RESUMEN
Antimicrobial peptides are an important component of the innate response in many species. Here we describe the isolation of the gene Dermcidin, which encodes an antimicrobial peptide that has a broad spectrum of activity and no homology to other known antimicrobial peptides. This protein was specifically and constitutively expressed in the sweat glands, secreted into the sweat and transported to the epidermal surface. In sweat, a proteolytically processed 47-amino acid peptide was generated that showed antimicrobial activity in response to a variety of pathogenic microorganisms. The activity of the peptide was maintained over a broad pH range and in high salt concentrations that resembled the conditions in human sweat. This indicated that sweat plays a role in the regulation of human skin flora through the presence of an antimicrobial peptide. This peptide may help limit infection by potential pathogens in the first few hours following bacterial colonization.
Asunto(s)
Antibacterianos/metabolismo , Péptidos , Glándulas Sudoríparas/metabolismo , Secuencia de Aminoácidos , Antibacterianos/farmacología , Mapeo Cromosómico , Humanos , Inmunohistoquímica , Hibridación in Situ , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacología , ARN Mensajero/biosíntesis , Sudor/química , Distribución TisularRESUMEN
A polymerase chain reaction (PCR)-based subtractive hybridization technique was used to identify transformation-related genes in malignant melanoma. Melanoma biopsies were compared with tissues of benign melanocytic naevi and 549 gene fragments were screened using arrayed filters. Thirty-eight clones were confirmed to be differentially expressed representing 30 different genes (18 melanoma-specific and 12 naevus-specific genes). To further confirm differential gene expression, Northern blot analyses with six of the 30 genes as probes were performed. All six were differentially expressed in benign and malignant melanocytic lesions, specifically dbpB/YB-1, 67-kDa laminin receptor, CAGH-3, 71-kDa heat shock protein and two unknown genes. The expression levels of these genes were then analysed in 50 different tissues to determine their overall expression profile. In conclusion, the technique of PCR-based subtractive hybridization in combination with arrayed filters allows detection of differences in gene expression even in tissues from which high-quality RNA is hard to isolate. The genes identified in this study are of interest because of their potential role in the pathogenesis of malignant melanoma.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Neoplasias Cutáneas/genética , Northern Blotting , Humanos , Hibridación in Situ/métodos , Melanoma/fisiopatología , Reacción en Cadena de la Polimerasa/métodos , ARN/genética , Neoplasias Cutáneas/fisiopatologíaRESUMEN
High-quality RNA is essential when analyzing expression patterns of tissues by the differential display technique. However, the isolation of intact RNA can be very difficult when tissues are used that contain many RNAses or that are hard to homogenize (e.g. skin samples). We describe an improved protocol for the extraction of high-quality RNA of snap-frozen biopsies from skin tissues that works in a reproducible and reliable manner. In addition, we developed a simplified non-radioactive differential display technique using RNA from small amounts of skin samples.
Asunto(s)
Colorimetría/métodos , ARN Neoplásico/aislamiento & purificación , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Neoplasias Cutáneas/química , Piel/química , Secciones por Congelación/métodos , Humanos , Melanoma/química , Nevo/química , ARN/análisis , ARN Neoplásico/análisisRESUMEN
BACKGROUND: The histogenesis of malignant rhabdoid tumor (RT) remains a matter of controversy. From published reports it appears that some extrarenal RT (ERRT) exhibit neural or neuroectodermal features. Systematic investigation of renal RT (RRT) for neural differentiation has not yet been published. In this study, two RRT and two ERRT were investigated by light and electron microscopy, immunocytochemistry, and cytogenetic analysis. RESULTS: All four cases exhibited similar morphology and a largely consistent immunohistochemical staining profile. Both tumor types showed immunoreactivity for various cytokeratins, epithelial membrane antigen (EMA), vimentin, neurofilaments, S100 protein, neuron specific enolase, Leu7, CD34, p53, MB2, MIC2, VS38c, laminin and fibronectin. Reactivity was usually confined to a small proportion of the tumor cells, especially in the case of antibodies against neurofilaments, NSE, S100 proteins Leu7, CD34 and p53. Only a few of the antibodies (anti-vimentin, MB2, MIC2, anti-EMA, and VS38c) stained the majority of the tumor cells. Chromosome analysis revealed a discrete aberration in 11p (most probably a paracentric inversion in 11p15) in the one RRT investigated. One ERRT had a normal karyotype, but the other one exhibited various structural abnormalities on chromosomes 18 and 22. CONCLUSIONS: The immunohistochemical investigations revealed many similarities between RRT and ERRT, and all four tumors investigated exhibited neuroectodermal differentiation. As far as histogenetic classification is concerned, our findings point in the direction of the group of tumors that includes Ewing's sarcoma and primitive neuroectodermal tumors.
