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1.
Nat Struct Mol Biol ; 31(7): 1018-1022, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38347147

RESUMO

ARID1B is a SWI/SNF subunit frequently mutated in human Coffin-Siris syndrome (CSS) and it is necessary for proliferation of ARID1A mutant cancers. While most CSS ARID1B aberrations introduce frameshifts or stop codons, the functional consequence of missense mutations found in ARID1B is unclear. We here perform saturated mutagenesis screens on ARID1B and demonstrate that protein destabilization is the main mechanism associated with pathogenic missense mutations in patients with Coffin-Siris Syndrome.


Assuntos
Proteínas de Ligação a DNA , Deformidades Congênitas da Mão , Deficiência Intelectual , Micrognatismo , Mutação de Sentido Incorreto , Estabilidade Proteica , Fatores de Transcrição , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Micrognatismo/genética , Deformidades Congênitas da Mão/genética , Deficiência Intelectual/genética , Anormalidades Múltiplas/genética , Face/anormalidades , Pescoço/anormalidades
2.
PLoS One ; 12(8): e0183679, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28837623

RESUMO

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the 'classical' (M1) and 'alternative' (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.


Assuntos
Polaridade Celular/genética , Macrófagos/citologia , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Humanos , Modelos Biológicos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
J Pharm Sci ; 102(7): 2128-35, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23695958

RESUMO

Protein aggregation, which takes place both in vivo and in vitro, is an important degradative pathway for all proteins. Protein aggregates have distinct physicochemical and biological properties that are important to study and characterize from the perspective of both fundamental and applied sciences. The size of protein aggregates varies across a huge range, spanning several orders of magnitude. Currently, protein aggregates larger than hundreds of nanometers in diameter are impossible to physically fractionate. Here, we present a new method to fractionate microscopic proteinaceous particles using preparative fluorescence-activated cell sorting technology.


Assuntos
Citometria de Fluxo/métodos , Imunoglobulina G/química , Humanos , Imunoglobulina G/isolamento & purificação , Luz , Tamanho da Partícula , Espalhamento de Radiação
4.
Methods Mol Biol ; 816: 55-66, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22130922

RESUMO

Osteocytes are the terminally differentiated cells of the osteoblastic lineage embedded within the mineralized bone matrix. T: hey have been identified as key players in mechanotransduction and in mineral and phosphate homeostasis. In addition, they appear to have a role in mediating bone formation, since they secrete the bone formation inhibitor sclerostin. In contrast to osteoblasts and osteoclasts, which reside on the bone surface, it has been difficult to isolate and analyze cellular and molecular properties of osteocytes due to their specific location inside the "hard" mineralized bone compartment. This chapter describes a method to isolate osteocytes from newborn mouse calvaria and adult mouse long bone, followed by immediate total RNA extraction allowing to selectively study osteocytic versus osteoblastic gene expression by quantitative real-time polymerase chain reaction (qPCR). The osteocyte-enriched cell fraction isolated by this method can further be purified by FACS and selectively expresses osteocytic marker genes, such as Dmp1 and Sost.


Assuntos
Fracionamento Celular/métodos , Separação Celular/métodos , Osteócitos/citologia , Osteócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Osso e Ossos/citologia , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Citometria de Fluxo/métodos , Expressão Gênica , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteoblastos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Crânio/citologia
5.
J Immunol ; 186(4): 2602-12, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21217016

RESUMO

The mechanism whereby IL-17 drives rheumatoid arthritis remains incompletely understood. We demonstrate that anti-IL-17 therapy in collagen-induced arthritis ameliorates bone damage by reducing the number of osteoclasts in joints. We found equal numbers of CD4(+) Th17 and IL-17 producing γδ T cells in the joints of arthritic mice, and in vitro, both populations similarly induced osteoclastogenesis. However, individual depletion and adoptive transfer studies revealed that in vivo, Th17 cells dominated with regard to bone destruction. Unlike γδ T cells, Th17 cells were found in apposition to tartrate-resistant acid phosphatase positive osteoclasts in subchondral areas of inflamed joints, a pattern reproduced in patient biopsies. This localization was caused by Ag-specific retention, because OVA-primed Th17 cells showed a γδ T cell-like diffuse distribution. Because IL-23, as produced by osteoclasts, enhanced T cell-mediated osteoclastogenesis, we propose that Ag-specific juxtaposition is key to foster the molecular cross talk of Th17 cells and osteoclasts, thus driving arthritic bone destruction.


Assuntos
Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Interleucina-17/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Células Th17/imunologia , Células Th17/patologia , Adulto , Idoso , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Cartilagem Articular/imunologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Comunicação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Técnicas de Cocultura , Colágeno Tipo II/administração & dosagem , Colágeno Tipo II/imunologia , Feminino , Humanos , Interleucina-17/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Osteoclastos/imunologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Células Th17/metabolismo
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