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1.
Nat Immunol ; 16(4): 354-65, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25729923

RESUMO

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.


Assuntos
Subunidade alfa de Receptor de Interleucina-18/imunologia , Interleucina-1/imunologia , Leucócitos Mononucleares/imunologia , Receptores de Interleucina-1/imunologia , Transdução de Sinais/imunologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-1/genética , Subunidade alfa de Receptor de Interleucina-18/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-18/genética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/imunologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/imunologia , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , c-Mer Tirosina Quinase
2.
Immunity ; 47(3): 498-509.e6, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28916264

RESUMO

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.


Assuntos
Imunidade Inata , Proteínas de Membrana/metabolismo , Transporte de RNA , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/metabolismo , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/imunologia , Linhagem Celular , Citoplasma , Proteína DEAD-box 58/metabolismo , Modelos Animais de Doenças , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/imunologia , Endossomos/metabolismo , Feminino , Expressão Gênica , Técnicas de Inativação de Genes , Herpes Simples/genética , Herpes Simples/imunologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Lisossomos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas de Transporte de Nucleotídeos , Ligação Proteica , Transporte Proteico , RNA Viral/genética , RNA Viral/metabolismo , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo
3.
J Immunol ; 205(2): 521-532, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32532837

RESUMO

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of ß2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Citoesqueleto/metabolismo , Integrina alfa3/metabolismo , Selectina L/metabolismo , Neutrófilos/fisiologia , Tetraspanina 25/metabolismo , Animais , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Migração Transendotelial e Transepitelial
4.
J Immunol ; 202(12): 3483-3492, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31061008

RESUMO

dsRNA is a common by-product of viral replication and acts as a potent trigger of antiviral immunity. SIDT1 and SIDT2 are closely related members of the SID-1 transmembrane family. SIDT2 functions as a dsRNA transporter and is required to traffic internalized dsRNA from endocytic compartments into the cytosol for innate immune activation, but the role of SIDT1 in dsRNA transport and in the innate immune response to viral infection is unclear. In this study, we show that Sidt1 expression is upregulated in response to dsRNA and type I IFN exposure and that SIDT1 interacts with SIDT2. Moreover, similar to SIDT2, SIDT1 localizes to the endolysosomal compartment, interacts with the long dsRNA analog poly(I:C), and, when overexpressed, enhances endosomal escape of poly(I:C) in vitro. To elucidate the role of SIDT1 in vivo, we generated SIDT1-deficient mice. Similar to Sidt2-/- mice, SIDT1-deficient mice produced significantly less type I IFN following infection with HSV type 1. In contrast to Sidt2-/- mice, however, SIDT1-deficient animals showed no impairment in survival postinfection with either HSV type 1 or encephalomyocarditis virus. Consistent with this, we observed that, unlike SIDT2, tissue expression of SIDT1 was relatively restricted, suggesting that, whereas SIDT1 can transport extracellular dsRNA into the cytoplasm following endocytosis in vitro, the transport activity of SIDT2 is likely to be functionally dominant in vivo.


Assuntos
Infecções por Cardiovirus/imunologia , Citoplasma/metabolismo , Vírus da Encefalomiocardite/fisiologia , Endossomos/metabolismo , Herpes Simples/imunologia , Herpesvirus Humano 1/fisiologia , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Animais , Células Cultivadas , DNA/imunologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Transporte de Nucleotídeos/genética , Poli I-C/imunologia , Transporte de RNA/genética
5.
PLoS Pathog ; 14(3): e1006945, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29601598

RESUMO

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity.


Assuntos
Apoptose , Membrana Celular/metabolismo , Gonorreia/patologia , Macrófagos/patologia , Mitocôndrias/patologia , Neisseria gonorrhoeae/patogenicidade , Porinas/metabolismo , Animais , Gonorreia/microbiologia , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Porinas/genética
6.
J Cell Sci ; 128(15): 2795-804, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26101352

RESUMO

Mitochondrial fission is important for organelle transport, quality control and apoptosis. Changes to the fission process can result in a wide variety of neurological diseases. In mammals, mitochondrial fission is executed by the GTPase dynamin-related protein 1 (Drp1; encoded by DNM1L), which oligomerizes around mitochondria and constricts the organelle. The mitochondrial outer membrane proteins Mff, MiD49 (encoded by MIEF2) and MiD51 (encoded by MIEF1) are involved in mitochondrial fission by recruiting Drp1 from the cytosol to the organelle surface. In addition, endoplasmic reticulum (ER) tubules have been shown to wrap around and constrict mitochondria before a fission event. Up to now, the presence of MiD49 and MiD51 at ER-mitochondrial division foci has not been established. Here, we combine confocal live-cell imaging with correlative cryogenic fluorescence microscopy and soft x-ray tomography to link MiD49 and MiD51 to the involvement of the ER in mitochondrial fission. We gain further insight into this complex process and characterize the 3D structure of ER-mitochondria contact sites.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Receptores Citoplasmáticos e Nucleares/genética , Animais , Apoptose/fisiologia , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Dinaminas , Imageamento Tridimensional , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Tomografia Computadorizada por Raios X
7.
Kidney Int ; 88(5): 1030-46, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26176828

RESUMO

Myeloperoxidase (MPO) is an important neutrophil lysosomal enzyme, a major autoantigen, and a potential mediator of tissue injury in MPO-ANCA-associated vasculitis (MPO-AAV) and glomerulonephritis. Here we examined MPO deposition in kidney biopsies from 47 patients with MPO-AAV. Leukocyte accumulation and fibrin deposition consistent with cell-mediated immunity was a major feature. Tubulointerstitial macrophage, CD4+ and CD8+ T-cell, and neutrophil numbers correlated with low presenting eGFR. MPO was not detected in kidneys from patients with minimal change or thin basement membrane disease, but was prominent in glomerular, periglomerular, and tubulointerstitial regions in MPO-AAV. Extracellular MPO released from leukocytes was pronounced in all MPO-AAV patients. Similar numbers of neutrophils and macrophages expressed MPO in the kidneys, but colocalization studies identified neutrophils as the major source of extracellular MPO. Extraleukocyte MPO was prominent in neutrophil extracellular traps in the majority of patients; most of which had traps in half or more glomeruli. These traps were associated with more neutrophils and more MPO within glomeruli. Glomerular MPO-containing macrophages generated extracellular trap-like structures. MPO also localized to endothelial cells and podocytes. The presence of the most active glomerular lesions (both segmental necrosis and cellular crescents) correlated with intraglomerular CD4+ cells and MPO+ macrophages. Thus, cellular and extracellular MPO may cause glomerular and interstitial injury.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos , Doenças Autoimunes/enzimologia , Armadilhas Extracelulares/enzimologia , Glomerulonefrite/enzimologia , Peroxidase/metabolismo , Idoso , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Células Dendríticas/enzimologia , Células Endoteliais/enzimologia , Líquido Extracelular/enzimologia , Feminino , Taxa de Filtração Glomerular , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Humanos , Glomérulos Renais/enzimologia , Glomérulos Renais/patologia , Macrófagos/enzimologia , Masculino , Neutrófilos/enzimologia , Podócitos/enzimologia
8.
J Biol Chem ; 288(38): 27584-27593, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23921378

RESUMO

Drp1 (dynamin-related protein 1) is recruited to both mitochondrial and peroxisomal membranes to execute fission. Fis1 and Mff are Drp1 receptor/effector proteins of mitochondria and peroxisomes. Recently, MiD49 and MiD51 were also shown to recruit Drp1 to the mitochondrial surface; however, different reports have ascribed opposing roles in fission and fusion. Here, we show that MiD49 or MiD51 overexpression blocked fission by acting in a dominant-negative manner by sequestering Drp1 specifically at mitochondria, causing unopposed fusion events at mitochondria along with elongation of peroxisomes. Mitochondrial elongation caused by MiD49/51 overexpression required the action of fusion mediators mitofusins 1 and 2. Furthermore, at low level overexpression when MiD49 and MiD51 form discrete foci at mitochondria, mitochondrial fission events still occurred. Unlike Fis1 and Mff, MiD49 and MiD51 were not targeted to the peroxisomal surface, suggesting that they specifically act to facilitate Drp1-directed fission at mitochondria. Moreover, when MiD49 or MiD51 was targeted to the surface of peroxisomes or lysosomes, Drp1 was specifically recruited to these organelles. Moreover, the Drp1 recruitment activity of MiD49/51 appeared stronger than that of Mff or Fis1. We conclude that MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and suggest that they provide specificity to the division of mitochondria.


Assuntos
Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Animais , Dinaminas/genética , GTP Fosfo-Hidrolases/genética , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Fatores de Alongamento de Peptídeos/genética , Peroxissomos/genética , Peroxissomos/metabolismo
9.
Methods Mol Biol ; 2080: 93-114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31745874

RESUMO

Fluorescence microscopy has become a powerful tool to investigate proteins in their natural environment. Well-established techniques like widefield and confocal fluorescence microscopy have commonly been used for decades to visualize biomolecules in single cells and tissue sections. Live cell microscopy allows for the investigation of biomolecular trafficking, and other specialized techniques, such as proximity ligation assays (PLA) and fluorescence lifetime imaging microscopy (FLIM), can be used to study interactions between biomolecules of interest. Finally, with the most recent rise of optical super-resolution microscopy, we can investigate target biomolecules in situ with unprecedented detail on the nanometer scale. Here, we discuss various optical microscopy techniques that have successfully been used to image MIF. We highlight applications, advantages, and limitations of each technique. The techniques described here can easily be adapted to investigate other target proteins, their localization, interaction partners, and mechanisms of action.


Assuntos
Proteínas de Transporte/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Microscopia de Fluorescência , Imagem Molecular , Mapeamento de Interação de Proteínas , Células Cultivadas , Processamento de Imagem Assistida por Computador , Fatores Inibidores da Migração de Macrófagos/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Microscopia Confocal , Imagem Molecular/métodos , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos
10.
Transl Res ; 216: 1-22, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31734267

RESUMO

Interleukin (IL)-22 activates STAT (signal transducer and activator of transcription) 3 and antiapoptotic and proproliferative pathways; but beyond this, the molecular mechanisms by which IL-22 promotes carcinogenesis are poorly understood. Characterizing the molecular signature of IL-22 in human DLD-1 colon carcinoma cells, we observed increased expression of 26 genes, including NNMT (nicotinamide N-methyltransferase, ≤10-fold) and CEA (carcinoembryonic antigen, ≤7-fold), both known to promote intestinal carcinogenesis. ERP27 (endoplasmic reticulum protein-27, function unknown, ≤5-fold) and the proinflammatory ICAM1 (intercellular adhesion molecule-1, ≤4-fold) were also increased. The effect on CEA was partly STAT3-mediated, as STAT3-silencing reduced IL-22-induced CEA by ≤56%. Silencing of CEA or NNMT inhibited IL-22-induced proliferation/migration of DLD-1, Caco-2, and SW480 colon carcinoma cells. To validate these results in primary tissues, we assessed IL-22-induced gene expression in organoids from human healthy colon and colon cancer patients, and from normal mouse small intestine and colon. Gene regulation by IL-22 was similar in DLD-1 cells and human and mouse healthy organoids. CEA was an exception with no induction by IL-22 in organoids, indicating the 3-dimensional organization of the tissue may produce signals absent in 2D cell culture. Importantly, augmentation of NNMT was 5-14-fold greater in human cancerous compared to normal organoids, supporting a role for NNMT in IL-22-mediated colon carcinogenesis. Thus, NNMT and CEA emerge as mediators of the tumor-promoting effects of IL-22 in the intestine. These data advance our understanding of the multifaceted role of IL-22 in the gut and suggest the IL-22 pathway may represent a therapeutic target in colon cancer.


Assuntos
Neoplasias do Colo/genética , Interleucinas/metabolismo , Organoides/patologia , Animais , Células CACO-2 , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Neoplasias do Colo/patologia , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Nicotinamida N-Metiltransferase/genética , Fator de Transcrição STAT3/metabolismo , Interleucina 22
11.
Cell Rep ; 23(9): 2782-2794, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29847806

RESUMO

The ß-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Detergentes/farmacologia , Proteínas de Escherichia coli/química , Biossíntese de Proteínas/efeitos dos fármacos , Multimerização Proteica , Estrutura Secundária de Proteína
12.
Artigo em Inglês | MEDLINE | ID: mdl-28611954

RESUMO

Gram-negative bacteria have a highly evolved cell wall with two membranes composed of complex arrays of integral and peripheral proteins, as well as phospholipids and glycolipids. In order to sense changes in, respond to, and exploit their environmental niches, bacteria rely on structures assembled into or onto the outer membrane. Protein secretion across the cell wall is a key process in virulence and other fundamental aspects of bacterial cell biology. The final stage of protein secretion in Gram-negative bacteria, translocation across the outer membrane, is energetically challenging so sophisticated nanomachines have evolved to meet this challenge. Advances in fluorescence microscopy now allow for the direct visualization of the protein secretion process, detailing the dynamics of (i) outer membrane biogenesis and the assembly of protein secretion systems into the outer membrane, (ii) the spatial distribution of these and other membrane proteins on the bacterial cell surface, and (iii) translocation of effector proteins, toxins and enzymes by these protein secretion systems. Here we review the frontier research imaging the process of secretion, particularly new studies that are applying various modes of super-resolution microscopy.


Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Membrana Celular/fisiologia , Bactérias Gram-Negativas/citologia , Sistemas de Translocação de Proteínas/fisiologia , Toxinas Bacterianas/metabolismo , Membrana Celular/química , Parede Celular/metabolismo , Lipopolissacarídeos , Imagem Óptica/métodos , Biogênese de Organelas , Transporte Proteico/fisiologia , Sistemas de Secreção Tipo I , Sistemas de Secreção Tipo II , Sistemas de Secreção Tipo III , Sistemas de Secreção Tipo IV , Sistemas de Secreção Tipo V , Sistemas de Secreção Tipo VI , Virulência
13.
Artigo em Inglês | MEDLINE | ID: mdl-28239420

RESUMO

BACKGROUND: Defining how epigenetic information is established in the germline during fetal development is key to understanding how epigenetic information is inherited and impacts on evolution and human health and disease. RESULTS: Here, we show that Polycomb Repressive Complex 2 is transiently localized in the nucleus of mouse fetal germ cells, while DNA methylation is removed from the germline. This coincides with significant enrichment of trimethylated lysine 27 on histone 3 near the nuclear lamina that is dependent on activity of the essential PRC2 catalytic proteins, Enhancer of Zeste 1 and/or 2. CONCLUSIONS: Combined, these data reveal a role for Polycomb Repressive Complex 2 and trimethylated lysine 27 on histone 3 during germline epigenetic programming that we speculate is required to repress target sequences while DNA methylation is removed.


Assuntos
Epigenômica , Histonas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Diferenciação Celular , Reprogramação Celular , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Feto/citologia , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Células Germinativas/metabolismo , Gônadas/metabolismo , Gônadas/patologia , Histonas/genética , Indóis/farmacologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Fator 3 de Transcrição de Octâmero/genética , Piridonas/farmacologia
14.
Nat Commun ; 8(1): 75, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28706202

RESUMO

Atherosclerosis is a major cause of mortality and morbidity, which is mainly driven by complications such as myocardial infarction and stroke. These complications are caused by thrombotic arterial occlusion localized at the site of high-risk atherosclerotic plaques, of which early detection and therapeutic stabilization are urgently needed. Here we show that near-infrared autofluorescence is associated with the presence of intraplaque hemorrhage and heme degradation products, particularly bilirubin by using our recently created mouse model, which uniquely reflects plaque instability as seen in humans, and human carotid endarterectomy samples. Fluorescence emission computed tomography detecting near-infrared autofluorescence allows in vivo monitoring of intraplaque hemorrhage, establishing a preclinical technology to assess and monitor plaque instability and thereby test potential plaque-stabilizing drugs. We suggest that near-infrared autofluorescence imaging is a novel technology that allows identification of atherosclerotic plaques with intraplaque hemorrhage and ultimately holds promise for detection of high-risk plaques in patients.Atherosclerosis diagnosis relies primarily on imaging and early detection of high-risk atherosclerotic plaques is important for risk stratification of patients and stabilization therapies. Here Htun et al. demonstrate that vulnerable atherosclerotic plaques generate near-infrared autofluorescence that can be detected via emission computed tomography.


Assuntos
Aterosclerose/diagnóstico por imagem , Artérias Carótidas/diagnóstico por imagem , Heme/metabolismo , Hemorragia/diagnóstico por imagem , Imagem Óptica/métodos , Placa Aterosclerótica/diagnóstico por imagem , Animais , Aterosclerose/sangue , Aterosclerose/patologia , Bilirrubina/sangue , Biliverdina/sangue , Biomarcadores/sangue , Biomarcadores/química , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Endarterectomia das Carótidas , Heme/química , Hemorragia/sangue , Hemorragia/patologia , Humanos , Camundongos , Imagem Óptica/instrumentação , Placa Aterosclerótica/sangue , Placa Aterosclerótica/patologia , Fatores de Risco , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos
15.
Sci Rep ; 6: 23229, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26987907

RESUMO

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.


Assuntos
DNA Mitocondrial/genética , Desenvolvimento Embrionário , Mitocôndrias/metabolismo , Oócitos/metabolismo , Sus scrofa/genética , Animais , Blastocisto/metabolismo , Meios de Cultura/química , Variações do Número de Cópias de DNA , Técnicas de Cultura Embrionária , Feminino , Fertilização in vitro , Perfilação da Expressão Gênica , Mitocôndrias/genética , Gravidez , Sus scrofa/embriologia , Suínos
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