Junction Defects Research Articles

Effects of Surface and Crystalline Defects on Reverse Characteristics of 4H-SiC Junction Barrier Schottky Diodes

To clarify the relationship between the dispersed reverse characteristics of 4H-SiC junction barrier Schottky (JBS) diodes and defects, we investigated the sensitivity of the reverse characteristics to surface and crystalline defects in 4H-SiC epitaxial layers. Strong correlations were obtained between the reverse characteristics of 4H-SiC JBS diodes and surface defects. Micropipes or particles reduced blocking voltage and carrot defects increased leakage current. Furthermore, the leakage current of 4H-SiC JBS diodes depends on the etch pit density of threading dislocations (TDs). Etch pits formed from TDs increased leakage current by about 10-9 A in samples without surface defects. In addition, hexagonal etch pits formed from unusual crystalline defects were observed; they also increased leakage current.

Claudin‐1‐ and ‐2‐based cytokine‐induced paracellular barrier impairment: prevention by the plant alkaloide, berberine

Cytokines mediate tight junction defects which contribute to diarrhea in inflammatory bowel diseases (IBD). Diarrhea has been treated in traditional Eastern medicine for a long time by the plant alkaloid berberine. We tested the hypothesis that berberine may have beneficial effects on TNFα-induced paracellular barrier defects. The effects of TNFα and berberine on confluent monolayers of HT-29/B6 human colon cells and on rat colon was analyzed in Ussing chambers in vitro. Localization and regulation of tight junction proteins was analyzed by Western blot and confocal laser scanning microscopy. TNFα caused a marked decrease of barrier properties in HT-29/B6 cells and in rat colon. Molecular analyses revealed a TNFα-induced removal of claudin-1 from the tight junction and an increased claudin-2 expression. Incubation with berberine prevented the TNFα effect. Blocker experiments in combination with phosphoblotting revealed that this effect was mediated via tyrosine kinase, pAkt, and NFκB pathways. Berberine alone inhibited phosphorylation of P42/P44 and phospho P38. TNFα-mediated impairment of paracellular barrier properties can be targeted directly by the plant alkaloid berberine. These results, obtained in colonic cell cultures as well as in colon epithelium, suggest a potential approach for therapeutic intervention in cytokine-mediated inflammatory diseases. (Supported by DFG FOR 721)

Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling

Spinal muscular atrophy (SMA), a devastating neurodegenerative disorder characterized by motor neuron loss and muscle atrophy, has been linked to mutations in the Survival Motor Neuron (SMN) gene. Based on an SMA model we developed in Drosophila, which displays features that are analogous to the human pathology and vertebrate SMA models, we functionally linked the fibroblast growth factor (FGF) signaling pathway to the Drosophila homologue of SMN, Smn. Here, we characterize this relationship and demonstrate that Smn activity regulates the expression of FGF signaling components and thus FGF signaling. Furthermore, we show that alterations in FGF signaling activity are able to modify the neuromuscular junction defects caused by loss of Smn function and that muscle-specific activation of FGF is sufficient to rescue Smn-associated abnormalities.

Neuromuscular Junction Defects in Mice with Mutation of dynein heavy chain 1

Disruptions in axonal transport have been implicated in a wide range of neurodegenerative diseases. Cramping 1 (Cra1/+) and Legs at odd angles (Loa/+) mice, with hypomorphic mutations in the dynein heavy chain 1 gene, which encodes the ATPase of the retrograde motor protein dynein, were originally reported to exhibit late onset motor neuron disease. Subsequent, conflicting reports suggested that sensory neuron disease without motor neuron loss underlies the phenotypes of Cra1/+ and Loa/+ mice. Here, we present behavioral and anatomical analyses of Cra1/+ mice. We demonstrate that Cra1/+ mice exhibit early onset, stable behavioral deficits, including abnormal hindlimb posturing and decreased grip strength. These deficits do not progress through 24 months of age. No significant loss of primary motor neurons or dorsal root ganglia sensory neurons was observed at ages where the mice exhibited clear symptomatology. Instead, there is a decrease in complexity of neuromuscular junctions. These results indicate that disruption of dynein function in Cra1/+ mice results in abnormal morphology of neuromuscular junctions. The time course of behavioral deficits, as well as the nature of the morphological defects in neuromuscular junctions, suggests that disruption of dynein function in Cra1/+ mice causes a developmental defect in synapse assembly or stabilization.

Tight junction defects in patients with atopic dermatitis

Atopic dermatitis (AD) is characterized by dry skin and a hyperactive immune response to allergens, 2 cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJs) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway. We evaluated the expression/function of the TJ protein claudin-1 in epithelium from AD and nonatopic subjects and screened 2 American populations for single nucleotide polymorphisms in the claudin-1 gene (CLDN1). Expression profiles of nonlesional epithelium from patients with extrinsic AD, nonatopic subjects, and patients with psoriasis were generated using Illumina's BeadChips. Dysregulated intercellular proteins were validated by means of tissue staining and quantitative PCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed by using a knockdown approach in primary human keratinocytes. Twenty-seven haplotype-tagging SNPs in CLDN1 were screened in 2 independent populations with AD. We observed strikingly reduced expression of the TJ proteins claudin-1 and claudin-23 only in patients with AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with T(H)2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging SNPs revealed associations with AD in 2 North American populations. Collectively, these data suggest that an impairment in tight junctions contributes to the barrier dysfunction and immune dysregulation observed in AD subjects and that this may be mediated in part by reductions in claudin-1.

TNFα-induced and berberine-antagonized tight junction barrier impairment via tyrosine kinase, Akt and NFκB signaling

TNFα-mediated tight junction defects contribute to diarrhea in inflammatory bowel diseases (IBDs). In our study, the signaling pathways of the TNFα 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 TNFα-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 TNFα 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 TNFα-induced barrier defects. This effect on resistance was confirmed in rat colon. TNFα removed claudin-1 from the tight junction and increased claudin-2 expression. Berberine prevented TNFα-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 NFκB 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.

Autoimmune mediated neuromuscular junction defects

This review summarizes the recent advances on pathogenesis of antibody-mediated disorders of the neuromuscular junction, and results of studies on clinical assessment and treatments. The incidence of myasthenia gravis, particularly in patients older than 50 years, is rising, and this is not solely due to improved disease recognition. It is uncertain how muscle specific tyrosine kinase (MuSK) antibody positive myasthenia gravis results in neuromuscular transmission failure since MuSK antibodies alter neuromuscular junction morphology without altering acetylcholine receptor numbers or turnover. Clinical tools have been developed that allow rapid and reliable disease assessment. The myasthenia gravis composite score addresses items commonly affected in myasthenia gravis, is sensitive to detect clinical change and helps guide the physician in therapy prescription. Immunosuppression remains the mainstay of myasthenia gravis treatment. Other therapies, such as rituximab, are increasingly prescribed for refractory myasthenia gravis, and drugs that inhibit complement are being explored in myasthenia gravis and Guillain-Barré syndrome (GBS). In Lambert-Eaton myasthenic syndrome (LEMS), SOX antibodies help distinguish between tumour and nontumour LEMS. Ganglioside complexes in GBS and Miller-Fisher syndrome are frequently present and are more pathogenic. Developments in serological assays, particularly of cell-based assays, are continuing to improve the diagnosis and investigation of these conditions. Learning more on pathogenicity has helped us to apply newer therapies.

A Modifier Screen for Bazooka/PAR-3 Interacting Genes in the Drosophila Embryo Epithelium

BackgroundThe development and homeostasis of multicellular organisms depends on sheets of epithelial cells. Bazooka (Baz; PAR-3) localizes to the apical circumference of epithelial cells and is a key hub in the protein interaction network regulating epithelial structure. We sought to identify additional proteins that function with Baz to regulate epithelial structure in the Drosophila embryo.Methodology/Principal FindingsThe baz zygotic mutant cuticle phenotype could be dominantly enhanced by loss of known interaction partners. To identify additional enhancers, we screened molecularly defined chromosome 2 and 3 deficiencies. 37 deficiencies acted as strong dominant enhancers. Using deficiency mapping, bioinformatics, and available single gene mutations, we identified 17 interacting genes encoding known and predicted polarity, cytoskeletal, transmembrane, trafficking and signaling proteins. For each gene, their loss of function enhanced adherens junction defects in zygotic baz mutants during early embryogenesis. To further evaluate involvement in epithelial polarity, we generated GFP fusion proteins for 15 of the genes which had not been found to localize to the apical domain previously. We found that GFP fusion proteins for Drosophila ASAP, Arf79F, CG11210, Septin 5 and Sds22 could be recruited to the apical circumference of epithelial cells. Nine of the other proteins showed various intracellular distributions, and one was not detected.Conclusions/SignificanceOur enhancer screen identified 17 genes that function with Baz to regulate epithelial structure in the Drosophila embryo. Our secondary localization screen indicated that some of the proteins may affect epithelial cell polarity by acting at the apical cell cortex while others may act through intracellular processes. For 13 of the 17 genes, this is the first report of a link to baz or the regulation of epithelial structure.

Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization.

Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC) is a multisystem disorder associated with abnormalities in polarized liver and kidney cells. Mutations in VPS33B account for most cases of ARC. We identified mutations in VIPAR (also called C14ORF133) in individuals with ARC without VPS33B defects. We show that VIPAR forms a functional complex with VPS33B that interacts with RAB11A. Knockdown of vipar in zebrafish resulted in biliary excretion and E-cadherin defects similar to those in individuals with ARC. Vipar- and Vps33b-deficient mouse inner medullary collecting duct (mIMDC-3) cells expressed membrane proteins abnormally and had structural and functional tight junction defects. Abnormal Ceacam5 expression was due to mis-sorting toward lysosomal degradation, but reduced E-cadherin levels were associated with transcriptional downregulation. The VPS33B-VIPAR complex thus has diverse functions in the pathways regulating apical-basolateral polarity in the liver and kidney.

Neuromuscular defects and breathing disorders in a new mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein leading to muscle paralysis and respiratory failure. In mouse, introducing the human SMN2 gene partially rescues Smn - / - embryonic lethality. However current models were either too severe or nearly unaffected precluding convenient drug testing for SMA. We report here new SMN2; Smn -/- lines carrying one to four copies of the human SMN2 gene. Mice carrying three SMN2 copies exhibited an intermediate phenotype with delayed appearance of motor defects and developmental breathing disorders reminiscent of those found in severe SMA patients. Although normal at birth, at 7 days of age respiratory rate was decreased and apnea frequency was increased in SMA mice in parallel with the appearance of neuromuscular junction defects in the diaphragm. With median survival of 15 days and postnatal onset of neurodegeneration, these mice could be an important tool for evaluating new therapeutics.

On the geometry control of magnetic devices: Impact of photo-resist profile, shadowing effect, and material properties

In this paper, we described how to control the magnetic junction critical dimension (CD) and profile defined by ion beam milling and its implication to device performance. The impact of standing waves on breaking the photo-resist and on the resultant junction defects have been highlighted. The ideal device profile and geometry are also discussed.

Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin

The alpha7beta1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of alpha7beta1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and alpha7 integrin (mdx/alpha7(-/-)), or dystrophin and utrophin (mdx/utr(-/-)), exhibit severe muscle pathology and die prematurely. To explore the contribution of the alpha7beta1 integrin and utrophin to muscle integrity and function, we generated mice lacking both alpha7 integrin and utrophin. Surprisingly, mice that lack both alpha7 integrin and utrophin (alpha7/utr(-/-)) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to alpha7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in alpha7/utr(-/-) skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The alpha7/utr(-/-) mouse, therefore, demonstrates the critical roles of alpha7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the alpha7beta1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.

Zebrafish survival motor neuron mutants exhibit presynaptic neuromuscular junction defects

Spinal muscular atrophy (SMA), a recessive genetic disease, affects lower motoneurons leading to denervation, atrophy, paralysis and in severe cases death. Reduced levels of survival motor neuron (SMN) protein cause SMA. As a first step towards generating a genetic model of SMA in zebrafish, we identified three smn mutations. Two of these alleles, smnY262stop and smnL265stop, were stop mutations that resulted in exon 7 truncation, whereas the third, smnG264D, was a missense mutation corresponding to an amino acid altered in human SMA patients. Smn protein levels were low/undetectable in homozygous mutants consistent with unstable protein products. Homozygous mutants from all three alleles were smaller and survived on the basis of maternal Smn dying during the second week of larval development. Analysis of the neuromuscular system in these mutants revealed a decrease in the synaptic vesicle protein, SV2. However, two other synaptic vesicle proteins, synaptotagmin and synaptophysin were unaffected. To address whether the SV2 decrease was due specifically to Smn in motoneurons, we tested whether expressing human SMN protein exclusively in motoneurons in smn mutants could rescue the phenotype. For this, we generated a transgenic zebrafish line with human SMN driven by the motoneuron-specific zebrafish hb9 promoter and then generated smn mutant lines carrying this transgene. We found that introducing human SMN specifically into motoneurons rescued the SV2 decrease observed in smn mutants. Our analysis indicates the requirement for Smn in motoneurons to maintain SV2 in presynaptic terminals indicating that Smn, either directly or indirectly, plays a role in presynaptic integrity.

Patología neuromuscular en cuidados intensivos

The spectrum of neuromuscular disease encountered in today's intensive care units (ICU) has evolved over the last few decades. However, in spite of many studies on neuromuscular disorders complicating critical illness as well as its epidemiology, etiology, treatment and prognosis, several key areas remain unclear. Two main groups are found among these neuromuscular abnormalities. The first group includes primary neuromuscular disorders present on admission to the ICU in which a possible etiology can be identified. Guillain-Barré syndrome and myasthenia gravis are two of the most common diseases admitted to ours units. In the second group, weakness is acquired in the ICU in the absence of preexisting neuromuscular disease. It is believed to reflect illnesses or treatments occurring in the ICU. Critical illness polyneuropathy (CIP) is the most clearly defined neuromuscular complication in this group. However, although we have better knowledge of its clinical, diagnosis, and prognosis features, its pathophysiological substrate has not been fully elucidated. Neuromuscular junction defects and specially myopathies, that frequently coexist with CIP, are the others main causes of acquired weakness in critically ill patients. Advances in understanding of these neuromuscular disorders could have an important impact in terms of developing effective preventive and therapeutic interventions that could help to improve the poor prognosis of these patients.

Testicular Cell Junction: A Novel Target for Male Contraception

Even though various contraceptive methods are widely available, the number of unwanted pregnancies is still on the rise in developing countries, pressurizing the already resource limited nations. One of the major underlying reasons is the lack of effective, low cost, and safe contraceptives for couples. During the past decade, some studies were performed using animal models to decipher if the Sertoli-germ cell junction in the testis is a target for male fertility regulation. Some of these study models were based on the use of hormones and/or chemicals to disrupt the hypothalamic-pituitary-testicular axis (e.g., androgen-based implants or pills) and others utilized a panel of chemical entities or synthetic peptides to perturb spermatogenesis either reversibly or non-reversibly. Among them, adjudin, a potential male contraceptive, is one of the compounds exerting its action on the unique adherens junctions, known as ectoplasmic specializations, in the testis. Since the testis is equipped with inter-connected cell junctions, an initial targeting of one junction type may affect the others and these accumulative effects could lead to spermatogenic arrest. This review attempts to cover an innovative theme on how male infertility can be achieved by inducing junction instability and defects in the testis, opening a new window of research for male contraceptive development. While it will still take much time and effort of intensive investigation before a product can reach the consumable market, these findings have provided hope for better family planning involving men.

Lamin A/C–mediated neuromuscular junction defects in Emery-Dreifuss muscular dystrophy

The LMNA gene encodes lamins A and C, two intermediate filament-type proteins that are important determinants of interphase nuclear architecture. Mutations in LMNA lead to a wide spectrum of human diseases including autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD), which affects skeletal and cardiac muscle. The cellular mechanisms by which mutations in LMNA cause disease have been elusive. Here, we demonstrate that defects in neuromuscular junctions (NMJs) are part of the disease mechanism in AD-EDMD. Two AD-EDMD mouse models show innervation defects including misexpression of electrical activity–dependent genes and altered epigenetic chromatin modifications. Synaptic nuclei are not properly recruited to the NMJ because of mislocalization of nuclear envelope components. AD-EDMD patients with LMNA mutations show the same cellular defects as the AD-EDMD mouse models. These results suggest that lamin A/C–mediated NMJ defects contribute to the AD-EDMD disease phenotype and provide insights into the cellular and molecular mechanisms for the muscle-specific phenotype of AD-EDMD.

Characterization and Classification of Zebrafish Brain Morphology Mutants

The mechanisms by which the vertebrate brain achieves its three-dimensional structure are clearly complex, requiring the functions of many genes. Using the zebrafish as a model, we have begun to define genes required for brain morphogenesis, including brain ventricle formation, by studying 16 mutants previously identified as having embryonic brain morphology defects. We report the phenotypic characterization of these mutants at several timepoints, using brain ventricle dye injection, imaging, and immunohistochemistry with neuronal markers. Most of these mutants display early phenotypes, affecting initial brain shaping, whereas others show later phenotypes, affecting brain ventricle expansion. In the early phenotype group, we further define four phenotypic classes and corresponding functions required for brain morphogenesis. Although we did not use known genotypes for this classification, basing it solely on phenotypes, many mutants with defects in functionally related genes clustered in a single class. In particular, Class 1 mutants show midline separation defects, corresponding to epithelial junction defects; Class 2 mutants show reduced brain ventricle size; Class 3 mutants show midbrain-hindbrain abnormalities, corresponding to basement membrane defects; and Class 4 mutants show absence of ventricle lumen inflation, corresponding to defective ion pumping. Later brain ventricle expansion requires the extracellular matrix, cardiovascular circulation, and transcription/splicing-dependent events. We suggest that these mutants define processes likely to be used during brain morphogenesis throughout the vertebrates. Anat Rec, 2009. (c) 2008 Wiley-Liss, Inc.

Spectral-domain optical coherence tomography images of inner/outer segment junctions and macular hole surgery outcomes

To evaluate the ability of spectral-domain optical coherence tomography (SD-OCT) images of the inner/outer segment (IS/OS) junction to predict macular hole surgery outcomes. Fifty-four eyes in 53 patients with idiopathic macular holes were studied. Postoperative visual acuity and SD-OCT images of the fovea were examined before and 12 months after surgery. The total area and the maximum length of the IS/OS junction defect were measured. The macular holes were successfully closed in all the eyes included in this series. The mean log MAR visual acuity improved significantly from 0.67 +/- 0.25 preoperatively to 0.16 +/- 0.22 postoperatively (p < 0.001). The mean total area and maximum length of the IS/OS junction defect at 12 months after surgery were 10,185 +/- 1,537 microm(2) and 100.5 +/- 10.4 microm respectively; these values were significantly negatively correlated with the postoperative visual acuity. SD-OCT is useful for quantitatively measuring IS/OS junction defects. The postoperative IS/OS junction may play an important role in visual recovery after macular hole surgery.

The neuroprotective factor Wlds fails to mitigate distal axonal and neuromuscular junction (NMJ) defects in mouse models of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common autosomal recessive neurodegenerative disorder in humans. Amongst the earliest signs of neurodegeneration are severe and progressive defects of the neuromuscular synapse. These defects, characterized by poor terminal arborization and immature motor endplates, presumably result in a loss of functional synapses. The slow Wallerian degeneration (Wlds) mutation in rodents has been shown to have a protective effect on mouse models of motor neuron disease by retarding axonal die-back and preventing neuromuscular synapse loss. In this study we tested the effects of the Wlds mutation on the disease phenotype of SMA model mice. Consistent with previous reports, the mutation slows axon and neuromuscular synapse loss following nerve injury in wild-type as well as in SMA mice. However, the synaptic defects found in severely affected SMA patients and model mice persist in the double (Wlds;SMA) mutants. No delay in disease onset was observed and survival was not significantly altered. Finally, Wlds had no effect on the striking phrenic nerve projection defects that we discovered in SMA model mice. Our results indicate that the reported protective effects of Wlds are insufficient to mitigate the neuromuscular phenotype due to reduced SMN protein, and that the mechanisms responsible for distal defects of the motor unit in SMA are unlikely to be similar to those causing neurodegeneration in genetic mutants such as the pmn mouse which is partially rescued by the Wlds protein.

Candidate Genes for Vesico-Ureteral Reflux

PURPOSE Vesico-ureteral reflux (VUR) is a complex, genetically heterogeneous developmental disorder characterized by the retrograde flow of urine from the bladder into the ureter. VUR is a major contributing factor to end-stage renal disease in paediatric patients. We aim to identify genetic factors contributing to VUR. MATERIAL AND METHODS In a collaborative study we investigated a man with a de novo translocation, who exhibits multiple congenital abnormalities, including severe bilateral VUR with ureterovesical junction defects. This translocation disrupts ROBO2, which encodes a transmembrane receptor for the SLIT ligand, and produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro. In addition, two novel ROBO2 intracellular missense variants were identified that segregate with CAKUT and VUR in two unrelated families. SLIT-ROBO signalling is involved in the development of the ureteric bud. To investigate the involvement of 12 other functional candidate genes (mostly involved in ureteric budding) and two reported loci in VUR, we performed a linkage study in four large, Dutch, multi-generational families with multiple affected individuals. RESULTS We were unable to detect linkage to any of the genes and loci and could exclude the GDNF, RET, SLIT2, SPRY1, PAX2, AGTR2, UPK1A and UPK3A genes and the 1p13 and 20p13 loci from linkage to VUR. CONCLUSIONS Mutations in ROBO2 contribute to the development of VUR in a small subset of patients. Our results provide further evidence that there appears to be genetic heterogeneity in VUR.