RESUMEN
Molecular mechanisms that maintain lineage integrity of helper T cells are largely unknown. Here we show histone deacetylases 1 and 2 (HDAC1 and HDAC2) as crucial regulators of this process. Loss of HDAC1 and HDAC2 during late T cell development led to the appearance of major histocompatibility complex (MHC) class II-selected CD4(+) helper T cells that expressed CD8-lineage genes such as Cd8a and Cd8b1. HDAC1 and HDAC2-deficient T helper type 0 (TH0) and TH1 cells further upregulated CD8-lineage genes and acquired a CD8(+) effector T cell program in a manner dependent on Runx-CBFß complexes, whereas TH2 cells repressed features of the CD8(+) lineage independently of HDAC1 and HDAC2. These results demonstrate that HDAC1 and HDAC2 maintain integrity of the CD4 lineage by repressing Runx-CBFß complexes that otherwise induce a CD8(+) effector T cell-like program in CD4(+) T cells.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Células TH1/inmunología , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Citocinas/metabolismo , Citotoxicidad Inmunológica/genética , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión ProteicaRESUMEN
The gaseous hormone ethylene is perceived in plants by membrane-bound receptors, the best studied of these being ETR1 from Arabidopsis. Ethylene receptors can mediate a response to ethylene concentrations at less than one part per billion; however, the mechanistic basis for such high-affinity ligand binding has remained elusive. Here we identify an Asp residue within the ETR1 transmembrane domain that plays a critical role in ethylene binding. Site-directed mutation of the Asp to Asn results in a functional receptor that has a reduced affinity for ethylene, but still mediates ethylene responses in planta. The Asp residue is highly conserved among ethylene receptor-like proteins in plants and bacteria, but Asn variants exist, pointing to the physiological relevance of modulating ethylene-binding kinetics. Our results also support a bifunctional role for the Asp residue in forming a polar bridge to a conserved Lys residue in the receptor to mediate changes in signaling output. We propose a new structural model for the mechanism of ethylene binding and signal transduction, one with similarities to that found in a mammalian olfactory receptor.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Receptores de Superficie Celular/metabolismo , Etilenos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Plants must rapidly and dynamically adapt to changes in their environment. Upon sensing environmental signals, plants convert them into cellular signals, which elicit physiological or developmental changes that allow them to respond to various abiotic and biotic cues. Because plants can be simultaneously exposed to multiple environmental cues, signal integration between plant cells, tissues, and organs is necessary to induce specific responses. Recently, CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides and their cognate CLAVATA-type receptors received increased attention for their roles in plant-environment interactions. CLE peptides are mobile signaling molecules, many of which are induced by a variety of biotic and abiotic stimuli. Secreted CLE peptides are perceived by receptor complexes on the surface of their target cells, which often include the leucine-rich repeat receptor-like kinase CLAVATA1. Receptor activation then results in cell-type and/or environment-specific responses. This review summarizes our current understanding of the diverse roles of environment-regulated CLE peptides in modulating plant responses to environmental cues. We highlight how CLE signals regulate plant physiology by fine-tuning plant-microbe interactions, nutrient homeostasis, and carbon allocation. Finally, we describe the role of CLAVATA receptors in the perception of environment-induced CLE signals and discuss how diverse CLE-CLAVATA signaling modules may integrate environmental signals with plant physiology and development.
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Interacción Gen-Ambiente , Transducción de Señal , Transporte Biológico , Carbono , PéptidosRESUMEN
Chronic rhinosinusitis (CRS) is a persistent nasal and paranasal sinus mucosa inflammation comprising two phenotypes, namely CRS with nasal polyps (CRSwNP) and without (CRSsNP). CRSwNP can be associated with asthma and hypersensitivity to non-steroidal anti-inflammatory drug (NSAID) in a syndrome known as NSAID-exacerbated respiratory disease (N-ERD). Furthermore, CRS frequently intertwines with respiratory allergies. This study investigated levels of 33 different nasal and serum cytokines and phenotypic characteristics of peripheral blood mononuclear cells (PBMCs) within cohorts of CRS patients (n = 24), additionally examining the influence of comorbid respiratory allergies by mass cytometry. N-ERD patients showed heightened type 2 nasal cytokine levels. Mass cytometry revealed increased activated naive B cell levels in CRSwNP and N-ERD, while resting naive B cells were higher in CRSsNP. Th2a cell levels were significantly elevated in allergic subjects, but not in CRS groups. In conclusion, there are distinct immunological features in PBMCs of CRS phenotypes and allergy.
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Hipersensibilidad , Pólipos Nasales , Rinitis , Rinosinusitis , Sinusitis , Humanos , Leucocitos Mononucleares , Enfermedad Crónica , CitocinasRESUMEN
BACKGROUND: Legumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood. RESULTS: We report a new hypernodulating mutant, defective in autoregulation, with disruption of a gene, DAR (Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. The dar-1 mutant produces ten-fold more nodules than wild type, similar to AON mutants with disrupted SUNN gene function. As in sunn mutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished in dar. Furthermore, dar-1 also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlike SUNN which functions in the shoot to control nodulation, DAR functions in the root. CONCLUSIONS: DAR encodes a membrane protein that is a member of a small protein family in M. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis. DAR gene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.
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Medicago truncatula , Micorrizas , Proteínas de Plantas , Nodulación de la Raíz de la Planta , Simbiosis , Medicago truncatula/genética , Medicago truncatula/microbiología , Medicago truncatula/fisiología , Micorrizas/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulación de la Raíz de la Planta/genética , Simbiosis/genética , Regulación de la Expresión Génica de las Plantas , Mutación , Genes de Plantas , Raíces de Plantas/microbiología , Raíces de Plantas/genética , Homeostasis , Nódulos de las Raíces de las Plantas/microbiología , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/metabolismoRESUMEN
INTRODUCTION: Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. METHODS: Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4+ T cells from patients with RA was established to quantify cell-cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell-cell interaction. RESULTS: Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4+ T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. CONCLUSION: Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.
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Artritis Reumatoide , Sinoviocitos , Humanos , Citocinas , Factor de Necrosis Tumoral alfa/farmacología , Membrana Sinovial/patología , Sinoviocitos/patología , Fibroblastos/patología , Células CultivadasRESUMEN
Climate warming has been suggested to impact high latitude grasslands severely, potentially causing considerable carbon (C) losses from soil. Warming can also stimulate nitrogen (N) turnover, but it is largely unclear whether and how altered N availability impacts belowground C dynamics. Even less is known about the individual and interactive effects of warming and N availability on the fate of recently photosynthesized C in soil. On a 10-year geothermal warming gradient in Iceland, we studied the effects of soil warming and N addition on CO2 fluxes and the fate of recently photosynthesized C through CO2 flux measurements and a 13 CO2 pulse-labeling experiment. Under warming, ecosystem respiration exceeded maximum gross primary productivity, causing increased net CO2 emissions. N addition treatments revealed that, surprisingly, the plants in the warmed soil were N limited, which constrained primary productivity and decreased recently assimilated C in shoots and roots. In soil, microbes were increasingly C limited under warming and increased microbial uptake of recent C. Soil respiration was increased by warming and was fueled by increased belowground inputs and turnover of recently photosynthesized C. Our findings suggest that a decade of warming seemed to have induced a N limitation in plants and a C limitation by soil microbes. This caused a decrease in net ecosystem CO2 uptake and accelerated the respiratory release of photosynthesized C, which decreased the C sequestration potential of the grassland. Our study highlights the importance of belowground C allocation and C-N interactions in the C dynamics of subarctic ecosystems in a warmer world.
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Carbono , Ecosistema , Pradera , Dióxido de Carbono , Nitrógeno , Plantas , SueloRESUMEN
Herein, we present, to our knowledge, the first spectroscopic characterization of the Cu(I) active site of the plant ethylene receptor ETR1. The x-ray absorption (XAS) and extended x-ray absorption fine structure (EXAFS) spectroscopies presented here establish that ETR1 has a low-coordinate Cu(I) site. The EXAFS resolves a mixed first coordination sphere of N/O and S scatterers at distances consistent with potential histidine and cysteine residues. This finding agrees with the coordination of residues C65 and H69 to the Cu(I) site, which are critical for ethylene activity and well conserved. Furthermore, the Cu K-edge XAS and EXAFS of ETR1 exhibit spectroscopic changes upon addition of ethylene that are attributed to modifications in the Cu(I) coordination environment, suggestive of ethylene binding. Results from umbrella sampling simulations of the proposed ethylene binding helix of ETR1 at a mixed quantum mechanics/molecular mechanics level agree with the EXAFS fit distance changes upon ethylene binding, particularly in the increase of the distance between H69 and Cu(I), and yield binding energetics comparable with experimental dissociation constants. The observed changes in the copper coordination environment might be the triggering signal for the transmission of the ethylene response.
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Cobre , Histidina , Sitios de Unión , Cobre/química , Cisteína/química , Etilenos , Espectroscopía de Absorción de Rayos X , Receptores de Superficie CelularRESUMEN
Type 1 innate lymphoid cells (ILC1) are tissue-resident lymphocytes that provide early protection against bacterial and viral infections. Discrete transcriptional states of ILC1 have been identified in homeostatic and pathological contexts. However, whether these states delineate ILC1 with different functional properties is not completely understood. Here, we show that liver ILC1 are heterogeneous for the expression of distinct effector molecules and surface receptors, including granzyme A (GzmA) and CD160, in mice. ILC1 expressing high levels of GzmA are enriched in the liver of adult mice, and represent the main hepatic ILC1 population at birth. However, the heterogeneity of GzmA and CD160 expression in hepatic ILC1 begins perinatally and increases with age. GzmA+ ILC1 differ from NK cells for the limited homeostatic requirements of JAK/STAT signals and the transcription factor Nfil3. Moreover, by employing Rorc(γt)-fate map (fm) reporter mice, we established that ILC3-ILC1 plasticity contributes to delineate the heterogeneity of liver ILC1, with RORγt-fm+ cells skewed toward a GzmA- CD160+ phenotype. Finally, we showed that ILC1 defined by the expression of GzmA and CD160 are characterized by graded cytotoxic potential and ability to produce IFN-γ. In conclusion, our findings help deconvoluting ILC1 heterogeneity and provide evidence for functional diversification of liver ILC1.
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Hígado/citología , Hígado/inmunología , Subgrupos Linfocitarios/citología , Linfocitos/citología , Animales , Antígenos CD/metabolismo , Proteínas Ligadas a GPI/metabolismo , Granzimas/metabolismo , Inmunidad Innata/inmunología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Receptores Inmunológicos/metabolismoRESUMEN
Arbuscular mycorrhizal (AM) symbiosis is accompanied by alterations to root cell metabolism and physiology, and to the pathways of orthophosphate (Pi) entry into the root, which increase with Pi delivery to cortical cells via arbuscules. How AM symbiosis influences the Pi content and Pi response dynamics of cells in the root cortex and epidermis is unknown. Using fluorescence resonance energy transfer (FRET)-based Pi biosensors, we mapped the relative cytosolic and plastidic Pi content of Brachypodium distachyon mycorrhizal root cells, analyzed responses to extracellular Pi and traced extraradical hyphae-mediated Pi transfer to colonized cells. Colonized cortical cells had a higher cytosolic Pi content relative to noncolonized cortical and epidermal cells, while plastidic Pi content was highest in cells at the infection front. Pi application to the entire mycorrhizal root resulted in transient changes in cytosolic Pi that differed in direction and magnitude depending on cell type and arbuscule status; cells with mature arbuscules showed a substantial transient increase in cytosolic Pi while those with collapsed arbuscules showed a decrease. Directed Pi application to extraradical hyphae resulted in measurable changes in cytosolic Pi of colonized cells 18 h after application. Our experiments reveal that cells within a mycorrhizal root vary in Pi content and Pi response dynamics.
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Técnicas Biosensibles , Brachypodium , Micorrizas , Brachypodium/genética , Brachypodium/metabolismo , Regulación de la Expresión Génica de las Plantas , Micorrizas/fisiología , Fosfatos/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Simbiosis/fisiologíaRESUMEN
Climate change is expected to increase the frequency and severity of droughts. These events, which can cause significant perturbations of terrestrial ecosystems and potentially long-term impacts on ecosystem structure and functioning after the drought has subsided are often called 'drought legacies'. While the immediate effects of drought on ecosystems have been comparatively well characterized, our broader understanding of drought legacies is just emerging. Drought legacies can relate to all aspects of ecosystem structure and functioning, involving changes at the species and the community scale as well as alterations of soil properties. This has consequences for ecosystem responses to subsequent drought. Here, we synthesize current knowledge on drought legacies and the underlying mechanisms. We highlight the relevance of legacy duration to different ecosystem processes using examples of carbon cycling and community composition. We present hypotheses characterizing how intrinsic (i.e. biotic and abiotic properties and processes) and extrinsic (i.e. drought timing, severity, and frequency) factors could alter resilience trajectories under scenarios of recurrent drought events. We propose ways for improving our understanding of drought legacies and their implications for subsequent drought events, needed to assess the longer-term consequences of droughts on ecosystem structure and functioning.
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Sequías , Ecosistema , Ciclo del Carbono , Cambio Climático , SueloRESUMEN
The development of flexible and reconfigurable sensors that can be readily tailored toward different molecular analytes constitutes a key goal and formidable challenge in biosensing. In this regard, synthetic nanopores have emerged as potent physical transducers to convert molecular interactions into electrical signals. Yet, systematic strategies to functionalize their surfaces with receptor proteins for the selective detection of molecular analytes remain scarce. Addressing these limitations, a general strategy is presented to immobilize nanobodies in a directional fashion onto the surface of track-etched nanopores exploiting copper-free click reactions and site-specific protein conjugation systems. The functional immobilization of three different nanobodies is demonstrated in ligand binding experiments with green fluorescent protein, mCherry, and α-amylase (α-Amy) serving as molecular analytes. Ligand binding is resolved using a combination of optical and electrical recordings displaying quantitative dose-response curves. Furthermore, a change in surface charge density is identified as the predominant molecular factor that underlies quantitative dose-responses for the three different protein analytes in nanoconfined geometries. The devised strategy should pave the way for the systematic functionalization of nanopore surfaces with biological receptors and their ability to detect a variety of analytes for diagnostic purposes.
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Técnicas Biosensibles , Nanoporos , Electricidad , ProteínasRESUMEN
Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial association of plants and fungi of the subphylum Glomeromycotina. Endosymbiotic AM fungi colonize the inner cortical cells of the roots, where they form branched hyphae called arbuscules that function in nutrient exchange with the plant. To support arbuscule development and subsequent bidirectional nutrient exchange, the root cortical cells undergo substantial transcriptional reprogramming. REDUCED ARBUSCULAR MYCORRHIZA1 (RAM1), previously studied in several dicot plant species, is a major regulator of this cortical cell transcriptional program. Here, we generated ram1 mutants and RAM1 overexpressors in a monocot, Brachypodium distachyon. The AM phenotypes of two ram1 lines revealed that RAM1 is only partly required to enable arbuscule development in B. distachyon Transgenic lines constitutively overexpressing BdRAM1 showed constitutive expression of AM-inducible genes even in the shoots. Following inoculation with AM fungi, BdRAM1-overexpressing plants showed higher arbuscule densities relative to controls, indicating the potential to manipulate the relative proportion of symbiotic interfaces via modulation of RAM1 However, the overexpressors also show altered expression of hormone biosynthesis genes and aberrant growth patterns, including stunted bushy shoots and poor seed set. While these phenotypes possibly provide additional clues about the scope of influence of BdRAM1, they also indicate that directed approaches to increase the density of symbiotic interfaces will require a more focused, potentially cell type specific manipulation of transcription factor gene expression.
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Brachypodium/genética , Brachypodium/microbiología , Glomeromycota/crecimiento & desarrollo , Glomeromycota/genética , Micorrizas/genética , Raíces de Plantas/genética , Simbiosis/genética , Regulación Fúngica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes Fúngicos , Micorrizas/crecimiento & desarrollo , Fenotipo , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Simbiosis/fisiología , Factores de TranscripciónRESUMEN
Photosynthesis and soil respiration represent the two largest fluxes of CO2 in terrestrial ecosystems and are tightly linked through belowground carbon (C) allocation. Drought has been suggested to impact the allocation of recently assimilated C to soil respiration; however, it is largely unknown how drought effects are altered by a future warmer climate under elevated atmospheric CO2 (eT_eCO2 ). In a multifactor experiment on managed C3 grassland, we studied the individual and interactive effects of drought and eT_eCO2 (drought, eT_eCO2 , drought × eT_eCO2 ) on ecosystem C dynamics. We performed two in situ 13 CO2 pulse-labeling campaigns to trace the fate of recent C during peak drought and recovery. eT_eCO2 increased soil respiration and the fraction of recently assimilated C in soil respiration. During drought, plant C uptake was reduced by c. 50% in both ambient and eT_eCO2 conditions. Soil respiration and the amount and proportion of 13 C respired from soil were reduced (by 32%, 70% and 30%, respectively), the effect being more pronounced under eT_eCO2 (50%, 84%, 70%). Under drought, the diel coupling of photosynthesis and SR persisted only in the eT_eCO2 scenario, likely caused by dynamic shifts in the use of freshly assimilated C between storage and respiration. Drought did not affect the fraction of recent C remaining in plant biomass under ambient and eT_eCO2 , but reduced the small fraction remaining in soil under eT_eCO2 . After rewetting, C uptake and the proportion of recent C in soil respiration recovered more rapidly under eT_eCO2 compared to ambient conditions. Overall, our findings suggest that in a warmer climate under elevated CO2 drought effects on the fate of recent C will be amplified and the coupling of photosynthesis and soil respiration will be sustained. To predict the future dynamics of terrestrial C cycling, such interactive effects of multiple global change factors should be considered.
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Carbono , Suelo , Dióxido de Carbono/análisis , Sequías , Ecosistema , Pradera , RespiraciónRESUMEN
Claviceps purpurea is an ergot fungus known for its neurotropic alkaloids, which have been identified as the main cause of ergotism, a livestock and human disease triggered by ergot consumption. Tetrahydroxanthone dimers, the so-called ergopigments, presumably also contribute to this toxic effect. Overexpression of the cluster-specific transcription factor responsible for the formation of these pigments in C. purpurea led to the isolation of three new metabolites (8-10). The new pigments were characterized utilizing HRMS, NMR techniques, and CD spectroscopy and shown to be xanthone dimers. Secalonic acid A and its 2,4'- and 4,4'-linked isomers were also isolated, and their absolute configuration was investigated. The contribution of secalonic acid A, its isomers, and new metabolites to the toxicity of C. purpurea was investigated in HepG2 and CCF-STTG1 cells. Along with cytotoxic properties, secalonic acid A was found to inhibit topoisomerase I and II activity.
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Claviceps/química , Xantenos/química , Células Hep G2 , Humanos , Estructura Molecular , Inhibidores de Topoisomerasa , XantonasRESUMEN
Cathelicidins have been reported to inhibit human papillomavirus infection in vitro; however, nothing is known about their activity in vivo. In this study, experimental skin infection with Mus musculus papillomavirus 1 resulted in robust development of cutaneous papillomas in cyclosporine A-treated C57BL/6J mice deficient for the murine cathelicidin-related antimicrobial peptide (CRAMP), in contrast to wild-type controls. Analysis of the underlying mechanisms revealed moderate disruption of virion integrity and lack of interference with viral entry and intracellular trafficking by a synthetic CRAMP peptide. Differences in the immune response to Mus musculus papillomavirus 1 infection were observed between CRAMP-deficient and wild-type mice. These included a stronger reduction in CD4+ and CD8+ T-cell numbers in infected skin, and lack of Mus musculus papillomavirus 1-specific neutralizing antibodies in response to cyclosporine A in the absence of endogenous CRAMP. CRAMP has modest direct anti-papillomaviral effects in vitro, but exerts protective functions against Mus musculus papillomavirus 1 skin infection and disease development in vivo, primarily by modulation of cellular and humoral immunity.
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Papiloma , Papillomaviridae , Animales , Péptidos Catiónicos Antimicrobianos , Catelicidinas , Ratones , Ratones Endogámicos C57BL , Papiloma/inducido químicamente , Papillomaviridae/genéticaRESUMEN
Nevadensin, an abundant polyphenol of basil, is reported to reduce alkenylbenzene DNA adduct formation. Furthermore, it has a wide spectrum of further pharmacological properties. The presented study focuses the impact of nevadensin on topoisomerases (TOPO) in vitro. Considering the DNA-intercalating properties of flavonoids, first, minor groove binding properties (IC50 = 31.63 µM), as well as DNA intercalation (IC50 = 296.91 µM) of nevadensin, was found. To determine potential in vitro effects on TOPO I and TOPO IIα, the relaxation and decatenation assay was performed in a concentration range of 1-500 µM nevadensin. A partial inhibition was detected for TOPO I at concentrations ≥ 100 µM, whereas TOPO IIα activity is only inhibited at concentrations ≥ 250 µM. To clarify the mode of action, the isolating in vivo complex of enzyme assay was carried out using human colon carcinoma HT29 cells. After 1 h of incubation, the amount of TOPO I linked to DNA was significantly increased by nevadensin (500 µM), why nevadensin was characterized as TOPO I poison. However, no effects on TOPO IIα were detected in the cellular test system. As a subsequent cellular response to TOPO I poisoning, a highly significant increase of DNA damage after 2 h and a decrease of cell viability after 48 h at the same concentration range were found. Furthermore, after 24 h of incubation a G2/M arrest was observed at concentrations ≥ 100 µM by flow cytometry. The analysis of cell death revealed that nevadensin induces the intrinsic apoptotic pathway via activation of caspase-9 and caspase-3. The results suggest that cell cycle disruption and apoptotic events play key roles in the cellular response to TOPO I poisoning caused by nevadensin in HT29 cells.
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Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/efectos de los fármacos , Flavonas/envenenamiento , Ciclo Celular/efectos de los fármacos , Neoplasias del Colon/enzimología , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Flavonas/administración & dosificación , Células HT29 , Humanos , Concentración 50 Inhibidora , Proteínas de Unión a Poli-ADP-Ribosa/efectos de los fármacos , Factores de TiempoRESUMEN
Asarone isomers are naturally occurring in Acorus calamus Linné, Guatteria gaumeri Greenman, and Aniba hostmanniana Nees. These secondary plant metabolites belong to the class of phenylpropenes (phenylpropanoids or alkenylbenzenes). They are further chemically classified into the propenylic trans- and cis-isomers α-asarone and ß-asarone and the allylic γ-asarone. Flavoring, as well as potentially pharmacologically useful properties, enables the application of asarone isomers in fragrances, food, and traditional phytomedicine not only since their isolation in the 1950s. However, efficacy and safety in humans are still not known. Preclinical evidence has not been systematically studied, and several pharmacological effects have been reported for extracts of Acorus calamus and propenylic asarone isomers. Toxicological data are rare and not critically evaluated altogether in the 21st century yet. Therefore, within this review, available toxicological data of asarone isomers were assessed in detail. This assessment revealed that cardiotoxicity, hepatotoxicity, reproductive toxicity, and mutagenicity as well as carcinogenicity were described for propenylic asarone isomers with varying levels of reliability. The toxicodynamic profile of γ-asarone is unknown except for mutagenicity. Based on the estimated daily exposure and reported adverse effects, officials restricted or published recommendations for the use of ß-asarone and preparations of Acorus calamus. In contrast, α-asarone and γ-asarone were not directly addressed due to a limited data situation.
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Derivados de Alilbenceno/toxicidad , Anisoles/toxicidad , Derivados de Alilbenceno/farmacocinética , Animales , Anisoles/farmacocinética , Carcinógenos/toxicidad , Cardiotoxicidad/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Humanos , Isomerismo , Reproducción/efectos de los fármacosRESUMEN
Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4+ T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4+ T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4+CCR6+ cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA.
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Artritis Reumatoide/etiología , Artritis Reumatoide/metabolismo , Susceptibilidad a Enfermedades , Histona Desacetilasa 1/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Artritis Reumatoide/patología , Biomarcadores , Colágeno/efectos adversos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Histona Desacetilasa 1/genética , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Noqueados , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
A nanopore-based CuII -sensing system is reported that allows for an ultrasensitive and selective detection of CuII with the possibility for a broad range of applications, for example in medical diagnostics. A fluorescent ATCUN-like peptide 5/6-FAM-Dap-ß-Ala-His is employed to selectively bind CuII ions in the presence of NiII and ZnII and was crafted into ion track-etched nanopores. Upon CuII binding the fluorescence of the peptide sensor is quenched, permitting the detection of CuII in solution. The ion transport characteristics of peptide-modified nanopore are shown to be extremely sensitive and selective towards CuII allowing to sense femtomolar CuII concentrations in human urine mimics. Washing with EDTA fully restores the CuII -binding properties of the sensor, enabling multiple repetitive measurements. The robustness of the system clearly has the potential to be further developed into an easy-to-use, lab-on-chip CuII -sensing device, which will be of great importance for bedside diagnosis and monitor of CuII levels in patients with copper-dysfunctional homeostasis.