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1.
Blood ; 119(19): 4527-31, 2012 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-22452982

RESUMEN

Autophagy is the process by which superfluous or damaged macromolecules or organelles are degraded by the lysosome. Pharmacologic and genetic evidence indicates that autophagy plays pleiotropic functions in cellular homeostasis, development, survival, and differentiation. The differentiation of human blood monocytes into macrophages is a caspase-dependent process when triggered ex vivo by colony stimulating factor-1. We show here, using pharmacologic inhibitors, siRNA approaches, and Atg7-/- mice, that autophagy initiated by ULK1 is required for proper colony stimulating factor-1-driven differentiation of human and murine monocytes. We also unravel a role for autophagy in macrophage acquisition of phagocytic functions. Collectively, these findings highlight an unexpected and essential role of autophagy during monocyte differentiation and acquisition of macrophage functions.


Asunto(s)
Autofagia/fisiología , Diferenciación Celular/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Proteína 7 Relacionada con la Autofagia , Homólogo de la Proteína 1 Relacionada con la Autofagia , Catepsina B/farmacología , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Macrófagos/fisiología , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/fisiología , Fagocitosis/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , ARN Interferente Pequeño/farmacología
2.
J Exp Med ; 204(9): 2075-87, 2007 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-17698589

RESUMEN

Cytolysis, interferon gamma and tumor necrosis factor (TNF) alpha secretion are major effector mechanisms of memory CD8+ T cells that are believed to be required for immunological protection in vivo. By using mutants of the intracellular bacterium Listeria monocytogenes, we found that none of these effector activities is sufficient to protect against secondary infection with wild-type (WT) bacteria. We demonstrated that CCL3 derived from reactivated memory CD8+ T cells is required for efficient killing of WT bacteria. CCL3 induces a rapid TNF-alpha secretion by innate inflammatory mononuclear phagocytic cells (MPCs), which further promotes the production of radical oxygen intermediates (ROIs) by both MPCs and neutrophils. ROI generation is the final bactericidal mechanism involved in L. monocytogenes clearance. These results therefore uncover two levels of regulation of the antibacterial secondary protective response: (a) an antigen-dependent phase in which memory CD8+ T cells are reactivated and control the activation of the innate immune system, and (b) an antigen-independent phase in which the MPCs coordinate innate immunity and promote the bactericidal effector activities. In this context, CCL3-secreting memory CD8+ T cells are able to mediate "bystander" killing of an unrelated pathogen upon antigen-specific reactivation, a mechanism that may be important for the design of therapeutic vaccines.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Quimiocinas CC/inmunología , Inmunidad/inmunología , Memoria Inmunológica/inmunología , Listeria monocytogenes/inmunología , Proteínas Inflamatorias de Macrófagos/inmunología , Fagocitos/inmunología , Factores de Necrosis Tumoral/inmunología , Animales , Proteínas Bacterianas/metabolismo , Efecto Espectador/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/microbiología , Linfocitos T CD8-positivos/parasitología , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/metabolismo , Citotoxicidad Inmunológica , Femenino , Inmunización , Interferón gamma/metabolismo , Leishmania major/inmunología , Listeriosis/inmunología , Proteínas Inflamatorias de Macrófagos/metabolismo , Ratones , Modelos Inmunológicos , Mutación/genética , Neutrófilos/metabolismo , Fagocitos/microbiología , Especies Reactivas de Oxígeno , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo
3.
PLoS Pathog ; 7(12): e1002457, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22241983

RESUMEN

Immunological memory is a hallmark of B and T lymphocytes that have undergone a previous encounter with a given antigen. It is assumed that memory cells mediate better protection of the host upon re-infection because of improved effector functions such as antibody production, cytotoxic activity and cytokine secretion. In contrast to cells of the adaptive immune system, innate immune cells are believed to exhibit a comparable functional effector response each time the same pathogen is encountered. Here, using mice infected by the intracellular bacterium Listeria monocytogenes, we show that during a recall bacterial infection, the chemokine CCL3 secreted by memory CD8+ T cells drives drastic modifications of the functional properties of several populations of phagocytes. We found that inflammatory ly6C+ monocytes and neutrophils largely mediated memory CD8+ T cell bacteriocidal activity by producing increased levels of reactive oxygen species (ROS), augmenting the pH of their phagosomes and inducing antimicrobial autophagy. These events allowed an extremely rapid control of bacterial growth in vivo and accounted for protective immunity. Therefore, our results provide evidence that cytotoxic memory CD8+ T cells can license distinct antimicrobial effector mechanisms of innate cells to efficiently clear pathogens.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad Celular , Memoria Inmunológica , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , Animales , Quimiocina CCL3/genética , Quimiocina CCL3/inmunología , Listeriosis/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados
4.
Nat Genet ; 32(3): 443-7, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12389029

RESUMEN

Mice that are homozygous with respect to the progressive motor neuronopathy (pmn) mutation (chromosome 13) develop a progressive caudio-cranial degeneration of their motor axons from the age of two weeks and die four to six weeks after birth. The mutation is fully penetrant, and expressivity does not depend on the genetic background. Based on its pathological features, the pmn mutation has been considered an excellent model for the autosomal recessive proximal childhood form of spinal muscular atrophy (SMA). Previously, we demonstrated that the genes responsible for these disorders were not orthologous. Here, we identify the pmn mutation as resulting in a Trp524Gly substitution at the last residue of the tubulin-specific chaperone e (Tbce) protein that leads to decreased protein stability. Electron microscopy of the sciatic and phrenic nerves of affected mice showed a reduced number of microtubules, probably due to defective stabilization. Transgenic complementation with a wildtype Tbce cDNA restored a normal phenotype in mutant mice. Our observations indicate that Tbce is critical for the maintenance of microtubules in mouse motor axons, and suggest that altered function of tubulin cofactors might be implicated in human motor neuron diseases.


Asunto(s)
Enfermedades de los Nervios Craneales/genética , Chaperonas Moleculares/genética , Mutación Missense , Secuencia de Aminoácidos , Animales , Axones/metabolismo , Northern Blotting , Células COS , Mapeo Cromosómico , Cruzamientos Genéticos , Análisis Mutacional de ADN , Vectores Genéticos , Células HeLa , Humanos , Hibridación in Situ , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Chaperonas Moleculares/fisiología , Datos de Secuencia Molecular , Mutación , ARN Mensajero/metabolismo , Factores de Tiempo , Transfección
5.
Development ; 136(21): 3647-55, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19820183

RESUMEN

The size of the mammalian body is determined by genetic and environmental factors differentially modulating pre- and postnatal growth. We now report a control of growth acting in the mouse from the first cleavages to the postnatal stages. It was evidenced by a hereditary epigenetic modification (paramutation) created by injection of a miR-124 microRNA into fertilized eggs. From the blastocyst to the adult, mouse pups born after microinjection of this miRNA showed a 30% increase in size. At the blastocyst stage, frequent duplication of the inner cell mass resulted in twin pregnancies. A role of sperm RNA as a transgenerational signal was confirmed by the giant phenotype of the progeny of transgenic males expressing miR-124 during spermiogenesis. In E2.5 to E8.5 embryos, increased levels of several transcripts with sequence homology to the microRNA were noted, including those of Sox9, a gene known for its crucial role in the progenitors of several adult tissues. A role in embryonic growth was confirmed by the large size of embryos expressing a Sox9 DNA transgene. Increased expression in the paramutants was not related to a change in miR-124 expression, but to the establishment of a distinct, heritable chromatin structure in the promoter region of Sox9. While the heritability of body size is not readily accounted for by Mendelian genetics, our results suggest the alternate model of RNA-mediated heritable epigenetic modifications.


Asunto(s)
Tamaño Corporal/genética , Epigénesis Genética , Ratones/embriología , MicroARNs/metabolismo , Factor de Transcripción SOX9/metabolismo , Animales , Masculino , Ratones/genética
6.
PLoS Pathog ; 6(10): e1001154, 2010 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-20976202

RESUMEN

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.


Asunto(s)
Membrana Celular/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/metabolismo , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Compartimento Celular/inmunología , Membrana Celular/inmunología , Células Cultivadas , Técnica del Anticuerpo Fluorescente/métodos , Antígenos de Histocompatibilidad Clase II/inmunología , Membranas Intracelulares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fagosomas/inmunología , Fagosomas/metabolismo
7.
J Cell Biol ; 177(2): 343-54, 2007 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-17438076

RESUMEN

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP-enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852-4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs. In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endosomas/metabolismo , Helicobacter pylori/química , Actinas/metabolismo , Citoesqueleto/metabolismo , Endocitosis , Glicosilfosfatidilinositoles/metabolismo , Células HeLa , Humanos , Transporte de Proteínas
8.
Nature ; 441(7092): 469-74, 2006 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-16724059

RESUMEN

Paramutation is a heritable epigenetic modification induced in plants by cross-talk between allelic loci. Here we report a similar modification of the mouse Kit gene in the progeny of heterozygotes with the null mutant Kit(tm1Alf) (a lacZ insertion). In spite of a homozygous wild-type genotype, their offspring maintain, to a variable extent, the white spots characteristic of Kit mutant animals. Efficiently inherited from either male or female parents, the modified phenotype results from a decrease in Kit messenger RNA levels with the accumulation of non-polyadenylated RNA molecules of abnormal sizes. Sustained transcriptional activity at the postmeiotic stages--at which time the gene is normally silent--leads to the accumulation of RNA in spermatozoa. Microinjection into fertilized eggs either of total RNA from Kit(tm1Alf/+) heterozygotes or of Kit-specific microRNAs induced a heritable white tail phenotype. Our results identify an unexpected mode of epigenetic inheritance associated with the zygotic transfer of RNA molecules.


Asunto(s)
Epigénesis Genética/genética , Herencia/genética , Mutación/genética , Proteínas Proto-Oncogénicas c-kit/genética , ARN/genética , ARN/metabolismo , Alelos , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Genotipo , Masculino , Ratones , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espermatozoides/metabolismo
9.
Infect Immun ; 79(6): 2396-403, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21402759

RESUMEN

The SecA2 auxiliary secretion system of Gram-positive bacteria promotes the export of virulence proteins essential for colonization of the host in the case of both Mycobacterium tuberculosis and Listeria monocytogenes, two intracellular bacteria causing diseases in humans. We and others have demonstrated that this secretion system is also linked to the onset of long-term CD8(+) T cell-mediated protective immunity in mice. In the case of L. monocytogenes, expression of SecA2 inside the cytosol of infected cells correlates with the generation of CCL3-secreting memory CD8(+) T cells that are required for protection against secondary challenge with wild-type (wt) L. monocytogenes. Since the SecA2 ATPase is well conserved among Gram-positive pathogenic bacteria, we hypothesized that SecA2 itself bears evolutionarily conserved motifs recognized by cytosolic pattern recognition receptors, leading to signaling events promoting the differentiation of CCL3(+) memory CD8(+) T cells. To test this possibility, we generated a stable L. monocytogenes chromosomal mutant that expressed a SecA2 ATPase bearing a mutated nucleotide binding site (NBS). Similarly to a SecA2 deletion mutant, the NBS mutant exhibited rough colonies, a bacterial chaining phenotype, an impaired protein secretion profile, and in vivo virulence in comparison to wt L. monocytogenes. Importantly, mice immunized with the SecA2 NBS mutant were not protected against secondary infection with wt L. monocytogenes and did not develop CCL3(+) memory CD8(+) T cells. NBS mutant and wt SecA2 proteins were expressed to comparable extents by bacteria, suggesting that SecA2 itself is unlikely to promote the induction of these cells. Rather, one or several of the SecA2 substrate proteins released inside the cytosol of infected cells may be involved.


Asunto(s)
Adenosina Trifosfatasas/fisiología , Proteínas Bacterianas/fisiología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Proteínas de Transporte de Membrana/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Western Blotting , Clonación Molecular , Femenino , Citometría de Flujo , Listeria monocytogenes/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Canales de Translocación SEC , Proteína SecA
10.
Blood ; 113(2): 347-57, 2009 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-18849489

RESUMEN

By presenting antigenic peptides on the cell surface, human leukocyte antigen (HLA) class I molecules are critical for immune defense. Their surface density determines, to a large extent, the level of CD8(+) T cell-dependent immune reactions; their loss is a major mechanism of immune escape. Therefore, powerful processes should regulate their surface expression. Here we document the mechanisms used by CD99 to mediate HLA class I modulation. Up-regulation of HLA class I by IFN-gamma requires CD99. In the trans Golgi network (TGN), and up to the cell surface, CD99 and HLA class I are physically associated via their transmembrane domain. CD99 also binds p230/golgin-245, a coiled-coil protein that recycles between the cytosol and buds/vesicles of the TGN and which plays a fundamental role in trafficking transport vesicles. p230/golgin-245 is anchored within TGN membranes via its Golgin-97, RanBP1, IMh1p, P230 (GRIP) domain and the overexpression of which leads to surface and intracellular down-modulation of HLA class I molecules.


Asunto(s)
Antígenos CD/inmunología , Autoantígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Moléculas de Adhesión Celular/inmunología , Aparato de Golgi/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas de la Membrana/inmunología , Regulación hacia Arriba/inmunología , Antígeno 12E7 , Antígenos CD/metabolismo , Antivirales/inmunología , Antivirales/farmacología , Autoantígenos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Moléculas de Adhesión Celular/metabolismo , Citosol/inmunología , Citosol/metabolismo , Aparato de Golgi/metabolismo , Antígenos HLA/biosíntesis , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Inmunidad Celular/fisiología , Interferón gamma/inmunología , Interferón gamma/farmacología , Células Jurkat , Proteínas de la Membrana/metabolismo , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/inmunología , Vesículas Transportadoras/inmunología , Vesículas Transportadoras/metabolismo , Regulación hacia Arriba/efectos de los fármacos
11.
J Cell Biol ; 173(5): 809-19, 2006 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-16754962

RESUMEN

The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.


Asunto(s)
ADP Ribosa Transferasas/farmacología , Proteínas Bacterianas/farmacología , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , ADP Ribosa Transferasas/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Interferencia de ARN/fisiología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Staphylococcus aureus/química , Staphylococcus aureus/enzimología , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo
12.
Proc Natl Acad Sci U S A ; 105(44): 16946-51, 2008 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-18974217

RESUMEN

Cytoplasmic coat proteins are required for cargo selection and budding of tubulovesicular transport intermediates that shuttle between intracellular compartments. To better understand the physical parameters governing coat assembly and coat-induced membrane deformation, we have reconstituted the Arf1-dependent assembly of the COPI coat on giant unilamellar vesicles by using fluorescently labeled Arf1 and coatomer. Membrane recruitment of Arf1-GTP occurs exclusively on disordered lipid domains and does not induce optically visible membrane deformation. In the presence of Arf1-GTP, coatomer self-assembles into weakly curved coats on membranes under high tension, while it induces extensive membrane deformation at low membrane tension. These deformations appear to have a composition different from the parental membrane because they are protected from phase transition. These findings suggest that the COPI coat is adapted to liquid disordered membrane domains where it could promote lipid sorting and that its mechanical effects can be tuned by membrane tension.


Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/fisiología , Proteína Coat de Complejo I/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/ultraestructura , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Guanosina Trifosfato/metabolismo , Estructura Terciaria de Proteína , Conejos , Liposomas Unilamelares/metabolismo
13.
J Cell Physiol ; 222(3): 648-57, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19957303

RESUMEN

It is well established that cells exposed to the limiting oxygen microenvironment (hypoxia) of tumors acquire resistance to chemotherapy, through mechanisms not fully understood. We noted that a large number of cell lines showed protection from apoptotic stimuli, staurosporine, or etoposide, when exposed to long-term hypoxia (72 h). In addition, these cells had unusual enlarged mitochondria that were induced in a HIF-1-dependent manner. Enlarged mitochondria were functional as they conserved their transmembrane potential and ATP production. Here we reveal that mitochondria of hypoxia-induced chemotherapy-resistant cells undergo a HIF-1-dependent and mitofusin-1-mediated change in morphology from a tubular network to an enlarged phenotype. An imbalance in mitochondrial fusion/fission occurs since silencing of not only the mitochondrial fusion protein mitofusin 1 but also BNIP3 and BNIP3L, two mitochondrial HIF-targeted genes, reestablished a tubular morphology. Hypoxic cells were insensitive to staurosporine- and etoposide-induced cell death, but the silencing of mitofusin, BNIP3, and BNIP3L restored sensitivity. Our results demonstrate that some cancer cells have developed yet another way to evade apoptosis in hypoxia, by inducing mitochondrial fusion and targeting BNIP3 and BNIP3L to mitochondrial membranes, thereby giving these cells a selective growth advantage.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos , Etopósido/farmacología , Mitocondrias/patología , Dilatación Mitocondrial , Neoplasias/patología , Estaurosporina/farmacología , Adenosina Trifosfato/metabolismo , Hipoxia de la Célula , Proliferación Celular/efectos de los fármacos , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Factores de Tiempo , Transfección , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
14.
Nature ; 426(6966): 563-6, 2003 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-14654841

RESUMEN

Protein coats deform flat lipid membranes into buds and capture membrane proteins to form transport vesicles. The assembly/disassembly cycle of the COPI coat on Golgi membranes is coupled to the GTP/GDP cycle of the small G protein Arf1. At the heart of this coupling is the specific interaction of membrane-bound Arf1-GTP with coatomer, a complex of seven proteins that forms the building unit of the COPI coat. Although COPI coat disassembly requires the catalysis of GTP hydrolysis in Arf1 by a specific GTPase-activating protein (ArfGAP1), the precise timing of this reaction during COPI vesicle formation is not known. Using time-resolved assays for COPI dynamics on liposomes of controlled size, we show that the rate of ArfGAP1-catalysed GTP hydrolysis in Arf1 and the rate of COPI disassembly increase over two orders of magnitude as the curvature of the lipid bilayer increases and approaches that of a typical transport vesicle. This leads to a model for COPI dynamics in which GTP hydrolysis in Arf1 is organized temporally and spatially according to the changes in lipid packing induced by the coat.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/química , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteína Coat de Complejo I/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Metabolismo de los Lípidos , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/ultraestructura , Aparato de Golgi/química , Guanosina Trifosfato/metabolismo , Hidrólisis , Membranas Intracelulares/química , Cinética , Lípidos/química , Liposomas/química , Liposomas/metabolismo , Modelos Biológicos , Conejos , Factores de Tiempo
15.
Int Urogynecol J ; 21(3): 261-70, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20052576

RESUMEN

INTRODUCTION AND HYPOTHESIS: Currently, most implants used for reinforcement in surgical treatment of pelvic floor disorders are knitted monofilament polypropylene (PP). While previously recognized as inert, PP is associated with high complication rates. Some recent literature suggests polyester prosthetics based on poly(ethylene terephthalate) (PET), which may be more inert in vivo. METHODS: A sample of 100 implants explanted from patients due to complications was examined to evaluate the relative degradation characteristics of PP and PET prosthetics. Histological, microscopic (scanning electron microscopy, SEM) and chemical analysis (Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC)) were conducted on these explants. RESULTS: Poly(ethylene terephtahlate) explants appeared to sustain less degradation in vivo than the PP explants observed in this cohort. CONCLUSIONS: This is the first study to evaluate synthetic implants used in a vaginal approach for pelvic floor reinforcement. The study provides evidence contrary to published literature characterizing PP as inert in such applications. Additionally, the study suggests the need for clinical trials comparatively investigating the performance of new types of monofilament prosthetics, such as those comprising PET.


Asunto(s)
Reacción a Cuerpo Extraño/etiología , Polipropilenos/efectos adversos , Complicaciones Posoperatorias/etiología , Mallas Quirúrgicas/efectos adversos , Remoción de Dispositivos , Femenino , Humanos , Microscopía Electrónica de Rastreo , Prolapso de Órgano Pélvico/cirugía , Estudios Prospectivos , Espectroscopía Infrarroja por Transformada de Fourier , Incontinencia Urinaria de Esfuerzo/cirugía
16.
Circ Res ; 101(2): 176-84, 2007 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-17556656

RESUMEN

Vessel occlusion is the most frequent cause for impairment of local blood flow within the brain resulting in neuronal damage and is a leading cause of disability and death worldwide. Polyunsaturated fatty acids and especially alpha-linolenic acid improve brain resistance against cerebral ischemia. The purpose of the present study was to evaluate the effects of polyunsaturated fatty acids and particularly alpha-linolenic acid on the cerebral blood flow and on the tone of vessels that regulate brain perfusion. alpha-Linolenic acid injections increased cerebral blood flow and induced vasodilation of the basilar artery but not of the carotid artery. The saturated fatty acid palmitic acid did not produce vasodilation. This suggested that the target of the polyunsaturated fatty acids effect was the TREK-1 potassium channel. We demonstrate the presence of this channel in basilar but not in carotid arteries. We show that vasodilations induced by the polyunsaturated fatty acid in the basilar artery as well as the laser-Doppler flow increase are abolished in TREK-1(-/-) mice. Altogether these data indicate that TREK-1 activation elicits a robust dilation that probably accounts for the increase of cerebral blood flow induced by polyunsaturated fatty acids such as alpha-linolenic acid or docosahexanoic acid. They suggest that the selective expression and activation of TREK-1 in brain collaterals could play a significant role in the protective mechanisms of polyunsaturated fatty acids against stroke by providing residual circulation during ischemia.


Asunto(s)
Isquemia Encefálica/metabolismo , Isquemia Encefálica/prevención & control , Cerebelo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Vasodilatadores/farmacología , Ácido alfa-Linolénico/farmacología , Animales , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Arterias Carótidas/fisiopatología , Cerebelo/irrigación sanguínea , Cerebelo/patología , Cerebelo/fisiopatología , Circulación Cerebrovascular/efectos de los fármacos , Flujometría por Láser-Doppler , Ratones , Ratones Noqueados , Canales de Potasio de Dominio Poro en Tándem/deficiencia , Ratas , Flujo Sanguíneo Regional/efectos de los fármacos
17.
Biochemistry ; 47(51): 13674-85, 2008 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-19035652

RESUMEN

NHE-1 is a ubiquitous, mitogen-activatable, mammalian Na+/H+ exchanger that maintains cytosolic pH and regulates cell volume. We have previously shown that the kinetics of NHE-1 positive cooperative activation by intracellular acidifications fit best with a Monod-Wyman-Changeux mechanism, in which a dimeric NHE-1 oscillates between a low- and a high-affinity conformation for intracellular protons. The ratio between these two forms, the allosteric equilibrium constant L0, is in favor of the low-affinity form, making the system inactive at physiological pH. Conversely the high-affinity form is stabilized by intracellular protons, resulting in the observed positive cooperativity. The aim of the present study was to investigate the kinetics and mechanism of NHE-1 regulation by osmotic shocks. We show that they modify the L0 parameter (865 +/- 95 and 3757 +/- 328 for 500 and 100 mOsM, respectively, vs 1549 +/- 57 in isotonic conditions).This results in an activation of NHE-1 by hypertonic shocks and, conversely, in an inhibition by hypotonic media. Quantitatively, this modulation of L0 follows an exponential distribution relative to osmolarity, that is, additive to the activation of NHE-1 by intracellular signaling pathways. These effects can be mimicked by the asymmetric insertion of amphiphilic molecules into the lipid bilayer. Finally, site-directed mutagenesis of NHE-1 shows that neither its association with membrane PIP2 nor its interaction with cortical actin are required for mechanosensation. In conclusion, NHE-1 allosteric equilibrium and, thus, its cooperative response to intracellular acidifications is extremely sensitive to modification of its membrane environment.


Asunto(s)
Regulación de la Expresión Génica , Presión Osmótica , Intercambiadores de Sodio-Hidrógeno/química , Animales , Proteínas de Transporte de Catión/metabolismo , Línea Celular , Cricetinae , Citosol/metabolismo , Fibroblastos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Microscopía Fluorescente , Modelos Biológicos , Isoformas de Proteínas , Transducción de Señal , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo
18.
J Clin Invest ; 110(9): 1329-37, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12417572

RESUMEN

Enteroaggregative Escherichia coli (EAEC) is a diarrheal pathogen defined by its characteristic aggregative adherence (AA) to HEp-2 cells in culture. We have previously shown that EAEC strains secrete a 10-kDa protein that is immunogenic in a human EAEC challenge model. We report here that this protein is encoded by a gene (called aap) lying immediately upstream of that encoding the AggR transcriptional activator, and that aap is under AggR control. The product of aap has a typical signal sequence and is secreted to the extracellular milieu, where it remains noncovalently attached to the surface of the bacterium. EAEC aap mutants aggregate more intensely than the wild-type parent in a number of assays, forming larger aggregates and fewer individual bacteria. Infection of colonic biopsies with wild-type EAEC strain 042 and its aap mutant revealed more dramatic autoagglutination of the mutant compared with the wild-type parent. Our data suggest that the aap gene product participates in formation of a surface coat that acts to disperse the bacteria, thus partially counteracting aggregation mediated by aggregative adherence fimbriae. We have therefore named the aap gene product "dispersin," and we propose that it may be representative of a functional class of colonization factors. Since dispersin is expressed in vivo, is highly immunogenic, and is present in most EAEC strains, it holds considerable promise as an EAEC immunogen.


Asunto(s)
Proteínas de Escherichia coli/análisis , Aglutinación , Adhesión Bacteriana , Mapeo Cromosómico , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , Microscopía Electrónica , Transactivadores/fisiología , Transcripción Genética
19.
Elife ; 52016 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-27458799

RESUMEN

When small phosphatidylcholine liposomes are added to perforated cells, they bind preferentially to the Golgi suggesting an exceptional avidity of this organelle for curved membranes without stereospecific interactions. We show that the cis golgin GMAP-210 accounts for this property. First, the liposome tethering properties of the Golgi resembles that of the amphipathic lipid-packing sensor (ALPS) motif of GMAP-210: both preferred small (radius < 40 nm) liposomes made of monounsaturated but not saturated lipids. Second, reducing GMAP-210 levels or redirecting its ALPS motif to mitochondria decreased liposome capture by the Golgi. Extensive mutagenesis analysis suggests that GMAP-210 tethers authentic transport vesicles via the same mechanism whereby the ALPS motif senses lipid-packing defects at the vesicle surface through its regularly spaced hydrophobic residues. We conclude that the Golgi uses GMAP-210 as a filter to select transport vesicles according to their size and bulk lipid composition.


Asunto(s)
Aparato de Golgi/metabolismo , Liposomas/química , Liposomas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas del Citoesqueleto , Análisis Mutacional de ADN , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/genética , Vesículas Transportadoras/metabolismo
20.
Microbes Infect ; 4(7): 693-8, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12067828

RESUMEN

Splenectomised squirrel monkeys (Saimiri sciureus) are increasingly being used as an experimental host for human malaria studies, notably for the assessment of candidate vaccines against Plasmodium falciparum blood-stage infection. Recently, S. sciureus monkeys in our primate-breeding colony were reported to be asymptomatic carriers of a putative Haemobartonella species. Patent haemobartonella infection is frequently activated following splenectomy, and may interfere with studies on the course of P. falciparum parasitaemia in these animals. Here, we show by 16S rRNA gene sequence analysis that this wall-less bacterium is not a rickettsia but, instead, is a haemotrophic mycoplasma. Haemotrophic mycoplasmas are a newly identified group of mycoplasmas that parasitise the surfaces of erythrocytes of a wide variety of vertebrate hosts.


Asunto(s)
Malaria Falciparum/complicaciones , Infecciones por Mycoplasma/complicaciones , Infecciones por Mycoplasma/veterinaria , Mycoplasma/aislamiento & purificación , Parasitemia/complicaciones , Saimiri/microbiología , Saimiri/parasitología , Animales , Modelos Animales de Enfermedad , Eritrocitos/microbiología , Eritrocitos/ultraestructura , Genes Bacterianos/genética , Enfermedades de los Monos/microbiología , Enfermedades de los Monos/parasitología , Mycoplasma/clasificación , Mycoplasma/genética , Mycoplasma/ultraestructura , Infecciones por Mycoplasma/tratamiento farmacológico , Infecciones por Mycoplasma/microbiología , Filogenia , Plasmodium falciparum , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Esplenectomía
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