RESUMEN
The cornified envelope, a structure unique to keratinocytes, is a hallmark of their terminal differentiation and plays an important role in epidermal barrier function. Cornified envelope is formed through the action of a membrane-associated transglutaminase, which covalently cross-links protein precursors into a highly insoluble network at the inner leaflet of the plasma membrane in granular keratinocytes and stratum corneum. Initial studies, using dansylcadaverine for enzyme-directed labeling of acyl-acceptor transglutaminase substrates in mouse epidermal homogenates identified a prominent 60-kDa substrate. Specific antibodies raised to this protein stained the cytoplasm of suprabasal cells of stratified squamous epithelia, whereas simple epithelia and nonepithelial tissues showed no staining. Immunoscreening of a cDNA expression library from adult mouse skin identified 18 positive clones. DNA sequencing of the largest clone (which hybridized to a keratinocyte-specific transcript of 2.0 kb) showed greater than 99.5% homology with mouse keratin 10. Immunoelectron microscopy using anti-S60 and another antibody to keratin 10 showed specific binding to cornified envelope associated filamentous structures. Proteolytic fragments of purified cornified envelope from mouse epidermis showed reactivity to anti-S60. These data show that mouse keratin 10 is tightly bound to cornified envelope and may be a cross-linked substrate. The tight binding of keratin filaments and CE suggests a mechanism by which they might interact to enhance the structural integrity of the stratum corneum.
Asunto(s)
Filamentos Intermedios/metabolismo , Queratinocitos/química , Queratinas/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Filamentos Intermedios/química , Ratones , Unión Proteica , Especificidad por Sustrato , Transglutaminasas/metabolismoRESUMEN
A side by side comparison of the Cell Dyn 3000 SL and Coulter STKS using identical samples was performed over a four month period in the Clinical Hematology Laboratory at the University of Cincinnati Medical Center. A total of 444 samples comprised of 114 sophomore medical students and 330 randomly selected clinical patients were compared in 20 hemogram parameters, and the ability of each instrument to generate differential suspect flags was analyzed. Correlation coefficients for leukocytes, erythrocytes, hemoglobin, hematocrit, and platelets were 0.99. Correlation coefficients for the Cell Dyn and STKS compared with a 600 cell manual differential were 0.98 and 0.89 for neutrophils, 0.96 and 0.87 for lymphocytes, and 0.72 and 0.48 for monocytes, respectively. Both instruments demonstrated high precision and accuracy by internal and external quality control standards. Each analyzer exhibited strength as a screening instrument for abnormal cell populations. The Coulter STKS had overall sensitivity of 75%, specificity of 94%, 25% false negatives, and 6% false positives. Sensitivity, specificity, false negative and positive rates for the Cell Dyn 3000 SL were 68%, 92%, 32%, and 8%, respectively. Based upon this extensive side by side comparison using an identical sample population, it has been concluded that although statistically significant systematic bias (p < 0.05) exists between the two instruments, both analyzers can adequately support the needs of the clinical hematology laboratory.