RESUMEN
Renal cell carcinoma (RCC) are frequently chemo- and radiation resistant. Thus, there is a need for identifying biological features of these cells that could serve as alternative therapeutic targets. We performed suppression subtractive hybridization (SSH) on patient-matched normal renal and RCC tissue to identify variably regulated genes. 11 genes were strongly up-regulated or selectively expressed in more than one RCC tissue or cell line. Screening of filters containing cancer-related cDNAs confirmed overexpression of 3 of these genes and 3 additional genes were identified. These 14 differentially expressed genes, only 6 of which have previously been associated with RCC, are related to tumour growth/survival (EGFR, cyclin D1, insulin-like growth factor-binding protein-1 and a MLRQ sub-unit homologue of the NADH:ubiquinone oxidoreductase complex), angiogenesis (vascular endothelial growth factor, endothelial PAS domain protein-1, ceruloplasmin, angiopoietin-related protein 2) and cell adhesion/motility (protocadherin 2, cadherin 6, autotaxin, vimentin, lysyl oxidase and semaphorin G). Since some of these genes were overexpressed in 80-90% of RCC tissues, it is important to evaluate their suitability as therapeutic targets.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Renales/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Northern Blotting , Adhesión Celular , Supervivencia Celular , Cartilla de ADN , ADN de Neoplasias/genética , Humanos , Neovascularización Patológica , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, alpha1-syntrophin, and the sarcoglycan complex. The precise role of alpha-dystrobrevin in skeletal muscle has not yet been determined. To study alpha-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and alpha-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Desmina/metabolismo , Proteínas Asociadas a la Distrofina , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas del Citoesqueleto/genética , ADN Complementario , Exones , Humanos , Proteínas de Filamentos Intermediarios/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Pruebas de PrecipitinaRESUMEN
Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.
Asunto(s)
Proteínas Contráctiles/metabolismo , Proteínas del Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Músculo Esquelético/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas Contráctiles/biosíntesis , Proteínas Contráctiles/genética , Proteínas del Citoesqueleto/genética , Distroglicanos , Filaminas , Humanos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos mdx , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Distrofias Musculares/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Conejos , Saccharomyces cerevisiae , Sarcoglicanos , Homología de Secuencia de AminoácidoRESUMEN
The direction of electron flow through nitrogenase is generally believed to be from the Fe protein to the P-clusters to the FeMo cofactor and then to substrate. In order to examine oxidation states of the P-clusters that might be involved in this pathway, we have constructed a form of the MoFe protein that contains a species called the MoFe cluster (Gavini, N., Ma, L., Watt, G., and Burgess, B.K. (1995) Biochemistry 33, 11842-11849) in place of FeMo cofactor. This MoFe cluster-containing protein was purified, and the presence of the cluster was confirmed by reisolation of the MoFe cluster followed by EPR spectroscopy. The protein does not reduce protons or acetylene, however, upon the addition of the Fe protein and MgATP, MgATP hydrolysis occurs at a rate 28% of the wild-type protein. As isolated in the presence of excess dithionite the MoFe cluster-containing protein is EPR silent. Upon addition of the Fe protein and MgATP a g = 1.94 EPR signal develops that integrates to about 1 spin per P-cluster. This signal only develops when both the Fe protein and MgATP are added and it arises from the P-clusters.
Asunto(s)
Molibdoferredoxina/química , Nitrogenasa/química , Azotobacter vinelandii/enzimología , Cromatografía DEAE-Celulosa , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Molibdoferredoxina/aislamiento & purificación , Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Especificidad por SustratoRESUMEN
We describe a direct bone marrow chromosome technique that was developed especially for use in studies of acute lymphoblastic leukemia (ALL). The features responsible for technical improvements include: the use of RPMI 1640 medium, supplemented with 30% fetal calf serum, to support cellular activity during both specimen transport and Colcemid treatment; the processing of only 0.1 ml of sedimented cells or less per centrifuge tube; the exposure of cells to Colcemid for a maximum of 25 min; control of the total time of exposure to hypotonic solution; the use of a steel wire as a stirring rod (fashioned to fit the centrifuge tube) for mixing cells; slide preparation by a specific edging-flaming technique; the natural aging of the slides to achieve optimal drying; and the use of a modified G-banding procedure that employs Wright's stain. This technique has been used in more than 350 cases of ALL and has consistently provided analyzable banded chromosomes, even in hyperdiploid cases with up to 91 chromosomes. It makes the previously recognized morphological difference between metaphases of residual normal cells and those from the leukemic clone less apparent. The edging-flaming technique of slide preparation is the most important component and is especially appropriate for spreading large numbers of chromosomes in ALL.