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1.
Proc Natl Acad Sci U S A ; 111(15): E1538-47, 2014 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-24706810

RESUMEN

Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the age-dependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form ∼3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained >95% viability, whereas the viability of starving, freely suspended (planktonic) cells decreased to <10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions.


Asunto(s)
Regulación Fúngica de la Expresión Génica/fisiología , Longevidad/fisiología , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Alginatos , Puntos de Control del Ciclo Celular/fisiología , Células Inmovilizadas/metabolismo , Células Inmovilizadas/fisiología , Citometría de Flujo , Ácido Glucurónico , Ácidos Hexurónicos , Análisis por Micromatrices , Reproducción/fisiología
2.
Am J Respir Cell Mol Biol ; 46(3): 290-8, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21960549

RESUMEN

It is widely held that exposure to pathogens such as fungi can be an agent of comorbidity, such as exacerbation of asthma or chronic obstructive pulmonary disease. Although many studies have examined allergic responses to fungi and their effects on pulmonary function, the possible pathologic implications of the early innate responses to fungal pathogens have not been explored. We examined early responses to the atypical fungus Pneumocystis in two common strains of mice in terms of overall immunological response and related pathology, such as cell damage and airway hyperresponsiveness (AHR). We found a strong strain-specific response in BALB/c mice that included recruitment of neutrophils, NK, NKT, and CD4 T cells. This response was accompanied by elevated indicators of lung damage (bronchoalveolar lavage fluid albumin and LDH) and profound AHR. This early response was absent in C57BL/6 mice, although both strains exhibited a later response associated with the clearance of Pneumocystis. We found that this AHR could not be attributed exclusively to the presence of recruited neutrophils, NKT, NK, or CD4 cells or to the actions of IFN-γ or IL-4. However, in the absence of STAT6 signaling, AHR and inflammatory cell recruitment were virtually absent. Gene expression analysis indicated that this early response included activation of several transcription factors that could be involved in pulmonary remodeling. These results show that exposure to a fungus such as Pneumocystis can elicit pulmonary responses that may contribute to morbidity, even without prior sensitization, in the context of certain genetic backgrounds.


Asunto(s)
Hiperreactividad Bronquial/metabolismo , Inmunidad Innata , Enfermedades Pulmonares Fúngicas/metabolismo , Pulmón/metabolismo , Infecciones por Pneumocystis/metabolismo , Factor de Transcripción STAT6/metabolismo , Albúminas/metabolismo , Animales , Antígenos CD1/genética , Antígenos CD1/metabolismo , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/microbiología , Hiperreactividad Bronquial/fisiopatología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/microbiología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Interferón gamma/deficiencia , Interferón gamma/genética , Interleucina-4/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Pulmón/fisiopatología , Enfermedades Pulmonares Fúngicas/genética , Enfermedades Pulmonares Fúngicas/inmunología , Enfermedades Pulmonares Fúngicas/microbiología , Enfermedades Pulmonares Fúngicas/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/microbiología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/microbiología , Infecciones por Pneumocystis/genética , Infecciones por Pneumocystis/inmunología , Infecciones por Pneumocystis/microbiología , Infecciones por Pneumocystis/fisiopatología , Receptores de Interleucina-4/deficiencia , Receptores de Interleucina-4/genética , Receptores de Interleucina-8B/deficiencia , Receptores de Interleucina-8B/genética , Factor de Transcripción STAT6/deficiencia , Factor de Transcripción STAT6/genética , Transducción de Señal , Especificidad de la Especie , Factores de Tiempo
3.
Am J Pathol ; 176(6): 2806-18, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20395428

RESUMEN

Immune-reconstitution after highly active antiretroviral therapy (HAART) is often incomplete, and some HIV-infected individuals fail to regenerate type-I interferon (IFN)-producing pDCs. We recently demonstrated that during Pneumocystis (PC) infection in CD4 T cell-competent mice the absence of type-I IFN signaling results in chronic pulmonary inflammation and fibrosis despite clearance. Because the mechanisms involved are poorly understood, we further characterized the role of type-I IFN signaling in immune responses to PC. We show that type-I IFN signaling around day 7 postinfection is critical to the outcome of inflammation. Microarray analysis of pulmonary CD11c(+) cells revealed that at day 7 post infection, wild-type cells up-regulated type-I IFN-responsive genes as well as SOCS1, which is a critical negative-regulator of type-I IFN and IFN-gamma signaling. This was associated with an eosinophilic lung inflammation, PC clearance, and complete restitution. However, pulmonary CD11c(+) cells from IFNAR(-/-) mice demonstrated increased tumor necrosis factor (TNF)-alpha production and lacked SOCS1-induction at day 7. This was followed by a transient lymphocytic and IFN-gamma response before switching to a chronic eosinophilic inflammation of the lung. Early neutralization of TNF-alpha did not prevent chronic inflammation in IFNAR(-/-) mice, but treatment with an anti-IFN-gamma antibody did. We propose that during PC lung infection type-I IFNs induce SOCS1-associated regulatory mechanisms, which prevent excessive IFN-gamma-mediated responses that cause chronic lung damage. Therefore, partial immune-reconstitution in AIDS, attributable to reduced type-I IFN actions, might disrupt regulatory aspects of inflammation, causing unexplained chronic pulmonary complications as seen in some patients during HAART.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Inflamación , Interferón Tipo I/inmunología , Interferón gamma/inmunología , Pulmón , Neumonía por Pneumocystis , Transducción de Señal/inmunología , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Antígeno CD11c/genética , Antígeno CD11c/inmunología , Perfilación de la Expresión Génica , Humanos , Inflamación/inmunología , Inflamación/microbiología , Inflamación/patología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Noqueados , Ratones SCID , Análisis por Micromatrices , Pneumocystis/inmunología , Neumonía por Pneumocystis/inmunología , Neumonía por Pneumocystis/microbiología , Neumonía por Pneumocystis/patología , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Factor de Necrosis Tumoral alfa/inmunología
4.
BMC Microbiol ; 11: 143, 2011 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-21693040

RESUMEN

BACKGROUND: Many chronic diseases, such as non-healing wounds are characterized by prolonged inflammation and respond poorly to conventional treatment. Bacterial biofilms are a major impediment to wound healing. Persistent infection of the skin allows the formation of complex bacterial communities termed biofilm. Bacteria living in biofilms are phenotypically distinct from their planktonic counterparts and are orders of magnitude more resistant to antibiotics, host immune response, and environmental stress. Staphylococcus aureus is prevalent in cutaneous infections such as chronic wounds and is an important human pathogen. RESULTS: The impact of S. aureus soluble products in biofilm-conditioned medium (BCM) or in planktonic-conditioned medium (PCM) on human keratinocytes was investigated. Proteomic analysis of BCM and PCM revealed differential protein compositions with PCM containing several enzymes involved in glycolysis. Global gene expression of keratinocytes exposed to biofilm and planktonic S. aureus was analyzed after four hours of exposure. Gene ontology terms associated with responses to bacteria, inflammation, apoptosis, chemotaxis, and signal transduction were enriched in BCM treated keratinocytes. Several transcripts encoding cytokines were also upregulated by BCM after four hours. ELISA analysis of cytokines confirmed microarray results at four hours and revealed that after 24 hours of exposure, S. aureus biofilm induced sustained low level cytokine production compared to near exponential increases of cytokines in planktonic treated keratinocytes. The reduction in cytokines produced by keratinocytes exposed to biofilm was accompanied by suppressed phosphorylation of MAPKs. Chemical inhibition of MAPKs did not drastically reduce cytokine production in BCM-treated keratinocytes suggesting that the majority of cytokine production is mediated through MAPK-independent mechanisms. CONCLUSIONS: Collectively the results indicate that S. aureus biofilms induce a distinct inflammatory response compared to their planktonic counterparts. The differential gene expression and production of inflammatory cytokines by biofilm and planktonic cultures in keratinocytes could have implications for the formation and persistence of chronic wounds. The formation of a biofilm should be considered in any study investigating host response to bacteria.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Citocinas/metabolismo , Regulación de la Expresión Génica , Queratinocitos/inmunología , Queratinocitos/microbiología , Transducción de Señal , Staphylococcus aureus/inmunología , Células Cultivadas , Medios de Cultivo Condicionados , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Perfilación de la Expresión Génica , Humanos , Fosforilación , Staphylococcus aureus/crecimiento & desarrollo
5.
Anal Biochem ; 391(2): 160-2, 2009 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-19406096

RESUMEN

We have developed a method for preparing high-quality total RNA from Ca-alginate-encapsulated Saccharomyces cerevisiae that is suitable for microarray analysis. Encapsulated cells were harvested from immobilized cell reactors and flash-frozen in liquid nitrogen. Following low-temperature mechanical disruption, cells were freed from Ca-alginate by reverse ionotropic gelation and purified by centrifugation, and then total RNA was extracted using hot acid phenol. The yield and quality of the RNA were consistently high; the RNA was free of contaminating alginate, and in microarray analysis it performed as well as RNA isolated from planktonic cells.


Asunto(s)
Alginatos/química , Calcio/química , Perfilación de la Expresión Génica , ARN de Hongos/aislamiento & purificación , Saccharomyces cerevisiae/genética , Ácido Glucurónico/química , Ácidos Hexurónicos/química
6.
Clin Cancer Res ; 14(20): 6564-73, 2008 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-18927297

RESUMEN

PURPOSE: The aim of this study was to show that novel photodynamic therapy (PDT) sensitizers can be activated by two-photon absorption in the near-IR region of the spectrum and to show, for the first time, that such activation can lead to tumor regressions at significant tissue depth. These experiments also evaluated effects of high-energy femtosecond pulsed laser irradiation on normal tissues and characterized the response of xenograft tumors to our PDT protocols. EXPERIMENTAL DESIGN: Human small cell lung cancer (NCI-H69), non-small cell lung cancer (A549), and breast cancer (MDA-MB-231) xenografts were induced in SCID mice. Irradiation of sensitized tumors was undertaken through the bodies of tumor-bearing mice to give a treatment depth of 2 cm. Posttreatment tumor regressions and histopathology were carried out to determine the nature of the response to these new PDT agents. Microarray expression profiles were conducted to assess the similarity of responses to single and two-photon activated PDT. RESULTS: Regressions of all tumor types tested were seen. Histopathology was consistent with known PDT effects, and no, or minimal, changes were noted in irradiated normal tissues. Cluster analysis of microarray expression profiling showed reproducible changes in transcripts associated with apoptosis, stress, oxygen transport, and gene regulation. CONCLUSIONS: These new PDT sensitizers can be used at a depth of 2 cm to produce excellent xenograft regressions. The tumor response was consistent with known responses to single-photon activated PDT. Experiments in larger animals are warranted to determine the maximal achievable depth of treatment.


Asunto(s)
Hematoporfirinas/uso terapéutico , Neoplasias/tratamiento farmacológico , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de la radiación , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Hematoporfirinas/síntesis química , Hematoporfirinas/química , Humanos , Rayos Infrarrojos , Rayos Láser , Masculino , Ratones , Ratones SCID , Neoplasias/metabolismo , Neoplasias/patología , Estrés Oxidativo/efectos de la radiación , Oxígeno/metabolismo , Fotones , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/química , Células Tumorales Cultivadas , Irradiación Corporal Total
7.
BMC Genomics ; 8: 3, 2007 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-17201921

RESUMEN

UNLABELLED: The low-shear microgravity environment, modeled by rotating suspension culture bioreactors called high aspect ratio vessels (HARVs), allows investigation in ground-based studies of the effects of microgravity on eukaryotic cells and provides insights into the impact of space flight on cellular physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) causes significant phenotypic changes of a select group of Saccharomyces cerevisiae genes associated with the establishment of cell polarity, bipolar budding, and cell separation. However, the mechanisms cells utilize to sense and respond to microgravity and the fundamental gene expression changes that occur are largely unknown. In this study, we examined the global transcriptional response of yeast cells grown under LSMMG conditions using DNA microarray analysis in order to determine if exposure to LSMMG results in changes in gene expression. RESULTS: LSMMG differentially changed the expression of a significant number of genes (1372) when yeast cells were cultured for either five generations or twenty-five generations in HARVs, as compared to cells grown under identical conditions in normal gravity. We identified genes in cell wall integrity signaling pathways containing MAP kinase cascades that may provide clues to novel physiological responses of eukaryotic cells to the external stress of a low-shear modeled microgravity environment. A comparison of the microgravity response to other environmental stress response (ESR) genes showed that 26% of the genes that respond significantly to LSMMG are involved in a general environmental stress response, while 74% of the genes may represent a unique transcriptional response to microgravity. In addition, we found changes in genes involved in budding, cell polarity establishment, and cell separation that validate our hypothesis that phenotypic changes observed in cells grown in microgravity are reflected in genome-wide changes. This study documents a considerable response to yeast cell growth in low-shear modeled microgravity that is evident, at least in part, by changes in gene expression. Notably, we identified genes that are involved in cell signaling pathways that allow cells to detect environmental changes, to respond within the cell, and to change accordingly, as well as genes of unknown function that may have a unique transcriptional response to microgravity. We also uncovered significant changes in the expression of many genes involved in cell polarization and bud formation that correlate well with the phenotypic effects observed in yeast cells when grown under similar conditions. These results are noteworthy as they have implications for human space flight.


Asunto(s)
Perfilación de la Expresión Génica , Saccharomyces cerevisiae/genética , Ingravidez , Análisis por Conglomerados , Regulación Fúngica de la Expresión Génica , Genómica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Saccharomyces cerevisiae/genética
8.
J Neuroimmunol ; 175(1-2): 19-30, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16631260

RESUMEN

IFN-gamma (IFN-gamma) has been shown to activate astrocytes to acquire immune functions. In this study the effect of IFN-gamma on murine astrocytes was investigated via microarray analysis. The activating effect of IFN-gamma on the astrocyte transcriptome showed predominance toward pathways involved in adaptive immunity, initiation of the immune response and innate immunity. Previously unknown astrocytic genes expressed included members of the p47 GTPases and guanine nucleotide binding protein (GBP) families. Down-regulatory effects of IFN-gamma stimulation were confined to pathways involved in growth regulation, cell differentiation and cell adhesion. This data supports the notion that astrocytes are an important immunocompetant cell in the brain and indicate that astrocytes may have a significant role in various infectious diseases such as Toxoplasmic Encephalitis and neurological diseases with an immunological component such as Alzheimer's and autoimmune disorders.


Asunto(s)
Astrocitos/inmunología , Astrocitos/metabolismo , Regulación de la Expresión Génica/inmunología , Genes MHC Clase II , Interferón gamma/fisiología , Análisis por Micromatrices/métodos , Animales , Células Cultivadas , Perfilación de la Expresión Génica/métodos , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunología
9.
PLoS One ; 8(9): e74647, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24058617

RESUMEN

Bile salts exhibit potent antibacterial properties, acting as detergents to disrupt cell membranes and as DNA-damaging agents. Although bacteria inhabiting the intestinal tract are able to resist bile's antimicrobial effects, relatively little is known about how bile influences virulence of enteric pathogens. Escherichia coli O157:H7 is an important pathogen of humans, capable of causing severe diarrhea and more serious sequelae. In this study, the transcriptome response of E. coli O157:H7 to bile was determined. Bile exposure induced significant changes in mRNA levels of genes related to virulence potential, including a reduction of mRNA for the 41 genes making up the locus of enterocyte effacement (LEE) pathogenicity island. Bile treatment had an unusual effect on mRNA levels for the entire flagella-chemotaxis regulon, resulting in two- to four-fold increases in mRNA levels for genes associated with the flagella hook-basal body structure, but a two-fold decrease for "late" flagella genes associated with the flagella filament, stator motor, and chemotaxis. Bile salts also caused increased mRNA levels for seventeen genes associated with iron scavenging and metabolism, and counteracted the inhibitory effect of the iron chelating agent 2,2'-dipyridyl on growth of E. coli O157:H7. These findings suggest that E. coli O157:H7 may use bile as an environmental signal to adapt to changing conditions associated with the small intestine, including adaptation to an iron-scarce environment.


Asunto(s)
Ácidos y Sales Biliares/farmacología , Escherichia coli O157/genética , Escherichia coli O157/patogenicidad , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/genética , Hierro/metabolismo , Hierro/farmacología , Adhesión Bacteriana/efectos de los fármacos , Adhesión Bacteriana/genética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Escherichia coli O157/efectos de los fármacos , Escherichia coli O157/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/efectos de los fármacos , Flagelos/genética , Islas Genómicas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Virulencia/efectos de los fármacos , Virulencia/genética
10.
PLoS One ; 7(7): e40973, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22808288

RESUMEN

Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.


Asunto(s)
Biopelículas/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Pirazinas/metabolismo , Pirazinas/farmacología , Staphylococcus aureus/fisiología , Apoptosis/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Medios de Cultivo Condicionados/farmacología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Metaboloma/efectos de los fármacos , Proteoma/metabolismo , Pirazinas/química , Staphylococcus aureus/efectos de los fármacos
11.
New Phytol ; 177(2): 333-349, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18028296

RESUMEN

To identify genes involved in the regulation and execution of leaf senescence and whole-plant nitrogen reallocation, near-isogenic barley germplasm divergent in senescence timing and protein concentration of mature grains was contrasted. Barley lines differing in allelic state at a major locus on chromosome six, controlling grain protein concentration, were obtained after four generations of backcrossing. Based on physiological data indicating major differences between low- and high-grain protein germplasm at 14-21 d past anthesis, the flag leaf and kernel transcriptomes of the low-protein parent and one high-protein near-isogenic line were compared at these time points, using the 22-k Barley1 Affymetrix microarray. Our data associate several genes with both known (based on sequence comparisons) and unknown functions with the senescence process. These include leucine-rich repeat transmembrane protein kinases, a glycine-rich RNA-binding protein with homology to AtGRP7 and a 'mother of FT/TF1' gene. Our data also indicate upregulation of genes coding for both plastidial and extraplastidial proteases in germplasm with accelerated leaf senescence. Functional characterization of candidate genes identified by this research may contribute to our understanding of the molecular network underlying leaf senescence and nitrogen reallocation.


Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hordeum/genética , Hordeum/metabolismo , Nitrógeno/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Alelos , Ciclo Celular , Genes de Plantas , Histonas/metabolismo , Hordeum/citología , Proteínas de Plantas/genética , Pliegue de Proteína , ARN de Planta/genética , ARN de Planta/metabolismo , Semillas/metabolismo , Azufre/metabolismo , Factores de Tiempo , Transcripción Genética
12.
J Immunol ; 180(10): 6854-67, 2008 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-18453606

RESUMEN

The fungal pathogen Aspergillus fumigatus is responsible for increasing numbers of fatal infections in immune-compromised humans. Alveolar macrophages (AM) are important in the innate defense against aspergillosis, but little is known about their molecular responses to fungal conidia in vivo. We examined transcriptional changes and superoxide release by AM from C57BL/6 and gp91(phox)(-/-) mice in response to conidia. Following introduction of conidia into the lung, microarray analysis of AM showed the transcripts most strongly up-regulated in vivo to encode chemokines and additional genes that play a critical role in neutrophil and monocyte recruitment, indicating that activation of phagocytes represents a critical early response of AM to fungal conidia. Of the 73 AM genes showing > or = 2-fold changes, 8 were also increased in gp91(phox)(-/-) mice by conidia and in C57BL/6 mice by polystyrene beads, suggesting a common innate response to particulate matter. Ingenuity analysis of the microarray data from C57BL/6 mice revealed immune cell signaling and gene expression as primary mechanisms of this response. Despite the well-established importance of phagocyte NADPH oxidase in resisting aspergillosis, we found no evidence of this mechanism in AM following introduction of conidia into the mouse lung using transcriptional, luminometry, or NBT staining analysis. In support of these findings, we observed that AM from C57BL/6 and gp91(phox)(-/-) mice inhibit conidial germination equally in vitro. Our results indicate that early transcription in mouse AM exposed to conidia in vivo targets neutrophil recruitment, and that NADPH oxidase-independent mechanisms in AM contribute to inhibition of conidial germination.


Asunto(s)
Aspergilosis/inmunología , Expresión Génica , Enfermedades Pulmonares Fúngicas/inmunología , Macrófagos Alveolares/metabolismo , NADPH Oxidasas/metabolismo , Animales , Aspergillus fumigatus , Líquido del Lavado Bronquioalveolar/citología , Ensayo de Inmunoadsorción Enzimática , Activación de Macrófagos/inmunología , Macrófagos Alveolares/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esporas Fúngicas/inmunología , Superóxidos/metabolismo , Transcripción Genética
13.
New Phytol ; 176(1): 56-69, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17803641

RESUMEN

Leaf senescence can be described as the dismantling of cellular components during a specific time interval before cell death. This has the effect of remobilizing N in the form of amino acids that can be relocalized to developing seeds. High levels of carbohydrates have previously been shown to promote the onset of the senescence process. Carbohydrate accumulation in barley (Hordeum vulgare) plants was induced experimentally by steam-girdling at the leaf base, occluding the phloem, and gene regulation under these conditions was investigated using the Affymetrix Barley GeneChip array and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Transcript levels of plastidial (aminopeptidases, cnd41) and vacuolar (thiol and serine) proteases clearly increase in girdled leaves. Of special interest are cnd41, a plastidial aspartyl peptidase that has been implicated in Rubisco degradation in tobacco; and cp-mIII, a highly upregulated carboxypeptidase. SAG12, hexokinases and other senescence-specific genes are also upregulated under these conditions. Applying a genomic approach to the innovative experimental system described here significantly enhances our knowledge of leaf proteolysis and whole-plant N recycling.


Asunto(s)
Metabolismo de los Hidratos de Carbono , Hordeum/metabolismo , Proteínas de Plantas/metabolismo , ARN Mensajero/metabolismo , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hordeum/genética , Hordeum/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Plastidios/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribulosa-Bifosfato Carboxilasa/metabolismo , Vacuolas/enzimología
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