RESUMO
Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130â¯kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83â¯kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples.
Assuntos
Acanthamoeba/imunologia , Anticorpos Monoclonais/biossíntese , Anticorpos Antiprotozoários/biossíntese , Lectina de Ligação a Manose/imunologia , Proteínas de Protozoários/imunologia , Acanthamoeba/química , Ceratite por Acanthamoeba/parasitologia , Animais , Antígenos de Protozoários/imunologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Hibridomas , Soros Imunes/sangue , Isotipos de Imunoglobulinas , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The peroxisome proliferator-activated receptor γ (PPARγ) is a central regulator of adipogenesis and modulates glucose and lipid metabolism. In this study, herpesvirus-associated ubiquitin-specific protease (HAUSP) was isolated as a binding partner of PPARγ. Both endogenous and exogenous PPARγ associated with HAUSP in co-immunoprecipitation analysis. HAUSP, but not the catalytically inactive HAUSP C223S mutant, increased the stability of both endogenous and exogenous PPARγ through its deubiquitinating activity. Site-directed mutagenesis experiments showed that the Lys(462) residue of PPARγ is critical for ubiquitination. HBX 41,108, a specific inhibitor of HAUSP, abolished the increase in PPARγ stability induced by HAUSP. In addition, knockdown of endogenous HAUSP using siRNA decreased PPARγ protein levels. HAUSP enhanced the transcriptional activity of both exogenous and endogenous PPARγ in luciferase activity assays. Quantitative RT-PCR analysis showed that HAUSP increased the transcript levels of PPARγ target genes in HepG2 cells, resulting in the enhanced uptake of glucose and fatty acids, and vice versa, upon siRNA knockdown of HAUSP. In vivo analysis using adenoviruses confirmed that HAUSP, but not the HAUSP C223S mutant, decreased blood glucose and triglyceride levels, which are associated with the increased expression of endogenous PPARγ and lipid accumulation in the liver. Our results demonstrate that the stability and activity of PPARγ are modulated by the deubiquitinating activity of HAUSP, which may be a target for the development of anti-diabetic drugs.
Assuntos
PPAR gama/metabolismo , Transcrição Gênica/fisiologia , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Adenoviridae , Substituição de Aminoácidos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Glicemia/genética , Glicemia/metabolismo , Células COS , Chlorocebus aethiops , Ácidos Graxos/sangue , Ácidos Graxos/genética , Técnicas de Silenciamento de Genes , Células HeLa , Células Hep G2 , Humanos , Indenos/farmacologia , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , PPAR gama/genética , Estabilidade Proteica , Pirazinas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transdução Genética , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/efeitos dos fármacosRESUMO
Stem cell therapy based on human mesenchymal stem cells (hMSCs) has shown great promise for various disease treatments. However, traditional stem cell-mediated therapy is limited due to their multipotent differentiation ability (uncontrolled spontaneous differentiation) and the difficulty in monitoring cells after implantation in vivo. Here, we report a new multi-functional stem cell nanotracer (M-NT) for directing controlled differentiation through gene delivery, as well as tracking stem cells with dual-modal imaging (optical and CT imaging). The M-NT was prepared through a facile surface modification process of â¼100 nm-sized gold nanoparticles with catechol-functionalized branched polyethylenimine (C-bPEI). The C-bPEI-functionalized M-NT exhibited greatly enhanced long-term colloidal stability in aqueous solution and a capability to complex with plasmid DNA (pDNA; i.e., pEGFP) through electrostatic interaction for gene delivery and transfection to control differentiation. M-NT/pEGFP complexes showed an enhanced transfection efficiency into hMSCs with low cytotoxicity compared with branched polyethylenimine/pDNA complexes. Accordingly, successful in vitro chondrogenic differentiation was achieved in hMSCs treated with M-NT/pSOX9 complexes. Finally, hMSCs transfected with M-NT/pEGFP complexes were transplanted into Balb/c nude mice and successfully visualized through dual-modal optical fluorescence and computed tomography (CT) imaging. We believe that this approach could represent a promising platform for genetic material-mediated direction of differentiation and cell tracking in stem cell therapy.