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.

• Klíčová slova
• advanced glycated end products, aging, diabetes, electron microscopy, nanomedicine, reconstructed human skin, ribose, skin absorption,
• MeSH
• bazální membrána účinky léků   ultrastruktura   MeSH
• buněčná diferenciace účinky léků   MeSH
• epidermis účinky léků   ultrastruktura   MeSH
• fibroblasty účinky léků   ultrastruktura   MeSH
• keratinocyty účinky léků   ultrastruktura   MeSH
• kůže účinky léků   ultrastruktura   MeSH
• lidé MeSH
• produkty pokročilé glykace metabolismus   MeSH
• ribosa farmakologie   MeSH
• škára účinky léků   ultrastruktura   MeSH
• stárnutí kůže účinky léků   MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• produkty pokročilé glykace MeSH
• ribosa 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
• dítě MeSH
• dospělí MeSH
• epidermolysis bullosa simplex genetika   patologie   MeSH
• fenotyp MeSH
• fluorescenční mikroskopie MeSH
• genetická predispozice k nemoci MeSH
• intermediární filamenta ultrastruktura   MeSH
• keratin-14 genetika   MeSH
• keratin-5 genetika   MeSH
• kůže ultrastruktura   MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace * MeSH
• předškolní dítě MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• keratin-14 MeSH
• keratin-5 MeSH
• KRT14 protein, human MeSH Prohlížeč
• KRT5 protein, human MeSH Prohlížeč

Papillomatous skin lesions from a green lizard (Lacerta viridis) were examined histologically, using electron microscopy and DNA was isolated from the lesions for herpes-viral DNA detection. Histology confirmed the lesions to be squamous epithelial papillomas. Using electron microscopy, no virus particles were detected. The specific sequence of herpesviral DNA-directed DNA polymerase (EC 2.7.7.7) was amplified using degenerate primers in a nested format. The 235-base-pair (bp) sequence was sequenced and compared with previously published DNA-directed DNA polymerase sequences from various reptile herpesviruses. The sequence from the green lizard showed significant similarity with sequence of fibropapilloma-associated turtle herpesviruses from sea turtles.

• MeSH
• DNA virů analýza   MeSH
• elektronová mikroskopie veterinární   MeSH
• Herpesviridae izolace a purifikace   MeSH
• herpetické infekce diagnóza   epidemiologie   patologie   veterinární   MeSH
• ještěři virologie   MeSH
• kůže patologie   ultrastruktura   virologie   MeSH
• papilom diagnóza   epidemiologie   patologie   veterinární   MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie nukleových kyselin MeSH
• želvy virologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Maďarsko epidemiologie   MeSH
• Názvy látek
• DNA virů MeSH

I have provided update to our two photon laser scanning microscope by adding new technique which enables us to simultaneously measured the second harmonic generation signals in the forward and backward directions; in the meantime, one can measure the two photon excitations fluorescence if the materials produce fluorescence. In the present work, the fascia muscles, muscles of pig and pig's skin were used. I found that these materials produced high second harmonic generation signal in both directions. These measurements show that the second harmonic generation strongly depends on the state of the polarization of the laser light and the orientation of the dipole moment in the molecules that interact with the laser light. It is therefore advantageous to control the laser's state of polarization, to maximize second harmonic generation. The novelty of this work is to establish new multi-functional technique by combing three platforms of laser scanning microscopy - the fluorescence microscopy, harmonic generation microscopy and polarizing microscopy in which one can use the second harmonic imaging to investigate the true architecture of the sensitive samples and the samples which do not produce auto-fluorescence. Moreover investigation of the new sample needs to look at all details of the true architecture of the sample. Thereby the sample will be exposed to the laser radiation more than the well-known sample, and that will cause photo-bleaching and photo-damage. Since the second harmonic generation does not undergo from photo-bleaching and photo-damage it will be the promising technique for investigating the sensitive and new samples. Then one can move to acquire fluorescence images after good investigation of the true architecture of the sample by the SH imaging.

• MeSH
• fascie chemie   ultrastruktura   MeSH
• konfokální mikroskopie metody   MeSH
• kůže chemie   ultrastruktura   MeSH
• mikroskopie fluorescenční multifotonová metody   MeSH
• polarizační mikroskopie metody   MeSH
• prasata MeSH
• svaly chemie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• alternativní sestřih genetika   MeSH
• degenerace retiny genetika   patologie   MeSH
• dítě MeSH
• elektroretinografie MeSH
• epitelové buňky ultrastruktura   MeSH
• fenotyp MeSH
• fibroblasty ultrastruktura   MeSH
• kationtové kanály TRP MeSH
• kationtové kanály TRPM genetika   MeSH
• kůže ultrastruktura   MeSH
• leukocyty ultrastruktura   MeSH
• lidé MeSH
• lyzozomy genetika   ultrastruktura   MeSH
• messenger RNA genetika   MeSH
• mukolipidózy genetika   patologie   MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• nemoci rohovky genetika   patologie   MeSH
• nemoci spojivky genetika   patologie   MeSH
• polymerázová řetězová reakce MeSH
• rohovkový epitel ultrastruktura   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kationtové kanály TRP MeSH
• kationtové kanály TRPM MeSH
• MCOLN1 protein, human MeSH Prohlížeč
• messenger RNA 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
• bodová mutace * MeSH
• elektronová mikroskopie MeSH
• fibroblasty fyziologie   ultrastruktura   MeSH
• kultivované buňky MeSH
• kůže ultrastruktura   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie ultrastruktura   MeSH
• molekulární biologie MeSH
• předškolní dítě MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH

Micromanipulation is a strong mechanical intervention into cellular integrity and induces large changes in the fine structure of the treated cells. Human diploid skin fibroblasts (KF1 and KF2 cell lines) were chosen as an experimental model. Special hatching needles were used for defined micromanipulation interventions (deformation of plasma membrane). Changes in cytoskeletal structures were visualized by using fluorescent and confocal microscopy. The actin cytoskeleton showed a more sensitive response to micromanipulation than microtubules. Characteristic changes in microfilaments, i.e., thickenings and knot formation, were visible in treated cells fixed immediately after micromanipulation and were the result of hatching-needle pressure on the plasma membrane as well as a reaction of actin filaments localized near the plasma membrane deformation. These direct changes and also other specific alterations in the actin filament network were detectable 14 to 16 h after treatment, but they were not observed when longer reparation intervals were used.

• MeSH
• aktiny ultrastruktura   MeSH
• buněčné linie MeSH
• cytoskelet ultrastruktura   MeSH
• fibroblasty ultrastruktura   MeSH
• fluorescenční mikroskopie MeSH
• konfokální mikroskopie MeSH
• kůže ultrastruktura   MeSH
• lidé MeSH
• mikrofilamenta ultrastruktura   MeSH
• mikromanipulace * MeSH
• mikrotubuly ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH

We present a unique case of a lipoma with widespread synovial metaplasia. A 52-year-old woman had a recurrence 1 year after excision of a subcutaneous lipoma of the neck. Histologically, the primary tumor was an ordinary lipoma. The recurrent tumor was a myxoid lipoma with synovial metaplasia. The synovial metaplastic process manifested as labyrinthlike clefts, which were lined by one or more synovial-like cell layers. Ultrastructurally, the synovial metaplastic cell had secretory, phagocytic, and fibroblastlike features.

• MeSH
• kůže patologie   ultrastruktura   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipom komplikace   patologie   ultrastruktura   MeSH
• lokální recidiva nádoru MeSH
• metaplazie komplikace   patologie   MeSH
• nádory kůže komplikace   patologie   ultrastruktura   MeSH
• synoviální membrána patologie   ultrastruktura   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

To verify the penetration of liposomes through skin, we have used liposomes with encapsulated protein G-gold conjugate in a gel vehicle. Skin samples were examined 2, 4, 24, 48, and 72 h after liposome application. Our findings show that the penetration of liposomes through skin depends mainly on their size. Liposomes up to 600 nm in diameter penetrate through skin rather easily, whereas liposomes 1000 nm and more in diameter remain interiorized in the stratum corneum. The main penetration of liposomes proceeds along the hair sheaths as indicated by larger amounts of free liposomes in the corium of guinea pigs.

• MeSH
• časové faktory MeSH
• elektronová mikroskopie metody   MeSH
• endocytóza MeSH
• keratinocyty metabolismus   MeSH
• králíci MeSH
• kůže účinky léků   metabolismus   ultrastruktura   MeSH
• lidé MeSH
• liposomy metabolismus   farmakokinetika   MeSH
• makrofágy metabolismus   MeSH
• morčata MeSH
• vitamin A farmakologie   MeSH
• vlasový folikul metabolismus   MeSH
• zlato metabolismus   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• morčata MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• liposomy MeSH
• vitamin A MeSH
• zlato MeSH

Storage granules (SGs) from ovine and canine models of Batten disease were found to be easily phagocytosed by four cell types studied. The cell types tested were human fibroblasts and peripheral monocytes (control and from a late infantile Batten disease patient), rat C6 cell line, and neonatal cardiomyocytes. The phagocytosed SGs elicited an increase in acid phosphatase activity which was localized in the phagolysosome. After phagocytosis SGs were followed for various times ranging from 7 to 21 days and were found to be of unchanged density (phase contrast), autofluorescence, and ultrastructural appearance. These findings point to their undergradability, or very low degree of degradability, in phagolysosomes in both normal or Batten cultured cells. The Batten disease SGs are not toxic and did not cause any adverse affect on the host cells. Either the normal clearance rate from lysosomes is too slow to be measured by this technique or subunit c accumulation in lysosomes need not result from a primary lysosomal protease defect. Subunit c may aggregate, because of the lack of some normally preventive factor, resulting in a physical barrier to the degradation of this highly apolar molecule.

• MeSH
• buněčné linie MeSH
• cytoplazmatická granula patologie   ultrastruktura   MeSH
• dospělí MeSH
• fagocytóza MeSH
• fibroblasty MeSH
• játra patologie   ultrastruktura   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• kůže patologie   ultrastruktura   MeSH
• lidé MeSH
• lyzozomy ultrastruktura   MeSH
• monocyty patologie   fyziologie   MeSH
• mozková kůra patologie   ultrastruktura   MeSH
• nemoci ovcí MeSH
• nemoci psů MeSH
• neuronální ceroidlipofuscinózy patologie   patofyziologie   veterinární   MeSH
• ovce MeSH
• pankreas patologie   ultrastruktura   MeSH
• psi MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• psi MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH