Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 46
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Molecules ; 26(4)2021 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-33673080

RESUMEN

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-ß-cyclodextrin (methyl-ß-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.


Asunto(s)
Lípidos de la Membrana/química , Microdominios de Membrana/química , Receptores LHRH/química , Colesterol/química , Colesterol/genética , Espectroscopía de Resonancia por Spin del Electrón , Células HEK293 , Humanos , Lípidos de la Membrana/genética , Microdominios de Membrana/genética , Microdominios de Membrana/ultraestructura , Dominios Proteicos/genética , Receptores LHRH/genética , Receptores LHRH/uso terapéutico , Receptores LHRH/ultraestructura , Transducción de Señal/genética
2.
Biochim Biophys Acta Biomembr ; 1860(6): 1350-1361, 2018 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-29551275

RESUMEN

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.


Asunto(s)
Exosomas/metabolismo , Colorantes Fluorescentes/metabolismo , Nanopartículas/metabolismo , Compuestos Orgánicos/metabolismo , Coloración y Etiquetado/métodos , Animales , Astrocitos/metabolismo , Centrifugación por Gradiente de Densidad , Exosomas/ultraestructura , Femenino , Citometría de Flujo , Colorantes Fluorescentes/análisis , Microscopía Confocal , Compuestos Orgánicos/análisis , Ratas , Ultracentrifugación
3.
J Neurovirol ; 23(5): 713-724, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28762184

RESUMEN

HIV-1 infection of the central nervous system causes HIV-associated neurocognitive disorders, even in aviremic patients. Although astrocyte malfunction was associated to these disorders, their implication is overshadowed by contributions of microglia and macrophages. Astrocytes are infected with HIV-1 in vivo and express a relevant amount of viral protein Nef. Nef was shown to stimulate its own release in exosomes from diverse cell types, which in turn have damaging effects on neighboring cells. Using immunoblotting and electron microscopy, we showed that human astrocytes expressing Nef.GFP similarly release Nef in exosomes. Importantly, Nef.GFP expression increases the secretion of exosomes from human astrocytes up to 5.5-fold, as determined by total protein content and nanoparticle tracking analysis. Protein analysis of exosomes and viruses separated on iodixanol gradient further showed that native or pseudotyped HIV-1-infected human astrocytes release exosomes, which contain Nef. Our results provide the basis for future studies of the damaging role of Nef-exosomes produced by HIV-infected astrocytes on the central nervous system.


Asunto(s)
Astrocitos/virología , Exosomas/virología , Infecciones por VIH/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Astrocitos/metabolismo , Línea Celular , Exosomas/metabolismo , VIH-1/metabolismo , Humanos
4.
Acta Chim Slov ; 64(3): 530-536, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28862291

RESUMEN

Determining the HIV-1 reservoir size in infected individuals is of great importance for improvement of their treatment. Plasma trans-activation response element (TAR) RNA has been suggested as one of the possible biomarkers. TAR RNA is produced during non-processive transcription in HIV-1 productively infected and latent T cells. Here, plasma samples and paired exosome samples of 55 subjects from the observational SCOPE cohort were analysed for the presence of TAR RNA. First, a PCR-based assay was optimized, which provided 100% specificity and 100% sensitivity in differentiating HIV-1 infected non-controllers from uninfected individuals. Next, TAR RNA was detected in the plasma of 63% of aviremic HIV-1-infected patients, who were either treated with antiretroviral therapy or were elite controllers. Although TAR RNA levels did not correlate with patient gender, age, CD4 levels, CD8 levels, they tended to correlate with CD4/CD8 ratio (P = 0.047). This study is the first to investigate plasma TAR RNA in a relatively large cohort of HIV-1-infected patients. We additionally show that the TAR RNA molecules in the plasma of aviremic patients are not limited to exosomes.


Asunto(s)
Infecciones por VIH/sangre , ARN/sangre , Elementos de Respuesta , Estudios de Cohortes , VIH-1 , Humanos
5.
Adv Exp Med Biol ; 892: 307-325, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26721280

RESUMEN

Fungi that tolerate very high environmental NaCl concentrations are good model systems to study mechanisms that enable them to endure osmotic and salinity stress. The whole genome sequences of six such fungal species have been analysed: Hortaea werneckii, Wallemia ichthyophaga and four Aureobasidium spp.: A. pullulans, A. subglaciale, A. melanogenum and A. namibiae. These fungi show different levels of halotolerance, with the presence of numerous membrane transport systems uncovered here that are believed to maintain physiological intracellular concentrations of alkali metal cations. Despite some differences, the intracellular cation contents of H. werneckii, A. pullulans and W. ichthyophaga remain low even under extreme extracellular salinities, which suggests that these species have efficient cation transport systems. We speculate that cation transporters prevent intracellular accumulation of Na(+), and thus avoid the toxic effects that such Na(+) accumulation would have, while also maintaining the high K(+)/Na(+) ratio that is required for the full functioning of the cell - another crucial task in high-Na(+) environments. This chapter primarily summarises the cation transport systems of these selected fungi, and it also describes other membrane transporters that might be involved in their mechanisms of halotolerance.


Asunto(s)
Basidiomycota/metabolismo , Proteínas Fúngicas/metabolismo , Saccharomycetales/metabolismo , Cloruro de Sodio/metabolismo , Basidiomycota/efectos de los fármacos , Basidiomycota/genética , Proteínas Fúngicas/genética , Expresión Génica , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Transporte Iónico , Presión Osmótica , Canales de Potasio/genética , Canales de Potasio/metabolismo , Saccharomycetales/efectos de los fármacos , Saccharomycetales/genética , Salinidad , Tolerancia a la Sal , Cloruro de Sodio/farmacología , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Estrés Fisiológico
6.
Fungal Genet Biol ; 74: 45-58, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25483129

RESUMEN

Although suggested, the involvement of the HOG pathway in adaptation processes in extremely halotolerant fungus Hortaea werneckii has never been specifically demonstrated. Here, we show that the H. werneckii HOG pathway is very robust, and that it includes two functionally redundant MAPK homologues, HwHog1A and HwHog1B, that show osmolyte-type-dependent phosphorylation. Inhibition of HwHog1 kinase activity with the ATP analogue BPTIP restricts H. werneckii colony growth at 3.0M NaCl, KCl and sorbitol, most likely due to restricted cell division. On the other hand, HwHog1-regulated transcription of a selected group of genes (HwSTL1, HwGUT2, HwOPI3, HwGDH1, HwUGP1, HwGPD1) is an osmolyte-specific process that is important for induction of gene transcription with high NaCl, for regulation of specific genes with high sorbitol, and has no role in KCl stressed cells. Survival of H. werneckii at moderate NaCl and KCl concentrations is not dependent on HwHog1 activity or the calcineurin pathway, and thus alternative mechanisms must exist. The HOG pathway described here is vital for the extreme osmotolerance of H. werneckii, and its regulation shows important differences from the homologue pathways characterised in other mesophilic and halotolerant fungi.


Asunto(s)
Adaptación Fisiológica , Ascomicetos/enzimología , Proteínas Fúngicas/fisiología , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/fisiología , Adaptación Fisiológica/genética , Ascomicetos/efectos de los fármacos , Ascomicetos/genética , Secuencia de Bases , Calcineurina/metabolismo , Simulación por Computador , Proteínas Fúngicas/genética , Redes y Vías Metabólicas/genética , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/genética , Datos de Secuencia Molecular , Concentración Osmolar , Cloruro de Potasio/farmacología , Cloruro de Sodio/farmacología , Sorbitol/farmacología
7.
Extremophiles ; 17(4): 623-36, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23712906

RESUMEN

The high-osmolarity glycerol (HOG) pathway is one of the several MAP kinase cascades in fungi. It is the main signal transduction system that is responsible for cellular stress responses, and has primarily been studied in the context of osmotic stress. In the present study, we provide the first insights into the HOG pathway of the obligatory halophilic basidiomycetous fungus Wallemia ichthyophaga, with the characterisation of its two Hog1-like kinases: WiHog1A and WiHog1B. These share high similarity to Hog1 kinase from Saccharomyces cerevisiae (ScHog1) at the level of amino-acid sequence. While WiHog1A could not optimally complement the function of ScHog1, WiHog1B was a fully functional Hog1-like kinase and could improve the halotolerance of the yeast, compared to the wild-type or the ScHog1-expressing hog1Δ strain. In W. ichthyophaga cells, Hog1 was constitutively phosphorylated under optimal osmotic conditions and dephosphorylated when the cells were challenged with hypo-osmolar or hyperosmolar stress. This pattern of phosphorylation kinetics is opposite to that of yeast. Transcriptional analysis of these two kinases in W. ichthyophaga shows that WiHOG1B is more responsive to changes in NaCl concentrations than WiHOG1A. Our identification and characterisation of these Hog1-like kinases from W. ichthyophaga confirm the existence of the HOG signalling pathway and its role in osmosensing in this halophilic fungus.


Asunto(s)
Basidiomycota/enzimología , Proteínas Fúngicas/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Secuencia de Aminoácidos , Basidiomycota/genética , Basidiomycota/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/genética , Datos de Secuencia Molecular , Mutación , Presión Osmótica , Fosforilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Nucleic Acids Res ; 39(18): 7908-19, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21724609

RESUMEN

Autoimmune regulator (AIRE) is a transcription factor that induces the expression of a large subset of otherwise strictly tissue restricted antigens in medullary thymic epithelial cells, thereby enabling their presentation to developing T cells for negative selection. Mutations in AIRE lead to autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare monogenetic disease. Although it has been reported that AIRE interacts with proteins involved in nuclear transport, DNA-damage response, chromatin remodeling, transcription and pre-mRNA-splicing, the precise mechanism of AIRE-induced tissue restricted antigen expression has remained elusive. In this study, we investigated an APECED patient mutation that causes the loss of the extreme C-terminus of AIRE and found that this mutant protein is transcriptionaly inactive. When tethered heterologously to DNA, this domain could stimulate transcription and splicing by itself. Moreover, the loss of this C-terminus disrupted interactions with the positive transcription elongation factor b (P-TEFb). Via P-TEFb, AIRE increased levels of RNA polymerase II on and enhanced pre-mRNA splicing of heterologous and endogenous target genes. Indeed, the inhibition of CDK9, the kinase subunit of P-TEFb, inhibited AIRE-induced pre-mRNA splicing of these genes. Thus, AIRE requires P-TEFb to activate transcription elongation and co-transcriptional processing of target genes.


Asunto(s)
Mutación , Poliendocrinopatías Autoinmunes/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Empalme del ARN , Factores de Transcripción/genética , Activación Transcripcional , Animales , Línea Celular , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/fisiología , Humanos , Ratones , Fosforilación , Estructura Terciaria de Proteína , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteína AIRE
9.
Traffic ; 11(1): 110-22, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19912576

RESUMEN

The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005;17:879-887). Extracellular Nef protein has deleterious effects on CD4(+) T cells (James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4(+) T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099-3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40-dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302-310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV-1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation-induced cell death of resting PBLs in vitro. Thus, HIV-infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4(+) T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS).


Asunto(s)
Apoptosis , Efecto Espectador , Linfocitos T CD4-Positivos/patología , Exosomas/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiología , Linfocitos T CD4-Positivos/virología , Membrana Celular/metabolismo , Exosomas/ultraestructura , Exosomas/virología , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , VIH-1/metabolismo , Células HeLa , Humanos , Immunoblotting , Células Jurkat , Luciferasas/genética , Microscopía Electrónica , Microscopía Fluorescente , Plásmidos , Transfección , Virión/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/biosíntesis , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo
10.
J Biol Chem ; 286(2): 1499-507, 2011 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-21071449

RESUMEN

The aim of our study was to investigate the association of desmosomal proteins with cholesterol-enriched membrane domains, commonly called membrane rafts, and the influence of cholesterol on desmosome assembly in epithelial Madin-Darby canine kidney cells (clone MDc-2). Biochemical analysis proved an association of desmosomal cadherin desmocollin 2 (Dsc2) in cholesterol-enriched fractions that contain membrane raft markers caveolin-1 and flotillin-1 and the novel raft marker ostreolysin. Cold detergent extraction of biotinylated plasma membranes revealed that ∼60% of Dsc2 associates with membrane rafts while the remainder is present in nonraft and cholesterol-poor membranes. The results of immunofluorescence microscopy confirmed colocalization of Dsc2 and ostreolysin. Partial depletion of cholesterol with methyl-ß-cyclodextrin disturbs desmosome assembly, as revealed by sequential recordings of live cells. Moreover, cholesterol depletion significantly reduces the strength of cell-cell junctions and partially releases Dsc2 from membrane rafts. Our data indicate that a pool of Dsc2 is associated with membrane rafts, particularly with the ostreolysin type of membrane raft, and that intact membrane rafts are necessary for desmosome assembly. Taken together, these data suggest cholesterol as a potential regulator that promotes desmosome assembly.


Asunto(s)
Adhesión Celular/fisiología , Colesterol/metabolismo , Desmocolinas/metabolismo , Desmosomas/fisiología , Microdominios de Membrana/fisiología , Animales , Caveolina 1/metabolismo , Línea Celular , Membrana Celular/fisiología , Perros , Proteínas Fúngicas/metabolismo , Proteínas Hemolisinas/metabolismo , Riñón/citología , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo
11.
Prog Mol Subcell Biol ; 53: 133-58, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22222830

RESUMEN

Solar salterns are constructed as shallow multi-pond systems for the production of halite through evaporation of seawater. The main feature of salterns is the discontinuous salinity gradient that provides a range of well-defined habitats with increasing salinities, from moderate to hypersaline. These present one of the most extreme environments, because of the low levels of biologically available water and the toxic concentrations of ions. Up to the year 2000, hypersaline environments were considered to be populated almost exclusively by prokaryotic microorganisms till fungi were reported to be active inhabitants of solar salterns. Since then, numerous fungal species have been described in hypersaline waters around the world. The mycobiota of salterns is represented by different species of the genus Cladosporium and the related meristematic melanized black yeasts, of non-melanized yeasts, of the filamentous genera Penicillium and Aspergillus and their teleomorphic forms (Eurotium and Emericella), and of the basidiomycetous genus Wallemia. Among these, two species became new model organisms for studying the mechanisms of extreme salt tolerance: the extremely halotolerant ascomycetous black yeast Hortaea werneckii and the obligate halophilic basidiomycete Wallemia ichthyophaga.


Asunto(s)
Salinidad , Cloruro de Sodio , Ascomicetos , Ecosistema , Hongos/clasificación , Tolerancia a la Sal , Agua de Mar/microbiología , Levaduras
12.
Fungal Genet Biol ; 48(5): 475-84, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21281727

RESUMEN

In Saccharomyces cerevisiae, the Sho1 protein is one of two potential osmosensors that can activate the kinase cascade of the HOG pathway in response to increased extracellular osmolarity. Two novel SHO1-like genes, HwSHO1A and HwSHO1B, have been cloned from the saltern-inhabiting, extremely halotolerant black yeast Hortaea werneckii. The HwSho1 protein isoforms are 93.8% identical in their amino-acid sequences, and have a conserved SH3 domain. When the HwSHO1 genes were transferred into S. cerevisae cells lacking the SHO1 gene, both of the HwSho1 isoforms fully complemented the function of the native S. cerevisiae Sho1 protein. Through microscopic and biochemical validation, we demonstrate that in S. cerevisiae, both of the HwSho1 proteins have characteristic subcellular localizations similar to the S. cerevisiae Sho1 protein, and they can both activate the HOG pathway under conditions of osmotic stress. To a lower extent, crosstalk to the mating pathway expressing HwSho1 proteins is conserved in the PBS2 deleted S. cerevisiae strain. These data show that the HwSho1 proteins from H. werneckii are true functional homologs of the Sho1 protein of S. cerevisiae.


Asunto(s)
Proteínas Fúngicas/metabolismo , Cloruro de Sodio/metabolismo , Levaduras/metabolismo , Secuencia de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Ósmosis , Filogenia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Levaduras/química , Levaduras/clasificación , Levaduras/genética
13.
Adv Appl Microbiol ; 77: 71-96, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22050822

RESUMEN

Hypersaline environments support substantial microbial communities of selected halotolerant and halophilic organisms, including fungi from various orders. In hypersaline water of solar salterns, the black yeast Hortaea werneckii is by far the most successful fungal representative. It has an outstanding ability to overcome the turgor loss and sodium toxicity that are typical for hypersaline environments, which facilitates its growth even in solutions that are almost saturated with NaCl. We propose a model of cellular responses to high salt concentrations that integrates the current knowledge of H. werneckii adaptations. The negative impact of a hyperosmolar environment is counteracted by an increase in the energy supply that is needed to drive the energy-demanding export of ions and synthesis of compatible solutes. Changes in membrane lipid composition and cell-wall structure maintain the integrity and functioning of the stressed cells. Understanding the salt responses of H. werneckii and other fungi (e.g., the halophilic Wallemia ichthyophaga) will extend our knowledge of fungal stress tolerance and promote the use of the currently unexploited biotechnological potential of fungi that live in hypersaline environments.


Asunto(s)
Hongos , Cloruro de Sodio , Aclimatación , Adaptación Fisiológica , Ascomicetos , Proteínas Fúngicas/genética , Hongos/metabolismo , Regulación Fúngica de la Expresión Génica , Filogenia , Cloruro de Sodio Dietético
14.
J Fungi (Basel) ; 7(11)2021 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-34829275

RESUMEN

Sensing and responding to changes in NaCl concentration in hypersaline environments is vital for cell survival. In this paper, we identified and characterized key components of the high-osmolarity glycerol (HOG) signal transduction pathway, which is crucial in sensing hypersaline conditions in the extremely halotolerant black yeast Hortaea werneckii and in the obligate halophilic fungus Wallemia ichthyophaga. Both organisms were isolated from solar salterns, their predominating ecological niche. The identified components included homologous proteins of both branches involved in sensing high osmolarity (SHO1 and SLN1) and the homologues of mitogen-activated protein kinase module (MAPKKK Ste11, MAPKK Pbs2, and MAPK Hog1). Functional complementation of the identified gene products in S. cerevisiae mutant strains revealed some of their functions. Structural protein analysis demonstrated important structural differences in the HOG pathway components between halotolerant/halophilic fungi isolated from solar salterns, salt-sensitive S. cerevisiae, the extremely salt-tolerant H. werneckii, and halophilic W. ichthyophaga. Known and novel gene targets of MAP kinase Hog1 were uncovered particularly in halotolerant H. werneckii. Molecular studies of many salt-responsive proteins confirm unique and novel mechanisms of adaptation to changes in salt concentration.

15.
Sci Rep ; 10(1): 21346, 2020 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-33288809

RESUMEN

Human plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation-sUC) and size- (size exclusion chromatography-SEC) based methods. According to nanoparticle tracking analysis (NTA), asymmetrical flow field-flow fractionation coupled to detectors (AF4-UV-MALS), miRNA quantification, transmission electron microscopy and enzyme-linked immunosorbent assay, enrichment of EVs from plasma with sUC method lead to high purity of EVs in the samples. High nanoparticle concentrations after SEC resulted from substantial contamination with lipoproteins and other aggregates of EV-like sizes that importantly affect downstream EV quantification. Additionally, sUC EV-enrichment method linked to quantification with NTA or AF4-UV-MALS is repeatable, as the relative standard deviation of EV size measured in independently processed samples from the same plasma source was 5.4% and 2.1% when analyzed by NTA or AF4-UV-MALS, respectively. In conclusion, the sUC EV-enrichment method is compatible with reliable measurement of concentration and size of EVs from plasma and should in the future be tested on larger cohorts in relation to different diseases. This is one of the first studies using AF4-UV-MALS to quantify EVs in blood plasma, which opens new possible clinical utility for the technique.


Asunto(s)
Biomarcadores/análisis , Vesículas Extracelulares/química , Plasma/química , Cromatografía en Gel , Femenino , Fraccionamiento de Campo-Flujo , Humanos , Masculino , Persona de Mediana Edad , Nanopartículas/química , Proteómica
16.
FEMS Yeast Res ; 9(2): 247-56, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19220869

RESUMEN

The black yeast-like fungus Hortaea werneckii is the predominant fungal species in salterns, and it is extremely halotolerant. The restructuring of the H. werneckii membrane lipid composition is one of its adaptations to high concentrations of salt, which is mainly achieved by increasing the unsaturation of its phospholipid fatty acids. Genes encoding three fatty acid-modifying enzymes, Delta(9)-, Delta(12)-desaturases and an elongase, have been identified in the genome of H. werneckii, each in two copies. Their transcription profiles show responsiveness to different salinity conditions, with the lowest expression at optimal salinity. Transcriptional responses to hyperosmotic and hypo-osmotic shock show substantial differences between cells exposed to osmotic shock and cells adapted to an osmotically stressful environment.


Asunto(s)
Acetiltransferasas/biosíntesis , Antifúngicos/farmacología , Ascomicetos/efectos de los fármacos , Ácido Graso Desaturasas/biosíntesis , Regulación Fúngica de la Expresión Génica , Sales (Química)/farmacología , Adaptación Fisiológica , Ascomicetos/enzimología , Ascomicetos/fisiología , Elongasas de Ácidos Grasos , Perfilación de la Expresión Génica , Presión Osmótica , Filogenia , Homología de Secuencia de Aminoácido , Estearoil-CoA Desaturasa
17.
Mycol Res ; 113(Pt 11): 1231-41, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19747974

RESUMEN

Extreme environments have for long been considered to be populated almost exclusively by prokaryotic organisms and therefore monopolized by bacteriologists. Solar salterns are natural hypersaline environments characterized by extreme concentrations of NaCl, often high concentrations of other ions, high uv irradiation and in some cases extremes in pH. In 2000 fungi were first reported to be active inhabitants of solar salterns. Since then many new species and species previously known only as food contaminants have been discovered in hypersaline environments around the globe. The eukaryotic microorganism most studied for its salt tolerance is Saccharomyces cerevisiae. However, S. cerevisiae is rather salt sensitive and not able to adapt to hypersaline conditions. In contrast, some species like Debaryomyces hansenii, Hortaea werneckii, and Wallemia ichthyophaga have been isolated globally from natural hypersaline environments. We believe that all three are more suitable model organisms to study halotolerance in eukaryotes than S. cerevisiae. Furthermore, they belong to different and distant taxonomic groups and have developed different strategies to cope with the same problems of ion toxicity and loss of water.


Asunto(s)
Hongos/fisiología , Tolerancia a la Sal/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hongos/ultraestructura , Regulación Fúngica de la Expresión Génica/fisiología , Homeostasis
18.
Fungal Genet Biol ; 45(6): 994-1007, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18343697

RESUMEN

We have investigated the mitochondrial responses to hyperosmotic environments of ionic (4.5 M NaCl) and non-ionic (3.0 M sorbitol) osmolytes in the most halo/osmo-tolerant black yeast, Hortaea werneckii. Adaptation to both types of osmolytes resulted in differential expression of mitochondria-related genes. Live-cell imaging has revealed a condensation of mitochondria in hyperosmotic media that depends on osmolyte type. In the hypersaline medium, this was accompanied by increased ATP synthesis and oxidative damage protection, whereas adaptation to the non-ionic osmolyte resulted in a decrease in ATP synthesis and lipid peroxidation level in mitochondria. A proteomic study of the mitochondria revealed preferential accumulation of energy metabolism enzymes in the hypersaline medium, and accumulation of protein chaperones in the non-ionic osmolyte. The HwBmh1/14-3-3 protein, localized to mitochondria in hypersaline conditions, and not at optimal salinity, suggesting its role in differential perception of ionic and non-ionic osmolytes in H. werneckii.


Asunto(s)
Ascomicetos/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Mitocondrias/genética , Cloruro de Sodio/farmacología , Sorbitol/farmacología , Ascomicetos/efectos de los fármacos , Ascomicetos/crecimiento & desarrollo , Ascomicetos/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Presión Osmótica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cloruro de Sodio/metabolismo , Sorbitol/metabolismo
19.
FEMS Microbiol Rev ; 42(3): 353-375, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29529204

RESUMEN

Hypersaline environments with salt concentrations up to NaCl saturation are inhabited by a great diversity of microorganisms belonging to the three domains of life. They all must cope with the low water activity of their environment, but different strategies exist to provide osmotic balance of the cells' cytoplasm with the salinity of the medium. One option used by many halophilic Archaea and a few representatives of the Bacteria is to accumulate salts, mainly KCl and to adapt the entire intracellular machinery to function in the presence of molar concentrations of salts. A more widespread option is the synthesis or accumulation of organic osmotic, so-called compatible solutes. Here, we review the mechanisms of osmotic adaptation in a number of model organisms, including the KCl accumulating Halobacterium salinarum (Archaea) and Salinibacter ruber (Bacteria), Halomonas elongata as a representative of the Bacteria that synthesize organic osmotic solutes, eukaryotic microorganisms including the unicellular green alga Dunaliella salina and the black yeasts Hortaea werneckii and the basidiomycetous Wallemia ichthyophaga, which use glycerol and other compatible solutes. The strategies used by these model organisms and by additional halophilic microorganisms presented are then compared to obtain an integrative picture of the adaptations to life at high salt concentrations in the microbial world.


Asunto(s)
Adaptación Fisiológica , Archaea/fisiología , Fenómenos Fisiológicos Bacterianos , Chlorophyta/fisiología , Hongos/fisiología , Salinidad , Modelos Biológicos
20.
PLoS One ; 13(1): e0191613, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29364927

RESUMEN

OBJECTIVE: To address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects. DESIGN: We optimized an HIV Nef enzyme-linked immunosorbent assay (ELISA) and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir in an HIV-infected cohort. METHODS: This study accessed 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort). We optimized an ELISA for detection of plasma Nef in HIV-negative subjects and HIV-infected non-controllers, and evaluated its utility to quantify plasma Nef levels in a cross-sectional study of ART-suppressed and elite controller HIV-infected subjects. RESULTS: Here, we describe the performance of an optimized HIV Nef ELISA. When we applied this assay to the study cohort we found that plasma Nef levels were correlated with plasma HIV RNA levels in untreated disease. However, we were able to detect Nef in plasma of approximately half of subjects on ART or with elite control, despite the lack of detectable plasma HIV RNA levels using standard assays. Plasma Nef levels were not consistently associated with CD4+ T-cell count, CD8+ T-cell count, self-reported nadir CD4+ T-cell count or the CD4+/CD8+ T-cell ratio in HIV-infected subjects. CONCLUSION: Since plasma HIV RNA levels are undetectable in virally suppressed subjects, it is reasonable to assume that viral protein expression in leaky reservoir, and not plasma virions, is the source of Nef accumulating in plasma. To examine this further, improvements of the assay sensitivity, by lowering the background through improvements in the quality of Nef antibodies, and detailed characterization of the HIV reservoirs are needed.


Asunto(s)
Productos del Gen nef/sangre , Infecciones por VIH/sangre , ARN Viral/sangre , Carga Viral , Adulto , Anciano , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA