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1.
J Biochem Mol Toxicol ; 24(1): 10-20, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20146380

RESUMO

In the epidermis, keratinocytes are involved in physical and first-line immune protection of the host. In this study, we analyzed the molecular responses to certain contact sensitizers (2,4-dinitrochlorobenzene and NiSO(4)) and irritants (sodium dodecyl sulfate and benzalkonium chloride) in cultured human keratinocytes from the bulge region of a plucked hair follicle (bulge-derived keratinocytes [BDKs]) and compared these molecular responses to those with the human monocytic leukemia cell line, THP-1. The BDKs, individually established without invasive biopsies, showed high reactivity to these stimulants. As a primary response to the contact sensitizers, the NRF2-mediated signaling pathway was upregulated in BDKs and THP-1. The expression of IL1B and IL8 genes was not induced by the irritants but by the sensitizers in THP-1. However, the expression of the IL1B and IL8 genes was induced at higher levels by the irritants in BDKs than by the sensitizers. Many genes orchestrating keratinocyte differentiation, including ID2, were significantly upregulated in response to the sensitizers in BDKs but not those in THP-1. The use of the ID2 gene to discriminate between sensitizers and irritants might be effective as a novel marker for application during in vitro sensitization with BDKs.


Assuntos
Diferenciação Celular , Folículo Piloso/citologia , Proteína 2 Inibidora de Diferenciação/metabolismo , Irritantes/toxicidade , Queratinócitos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Adulto , Apoptose/efeitos dos fármacos , Biomarcadores , Linhagem Celular Tumoral , Células Cultivadas , Dermatite de Contato/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Interleucinas/genética , Interleucinas/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos
2.
J Hum Genet ; 54(7): 403-8, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19557015

RESUMO

Basal cell nevus syndrome (BCNS or Gorlin syndrome, OMIM: 109400) is a rare autosomal dominant disorder with high penetrance. It is characterized by developmental anomalies and predisposition to tumors (for example, basal cell carcinoma (BCC) and medulloblastoma). PTCH1, the human homolog of the Drosophila patched gene, was identified as a gene responsible for BCNS. The PTCH1 protein is a Hedgehog (Hh) protein receptor and is pivotal for early development, stem cell maintenance and/or differentiation. We analyzed the six Japanese families with BCNS and identified six germline mutations in the PTCH1 gene. One family had a nonsense mutation (c.1196G>A), one had a 1-bp deletion (c.2029delA), two had 2-bp deletions (c.239_240delGA and c.1670_1671delCA) and one had a 58-bp duplication (c.1138_1195dup). They caused premature termination, resulting in the truncation of the PTCH1 protein. Analysis of a high-density single nucleotide polymorphism (SNP) mapping array showed a large approximately 1.2-Mb deletion, including the PTCH1 gene in one allele, in a family in which PTCH1 mutations were not identified at the sequence level. These data indicated that all the six families who were diagnosed with BCNS had mutations in the PTCH1 gene and that a single copy of a PTCH1 mutation causes BCNS.


Assuntos
Síndrome do Nevo Basocelular/genética , Mutação em Linhagem Germinativa/genética , Receptores de Superfície Celular/genética , Adulto , Povo Asiático/genética , Sequência de Bases , Criança , Pré-Escolar , Pontos de Quebra do Cromossomo , Feminino , Humanos , Japão , Masculino , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Patched , Receptor Patched-1 , Linhagem , Polimorfismo de Nucleotídeo Único/genética
3.
Int J Oncol ; 34(5): 1191-9, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19360332

RESUMO

Human keratinocyte strains derived from the bulge region of plucked human follicles were successfully established from all 43 donors (age 24-76) regardless of the age and gender. The total cell number, number of population doublings and population doubling time were similar among the strains. These bulge-derived keratinocytes, BDKs, expressed keratin family genes specific to basal cell layers of the epidermis. They also expressed CD34, one of the bulge stem cell marker genes. The growth behavior and positivity of CD34 indicate that BDKs contain stem cells. BDKs were cultured until confluency or treated with CaCl2 to induce differentiation. Morphology and expression of keratin family genes in BDKs before and after differentiation induction with CaCl2 were similar to those of epidermal keratinocytes obtained from skin biopsies (NHEKs). However, expression levels of keratin-10, a prickle cell layer marker, in CaCl2-treated BDKs were lower than those in CaCl2-treated NHEKs. Higher expression of integrin-alpha6, a basal cell layer marker, was also noted in BDKs than in NHEKs after differentiation induction. Expression of stem cell marker genes other than CD34, including CD200, Sox2 and NANOG, was about the same at confluency in both cells, but significantly higher in BDKs than NHEKs after differentiation. These results indicate that BDKs were more refractory to differentiation than NHEKs. We then examined Wnt signaling inhibitor genes, DKK-3 and WIF-1 that function as tumor suppressors. DKK-3 expression decreased in both BDKs and NHEKs after CaCl2-induced differentiation. Expression of WIF-1 decreased 50% in BDKs one day after CaCl2 treatment and remained low, but was induced 1.7 times in NHEKs one day after CaCl2 treatment and further induced thereafter (>2.5 times), suggesting that WIF-1 may be involved in maintaining the differentiation-refractory status of BDKs. Since cancer stem cells in the skin have been reported to be similar to bulge-derived stem cells, our BDK strains may be of use in studying characteristics of cancer stem cells of the epidermis.


Assuntos
Diferenciação Celular/fisiologia , Folículo Piloso/citologia , Queratinócitos/fisiologia , Adulto , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/fisiologia , Idoso , Biópsia , Cálcio/farmacologia , Técnicas de Cultura de Células/métodos , Proliferação de Células , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Perfilação da Expressão Gênica , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Pele/patologia , Adulto Jovem
4.
Reproduction ; 132(3): 455-64, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16940286

RESUMO

The zona pellucida, an extracellular matrix surrounding mammalian oocytes, is composed of three or four glycoproteins. It is well known that the zona pellucida plays several critical roles during fertilization, but there is little knowledge about its formation. The purpose of this study is to examine whether a pig zona pellucida glycoprotein 2 (pZP2) would assemble with mouse zona pellucida. A transgene construct was prepared by placing a minigene encoding pZP2 downstream from the promoter of mouse ZP2. The result showed that the transgenic protein was synthesized in growing oocytes but not incorporated into the zona pellucida. Furthermore, the pZP2 transgene did not rescue the phenotype in ZP2-knockout zona-deficient mice. These results indicate that pZP2 does not participate in mouse zona pellucida formation and the zona pellucida is constituted from its component proteins in a molecular species-specific manner between mice and pigs.


Assuntos
Proteínas do Ovo/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Zona Pelúcida/metabolismo , Animais , Northern Blotting/métodos , Proteínas do Ovo/análise , Proteínas do Ovo/genética , Feminino , Engenharia Genética , Imuno-Histoquímica/métodos , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Oogênese/fisiologia , Ovário/citologia , Ovário/fisiologia , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/genética , Proteínas Recombinantes/farmacologia , Suínos , Transgenes , Glicoproteínas da Zona Pelúcida
5.
Prenat Diagn ; 23(9): 743-6, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12975786

RESUMO

OBJECTIVES: Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of tissue nonspecific alkaline phosphatase (TNSALP) activity. This disorder is caused by various mutations in the TNSALP gene. We report here hypophosphatasia in two siblings, both of them severely affected by the perinatal (lethal) type. METHODS: We diagnosed the first infant by clinical and radiologic manifestations, and laboratory findings. Laboratory findings were characterized by deficiency of serum alkaline phosphatase. Both parents and the second infant were then analyzed by molecular techniques. RESULTS: The radiograph of the first infant showed severe hypomineralization of the skeleton. Molecular analysis of the second infant showed that this condition was caused by a homozygous single T nucleotide deletion at cDNA number 1559 (1559delT). Both parents were heterozygous carriers for this mutation, although they were not consanguineous. CONCLUSION: This mutation has been frequently found in Japanese hypophosphatasia patients, but this is the first observation of a homozygous deletion. This report shows that homozygosity for the 1559delT mutation of the TNSALP gene results in a severe lethal phenotype.


Assuntos
Fosfatase Alcalina/genética , Aconselhamento Genético , Hipofosfatasia/diagnóstico , Hipofosfatasia/genética , Complicações na Gravidez/diagnóstico , Diagnóstico Pré-Natal , Anormalidades Múltiplas/diagnóstico por imagem , Anormalidades Múltiplas/embriologia , Adulto , Fosfatase Alcalina/deficiência , Diagnóstico Diferencial , Evolução Fatal , Feminino , Humanos , Recém-Nascido , Masculino , Mutação/genética , Linhagem , Gravidez , Radiografia , Ultrassonografia Pré-Natal
6.
Prenat Diagn ; 24(1): 45-9, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14755409

RESUMO

Hypochondroplasia is an autosomal dominant skeletal dysplasia expressing postnatal onset of short stature with mild rhizomelic shortening of the limbs. This manifestation leads to restricted prenatal diagnosis of the disorder. We report here on a sporadic case of a hypochondroplastic baby, whose prenatal sonographic measurements were serially recorded from 19 weeks of gestation. Mild shortening of the limbs became manifest after 26 weeks of gestation. Biparietal diameter was within the normal range throughout gestation. Both parents were of average stature. A tentative diagnosis of a nonlethal short-limb skeletal dysplasia was made. At birth, the clinical manifestations of the neonate were not characteristic, but the radiographic features raised the possibility of hypochondroplasia. Molecular analyses revealed a C to G mutation at nucleotide 1659 of the fibroblast growth factor receptor 3 (FGFR3) gene, a common mutation in hypochondroplasia.


Assuntos
Osteocondrodisplasias/diagnóstico , Diagnóstico Pré-Natal , Proteínas Tirosina Quinases , Adulto , Diagnóstico Diferencial , Feminino , Testes Genéticos , Idade Gestacional , Humanos , Recém-Nascido , Masculino , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/embriologia , Osteocondrodisplasias/genética , Gravidez , Terceiro Trimestre da Gravidez , Radiografia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/genética
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