RESUMEN
Scurfy (sf), is an X-linked recessive lethal mutation that occurs spontaneously in the C3H mouse. The disease is characterized by lymphoid and hematopoietic dysfunction. Affected males are of small stature and exhibit scaliness and crusting of the eyelids, ears, tail, and feet, marked splenomegaly, moderate hepatomegaly, enlarged lymph nodes, and atrophy of the thymus. The average lifespan of the affected hemizygous males (sf/y) is 24 +/- 0.7 days. Total cellular proteins were extracted from pooled samples of thymus and spleen obtained from combined litters of mice. Tissue-specific protein profiles characteristic of either sf mutant or normal mice were analyzed by two dimensional polyacrylamide gel electrophoresis (2DPAGE) at different stages of the phenotypic expression of the sf mutation, to identify changes in protein patterns that might be associated with the progression of the disease. The resultant gels were silver stained, digitized, and analyzed, by image analysis utilizing a pipelined image processor connected to a host computer. At 14 +/- 1 days of age, protein patterns from sf mutant and normal mice control organs showed considerable homogeneity, although there were proteins identified unique to the sf mutant and to the normal controls. At 20 +/- 1 days of age, the pattern differences between the sf mutant and normal control increased markedly. Differences were expressed as the percent of proteins that were unique to either the sf mutant or the normal control from the total number of each type. The percent of proteins that increased or decreased in the three organs utilized in this study ranged between 21%-39% at 14 days and were between 25%-54% at 20 days. Differences in protein expression between the normal and sf mutant as the disorder progressed for each of the three tissues examined. In addition, thymus protein profiles from 9 day old littermates that were phenotypically normal but genotypically unknown were evaluated to determine if marker proteins could be identified for the sf mutation. Limited protein changes were noted at relative molecular weights of 66, 60, 54, 39, 37, 33, 25, 23, 27, and 11 kDa. These data suggest that the sf mutation follows a trackable pattern of protein expression and repression different than the normal control C3H mouse. Several potential marker proteins associated with the sf mutation were identified in 9 day thymus prior to the phenotypic expression of the disease. These putative biomarkers may be useful for characterizing the sf mutation and the mutant may act a possible model the Wiskott-Aldrich syndrome (WAS).
Asunto(s)
Anomalías Múltiples/genética , Electroforesis en Gel Bidimensional , Trastornos Linfoproliferativos/genética , Ratones Mutantes/genética , Proteínas/análisis , Vísceras/química , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Factores de Edad , Animales , Biomarcadores , Densitometría , Modelos Animales de Enfermedad , Femenino , Genes Letales , Genes Recesivos , Heterocigoto , Procesamiento de Imagen Asistido por Computador , Focalización Isoeléctrica , Trastornos Linfoproliferativos/metabolismo , Trastornos Linfoproliferativos/patología , Masculino , Ratones , Ratones Endogámicos C3H/genética , Tinción con Nitrato de Plata , Timo/química , Timo/patología , Síndrome de Wiskott-Aldrich , Cromosoma XRESUMEN
Total cellular proteins from mouse C3H10T1/2 fibroblasts were compared by two-dimensional (2-D) gel electrophoresis after radiolabeling with [35S]methionine (35S-Met) or 14C-amino acids (14C-AA). 35S-Met labeling of protein was three to four times greater than 14C-AA incorporation over a 24 h period. Automated comparative analysis of replicate fluorographs after 6, 12, and 24 h of labeling showed considerable homology between radiolabeling methods. More than 88% percent of 35S-Met and 14C-AA-labeled proteins were common at each time point. However, the total number of 35S-Met-labeled proteins dropped from 6 to 24 h while the number of 14C-AA-labeled proteins increased. Additionally, twenty-one proteins were uniquely labeled by 14C-AA that were not detectable by 35S-Met over the labeling period. Densitometric analysis showed that several 35S-Met and 14C-AA-labeled proteins exhibited time-related differences in radiolabel incorporation while most proteins remained relatively constant. Protein patterns of silver-stained gels from 6 to 24 h were highly registered and showed few qualitative differences. Proteins detected in radiolabeled gels were generally, but not always, found in silver-stained gels. Thus, 35S-Met appears better suited for short-term radiolabeling of cellular protein while more comprehensive labeling of protein occurs with 14C-AA during prolonged incubation of cell cultures under present experimental conditions.