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1.
J Invest Dermatol ; 139(9): 1867-1875.e7, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30905808

RESUMO

Atopic dermatitis is a common inflammatory skin disease caused by the interaction of genetic and environmental factors. By allelic copy number analysis at missense single-nucleotide polymorphisms on 26 genes with copy number variation, we identified a significant association between atopic dermatitis and human KPRP. Human KPRP expression, which was localized to the upper granular layer of epidermis, was significantly decreased in atopic dermatitis compared with normal skin. KPRP was histologically colocalized with loricrin and was mainly detected in cytoskeleton fractions of human keratinocytes. To further investigate the role of KPRP in skin, Kprp-knockout mice were generated. Heterozygous knockout (Kprp+/-) mice exhibited reduced KPRP expression to level a similar to that of human AD lesional skin. Kprp+/- mice showed abnormal desmosome structure and detachment of lower layers of the stratum corneum. Percutaneous inflammation by topical application of croton oil or oxazolone was enhanced, and epicutaneous immunization with ovalbumin induced a high level of IgE in Kprp+/- mice. Our study, started from allelic copy number analysis in human AD, identified the importance of KPRP, the decrease of which leads to barrier dysfunction.


Assuntos
Proteínas do Citoesqueleto/genética , Dermatite Atópica/genética , Epiderme/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Queratinócitos/patologia , Proteínas/genética , Adjuvantes Imunológicos/administração & dosagem , Animais , Estudos de Casos e Controles , Óleo de Cróton/imunologia , Proteínas do Citoesqueleto/deficiência , Variações do Número de Cópias de DNA , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Desmossomos/patologia , Desmossomos/ultraestrutura , Modelos Animais de Doenças , Epiderme/efeitos dos fármacos , Epiderme/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Oxazolona/imunologia , Proteínas/metabolismo , Perda Insensível de Água/genética
2.
J Invest Dermatol ; 135(1): 192-201, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25084053

RESUMO

By means of morphological and biochemical criteria, we here provide evidence for the localization and function of premature ovarian failure, 1B (POF1B) in desmosomes. In monolayers of Caco-2 intestinal cells and in stratified HaCaT keratinocytes, endogenous POF1B colocalized with desmoplakin at desmosome plaques and in cytoplasmic particles aligned along intermediate filaments (IFs). POF1B predominantly co-fractionated with desmosomes and IF components and exhibited properties characteristic of desmosomes (i.e., detergent insolubility and calcium independence). The role of NH2 and COOH domains in the association of POF1B with desmosomes and IFs was revealed by transient expression of the truncated protein in Caco-2 cells and in cells lacking desmosomes. The function of POF1B in desmosomes was investigated in HaCaT keratinocytes stably downregulated for POF1B expression. Transmission electron microscopy analysis revealed a decrease in desmosome number and size, and desmosomes of the downregulated keratinocytes displayed weak electron-dense plaques. Desmosome alterations were associated with defects in cell adhesion, as revealed by the reduced resistance to mechanical stress in the dispase fragmentation assay. Moreover, desmosome localization of POF1B was restricted to granular layers in human healthy epidermis, whereas it largely increased in hyperproliferative human skin diseases, thus demonstrating the localization of POF1B also in desmosomes of multistratified epithelia.


Assuntos
Desmossomos/metabolismo , Queratinócitos/metabolismo , Insuficiência Ovariana Primária/metabolismo , Proteínas/metabolismo , Dermatopatias/metabolismo , Células CACO-2 , Cálcio/metabolismo , Adesão Celular/fisiologia , Proliferação de Células , Citoplasma/metabolismo , DNA Complementar/metabolismo , Desmoplaquinas/metabolismo , Desmossomos/ultraestrutura , Células Epidérmicas , Epiderme/metabolismo , Feminino , Humanos , Intestinos/citologia , Queratinócitos/citologia , Proteínas dos Microfilamentos , Microscopia Eletrônica de Transmissão , Insuficiência Ovariana Primária/patologia , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Dermatopatias/patologia , Estresse Mecânico
3.
Microsc Microanal ; 11(4): 283-92, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16079012

RESUMO

Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4-5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37 degrees C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 mug/ml), transferrin (10 mug/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.


Assuntos
Cálcio/farmacologia , Filamentos Intermediários/ultraestrutura , Queratinas/análise , Timo/química , Animais , Células Cultivadas , Desmossomos/ultraestrutura , Células Epiteliais/química , Células Epiteliais/ultraestrutura , Imuno-Histoquímica , Microscopia Eletrônica , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos WF , Timo/citologia , Timo/ultraestrutura
4.
Morfologiia ; 123(1): 41-5, 2003.
Artigo em Russo | MEDLINE | ID: mdl-12741109

RESUMO

The effect of highly specific and selective actin-polymerizing and labelling agent, phalloidin, on electrotonic conductivity and structure of the mixed synapses of goldfish Mauthner neurons (MN) was studied. It was shown that the paired subthreshold electrostimulation of afferent input against a background of phalloidin application resulted in the average 80% increase of the amplitude of MN response to the second stimulus. In control group it increased by only 10% and was observed only after suprathreshold stimulation, while subthreshold stimuli were ineffective. We interpret these data as the manifestation of increased conductivity of the mixed synapses, induced by actin polymerization. At the ultrastructural level, phalloidin application at MN and their mixed synapses increased the size and number of actin-containing desmosome-like junctions, as well as the number of fibrillar bridges crossing their cleft. Using the phalloidin-colloid gold marker, the actin nature of these bridges was demonstrated. Interdependent morpho-functional changes found in the mixed synapses, provide the indication of actin involvement in the conduction of electrotonic signal through the mixed synapse. The bridges crossing the cleft of desmosome-like junction could be the structural substrate of this process.


Assuntos
Actinas/fisiologia , Carpa Dourada/fisiologia , Neurônios Aferentes/fisiologia , Sinapses/fisiologia , Actinas/metabolismo , Animais , Desmossomos/fisiologia , Desmossomos/ultraestrutura , Condutividade Elétrica , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Condução Nervosa/efeitos dos fármacos , Condução Nervosa/fisiologia , Neurônios Aferentes/ultraestrutura , Faloidina/farmacologia , Sinapses/ultraestrutura
5.
Circ Res ; 90(3): e40, 2002 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-11861428

RESUMO

Recent progress in cell transplantation therapy to repair impaired hearts has encouraged further attempts to bioengineer 3-dimensional (3-D) heart tissue from cultured cardiomyocytes. Cardiac tissue engineering is currently pursued utilizing conventional technology to fabricate 3-D biodegradable scaffolds as a temporary extracellular matrix. By contrast, new methods are now described to fabricate pulsatile cardiac grafts using new technology that layers cell sheets 3-dimensionally. We apply novel cell culture surfaces grafted with temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), from which confluent cells detach as a cell sheet simply by reducing temperature without any enzymatic treatments. Neonatal rat cardiomyocyte sheets detached from PIPAAm-grafted surfaces were overlaid to construct cardiac grafts. Layered cell sheets began to pulse simultaneously and morphological communication via connexin43 was established between the sheets. When 4 sheets were layered, engineered constructs were macroscopically observed to pulse spontaneously. In vivo, layered cardiomyocyte sheets were transplanted into subcutaneous tissues of nude rats. Three weeks after transplantation, surface electrograms originating from transplanted grafts were detected and spontaneous beating was macroscopically observed. Histological studies showed characteristic structures of heart tissue and multiple neovascularization within contractile tissues. Constructs transplanted into 3-week-old rats exhibited more cardiomyocyte hypertrophy and less connective tissue than those placed into 8-week-old rats. Long-term survival of pulsatile cardiac grafts was confirmed up to 12 weeks. These results demonstrate that electrically communicative pulsatile 3-D cardiac constructs were achieved both in vitro and in vivo by layering cardiomyocyte sheets. Cardiac tissue engineering based on this technology may prove useful for heart model fabrication and cardiovascular tissue repair. The full text of this article is available at http://www.circresaha.org.


Assuntos
Técnicas de Cultura/métodos , Ventrículos do Coração/citologia , Ventrículos do Coração/transplante , Miocárdio/citologia , Temperatura , Citoesqueleto de Actina/ultraestrutura , Fatores Etários , Animais , Animais Recém-Nascidos , Mapeamento Potencial de Superfície Corporal , Comunicação Celular/fisiologia , Células Cultivadas , Técnicas de Cultura/instrumentação , Procedimentos Cirúrgicos Dermatológicos , Desmossomos/ultraestrutura , Técnicas Eletrofisiológicas Cardíacas , Sobrevivência de Enxerto/fisiologia , Sistema de Condução Cardíaco/fisiologia , Injeções Subcutâneas , Masculino , Contração Miocárdica/fisiologia , Ratos , Ratos Nus , Ratos Wistar , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Transplante de Tecidos/métodos
6.
Biophys Chem ; 50(1-2): 97-112, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-8011944

RESUMO

Desmosomes are intercellular junctions that function in cell-cell adhesion and attachment of intermediate filaments (IF) to the cell surface. Desmogleins and desmocollins are the major components of the transmembrane adhesion complex, whereas desmoplakins (DPs) are the most prominent components of the cytoplasmic plaque. Based on sequence similarity, desmogleins and desmocollins are related to the calcium-dependent homophilic adhesion molecules known as cadherins. Like the classical cadherins, the desmosomal cadherins contain four homologous extracellular domains bearing putative calcium-binding sites, a single transmembrane spanning domain, and a C-terminal cytoplasmic tail. Molecules in the desmoglein subclass contain a unique C-terminal extension within which is found a repeating motif that is predicted to form two beta-strands and two turns. Stable cell lines expressing desmoglein 1 have been generated from normally non-adherent L cell fibroblasts, to study the contribution of this cadherin to desmosomal adhesion. The predicted sequence of desmoplakin (DP) I suggests it will form homodimers comprising a central alpha-helical coiled-coil rod and two globular end domains. The C-terminus contains three regions with significant homology, each of which is made up of a 38-residue motif also found in two other molecules involved in organization of IF, bullous pemphigoid antigen and plectin. Ectopically expressed polypeptides including the C-terminus of DP I specifically align with keratin and vimentin IF in cultured cells, whereas those lacking this domain do not align with IF. The last 68 amino acids of DP are required for alignment along keratin but not vimentin IF, and residues 48-68 from the C-terminal end are critical for this interaction. These results suggest that the C-terminus of DP plays a role in the attachment of IF to the desmosome and that a specific site is necessary for interaction with keratin IF. A sequence at the most N-terminal end of DP appears to be required for efficient incorporation into the desmosomal plaque. Interestingly, this region has not been reported to be present in the homologous bullous pemphigoid antigen or plectin molecules and may represent a desmosomal targeting sequence.


Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteínas do Citoesqueleto/ultraestrutura , Desmossomos/fisiologia , Desmossomos/ultraestrutura , Animais , Caderinas/genética , Caderinas/fisiologia , Bovinos , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , DNA Complementar/análise , Desmocolinas , Desmogleína 1 , Desmogleínas , Desmoplaquinas , Junções Comunicantes/fisiologia , Células L , Proteínas de Membrana/fisiologia , Proteínas de Membrana/ultraestrutura , Camundongos , Relação Estrutura-Atividade
7.
Exp Cell Res ; 207(1): 19-32, 1993 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8319770

RESUMO

Gap junctional intercellular communication (GJIC), desmosomes, and cell movement were evaluated in a rat ovarian epithelial cell model system which consisted of an immortalized clonal cell line (SIGC), a pSV3neotransfected clonal derivative (SV-SIGC), and a nude mouse SV-SIGC-tumor-derived cell line (T-SV-SIGC). Complementary ultrastructural, indirect immunofluorescence, and Western blot data identified a relatively small loss of desmosomes and associated cytokeratins in SV-SIGC compared to SIGC but a near total loss in T-SV-SIGC. SIGC and SV-SIGC migrated outward from monolayer-coated microcarrier beads as epithelial sheets, whereas in T-SV-SIGC there was dissociation and migration of individual fibroblastoid cells. GJIC was assessed by fluorescence recovery after photobleaching (gap FRAP) and equations based on Fick's first law of diffusion were derived to quantitatively compare GJIC of systems with different recovery equilibria after photobleaching. Taken together the data suggested that GJIC in SIGC was quantitatively reduced to similar levels by various conditions associated with reduced cell-cell adhesiveness including transformation to T-SV-SIGC, mitosis, and culture in low calcium medium. These results supported linkage between changes in desmosomal adhesiveness, cell movement, and GJIC.


Assuntos
Comunicação Celular/efeitos dos fármacos , Desmossomos , Junções Intercelulares/metabolismo , Animais , Cálcio , Adesão Celular , Linhagem Celular , Linhagem Celular Transformada , Células Clonais , Meios de Cultura , Desmossomos/química , Desmossomos/ultraestrutura , Difusão , Feminino , Corantes Fluorescentes , Junções Intercelulares/ultraestrutura , Matemática , Metástase Neoplásica/fisiopatologia , Ratos
8.
Biull Eksp Biol Med ; 106(8): 231-3, 1988 Aug.
Artigo em Russo | MEDLINE | ID: mdl-3416060

RESUMO

A study was made of the ultrastructural changes of epidermocytes cultured on hypocalcic media (Ca2+ content below 0.08 mM) in comparison with epidermocytes, cultured on media with standard Ca2+ content (1-2 mM). A detailed description of methods for epidermocyte cultivation and data of electron microscopic cell research is presented. It has been established that the use of hypocalcic nutrient medium accelerates the process of cultured epithelial sheet formation for grafting in skin defects. However, before removing a sheet from the cultural vessel bottom the nutrition media should be supplemented with Ca2+ to strengthen intercellular coupling of epidermocytes. Thus, Ca2+ addition to the medium can regulate the generation of desmosomes and the formation of the monolayer of stratified sheet of the cultured epithelium.


Assuntos
Cálcio/metabolismo , Meios de Cultura/metabolismo , Epiderme/ultraestrutura , Cálcio/administração & dosagem , Diferenciação Celular , Células Cultivadas , Desmossomos/ultraestrutura , Epiderme/metabolismo , Humanos , Microscopia Eletrônica , Fatores de Tempo
9.
In Vitro Cell Dev Biol ; 24(6): 593-600, 1988 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2455705

RESUMO

The recognized need for epithelial cell culture models for cystic fibrosis (CF) research has resulted in ongoing efforts to improve normal and CF submandibular duct cell culture capabilities. The duct is most likely the site of the CF defect in this and other exocrine glands. In a previous report conditions required for the successful primary explant culture of normal and CF submandibular glands were outlined; however, terminal keratinization and involution of these cultures were recognized as severe limiting factors to their utilization in CF research. This report explores the effects of calcium concentrations in the medium, growth factor supplements, and matrix components on growth and differentiation of these cultures. Results of the study further confirm the ductal origin of cells in the outgrowth and demonstrate that progressive keratinization is initiated only after cells proliferate beyond the environment of the explant fragment. Keratinization with subsequent multilayering, desmosome formation, and involution in the cell outgrowth are governed in degree by the calcium concentration of the growth medium. Upon reduction of medium calcium to 0.1 mM concentration, the cells proliferate as a monolayer and subculture through 8 to 9 passages and retain the capacity to undergo ductlike differentiation.


Assuntos
Cálcio/farmacologia , Fibrose Cística/patologia , Glândula Submandibular/ultraestrutura , Sangue , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Células Cultivadas , Colágeno , Meios de Cultura , Desmossomos/ultraestrutura , Humanos , Imuno-Histoquímica , Queratinas/metabolismo , Laminina , Microscopia Eletrônica , Microvilosidades/ultraestrutura , Organoides/ultraestrutura , Glândula Submandibular/efeitos dos fármacos
10.
Anat Rec ; 214(1): 76-81, 1986 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-3954061

RESUMO

The ultrastructure of the hypothalamic intraventricular blood vessels is studied in two fish: Salmo gairdneri, a primitive teleost and Gambusia affinis, a modern teleost. Morphological differences found between vessels belonging to the lateral and medial recesses in Salmo are described as well as contrasted with those of Gambusia. The endothelial cells are joined by elaborate tight junctions in both teleosts and also by desmosomes in Gambusia. These vessels are continuous capillaries with pericytes which lack junctional complexes. The perivascular cells of the larger vessels display morphological characteristics of myoid cells.


Assuntos
Peixes/anatomia & histologia , Hipotálamo Posterior/irrigação sanguínea , Hipotálamo/irrigação sanguínea , Animais , Vasos Sanguíneos/ultraestrutura , Capilares/ultraestrutura , Desmossomos/ultraestrutura , Endotélio/ultraestrutura , Junções Intercelulares/ultraestrutura , Microscopia Eletrônica , Especificidade da Espécie , Truta/anatomia & histologia
11.
J Anat ; 130(Pt 1): 55-61, 1980 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7364663

RESUMO

Adult hamsters were used for this electron microscopic study of the hypothalamic region. Specialized contacts between astrocytes and astrocytes, and between astrocytes and other cellular elements, are described and illustrated. The specialized inter-astrocytic junctions occur primarily in perivascular and subpial regions, but also in areas of high synaptic density. The junctions between astrocytic processes are of hemidesmosomal type. Astrocytes are connected to oligodendroglial cells by means of desmosomes, and to neuronal processes by means of zonulae occludens. The functional significance of these arrangements is discussed.


Assuntos
Astrócitos/ultraestrutura , Hipotálamo/ultraestrutura , Junções Intercelulares/ultraestrutura , Animais , Cricetinae , Desmossomos/ultraestrutura , Feminino , Masculino , Microscopia Eletrônica , Oligodendroglia/ultraestrutura
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