RESUMO
Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Queratinas/imunologia , Hepatopatias/imunologia , Animais , Encéfalo/citologia , Química Encefálica , Linhagem Celular , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cricetinae , Inibidores Enzimáticos/farmacologia , Epitopos/genética , Epitopos/imunologia , Células HT29 , Humanos , Immunoblotting , Imuno-Histoquímica , Queratina-18 , Queratina-8 , Queratinas/análise , Queratinas/genética , Rim/química , Rim/citologia , Fígado/química , Fígado/citologia , Hepatopatias/genética , Hepatopatias/metabolismo , Camundongos , Mutação/genética , Ácido Okadáico/farmacologia , Fosforilação/efeitos dos fármacos , Coelhos , Serina/genética , Serina/imunologia , Serina/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transfecção , Vacinação , Vanadatos/farmacologiaRESUMO
Apoptosis is a critical process in normal mammary gland development and the rapid clearance of apoptotic cells prevents tissue injury associated with the release of intracellular antigens from dying cells. Milk fat globule-EGF-factor 8 (Mfge8) is a milk glycoprotein that is abundantly expressed in the mammary gland epithelium and has been shown to facilitate the clearance of apoptotic lymphocytes by splenic macrophages. We report that mice with disruption of Mfge8 had normal mammary gland development until involution. However, abnormal mammary gland remodeling was observed postlactation in Mfge8 mutant mice. During early involution, Mfge8 mutant mice had increased numbers of apoptotic cells within the mammary gland associated with a delay in alveolar collapse and fat cell repopulation. As involution progressed, Mfge8 mutants developed inflammation as assessed by CD45 and CD11b staining of mammary gland tissue sections. With additional pregnancies, Mfge8 mutant mice developed progressive dilatation of the mammary gland ductal network. These data demonstrate that Mfge8 regulates the clearance of apoptotic epithelial cells during mammary gland involution and that the absence of Mfge8 leads to inflammation and abnormal mammary gland remodeling.
Assuntos
Antígenos de Superfície/genética , Glândulas Mamárias Animais/fisiologia , Proteínas do Leite/genética , Animais , Apoptose , Técnicas de Cultura de Células , Células Epiteliais/fisiologia , Feminino , Heterozigoto , Homozigoto , Humanos , Inflamação , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Floor of the mouth squamous cell carcinomas exhibit many characteristics that suggest they represent a distinct biological subset within head and neck tumors. The features of preinvasive lateral intraepithelial spread, high rate of conversion of intraepithelial neoplasia to invasive carcinoma, and high incidence of occult metastases, suggest the importance of motility-associated proteins in the pathogenesis of these lesions. Two such proteins, tenascin and beta 6 integrin, are generally overexpressed in squamous carcinomas, and may play a central role in the invasive process of floor of the mouth lesions. The purpose of this study was to evaluate in situ and invasive squamous cell carcinomas from the floor of the mouth for the expression of tenascin and beta 6 integrin. Twenty lesions each of floor of the mouth in situ carcinomas and squamous cell carcinomas, and 10 normal controls were stained for tenascin and beta 6 using a standard immunohistochemical protocol for formalin-fixed specimens. Sections were assessed for staining intensity, pattern, and co-localization. Tenascin was highly expressed at the keratinocyte-connective tissue interface of both in situ and invasive carcinomas. beta 6 was expressed in basal keratinocytes of all in situ and invasive lesions, but was not evident in any of the control epithelia. There was no significant difference in staining of in situ and invasive carcinomas, but there was a significant difference in staining between these lesions and controls. Staining was colocalized in serial sections, supporting a receptor-ligand relationship. Both tenascin and beta 6 were weakly expressed in dysplastic areas adjacent to carcinomas suggesting that changes in the expression of these proteins occurs prior to the invasive phenotype. We conclude that tenascin and beta 6 are overexpressed in in situ and invasive floor of the mouth carcinomas, but that transgression of the basement membrane by neoplastic epithelial cells requires additional changes to the keratinocyte molecular profile.
