Aging depicts one of the major challenges in pharmacology owing to its complexity and heterogeneity. Thereby, advanced glycated end-products modify extracellular matrix proteins, but the consequences on the skin barrier function remain heavily understudied. Herein, we utilized transmission electron microscopy for the ultrastructural analysis of ribose-induced glycated reconstructed human skin (RHS). Molecular and functional insights substantiated the ultrastructural characterization and proved the relevance of glycated RHS beyond skin aging. In particular, electron microscopy mapped the accumulation and altered spatial orientation of fibrils and filaments in the dermal compartment of glycated RHS. Moreover, the epidermal basement membrane appeared thicker in glycated than in non-glycated RHS, but electron microscopy identified longitudinal clusters of the finest collagen fibrils instead of real thickening. The stratum granulosum contained more cell layers, the morphology of keratohyalin granules decidedly differed, and the stratum corneum lipid order increased in ribose-induced glycated RHS, while the skin barrier function was almost not affected. In conclusion, dermal advanced glycated end-products markedly changed the epidermal morphology, underlining the importance of matrix⁻cell interactions. The phenotype of ribose-induced glycated RHS emulated aged skin in the dermis, while the two to three times increased thickness of the stratum granulosum resembled poorer cornification.
- MeSH
- Basement Membrane drug effects ultrastructure MeSH
- Cell Differentiation drug effects MeSH
- Epidermis drug effects ultrastructure MeSH
- Fibroblasts drug effects ultrastructure MeSH
- Keratinocytes drug effects ultrastructure MeSH
- Skin drug effects ultrastructure MeSH
- Humans MeSH
- Glycation End Products, Advanced metabolism MeSH
- Ribose pharmacology MeSH
- Dermis drug effects ultrastructure MeSH
- Skin Aging drug effects MeSH
- Microscopy, Electron, Transmission MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Epidermolysis bullosa simplex (EBS) is an inherited skin disorder caused by mutations in the keratin 5 (KRT5) and keratin 14 (KRT14) genes, with fragility of basal keratinocytes leading to epidermal cytolysis and blistering. OBJECTIVES: In this study, we characterized mutations in KRT5 and KRT14 genes in patients with EBS and investigated their possible structure-function correlations. MATERIALS AND METHODS: Mutations were characterized using polymerase chain reaction (PCR) and DNA sequencing. Further, to explore possible correlations with function, the structural effects of the mutations in segment 2B of KRT5 and KRT14 and associated with EBS in our patients, as well as those reported previously, were modelled by molecular dynamics with the aid of the known crystal structure of the analogous segment of human vimentin. RESULTS: We have identified mutations in the KRT5 and KRT14 genes in 16 of 23 families affected by EBS in the Czech Republic. Eleven different sequence variants were found, of which four have not been reported previously. Novel mutations were found in two patients with the EBS-Dowling-Meara variant (EBS-DM) [KRT14-p.Ser128Pro and KRT14-p.Gln374_Leu387dup(14)] and in three patients with localized EBS (KRT14-p.Leu136Pro and KRT5-p.Val143Ala). Molecular dynamics studies show that the mutations p.Glu411del and p.Ile467Thr perturb the secondary alpha-helical structure of the mutated polypeptide chain, the deletion p.Glu411del in KRT14 has a strong but only local influence on the secondary structure of KRT14, and the structural impact of the mutation p.Ile467Thr in KRT5 is spread along the helix to the C-terminus. In all the other point mutations studied, the direct structural impact was significantly weaker and did not destroy the alpha-helical pattern of the secondary protein structure. The changes of 3-D structure of the KRT5/KRT14 dimer induced by the steric structural impact of the single point mutations, and the resulting altered inter- and intramolecular contacts, are spread along the protein helices to the protein C-terminus, but the overall alpha-helical character of the secondary structure is not destroyed and the atomic displacements induced by mutations cause only limited-scale changes of the quaternary structure of the dimer. CONCLUSIONS: The results of molecular modelling show relationships between patients' phenotypes and the structural effects of individual mutations.
- MeSH
- Child MeSH
- Adult MeSH
- Epidermolysis Bullosa Simplex genetics pathology MeSH
- Phenotype MeSH
- Microscopy, Fluorescence MeSH
- Genetic Predisposition to Disease MeSH
- Intermediate Filaments ultrastructure MeSH
- Keratin-14 genetics MeSH
- Keratin-5 genetics MeSH
- Skin ultrastructure MeSH
- Humans MeSH
- Models, Molecular MeSH
- Mutation MeSH
- Child, Preschool MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Humans MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Dermatology MeSH
- Dermoscopy MeSH
- Skin anatomy & histology ultrastructure MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Interview MeSH
PURPOSE: To confirm and define a molecular basis for a case of mucolipidosis type IV (ML IV) with an extremely atypical phenotype pattern. DESIGN: Observational case report of a patient with ML IV with disease progression restricted to ocular symptoms. METHODS: Complete ophthalmologic and neurologic examination. Ultrastructural examination of white blood cells, skin, conjunctiva, and corneal epithelium. The MCOLN1 gene was sequenced from cDNA and the proportion of splicing variants were assessed by quantitative allele-specific polymerase chain reaction. RESULTS: Absence of any neurological abnormalities. Retinal pathologic features were the main cause of visual disability: low visual acuity and cloudy corneas since 2 years of age, progressive decrease in visual acuity since the age of 9 years. Ultrastructural examination showed storage lysosomes filled with either concentric membranes or lucent precipitate in corneal and conjunctive epithelia and in vascular endothelium. Cultured fibroblasts were free of any autofluorescence. Sequencing of the MCOLN1 gene identified compound heterozygosity for D362Y and A-->T transition leading to the creation of a novel donor splicing site and a 4-bp deletion from exon 13 at the mRNA level. Both normal and pathologic splice forms were detected in skin fibroblasts and leukocytes, with the normal form being more abundant. CONCLUSIONS: The case of this patient with ML IV is unique and is characterized by a curious lack of generalized symptoms. In this patient, the disorder was limited to the eyes and appeared without the usual psychomotor deterioration. The resulting phenotype is the mildest seen to date.
- MeSH
- Alternative Splicing genetics MeSH
- Retinal Degeneration genetics pathology MeSH
- Child MeSH
- Electroretinography MeSH
- Epithelial Cells ultrastructure MeSH
- Phenotype MeSH
- Fibroblasts ultrastructure MeSH
- Financing, Organized MeSH
- TRPM Cation Channels genetics MeSH
- Skin ultrastructure MeSH
- Leukocytes ultrastructure MeSH
- Humans MeSH
- Lysosomes genetics ultrastructure MeSH
- RNA, Messenger genetics MeSH
- Mucolipidoses genetics pathology MeSH
- Mutation MeSH
- DNA Mutational Analysis MeSH
- Corneal Diseases genetics pathology MeSH
- Conjunctival Diseases genetics pathology MeSH
- Polymerase Chain Reaction MeSH
- Epithelium, Corneal ultrastructure MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Case Reports MeSH
Mitochondrial disorders represent a heterogeneous group of multisystem diseases with extreme variability in clinical phenotype. The diagnosis of mitochondrial disorders relies heavily on extensive biochemical and molecular analyses combined with morphological studies including electron microscopy. Although muscle is the tissue of choice for electron microscopic studies, the authors investigated cultivated human skin fibroblasts (HSF) harboring 3 different pathologic mtDNA mutations: 3243A > G, 8344A > G, 8993T > G. They addressed to the possibility of whether mtDNA mutations influence mitochondrial morphology in HSF and if ultrastructural changes of mitochondria may be used for differential diagnostics of mitochondrial disorders caused by mtDNA mutations. Ultrastructural analysis of patients' HSF revealed a heterogeneous mixture of mainly abnormal, partially swelling mitochondria with unusual and sparse cristae. The most characteristic cristal abnormalities were heterogeneity in size and shapes or their absence. Typical filamentous and branched mitochondria with numerous cristae as appeared in control HSF were almost not observed. In all lines of cultured HSF with various mtDNA mutations, similar ultrastructural abnormalities and severely changed mitochondrial interior were found, although no alterations in function and amount of OXPHOS were detected by routinely used biochemical methods in two lines of cultured HSF. This highlights the importance of morphological analysis, even in cultured fibroblasts, in diagnostics of mitochondrial disorders.
- MeSH
- Point Mutation MeSH
- Financing, Organized MeSH
- Cells, Cultured MeSH
- Skin ultrastructure MeSH
- Middle Aged MeSH
- Humans MeSH
- DNA, Mitochondrial physiology ultrastructure MeSH
- Mitochondria ultrastructure MeSH
- Molecular Biology MeSH
- Child, Preschool MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Child, Preschool MeSH
- Publication type
- Case Reports MeSH
- MeSH
- Cytoskeleton ultrastructure MeSH
- Skin ultrastructure MeSH
- Humans MeSH
- Micromanipulation MeSH
- Check Tag
- Humans MeSH
- MeSH
- Actinomycetales ultrastructure MeSH
- Adult MeSH
- Microscopy, Electron MeSH
- Actinomycetales Infections microbiology pathology MeSH
- Skin Diseases, Infectious microbiology pathology MeSH
- Rabbits MeSH
- Skin microbiology ultrastructure MeSH
- Middle Aged MeSH
- Humans MeSH
- Microscopy, Electron, Scanning MeSH
- Animals MeSH
- Check Tag
- Adult MeSH
- Rabbits MeSH
- Middle Aged MeSH
- Humans MeSH
- Animals MeSH
