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Acta Dermato-venereologica May 2017Linear IgG deposits along the basement membrane of adnexa has been proposed to be useful in the diagnosis of bullous pemphigoid (BP), but no controlled studies have been...
Linear IgG deposits along the basement membrane of adnexa has been proposed to be useful in the diagnosis of bullous pemphigoid (BP), but no controlled studies have been performed. This study evaluated linear IgG fluorescence of the basement membrane of sweat gland ducts (SGD) and other adnexa in perilesional biopsies from patients with BP (n = 64) and controls (n = 82), using direct immunofluorescence microscopy. Fluorescence intensity was graded semi-quantitatively. Positive SGDs were found in 58 (90.6%) patients with BP and 44 (53.7%) controls, a statistically significant difference (p < 0.0001). The sensitivity of positive SGDs for BP was high (90.6%), but the specificity was low (46.3%). Only strong fluorescence intensity was associated with high specificity. In conclusion, positive SGDs in direct immunofluorescence microscopy are highly sensitive for BP; however, only strong fluorescence has acceptable specificity. Weak positivity of SGDs without linear fluorescence of the epidermal basement membrane may not be sufficiently specific for BP.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Autoantibodies; Autoantigens; Basement Membrane; Biomarkers; Case-Control Studies; Child; Dystonin; Female; Humans; Immunoglobulin G; Luminescent Measurements; Male; Microscopy, Fluorescence; Middle Aged; Non-Fibrillar Collagens; Pemphigoid, Bullous; Predictive Value of Tests; Reproducibility of Results; Sweat Glands; Turkey; Young Adult; Collagen Type XVII
PubMed: 28093596
DOI: 10.2340/00015555-2615 -
Genetics Sep 2016Dystonia musculorum is a neurodegenerative disorder caused by a mutation in the dystonin gene. It has been described in mice and humans where it is called hereditary...
Dystonia musculorum is a neurodegenerative disorder caused by a mutation in the dystonin gene. It has been described in mice and humans where it is called hereditary sensory autonomic neuropathy. Mutated mice show severe movement disorders and die at the age of 3-4 weeks. This study describes the discovery and molecular, clinical, as well as pathological characterization of a new spontaneously occurring mutation in the dystonin gene in C57BL/6N mice. The mutation represents a 40-kb intragenic deletion allele of the dystonin gene on chromosome 1 with exactly defined deletion borders. It was demonstrated by Western blot, mass spectrometry, and immunohistology that mice with a homozygous mutation were entirely devoid of the dystonin protein. Pathomorphological lesions were restricted to the brain stem and spinal cord and consisted of swollen, argyrophilic axons and dilated myelin sheaths in the white matter and, less frequently, total chromatolysis of neurons in the gray matter. Axonal damage was detected by amyloid precursor protein and nonphosphorylated neurofilament immunohistology. Axonopathy in the central nervous system (CNS) represents the hallmark of this disease. Mice with the dystonin mutation also showed suppurative inflammation in the respiratory tract, presumably due to brain stem lesion-associated food aspiration, whereas skeletal muscles showed no pathomorphological changes. This study describes a novel mutation in the dystonin gene in mice leading to axonopathy in the CNS. In further studies, this model may provide new insights into the pathogenesis of neurodegenerative diseases and may elucidate the complex interactions of dystonin with various other cellular proteins especially in the CNS.
Topics: Alleles; Animals; Axons; Central Nervous System; Dystonic Disorders; Dystonin; Female; Gene Deletion; Male; Mice; Mice, Inbred C57BL; Mutation; Nerve Tissue Proteins; Neurons
PubMed: 27401753
DOI: 10.1534/genetics.116.186932 -
Actas Dermo-sifiliograficas Oct 2016
Review
Topics: Adult; Autoantibodies; Autoantigens; Azathioprine; Biopsy; Diagnosis, Differential; Drug Therapy, Combination; Dystonin; Erythema Multiforme; Female; Humans; Immunosuppressive Agents; Non-Fibrillar Collagens; Pemphigoid, Bullous; Prednisone; Collagen Type XVII
PubMed: 27048923
DOI: 10.1016/j.ad.2016.02.008 -
PloS One 2016The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as...
The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.
Topics: Actin Cytoskeleton; Actins; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Cytoskeleton; Dystonin; Epithelial Cells; Hemidesmosomes; Humans; Integrin beta4; Intermediate Filament Proteins; Intermediate Filaments; Keratinocytes; Keratins; Nerve Tissue Proteins; Plectin; Protein Binding
PubMed: 27007410
DOI: 10.1371/journal.pone.0149106 -
PloS One 2016Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and...
Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as was myelin basic protein expression in both cerebral cortex and spinal cord. Together these data suggest that, unlike Schwann cells, oligodendrocytes do not have an intrinsic requirement for neuronal dystonin for differentiation and myelination.
Topics: Animals; Apoptosis; Carrier Proteins; Cell Differentiation; Cell Movement; Cell Proliferation; Cell Shape; Cytoskeletal Proteins; Dystonin; Mice; Myelin Sheath; Nerve Tissue Proteins; Neurons; Oligodendroglia; RNA, Messenger; Rats, Sprague-Dawley; Stem Cells
PubMed: 26886550
DOI: 10.1371/journal.pone.0149201 -
Annals of Dermatology Dec 2015Epidermolysis bullosa (EB) comprises a collection of clinically diverse inherited blistering diseases that affect the skin and, in some subtypes, mucous membranes and... (Review)
Review
Epidermolysis bullosa (EB) comprises a collection of clinically diverse inherited blistering diseases that affect the skin and, in some subtypes, mucous membranes and other organs. Currently classified into four main subtypes (EB simplex, junctional EB, dystrophic EB, and Kindler syndrome, mainly based on the level of skin cleavage), the spectrum of EB extends to more than 30 clinical subtypes with pathogenic mutations in at least 18 distinct genes. This review focuses on three recent additions to variants of EB: all are autosomal recessive, and result from mutations in either DST-e (coding for epidermal dystonin, also known as the 230 kDa bullous pemphigoid antigen, BP230), EXPH5 (coding for exophilin-5, also known as Slac2-b), or ITGA3 (coding for the integrin alpha-3 subunit). Each of these new forms of EB is reviewed with respect to the initial gene discovery, clinical features, the current mutation database, and skin pathology. Awareness of these recently described forms of EB is helpful in the clinical evaluation of patients with EB and in defining genotype-phenotype correlation for inherited blistering skin diseases.
PubMed: 26719633
DOI: 10.5021/ad.2015.27.6.658 -
Cell & Bioscience 2015Under inflammatory conditions or during tumor progression macrophages acquire distinct phenotypes, with factors of the microenvironment such as hypoxia and transforming...
BACKGROUND
Under inflammatory conditions or during tumor progression macrophages acquire distinct phenotypes, with factors of the microenvironment such as hypoxia and transforming growth factor β (TGFβ) shaping their functional plasticity. TGFβ is among the factors causing alternative macrophage activation, which contributes to tissue regeneration and thus, resolution of inflammation but may also provoke tumor progression. However, the signal crosstalk between TGFβ and hypoxia is ill defined.
RESULTS
Exposing human primary macrophages to TGFβ elicited a rapid SMAD2/SMAD3 phosphorylation. This early TGFβ-signaling remained unaffected by hypoxia. However, with prolonged exposure periods to TGFβ/hypoxia the expression of SMAD2 declined because of decreased protein stability. In parallel, hypoxia increased mRNA and protein amount of the calpain regulatory subunit, with the further notion that TGFβ/hypoxia elicited calpain activation. The dual specific proteasome/calpain inhibitor MG132 and the specific calpain inhibitor 1 rescued SMAD2 degradation, substantiating the ability of calpain to degrade SMAD2. Decreased SMAD2 expression reduced TGFβ transcriptional activity of its target genes thrombospondin 1, dystonin, and matrix metalloproteinase 2.
CONCLUSIONS
Hypoxia interferes with TGFβ signaling in macrophages by calpain-mediated proteolysis of the central signaling component SMAD2.
PubMed: 26146544
DOI: 10.1186/s13578-015-0026-x -
Autophagy 2015A homozygous mutation in the DST (dystonin) gene causes a newly identified lethal form of hereditary sensory and autonomic neuropathy in humans (HSAN-VI). DST loss of...
A homozygous mutation in the DST (dystonin) gene causes a newly identified lethal form of hereditary sensory and autonomic neuropathy in humans (HSAN-VI). DST loss of function similarly leads to sensory neuron degeneration and severe ataxia in dystonia musculorum (Dst(dt)) mice. DST is involved in maintaining cytoskeletal integrity and intracellular transport. As autophagy is highly reliant upon stable microtubules and motor proteins, we assessed the influence of DST loss of function on autophagy using the Dst(dt-Tg4) mouse model. Electron microscopy (EM) revealed an accumulation of autophagosomes in sensory neurons from these mice. Furthermore, we demonstrated that the autophagic flux was impaired. Levels of LC3-II, a marker of autophagosomes, were elevated. Consequently, Dst(dt-Tg4) sensory neurons displayed impaired protein turnover of autophagosome substrate SQTSM1/p62 and of polyubiquitinated proteins. Interestingly, in a previously described Dst(dt-Tg4) mouse model that is partially rescued by neuronal specific expression of the DST-A2 isoform, autophagosomes, autolysosomes, and damaged organelles were reduced when compared to Dst(dt-Tg4) mutant mice. LC3-II, SQTSM1, polyubiquitinated proteins and autophagic flux were also restored to wild-type levels in the rescued mice. Finally, a significant decrease in DNAIC1 (dynein, axonemal, intermediate chain 1; the mouse ortholog of human DNAI1), a member of the DMC (dynein/dynactin motor complex), was noted in Dst(dt-Tg4) dorsal root ganglia and sensory neurons. Thus, DST-A2 loss of function perturbs late stages of autophagy, and dysfunctional autophagy at least partially underlies Dst(dt) pathogenesis. We therefore conclude that the DST-A2 isoform normally facilitates autophagy within sensory neurons to maintain cellular homeostasis.
Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagy; Carrier Proteins; Cytoskeletal Proteins; Dynactin Complex; Dystonia; Dystonin; Heat-Shock Proteins; Mice, Transgenic; Microtubule-Associated Proteins; Microtubules; Nerve Tissue Proteins; Phagosomes; Sensory Receptor Cells; Sequestosome-1 Protein
PubMed: 26043942
DOI: 10.1080/15548627.2015.1052207 -
PloS One 2014BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the...
BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5' end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3' end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts.
Topics: Actin Cytoskeleton; Alternative Splicing; Animals; Carrier Proteins; Cell Line; Cell Movement; Cytoskeletal Proteins; Cytoskeleton; Dystonin; Endocytosis; Golgi Apparatus; Mice; Microtubule-Associated Proteins; Microtubules; Molecular Sequence Data; Myoblasts; Nerve Tissue Proteins; Protein Isoforms
PubMed: 25244344
DOI: 10.1371/journal.pone.0107535 -
RNA (New York, N.Y.) Oct 2014IRBIS is a computational pipeline for detecting conserved complementary regions in unaligned orthologous sequences. Unlike other methods, it follows the... (Comparative Study)
Comparative Study
IRBIS is a computational pipeline for detecting conserved complementary regions in unaligned orthologous sequences. Unlike other methods, it follows the "first-fold-then-align" principle in which all possible combinations of complementary k-mers are searched for simultaneous conservation. The novel trimming procedure reduces the size of the search space and improves the performance to the point where large-scale analyses of intra- and intermolecular RNA-RNA interactions become possible. In this article, I provide a rigorous description of the method, benchmarking on simulated and real data, and a set of stringent predictions of intramolecular RNA structure in placental mammals, drosophilids, and nematodes. I discuss two particular cases of long-range RNA structures that are likely to have a causal effect on single- and multiple-exon skipping, one in the mammalian gene Dystonin and the other in the insect gene Ca-α1D. In Dystonin, one of the two complementary boxes contains a binding site of Rbfox protein similar to one recently described in Enah gene. I also report that snoRNAs and long noncoding RNAs (lncRNAs) have a high capacity of base-pairing to introns of protein-coding genes, suggesting possible involvement of these transcripts in splicing regulation. I also find that conserved sequences that occur equally likely on both strands of DNA (e.g., transcription factor binding sites) contribute strongly to the false-discovery rate and, therefore, would confound every such analysis. IRBIS is an open-source software that is available at http://genome.crg.es/~dmitri/irbis/.
Topics: Animals; Base Sequence; Caenorhabditis elegans; Conserved Sequence; Drosophila melanogaster; Exons; Genes; Humans; Introns; Molecular Sequence Data; RNA Splicing; RNA, Small Nucleolar; Sequence Homology, Nucleic Acid; Software
PubMed: 25142064
DOI: 10.1261/rna.045088.114