : Following administration of various concentrations of PAN and antioxidants we observed the changes of podocyte cytoskeletons in cultured rat glomerular epithelial cells (GEpC) by method of scanning electron microscope, reactive oxyten species (ROS) analysis, permeability assay, confocal microscope, and Western blot assay. Results : PAN not only induced the ultrastructural changes of GEpC, such as shortening and fusion of microvilli, but also separated the intercellular gaps and linear ZO-1. PAN induced oxidative stresses in time and dose dependent manners and increases of intercellular permeability in anti-oxidants inhibitable manners. High concentration of PAN induced not only actin polymerization and disorganization, but also the conglomerulation and internal dislocation of α-actinin protein. The intensities of fluorescences of ZO-1 protein were diminished and internalized by PAN in a dose-dependent manner, which were inhibited by anti anti-oxidants. Conclusion : PAN induced the changes of podocytes cytoskeleton and junctional barriers by way of increasing ROS in GEpC that resulted in increasing their permeability in a antioxidatn-inhibitable manner. Glomerular hyperpermeability induced by PAN mediateing through oxidative stresses is thought to take part in the mechanism of proteinuria in nephrotic syndrome.

A seamless trespass: germ cell migration across the seminiferous epithelium during spermatogenesis

During spermatogenesis, preleptotene spermatocytes traverse the blood–testis barrier (BTB) in the seminiferous epithelium, which is reminiscent of viral pathogens breaking through the tight junctions of host epithelial cells. The process also closely resembles the migration of leukocytes across endothelial tight junctions to reach inflammation sites. Cell adhesion molecules of the immunoglobulin superfamily (e.g., JAM/CAR/nectin) participate in germ cell migration by conferring transient adhesion between Sertoli and germ cells through homophilic and heterophilic interactions. The same molecules also comprise the junctional complexes at the BTB. Interestingly, JAM/CAR/nectin molecules mediate virus uptake and leukocyte transmigration in strikingly similar manners. It is likely that the strategy used by viruses and leukocytes to break through junctional barriers is used by germ cells to open up the inter–Sertoli cell junctions. In associating these diverse cellular events, we highlight the “guiding” role of JAM/CAR/nectin molecules for germ cell passage. Knowledge on viral invasion and leukocyte transmigration has also shed insights into germ cell movement during spermatogenesis.

A Sertoli Cell-Specific Knockout of Connexin43 Prevents Initiation of Spermatogenesis

The predominant testicular gap junctional protein connexin43 (cx43) is located between neighboring Sertoli cells (SCs) and between SCs and germ cells. It is assumed to be involved in testicular development, cell differentiation, initiation, and maintenance of spermatogenesis with alterations of its expression being correlated with various testicular disorders. Because total disruption of the cx43 gene leads to perinatal death, we generated a conditional cx43 knockout (KO) mouse using the Cre/loxP recombination system, which lacks the cx43 gene solely in SCs (SCCx43KO), to evaluate the SC-specific functions of cx43 on spermatogenesis in vivo. Adult SCCx43KO(-/-) mice showed normal testis descent and development of the urogenital tract, but testis size and weight were drastically lower compared with heterozygous and wild-type littermates. Histological analysis and quantitation of mRNA expression of germ cell-specific marker genes revealed a significant reduction in the number of spermatogonia but increased SC numbers/tubule with only a few tubules left showing normal spermatogenesis. Thus, SC-specific deletion of cx43 mostly resulted in an arrest of spermatogenesis at the level of spermatogonia or SC-only syndrome and in intratubular SC clusters. Our data demonstrate for the first time that cx43 expression in SCs is an absolute requirement for normal testicular development and spermatogenesis.

Size‐selective junctional barrier and Ca2+‐independent cell adhesion in the testis of Cynops pyrrhogaster: Expression and Function of Occludin

In urodeles which has testicular structure different from that in mammals, blood-testis barrier was reported to exist like in mammals. However, molecular and functional analyses of the components of the blood-testis barrier in urodeles have not been reported yet. Toward elucidation of the barrier functions and their molecular components in newt testis, we aimed to isolate occludin cDNAs and obtained two kinds of occludin partial cDNAs (occludin 1 and 2) encoding the putative second extracellular loop. Immunoblot and immunofluorescence studies using antibodies against peptides each corresponding to a part of the second extracellular loop of occludin 1 and 2, and those against beta-catenin and zonula occludens-1 (ZO-1) showed that occludin, as well as beta-catenin and ZO-1, was expressed not only in Sertoli cells but also in germ cells throughout all the stages from spermatogonia to elongate spermatids. Tracer experiments revealed a size-selective barrier which allows small molecules ( approximately 500 Da) to get into cysts through Sertoli cells' barrier, but not larger ones (>1.9 kDa) in the stages from spermatogonia to almost mature sperm. No occludin peptides corresponding to a part of the second extracellular loop destroyed the junctional barrier, while both the peptides and antibodies significantly inhibited reaggregation of the dissociated testicular cells which was to a large extent Ca(2+)-independent. These results indicate that the second extracellular loop of occludin is involved in cell adhesion rather than in size-selective barrier in newt testis, though the possibility cannot be excluded that the peptides were not long enough to inhibit the barrier function.

Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells

AbstractRecent evidence from this laboratory indicated that reduced expression of caveolin-1 accompanied the diminished expression of tight junction (TJ)–associated proteins occludin and zonula occludens-1 (ZO-1) following stimulation of brain microvascular endothelial cells (BMECs) with the chemokine CCL2 (formerly called MCP-1). Because attenuated caveolin-1 levels have also been correlated with heightened permeability of other endothelia, the objective of this study was to test the hypothesis that reduced caveolin-1 expression is causally linked to the action of CCL2 on BMEC junctional protein expression and barrier integrity. This was achieved using adenovirus to nondestructively deliver caveolin-1 siRNA (Ad-siCav-1) to BMEC monolayers, which model the blood-brain barrier (BBB). Treatment with siRNA reduced the caveolin-1 protein level as well as occludin and ZO-1. Additionally, occludin exhibited dissociation from the cytoskeletal framework. These changes were attended by comparable alterations in adherens junction (AJ)–associated proteins, VE-cadherin and β-catenin, increased BMEC paracellular permeability, and facilitated the ability of CCL2 to stimulate monocytic transendothelial migration. Furthermore, treating BMECs with cavtratin, a synthetic cell-permeable peptide encoding the caveolin-1 scaffolding domain, antagonized effects of both Ad-siCav-1 and CCL2. These results collectively highlight caveolin-1 loss as a critical step in CCL2-induced modulation of BMEC junctional protein expression and integrity, and possibly serve a crucial role in regulating inflammation at the BBB.

Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis

Products generated from lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TGRL) are reported to increase endothelial layer permeability. We hypothesize that these increases in permeability result from the active rearrangement and dissolution of the junctional barrier in human aortic endothelial cells, as well as induction of the apoptotic cascade. Human aortic endothelial cells were treated with TGRL lipolysis products generated from coincubation of human TGRL plus lipoprotein lipase. Measurement of transendothelial electrical resistance demonstrated a time-dependent decrease in endothelial barrier function in response to TGRL lipolysis products. Immunofluorescent localization of zonula occludens-1 (ZO-1) showed radial rearrangement along cell borders after 1.5 h of treatment with lipolysis products. A concurrent redistribution of F-actin from the cell body to the cell margins was observed via rhodamine phalloidin staining. Immunofluorescent imaging for occludin and vascular endothelial cadherin showed that these proteins relocalize as well, although these changes are less prominent than for ZO-1. Western analysis of cells exposed to lipolysis products for 3 h revealed the fragmentation of ZO-1, a reduction in occludin, and no change of vascular endothelial cadherin. Lipolysis products also increased caspase-3 activity and induced nuclear fragmentation. Treatments did not cause oncosis in cells at any point during the incubation. These results demonstrate that TGRL lipolysis products play an important role in the regulation of endothelial permeability, the organization of the actin cytoskeleton, the localization and expression of junctional proteins, especially ZO-1, and the induction of apoptosis.

Modulation of tight junction properties relevant to fluid transport across rabbit corneal endothelium

Paracellular junctions could play an important role in corneal endothelial fluid transport. In this study we explored the effects of different reagents on the tight junctional barrier by assessing the translayer specific electrical resistance (TER) across rabbit corneal endothelial preparations and cultured rabbit corneal endothelial cells' (CRCEC) monolayers, the paracellular permeability (Papp) for fluorescein isothiocyanate (FITC) dextrans across CRCEC, and fluid transport across de-epithelialized rabbit corneal endothelial preparations. Palmitoyl carnitine (PC), poly- l-lysine (PLL), adenosine triphosphate (ATP), and dibutyryl adenosine 3′,5′-cyclic monophosphate (dB-cAMP) were used to modulate corneal endothelial fluid transport and tight junctions (TJs). After seeding, the TER across CRCEC reached maximal values (29.2 ± 1.0 Ω cm 2) only after the 10th day. PC (0.1 mM) caused decreases both in TER (by 40%) and fluid transport (swelling rate: 18.5 ± 0.3 μm/h), and an increase in Papp. PLL resulted in increased TER rose and Papp but decreased fluid transport (swelling rate: 10 ± 0.3 μm/h). dB-cAMP (0.1 mM) and ATP (0.1 mM) decreased TER by 16% and 6%, increased Papp slightly, and stimulated fluid transport; the rates of de-swelling (in μm/h) were −5.4 ± 0.3 and −12.1 ± 0.4, respectively. PC might cause the junctions to open up unspecifically and thus increase passive leak. PLL is a known junctional charge modifier that may be adding steric hindrance to the tight junctions. The results with dB-cAMP and ATP are consistent with fluid transport via the paracellular route.

JAM-C Regulates Tight Junctions and Integrin-mediated Cell Adhesion and Migration

Junctional Adhesion Molecules (JAMs) have been described as major components of tight junctions in endothelial and epithelial cells. Tight junctions are crucial for the establishment and maintenance of cell polarity. During tumor development, they are remodeled, enabling neoplastic cells to escape from constraints imposed by intercellular junctions and to adopt a migratory behavior. Using a carcinoma cell line we tested whether JAM-C could affect tight junctions and migratory properties of tumor cells. We show that transfection of JAM-C improves the tight junctional barrier in tumor cells devoid of JAM-C expression. This is dependent on serine 281 in the cytoplasmic tail of JAM-C because serine mutation into alanine abolishes the specific localization of JAM-C in tight junctions and establishment of cell polarity. More importantly, the same mutation stimulates integrin-mediated cell migration and adhesion via the modulation of beta1 and beta3 integrin activation. These results highlight an unexpected function for JAM-C in controlling epithelial cell conversion from a static, polarized state to a pro-migratory phenotype.

Oxidized LDL specifically promotes the initiation of monocyte invasion during transendothelial migration with upregulated PECAM-1 and downregulated VE-cadherin on endothelial junctions

It is poorly understood how oxidized LDL (oxLDL) promotes monocyte dynamics in transendothelial migration (TEM) in atherogenesis. We developed an in vitro 3D-live-single cell TEM assay system with subendothelial oxLDL embedded in ultra-thin collagen gels, mimicking subendothelial oxLDL accumulation in vivo. With dividing monocyte dynamics into three stages (1: adhesion on endothelium, 2: invasion and 3: complete transmigration below endothelium), we analyzed the stage transition dynamics of individual living human monocytes. OxLDL did not enhance initial monocyte adhesion to endothelium (stage 1), but it specifically primed adherent monocytes to start invasion (stage 1 → 2). Once invasion started, it had no effect thereafter on monocyte stage transition (stage 2 → 3). OxLDL upregulated PECAM-1 and downregulated VE-cadherin on endothelial junctions without monocyte addition, both of which could promote monocyte entry by enhanced homophilic binding to monocyte PECAM-1, and by disrupted junctional barrier, respectively. Meanwhile, monocyte speed at neither locomotion on endothelium (stage 1) nor subendothelial migration (stage 3) was altered by oxLDL. These data indicate that before monocyte adhesion, endothelial junctions changed their conformation to more monocyte-acceptable state in response to oxLDL, resulting the stage-specific promotion of monocyte TEM (stage 1 → 2; initiation of invasion) with no enhancement of its initial adhesion or migration speed.

The role of caspase-3 in lipopolysaccharide-mediated disruption of intestinal epithelial tight junctions

The mechanisms responsible for microbially induced epithelial apoptosis and increased intestinal permeability remain unclear. This study assessed whether purified bacterial lipopolysaccharide (LPS) increases epithelial apoptosis and permeability and whether these changes are dependent on caspase-3 activation. In nontumorigenic epithelial monolayers, Escherichia coli O26:B6 LPS increased apoptosis, as shown by nuclear breakdown, caspase-3 activation, and PARP cleavage, and induced disruption of tight junctional ZO-1. Apical, but not basolateral, exposure to LPS increased epithelial permeability. Addition of a caspase-3 inhibitor abolished the effects of LPS. The findings describe a novel mechanism whereby apical LPS may disrupt epithelial tight junctional ZO-1 and barrier function in a caspase-3-dependent fashion.

Immunohistochemical study of serum albumin in normal and cadmium-treated mouse testis organs by “in vivo cryotechnique”

The in vivo injection of cadmium (Cd) was reported to induce blood-testis barrier disruption, and assumed to be an experimental model to examine junctional structures in seminiferous tubules. The purpose of this study is to investigate time-dependent changes of albumin permeability in the normal or Cd-treated mouse testis by our "in vivo cryotechnique" with immunohistochemistry, reflecting tight junctional (TJ) barriers of Sertoli cells. The albumin in the seminiferous tubules was firstly immobilized by the cryotechnique, in which normal blood circulation was always kept. The cryofixed testicular tissues were then processed for freeze-substitution, and embedded in the paraffin wax. Serial sections were immunostained by anti-mouse albumin antibody with peroxidase immunostaining, and also stained with hematoxylin-eosine (HE) for morphological observation. In normal seminiferous tubules, the immunoreaction products were localized around peritubular myoid cells and between Leydig cells, as well as in blood vessels. They were also localized as arch-like patterns around some spermatogonia in basal compartments of seminiferous tubules. Twenty-four and 48 hrs after Cd-treatment, some enlarged spaces and vesicular formations in the seminiferous epithelium were observed on the HE-stained sections. The albumin immunolocalization was detected not only in the basal compartments, but also in the adluminal compartments between Sertoli cells and germ cells. Thus, the structural disruptions of inter-Sertoli TJ barriers could be clearly demonstrated by the "in vivo cryotechnique".

724 単球の内皮下浸潤プロセスに及ぼす酸化LDLの影響 : 内皮細胞間接着タンパクの発現変化を介した影響(S08-1 細胞の構造とメカノバイオロジー(1),S08 細胞の構造とメカノバイオロジー)

The effects of oxidized LDL (oxLDL) on monocytes transendothelial migration (TEM) were investigated with an in vitro 3D-live-cell TEM assay system. Nevertheless, oxLDL did not enhance initial monocyte adhesion to endothelium and monocyte locomotion speed on EC's, total number of migrated monocytes was increased with oxLDL. OxLDL upregulated PECAM-1 and downregulated VE-cadherin on endothelial junctions without monocyte addition, both of which could promote monocyte entry by enhanced homophilic binding to monocyte PECAM-1, and by disrupted junctional barrier, respectively. These data indicate that before monocyte adhesion, endothelial junctions changed their conformation to more monocyte-acceptable state response to oxLDL, resulting the stage-specific promotion of monocyte TEM (stage initiation of invasion) with no enhancement of its initial adhesion or migration speed.

Protein/Protein Interactions (PDZ) in Proximal Tubules

IntroductionEssentially all cellular functions rely on the correctspatial organization of proteins. In epithelial cells,this is manifested by a separation of the plasmamembrane into an apical and a basolateral area,which is required for vectorial transport of solutes,and by the formation of junctional complexes toguarantee barrier function. In this article, we sum-marize the current knowledge of PDZ protein-basedmultimeric protein complexes in renal proximaltubular (PT) cells (for definition of the PDZ domain,seebelow), which are involved in the organization ofthe brush border membrane, the basolateral mem-brane and the tight junction of PT cells. In addition,we also consider a fourth subcellular structure, thesubapical compartment (SAC). This compartmenthas been ascribed to the recycling of endocytosedproteins and also to the routing of endocytosedmaterial to the lysosomes.In renal proximal tubules, the majority of filteredsolutes and water are reabsorbed by transepithelialtransport processes and by paracellular pathways[68]. In addition, PT-cells exhibit an exceptionallyhigh rate of endocytosis to recover small peptides andhormones. On a morphological basis, three differentdistinct epithelial cells can be distinguished along theproximal tubules. In the S1 segment, brush bordersare formed by long microvilli, whereas the appear-ance of the microvilli in the S2 and S3 segments is lesspronounced and varies among different species [37].Although this longitudinal morphological heteroge-neity of PT cells is also reflected by functional dif-ferences, in this article, we will not distinguish amongproximal tubular segments, i.e., the PDZ-based pro-tein complexes discussed below are assumed to be ofuniform nature along the entire proximal tubule.ThePDZmoduleNumerous protein domains have been described thatare implicated in protein-protein interactions (see e.g.,www.mshri.on.ca). Based on sequence similarities be-tween the post-synaptic density protein PSD-95, theDrosophilajunctional protein Disc-large and the tightjunctional protein ZO1, one such domain was definedas PDZ. PDZ proteins, among other functions, areprimarily thought to organize large functional unitssuch as synapses [55] or tight junctions [19]. Thecharacteristics of PDZ domains have been reviewedrecently[14,15,28,55]andaresummarizedasfollows:In mammals, over 400 different PDZ proteinshave been identified and grouped into three majorfamilies according to their domain organization:Afirst family encompasses proteins, which only containPDZ domains. The second, MAGUK family (mem-brane-associated guanylate kinases), contains one ormore PDZ domains besides a GuK domain (gua-nylate kinase domain) and a SH3 domain. A thirdfamily was defined containing proteins of multiplePDZ domains together with a variable number ofother protein domains.The PDZ domain comprises between 80 and 90amino-acid residues of which the three-dimensionalstructure has been basically resolved (see http://smart.enbl-heidelberg.de). The building principle is asandwich structure of 6 b-strands and two a-helicesthat form a hydrophobic cleft into which a short pep-tide can be accommodated. As an example, the struc-tural aspect of the CFTR-NHERF interaction hasbeen discussed in detail [38]. In most cases, PDZdomainsbindtoaC-terminalamino-acidmotif.Threedifferent classes of PDZ binding motifs have beenrecognized. They all include the last four C-terminalamino acids, whose characteristics of interaction canbe modulated by more upstream amino acids [55].Besides the classical canonical determinants, addi-tional PDZ binding motifs have been described and

Update on the immunologic basis of Helicobacter pylori gastritis

Helicobacter pylori is a ubiquitous bacterial pathogen that has evolved to chronically infect the gastric mucosal surface, evade host immune clearance, and cause peptic ulcer disease or gastric neoplasia in a significant minority of infected individuals. Understanding the colonization, persistence, and virulence determinants of the bacterium as well as the innate and adaptive immune responses of the host are critically important if we are to develop novel treatment strategies for eradication of infection and prevention of H. pylori-induced gastroduodenal disease. Substantial progress has been made in understanding the role of CagA in altering gastric epithelial cell signaling pathways. Intracellular CagA has been shown to activate the Ras/MEK/ERK mitogen activated protein kinase cascade and to associate with epithelial tight-junction scaffolding protein ZO-1. CagA was shown to regulate cellular responses and possibly contribute to gastritis by phosphorylation-dependent pathways. A phosphorylation-independent mechanism for CagA intracellular effects has also been proposed. The potential for CagA to disrupt the apical junctional barrier and for the outer membrane protein oipA to promote IL-8 secretion and gastric inflammation have also been explored. A number of different mechanisms by which H. pylori escapes and evades host immune attack to cause chronic indolent inflammation have been uncovered. Meanwhile, the examination and development of new adjuvants and vaccine delivery mechanisms to induce mucosal immune responses against key bacterial antigens has been a continuing focus of investigation in both animal and human studies. H. pylori induces gastritis through production of a variety of antigens, virulence factors, and soluble mediators. The bacterium also dysregulates, disarms, and evades host immune responses to maintain chronic colonization of the gastric mucosa. Understanding the mechanisms of its growth and survival in the human stomach are essential for the development of an effective vaccine and other novel eradication strategies.