RESUMEN
Although protein recapture and catabolism is known as a key function of kidney proximal tubular cells (PTCs), to date, no single protease has been shown to be required. Asparagine endopeptidase (AEP) is an unusually specific endosomal and lysosomal cysteine protease, expressed at high levels in the PTCs of the mammalian kidney. We report that mice lacking AEP accumulate a discrete set of proteins in their PTC endosomes and lysosomes, which indicates a defect in the normal catabolism of proteins captured from the filtrate. Moreover, the mice develop progressive kidney pathology, including hyperplasia of PTCs, interstitial fibrosis, development of glomerular cysts, and renal pelvis dilation. By 6 mo of age, the glomerular filtration rate in AEP-null mice dropped by almost a factor of 2, and the mice developed proteinuria. We also show that EGF receptor levels are significantly higher in AEP-null PTCs, which likely explains the hyperplasia, and we show that chemical inhibition of AEP activity suppresses down-regulation of the EGF receptor in vitro. Thus, AEP is required for normal protein catabolism by PTCs, and its loss induces proliferative and other abnormalities in the murine kidney, at least in part through defective regulation of the EGF receptor.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Riñón/metabolismo , Riñón/patología , Animales , Cisteína Endopeptidasas/genética , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Receptores ErbB/genética , Receptores ErbB/metabolismo , Tasa de Filtración Glomerular/genética , Tasa de Filtración Glomerular/fisiología , Immunoblotting , Inmunohistoquímica , Focalización Isoeléctrica , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Lectinas/metabolismo , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Microscopía Fluorescente , Resistina/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Espectrometría de Masas en Tándem , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
To directly map the position of promoter DNA within the RNA polymerase II (Pol II) transcription preinitiation complex (PIC), FeBABE was tethered to specific sites within the HIS4 promoter and used to map exposed surfaces of Pol II and the general transcription factors in proximity to DNA. Our results distinguish between previously proposed models for PIC structure and demonstrate that downstream promoter DNA is positioned over the central cleft of Pol II, with DNA upstream of TATA extending toward the Pol II subunit Rpb3. Also mapped were segments of TFIIB, TFIIE, TFIIF and TFIIH in proximity to promoter DNA. DNA downstream of the transcription bubble maps to a path between the two helicase subdomains of the TFIIH subunit Rad25 (also called XPB). Together, our results show how the general factors and Pol II converge on promoter DNA within the PIC.
Asunto(s)
ADN de Hongos/genética , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Polimerasa II/química , ADN Polimerasa II/genética , ADN de Hongos/química , Radical Hidroxilo , Modelos Moleculares , Conformación de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/genéticaRESUMEN
The innate and adaptive immune system utilise endocytic protease activity to promote functional immune responses. Cysteine and aspartic proteases (cathepsins) constitute a subset of endocytic proteases, the immune function of which has been described extensively. Although historically these studies have focused on their role in processes such as antigen presentation and zymogen processing within the endocytic compartment, recent discoveries have demonstrated a critical role for these proteases in other intracellular compartments, and within the extracellular milieu. It has also become clear that their pattern of expression and substrate specificities are more diverse than was first envisaged. Here, we discuss recent advances addressing the role of lysosomal proteases in various aspects of the immune response. We pay attention to reports demonstrating cathepsin activity outside of its canonical endosome/lysosome microenvironment.
Asunto(s)
Fenómenos del Sistema Inmunológico/fisiología , Sistema Inmunológico/enzimología , Lisosomas/enzimología , Péptido Hidrolasas/inmunología , Animales , HumanosRESUMEN
One of the great mysteries of the nucleolus surrounds its disappearance during mitosis and subsequent reassembly at late mitosis. Here, the relative dynamics of nucleolar disassembly and reformation were dissected using quantitative 4D microscopy with fluorescent protein-tagged proteins in human stable cell lines. The data provide a novel insight into the fates of the three distinct nucleolar subcompartments and their associated protein machineries in a single dividing cell. Before the onset of nuclear envelope (NE) breakdown, nucleolar disassembly started with the loss of RNA polymerase I subunits from the fibrillar centers. Dissociation of proteins from the other subcompartments occurred with faster kinetics but commenced later, coincident with the process of NE breakdown. The reformation pathway also follows a reproducible and defined temporal sequence but the order of reassembly is shown not to be dictated by the order in which individual nucleolar components reaccumulate within the nucleus after mitosis.
Asunto(s)
Nucléolo Celular/metabolismo , Cinética , Mitosis , Colorantes Fluorescentes , Células HeLa , Humanos , Imagenología Tridimensional , Inmunohistoquímica , Modelos Biológicos , Membrana Nuclear/metabolismo , Región Organizadora del Nucléolo/metabolismo , Pruebas de Precipitina , ARN Polimerasa I/metabolismoRESUMEN
Violence in the health- and social-care workplace remains unexplored, with a knowledge base which is often ambiguous or incomplete. However, the issue has attracted increasing attention over the last two decades as indicated by an expanding range of policy initiatives and growing research literature. Additionally, a proliferation of training programmes for healthcare staff has appeared. This paper will explore the reasons for an observed tendency for interventions to focus on training as the primary response, which suggests a misperception of the problem of violence as principally a function of interpersonal conflict. It argues that a radical cultural shift is needed, which recognises the organizational and societal roots of violence, and that adopts and applies the principles of a public health approach.
Asunto(s)
Exposición Profesional/prevención & control , Salud Laboral , Violencia/prevención & control , Servicios de Salud Comunitaria/métodos , Relaciones Comunidad-Institución , Humanos , Cultura Organizacional , Práctica de Salud Pública , Desarrollo de Personal/métodos , Reino UnidoRESUMEN
Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIα in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIα is a component of the initiation-competent RNA polymerase Iß complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIα functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasa I/genética , Transcripción Genética , ADN/metabolismo , Activación Enzimática , Hidrólisis , Regiones Promotoras GenéticasRESUMEN
BACKGROUND: Research consistently suggests nurses working in mental health settings are more likely to be assaulted than nurses in other settings. AIMS: Belated recognition of the issue in terms of social policy (Elston et al. 2006) has been accompanied by an as yet unexamined contest between conflicting 'frames' of the problem, which this paper seeks to make transparent. METHOD: Frame analysis. RESULTS: Two distinct 'master' frames are discussed: the 'individualizing' and the 'co-creationist'. CONCLUSIONS: The influence of these frames has influenced the nature of responses to the problem but the recent dominance of the individualizing frame is being challenged by the emergence, or perhaps re-emergence, of co-creationism.