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1.
Comput Biol Med ; 100: 17-26, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29960146

RESUMO

Because of variable inconvenient living conditions in some places around the world, it is difficult to collect reliable physiological data for ostriches. Therefore, this study aims to provide a comprehensive in silico insight for the nature of polymorphism of important genetic loci that are related to physiological and reproductive traits. Sixty-nine mature ostriches ranging over half of Iraq were screened. Six exonic genetic loci, including cytochrome c oxidase I (COX1), cytochrome b (CYTB), secretogranin V (SCG5), feather keratin 2-like (FK2), prolactin (PRL) and placenta growth factor (PGF) were genotyped by PCR-single stranded conformation polymorphism (SSCP). Thirty-six novel SNPs, including seventeen nonsynonymous (ns) SNPs, were observed. Several computational software programs were utilized to assess the extent of the nsSNPs on their corresponding proteins structure, function and stability. The results showed several deleterious functional and stability changes in almost all the proteins studied. The total severity of each missense mutation was evaluated and compared with other nsSNPs accumulatively. It is evident from the extensive cumulative in silico computation that both p.E34D and p.E60K in PGF have the highest deleterious effect. The cumulative predictions from the present study are an impressive guide for the genotypes of African ostriches, which bypassed the expensive protocols for wet laboratory screening, to identify the effects of variants. To the best of our knowledge, this is the first investigation of its kind on the analyses and prediction outcome of missense mutations in African ostrich populations. The highly deleterious nsSNPs in the placenta growth factor are possible adaptive mutations which might be associated with adaptation in extreme and new environments. The flow and protocol of the computational predictions can be extended for various wild animals to identify the molecular nature of adaptations.


Assuntos
Adaptação Fisiológica , Proteínas Aviárias/genética , Ciclo-Oxigenase 1/genética , Citocromos b/genética , Queratina-2/genética , Mutação de Sentido Incorreto , Proteína Secretora Neuroendócrina 7B2/genética , Fator de Crescimento Placentário/genética , Polimorfismo de Nucleotídeo Único , Prolactina/genética , Struthioniformes/genética , África , Animais , Genética Populacional
3.
Dev Neurobiol ; 77(11): 1308-1320, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28719101

RESUMO

The olfactory epithelium (OE) has the remarkable capability to constantly replace olfactory receptor neurons (ORNs) due to the presence of neural stem cells (NSCs). For this reason, the OE provides an excellent model to study neurogenesis and neuronal differentiation. In the present work, we induced neuronal degeneration in the OE of Xenopus laevis larvae by bilateral axotomy of the olfactory nerves. We found that axotomy induces specific- neuronal death through apoptosis between 24 and 48h post-injury. In concordance, there was a progressive decrease of the mature-ORN marker OMP until it was completely absent 72h post-injury. On the other hand, neurogenesis was evident 48h post-injury by an increase in the number of proliferating basal cells as well as NCAM-180- GAP-43+ immature neurons. Mature ORNs were replenished 21 days post-injury and the olfactory function was partially recovered, indicating that new ORNs were integrated into the olfactory bulb glomeruli. Throughout the regenerative process no changes in the expression pattern of the neurotrophin Brain Derivate Neurotrophic Factor were observed. Taken together, this work provides a sequential analysis of the neurodegenerative and subsequent regenerative processes that take place in the OE following axotomy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1308-1320, 2017.


Assuntos
Axotomia , Degeneração Neural/etiologia , Degeneração Neural/patologia , Mucosa Olfatória/patologia , Traumatismos do Nervo Olfatório/patologia , Regeneração/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Caspase 3/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica/fisiologia , Queratina-2/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteína de Marcador Olfatório/metabolismo , Traumatismos do Nervo Olfatório/etiologia , Recuperação de Função Fisiológica/fisiologia , Olfato/fisiologia , Fatores de Tempo , Xenopus laevis
4.
Anim Sci J ; 88(8): 1189-1197, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28026086

RESUMO

Improper or delayed pregnancy diagnosis has significant impact over animal production, particularly in buffaloes which inherently suffer from several reproductive inefficiencies. Thus the present study has undertaken to identify serum protein markers pertaining to early pregnancy diagnosis in buffaloes. Serum samples were collected from 10 pregnant Murrah Buffalo heifers at weekly intervals from days 0-35 post-artificial insemination and from 12 inseminated non-pregnant cyclic buffalo heifers on days 0, 7, 14 and 21. Two-dimensional gel electrophoresis and densitometric analysis revealed the presence of five protein spots showing average density fold change of ≥4 during early pregnancy. Mass spectrometry analysis identified these up-regulated proteins as anti-testosterone antibody light chain, apolipoprotein A-II precursor, serum amyloid A, cytokeratin type II, component IV isoform 1, which are have established roles in embryogenesis, but over-expression of the fifth identified protein immunoglobulin lambda light chain in pregnancy has been elucidated as a novel finding in the current study. Further, with bioinformatics analysis, potential antigenic B-cell epitopes were predicted for all these five proteins. An antibody cocktail-based approach involving antibodies against all these five up-regulated entire proteins or their epitopes could be developed for early detection of pregnancy in buffaloes. © 2016 Japanese Society of Animal Science.


Assuntos
Anticorpos/sangue , Búfalos , Testes de Gravidez/veterinária , Prenhez , Animais , Apolipoproteína A-II/sangue , Biomarcadores/sangue , Complemento C4 , Eletroforese em Gel Bidimensional , Epitopos de Linfócito B/sangue , Feminino , Queratina-2/sangue , Espectrometria de Massas , Gravidez , Testes de Gravidez/métodos , Precursores de Proteínas/sangue , Proteína Amiloide A Sérica , Testosterona/imunologia
5.
Tumour Biol ; 37(9): 12423-12440, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27324070

RESUMO

Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and carcinogenesis may prove crucial in developing novel therapeutics that specifically target cancer cells.


Assuntos
Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Flavonoides/farmacologia , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Piperidinas/farmacologia , Actinas/análise , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Fibroblastos/efeitos dos fármacos , Flavonoides/uso terapêutico , Queratina-2/análise , Neoplasias Pulmonares/patologia , Camundongos , Piperidinas/uso terapêutico
6.
Acta Histochem ; 118(5): 505-12, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27265811

RESUMO

In order to investigate the effects of the keratin 2 (KRT2) on alpaca melanocyte in vivo and vitro, the immunohistochemistry (IHC), quantitative real-time PCR (qPCR), Western blot, and alpaca melanocytes transfection methods were used. The results showed that mRNA and protein expression of KRT2 was highly expressed in brown skin in comparison with that in white skin. Moreover, we found that KRT2 was expressed in alpaca melanocytes in vitro by immunocytochemistry. After transfection with KRT2 in alpaca melanocytes, the relative mRNA and protein expression of KRT2, microphthalmia-associtated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) in alpaca skin melanocytes was increased with significant differences; a further result was the increase of melanin production. The results suggested that KRT2 functions in alpaca hair color formation, which offered an essential theoretical basis for further exploration of the role of melanogenesis.


Assuntos
Queratina-2/metabolismo , Melaninas/biossíntese , Pele/metabolismo , Animais , Camelídeos Americanos , Expressão Gênica , Cor de Cabelo , Queratina-2/genética , Melanócitos/metabolismo , Pele/citologia , Pigmentação da Pele
7.
Acta Derm Venereol ; 96(4): 473-8, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26581228

RESUMO

Twenty-six families with keratinopathic ichthyoses (epidermolytic ichthyosis, superficial epidermolytic ichthyosis or congenital reticular ichthyosiform erythroderma) were studied. Epidermolytic ichthyosis is caused by mutations in the genes KRT1 or KRT10, mutations in the gene KRT2 lead to superficial epidermolytic ichthyosis, and congenital reticular ichthyosiform erythroderma is caused by frameshift mutations in the genes KRT10 or KRT1, which lead to the phenomenon of revertant mosaicism. In this study mutations were found in KRT1, KRT2 and KRT10, including 8 mutations that are novel pathogenic variants. We report here the first case of a patient with congenital reticular ichthyosiform erythroderma carrying a mutation in KRT10 that does not lead to an arginine-rich reading frame. Novel clinical features found in patients with congenital reticular ichthyosiform erythroderma are described, such as mental retardation, spasticity, facial dysmorphisms, symblepharon and malposition of the 4th toe.


Assuntos
Hiperceratose Epidermolítica/genética , Ictiose Lamelar/genética , Queratina-10/genética , Queratina-1/genética , Queratina-2/genética , Mutação , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Hereditariedade , Humanos , Hiperceratose Epidermolítica/diagnóstico , Ictiose Lamelar/diagnóstico , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Fatores de Risco , Índice de Gravidade de Doença , Adulto Jovem
8.
J Dermatol Sci ; 81(1): 10-6, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26603179

RESUMO

BACKGROUND: K1 and K2 are the main type II keratins in the suprabasal epidermis where each of them heterodimerizes with the type I keratin K10 to form intermediate filaments. In regions of the ears, tail, and soles of the mouse, only K2 is co-expressed with K10, suggesting that these keratins suffice to form a mechanically resilient cytoskeleton. OBJECTIVE: To determine the effects of the suppression of both main keratins, K2 and K10, in the suprabasal plantar epidermis of the mouse. METHODS: Krt2(-/-) Krt10(-/-) mice were generated by crossing Krt2(-/-) and Krt10(-/-) mice. Epidermal morphology of soles of hind-paws was examined macroscopically and histologically. Immunofluorescence analysis and quantitative PCR analysis were performed to analyze the expression of keratins in sole skin of wildtype and Krt2(-/-) Krt10(-/-) mice. Highly abundant proteins of the sole stratum corneum were determined by electrophoretic and chromatographic separation and subsequent mass spectrometry. RESULTS: K2 and K10 are the most prominent suprabasal keratins in normal mouse soles with the exception of the footpads where K1, K9 and K10 predominate. Mice lacking both K2 and K10 were viable and developed epidermal acanthosis and hyperkeratosis in inter-footpad epidermis of the soles. The expression of keratins K1, K9 and K16 was massively increased at the RNA and protein levels in the soles of Krt2(-/-) Krt10(-/-) mice. CONCLUSIONS: This study demonstrates that the loss of the main cytoskeletal components of plantar epidermis, i.e. K2 and K10, can be only partly compensated by the upregulation of other keratins. The thickening of the epidermis in the soles of Krt2(-/-) Krt10(-/-) mice may serve as a model for pathomechanistic aspects of palmoplantar keratoderma.


Assuntos
Epiderme/fisiologia , Queratina-10/fisiologia , Queratina-2/fisiologia , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Epiderme/anatomia & histologia , Extremidades , Humanos , Queratina-1/genética , Queratina-1/metabolismo , Queratina-10/genética , Queratina-16/genética , Queratina-16/metabolismo , Queratina-2/deficiência , Queratina-2/genética , Queratina-9/genética , Queratina-9/metabolismo , Ceratodermia Palmar e Plantar/genética , Ceratodermia Palmar e Plantar/metabolismo , Ceratodermia Palmar e Plantar/patologia , Ceratose/genética , Ceratose/metabolismo , Ceratose/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima
9.
Toxicol In Vitro ; 30(1 Pt B): 462-75, 2015 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-26391144

RESUMO

The moisturizing and potentially protective properties of the organic osmolyte betaine (trimethylglycine) have made it an attractive component for skin care products. Its wide use despite the lack of comprehensive studies addressing its specific effects in skin led us to characterize the molecular targets of betaine in keratinocytes and to explore, whether it modifies the effects of acute UVB exposure. Genome-wide expression analysis was performed on organotypic cultures of rat epidermal keratinocytes, treated either with betaine (10mM), UVB (30 mJ/cm(2)) or their combination. Results were verified with qRT-PCR, western blotting and immunohistochemistry. Additionally, cell proliferation and differentiation were analyzed. Among the 89 genes influenced by betaine, the differentiation marker keratin 2 showed the highest upregulation, which was also confirmed at protein level. Expression of Egr1, a transcription factor, and Purkinje cell protein 4, a regulator of Ca(2+)/calmodulin metabolism, also increased, while downregulated genes included several ion-channel components, such as Fxyd2. Bioinformatics analyses suggest that genes modulated by betaine are involved in DNA replication, might counteract UV-induced processes, and include many targets of transcription factors associated with cell proliferation and differentiation. Our results indicate that betaine controls unique gene expression pathways in keratinocytes, including some involved in differentiation.


Assuntos
Betaína/farmacologia , Queratina-2/genética , Queratinócitos/efeitos dos fármacos , Animais , Linhagem Celular , Estudo de Associação Genômica Ampla , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , RNA Mensageiro/análise , Ratos , Raios Ultravioleta
11.
Free Radic Biol Med ; 73: 75-83, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24838180

RESUMO

The nitration of proteins results from the vigorous production of reactive nitrogen species in inflammatory disease. We previously reported the proteomic analysis of nitrated tryptophan residues in in vitro model cells for inflammatory diseases using a 6-nitrotryptophan-specific antibody. In this paper, we applied this method to the analysis of a disease model animal and identified the 6-nitrotryptophan-containing proteins in the skin of atopic dermatitis model mice (AD-NC/Nga mice). We found three nitrotryptophan-containing proteins, namely, carbonic anhydrase III (CAIII), α-enolase (α-ENO), and cytoskeletal keratin type II (KTII), and identified the positions of the nitrotryptophan residues in their amino acid sequences: Trp47 and Trp123 in CAIII, Trp365 in α-ENO, and Trp221 in KTII. Among these, the nitration of CAIII was increased not only in the lesional skin of AD-NC/Nga mice but also in the mice that did not present any symptoms. The in vitro nitration of purified CAIII by peroxynitrite reduced its CO2 hydratase activity in a dose-dependent manner. In addition, we found that CAIII was induced during the differentiation of normal human epidermal keratinocytes. Furthermore, we found the presence of CAIII and the formation of 6-nitrotryptophan-containing proteins in both the lesional and the nonlesional sections of the skin of patients with atopic dermatitis through immunohistochemical staining. This study provides the first demonstration of the formation of 6-nitrotryptophan in human tissues and disease.


Assuntos
Anidrase Carbônica III/metabolismo , Dermatite Atópica/patologia , Queratina-2/metabolismo , Fosfopiruvato Hidratase/metabolismo , Triptofano/análogos & derivados , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/patologia , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Ácido Peroxinitroso/química , Ratos , Espécies Reativas de Nitrogênio/química , Pele/patologia , Triptofano/química , Triptofano/imunologia , Triptofano/metabolismo
12.
J Invest Dermatol ; 134(10): 2579-2588, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24751727

RESUMO

Keratin K2 is one of the most abundant structural proteins of the epidermis; however, its biological significance has remained elusive. Here we show that suprabasal type II keratins, K1 and K2, are expressed in a mutually exclusive manner at different body sites of the mouse, with K2 being confined to the ear, sole, and tail skin. Deletion of K2 caused acanthosis and hyperkeratosis of the ear and the tail epidermis, corneocyte fragility, increased transepidermal water loss, and local inflammation in the ear skin. The loss of K2 was partially compensated by upregulation of K1 expression. However, a significant portion of K2-deficient suprabasal keratinocytes lacked a regular cytoskeleton and developed massive aggregates of the type I keratin, K10. Aggregate formation, but not hyperkeratosis, was suppressed by the deletion of both K2 and K10, whereas deletion of K10 alone caused clumping of K2 in ear skin. Taken together, this study demonstrates that K2 is a necessary and sufficient binding partner of K10 at distinct body sites of the mouse and that unbalanced expression of these keratins results in aggregate formation.


Assuntos
Dermatite/metabolismo , Hiperceratose Epidermolítica/metabolismo , Queratina-10/metabolismo , Queratina-2/deficiência , Queratina-2/metabolismo , Dermatopatias/metabolismo , Animais , Dermatite/genética , Dermatite/patologia , Modelos Animais de Doenças , Orelha , , Hiperceratose Epidermolítica/genética , Hiperceratose Epidermolítica/patologia , Queratina-1/metabolismo , Queratina-10/genética , Queratina-2/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pele/metabolismo , Pele/patologia , Dermatopatias/genética , Dermatopatias/patologia , Cauda
13.
J Neurosci Res ; 92(2): 206-17, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24254769

RESUMO

ß-Amyloid (Aß) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2-coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac-tau) and tau phosphorylation (P-tau) by inhibiting activation of P300 and GSK3ß. Aß was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aß accumulation was accompanied by increased Ac-tau and P-tau levels. Concomitantly, these cells showed increased P300 and GSK3ß P-Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3-30 µM) and resveratrol (20 µM). Moreover, decreased expression of SIRT1 and its activity by Aß were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 µM, PKA inhibitor), TBCA (20 µM, inhibitor of CK2), and sirtinol (20 µM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP-dependent protein kinase-linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau-related neurodegeneration in the AD brain.


Assuntos
Queratina-2/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Sirtuína 1/biossíntese , Tauopatias/metabolismo , Tetrazóis/farmacologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Western Blotting , Linhagem Celular , Cilostazol , Imunofluorescência , Humanos , Camundongos , Neurônios/metabolismo , Transfecção , Proteínas tau/metabolismo
14.
Pediatr Dermatol ; 30(4): 469-72, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22612346

RESUMO

Superficial epidermolytic ichthyosis (SEI), previously known as ichthyosis bullosa of Siemens, is a rare genetic skin condition, characterized by blisters and hyperkeratosis. It can be easily confused with epidermolytic hyperkeratosis, known now as epidermolytic ichthyosis, and genetic testing can be helpful in differentiating between the two conditions. We describe two children with SEI confirmed by genetic testing, including one with a novel mutation. We also describe other affected family members with SEI.


Assuntos
Hiperceratose Epidermolítica/diagnóstico , Hiperceratose Epidermolítica/genética , Ictiose Bolhosa de Siemens/diagnóstico , Ictiose Bolhosa de Siemens/genética , Queratina-2/genética , Pré-Escolar , Diagnóstico Diferencial , Saúde da Família , Feminino , Genes Dominantes , Heterozigoto , Humanos , Masculino
15.
J Proteomics ; 75(2): 435-49, 2011 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-21884835

RESUMO

Keratins are the main constituent of human skin and have been identified as major oxidative target proteins. However, there has been a lack of studies aimed at identifying the oxidation sites of keratins because of the difficulties associated with their insolubility and handling. Here, we introduce a mass spectrometry (MS)-based proteomic methodology to screen oxidative modifications in human skin keratins. Human skin proteins were obtained non-invasively by tape stripping and solubilized in SDS buffer, followed by purification and digestion using the modified filter-aided sample preparation method. The tryptic peptides were then analyzed by MALDI-TOF/MS, LC-ESI/MS, and MS/MS. PMF analyses have identified keratins K1 and K10 as the major proteins of human skin. Met(259), Met(262), Met(296), and Met(469), located in the α-helical rod domain of K1, were the most susceptible sites to oxidation induced by hydrogen peroxide in vitro and in vivo. Our results indicate a potential use of the identified methionine residues as biomarkers of oxidative skin damage. The present methodology is the first MS-based approach to detecting oxidative modifications in keratins obtained directly from human skin and can be easily applied to the monitoring of other keratin modifications in various skin conditions.


Assuntos
Queratinas/análise , Metionina/química , Pele/química , Sequência de Aminoácidos , Artefatos , Humanos , Peróxido de Hidrogênio/química , Queratina-1/química , Queratina-10/química , Queratina-2/química , Queratina-9/química , Queratinas/química , Queratinas/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Oxirredução , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem , Tripsina/metabolismo
16.
Exp Dermatol ; 19(7): 674-81, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20456496

RESUMO

Disorders of keratinization are often treated with vitamin A derivatives (retinoids) which affect keratinocyte differentiation, including keratin (KRT) gene expression. In vivo, suprabasal keratinocytes normally express only keratin (K) 1, K2 and K10, but after topical application of all-trans retinoic acid (ATRA), the granular cells will additionally express K4 and K13, i.e. keratins normally present in oral mucosa and in cultured epidermal keratinocytes. To learn more about the retinoid regulation of keratin expression under in vivo-like conditions, we cultured keratinocytes on de-epidermized dermis in only 0.5% serum. These cells produce a normal-looking epidermis that expresses high mRNA levels of KRT1, KRT2 and KRT10, but minimal amounts of KRT4 and KRT13. Addition of ATRA to the medium for 48 h caused a dose-dependent increase in KRT4/KRT13 and a down-regulation of KRT2 mRNA. An increase in K4 protein was also found. The response was greater than the up-regulation of another retinoid-regulated gene, CRABPII. By studying 10 retinoids with different affinities for the retinoic acid receptors (RAR) and retinoid X receptors (RXR) isoforms, the reciprocal expression of KRT2 and KRT4/KRT13 could be connected with agonists for RARalpha. Two of these agonists, CD336/Am580 and CD2081, altered the expression profile with similar potency as the pan-RAR agonists ATRA and CD367. Co-addition of a pan-RAR antagonist (CD3106/AGN193109) markedly inhibited the induction of KRT4/KRT13 expression, whereas the down-regulation of KRT2 was less affected. In conclusion, RARalpha agonists elicit a reciprocal modulation of KRT2 and KRT4/KRT13 expression in human epidermis, but whether or not the keratin genes also possess RARalpha-specific regulatory elements is still unclear.


Assuntos
Queratinas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Retinoides/metabolismo , Retinoides/farmacologia , Pele/efeitos dos fármacos , Pele/metabolismo , Benzoatos/metabolismo , Benzoatos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Queratina-13/genética , Queratina-13/metabolismo , Queratina-2/genética , Queratina-2/metabolismo , Queratina-4/genética , Queratina-4/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptor alfa de Ácido Retinoico , Receptores X Retinoide/metabolismo , Tetra-Hidronaftalenos/metabolismo , Tetra-Hidronaftalenos/farmacologia , Tretinoína/metabolismo , Tretinoína/farmacologia
17.
J Invest Dermatol ; 130(9): 2286-94, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20445547

RESUMO

Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.


Assuntos
Epiderme , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos , Raios Ultravioleta/efeitos adversos , Apoptose/fisiologia , Apoptose/efeitos da radiação , Diferenciação Celular/fisiologia , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Difusão , Células Epidérmicas , Epiderme/metabolismo , Epiderme/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Corantes Fluorescentes/farmacocinética , Humanos , Isoquinolinas/farmacocinética , Queratina-1/metabolismo , Queratina-10/metabolismo , Queratina-2/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Queratinas/metabolismo , Metabolismo dos Lipídeos , Microscopia Eletrônica , Técnicas de Cultura de Órgãos , Permeabilidade , RNA Interferente Pequeno , Solubilidade , Ácido Urocânico/metabolismo
18.
J Invest Dermatol ; 130(5): 1268-78, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20043016

RESUMO

Cholesterol is organized in distinctive liquid-ordered micro-domains within biological membranes called lipid rafts. These micro-domains direct multiple physiological functions in mammalian cells by modulating signaling processes. Recent findings suggest a role for lipid rafts in cellular processes in human keratinocytes such as early differentiation and apoptosis. However, research of lipid rafts is hindered by technological limitations in visualizing dynamic cholesterol organization in plasma membranes. This study addresses a real-time, non-invasive method for the long-term observation of cholesterol reorganization in plasma membranes. In addition, this study also addresses the dynamic process of cholesterol depletion and repletion in primary human keratinocytes. Cholesterol reorganization was measured by observed changes in cellular impedance. Disruption of lipid rafts with low concentrations of methyl-beta-cyclodextrin (MbetaCD) resulted in an increase in the proliferative capacity of keratinocytes, which was assessed using real-time proliferation curves and adenosine triphosphate (ATP)-based proliferation assays. Quantitative PCR showed a concomitant decrease in messenger RNA (mRNA) expression of the early differentiation markers keratins 1 and 10. Conversely, specific cholesterol reintegration led to a 4.5-fold increase in keratin 2 mRNA expression, a marker for late keratinocyte differentiation, whereas depletion resulted in a significant downregulation. These findings imply a strictly controlled mechanism for the regulation of membrane cholesterol composition in both early and terminal keratinocyte differentiation. The impedance-based method that this study addresses further enhances our understanding of how physiological processes in keratinocytes are controlled by membrane cholesterol.


Assuntos
Colesterol/metabolismo , Células Epidérmicas , Queratinócitos/citologia , Queratinócitos/metabolismo , Microdomínios da Membrana/metabolismo , Cálcio/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Impedância Elétrica , Filipina/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Queratina-1/genética , Queratina-10/genética , Queratina-2/genética , Microdomínios da Membrana/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , beta-Ciclodextrinas/farmacologia
19.
Arch Dermatol Res ; 301(7): 475-85, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19294396

RESUMO

The biosynthesis of retinoic acid (RA) from retinol is controlled by several enzymes, e.g. dehydrogenases (RalDH2, RoDH-4) and retinol-esterifying enzyme (LRAT), whereas its degradation mainly involves CYP26 enzymes. In keratinocytes, RA activates the nuclear retinoid-receptors inducing the transcription of many genes. Here, we examined the effects of RA and the CYP26 inhibitors, liarozole and talarozole, on retinoid metabolism and RA-regulated genes in organotypic epidermis. RA induced the expression of CYP26 enzymes already after 8 h, whereas LRAT exhibited a later response and peaked at 48 h, indicating a feedback induction of retinol esterification. In line with a reduced biosynthesis of RA from retinol after exogenous RA, the expression of RDH16 reduced 80% in response to exogenous RA. The mRNA expression of RA-regulated genes (KRT2, KRT4, CRABPII and HBEGF) was altered within 24 h after RA exposure. In contrast, the CYP26 inhibitors caused only minor effects, except for a clear-cut induction of CYP26A1 only when combined with minute amounts of exogenous RA. Cellular accumulation of exogenous [3H]RA was higher after talarozole than after liarozole, probably indicating a greater CYP26-inhibitory potency of the former drug. The present study shows that CYP26A1 expression is extremely sensitive to both exogenous RA and increased endogenous RA levels, i.e. due to CYP26 inhibition, and thus an excellent biomarker for retinoid signalling in organotypic epidermis.


Assuntos
Aciltransferases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Epiderme/metabolismo , Queratinócitos/metabolismo , Retinal Desidrogenase/metabolismo , Tretinoína/farmacologia , Aciltransferases/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Antagonistas de Androgênios/farmacologia , Benzotiazóis/farmacologia , Biomarcadores/metabolismo , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450 , Epiderme/patologia , Retroalimentação Fisiológica , Regulação Enzimológica da Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratina-2/genética , Queratina-2/metabolismo , Queratina-4/genética , Queratina-4/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Retinal Desidrogenase/genética , Ácido Retinoico 4 Hidroxilase , Tretinoína/metabolismo , Triazóis/farmacologia , Vitamina A/metabolismo
20.
J Am Acad Dermatol ; 57(2): 285-91, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17553594

RESUMO

BACKGROUND: Circumscribed palmar or plantar hypokeratosis (CPH) is a rare skin disorder only recently described. OBJECTIVE: To determine the diagnostic features and to provide insight into the pathogenesis of CPH, with analysis of two new Japanese cases. METHODS: Dermoscopy, immunohistochemistry, electron microscopy, polymerase chain reaction amplification for human papillomavirus (HPV) DNA and 16S microbial rRNA gene profiling were conducted. RESULTS: Dermoscopy showed characteristic features using both dry and jelly immersion observation; step-like desquamation and a homogeneous erythema with regularly distributed whitish spots. Immunohistochemistry revealed strong staining with anti-pankeratin antibody (AE1+AE3) and anti-keratin 16 antibody, and decreased expression of keratin 2e. EM revealed a breakage of the corneocytes within their cytoplasm, but structures for cell attachment were intact. HPV and lesion-specific bacteria were not detected. LIMITATIONS: The number of cases analyzed was two. CONCLUSION: Hyperproliferative epidermal state along with enhanced corneocyte fragility may account for the unique features in CPH.


Assuntos
Dermatoses da Mão/metabolismo , Queratina-2/metabolismo , Queratinas/metabolismo , Ceratose/metabolismo , Idoso , Bactérias/genética , DNA Viral/análise , Feminino , Dermatoses da Mão/microbiologia , Dermatoses da Mão/patologia , Humanos , Imuno-Histoquímica/métodos , Ceratose/microbiologia , Ceratose/patologia , Microscopia Eletrônica , Pessoa de Meia-Idade , Papillomaviridae/genética , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/análise , Coloração e Rotulagem
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