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1.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-33800795

RESUMEN

Plant growth and development are challenged by biotic and abiotic stresses including salinity and heat stresses. For Populus simonii × P. nigra as an important greening and economic tree species in China, increasing soil salinization and global warming have become major environmental challenges. We aim to unravel the molecular mechanisms underlying tree tolerance to salt stress and high temprerature (HT) stress conditions. Transcriptomics revealed that a PsnNAC036 transcription factor (TF) was significantly induced by salt stress in P. simonii × P. nigra. This study focuses on addressing the biological functions of PsnNAC036. The gene was cloned, and its temporal and spatial expression was analyzed under different stresses. PsnNAC036 was significantly upregulated under 150 mM NaCl and 37 °C for 12 h. The result is consistent with the presence of stress responsive cis-elements in the PsnNAC036 promoter. Subcellular localization analysis showed that PsnNAC036 was targeted to the nucleus. Additionally, PsnNAC036 was highly expressed in the leaves and roots. To investigate the core activation region of PsnNAC036 protein and its potential regulatory factors and targets, we conducted trans-activation analysis and the result indicates that the C-terminal region of 191-343 amino acids of the PsnNAC036 was a potent activation domain. Furthermore, overexpression of PsnNAC036 stimulated plant growth and enhanced salinity and HT tolerance. Moreover, 14 stress-related genes upregulated in the transgenic plants under high salt and HT conditions may be potential targets of the PsnNAC036. All the results demonstrate that PsnNAC036 plays an important role in salt and HT stress tolerance.


Asunto(s)
Genes de Plantas , Respuesta al Choque Térmico/genética , Proteínas de Plantas/fisiología , Populus/genética , Estrés Salino/genética , Plantas Tolerantes a la Sal/genética , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Clorofila/biosíntesis , Cruzamientos Genéticos , Regulación de la Expresión Génica de las Plantas , Calor , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Populus/fisiología , Regiones Promotoras Genéticas/genética , Salinidad , Plantas Tolerantes a la Sal/crecimiento & desarrollo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Fracciones Subcelulares/metabolismo , Tabaco/genética , Tabaco/metabolismo , Factores de Transcripción/genética , Activación Transcripcional
2.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33809440

RESUMEN

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.


Asunto(s)
Acilcoenzima A/metabolismo , Aciltransferasas/genética , Proteínas de Arabidopsis/genética , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Autofagia/genética , Biomarcadores/metabolismo , Aciltransferasas/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/metabolismo , Autofagosomas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica de las Plantas , Células del Mesófilo/metabolismo , Células del Mesófilo/ultraestructura , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/metabolismo
3.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-33669866

RESUMEN

Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape.


Asunto(s)
Brassica napus/genética , Genes Letales , Complejo de Proteína del Fotosistema II/metabolismo , Mapeo Físico de Cromosoma , Proteínas de Plantas/genética , Brassica napus/ultraestructura , Cromosomas de las Plantas/genética , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Anotación de Secuencia Molecular , Mutación/genética , Fenotipo , Filogenia , Proteínas de Plantas/metabolismo , Fracciones Subcelulares/metabolismo , Transformación Genética
4.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-33669494

RESUMEN

Campylobacter concisus is a human-pathogenic bacterium of the gastrointestinal tract. This study aimed at the contribution of the mucosal immune system in the context of intestinal epithelial barrier dysfunction induced by C. concisus. As an experimental leaky gut model, we used in vitro co-cultures of colonic epithelial cell monolayers (HT-29/B6-GR/MR) with M1-macrophage-like THP-1 cells on the basal side. Forty-eight hours after C. concisus infection, the decrease in the transepithelial electrical resistance in cell monolayers was more pronounced in co-culture condition and 22 ± 2% (p < 0.001) higher than the monoculture condition without THP-1 cells. Concomitantly, we observed a reduction in the expression of the tight junction proteins occludin and tricellulin. We also detected a profound increase in 4 kDa FITC-dextran permeability in C. concisus-infected cell monolayers only in co-culture conditions. This is explained by loss of tricellulin from tricellular tight junctions (tTJs) after C. concisus infection. As an underlying mechanism, we observed an inflammatory response after C. concisus infection through pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) released from THP-1 cells in the co-culture condition. In conclusion, the activation of subepithelial immune cells exacerbates colonic epithelial barrier dysfunction by C. concisus through tricellulin disruption in tTJs, leading to increased antigen permeability (leaky gut concept).


Asunto(s)
Campylobacter/inmunología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Apoptosis , Línea Celular , Supervivencia Celular , Técnicas de Cocultivo , Impedancia Eléctrica , Células Epiteliales/patología , Humanos , Inflamación/patología , Intestinos/microbiología , Intestinos/patología , Macrófagos/metabolismo , Modelos Biológicos , Ocludina/metabolismo , Fracciones Subcelulares/metabolismo , Uniones Estrechas/metabolismo
5.
Nat Commun ; 12(1): 1392, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33654102

RESUMEN

Rice grains typically contain high levels of toxic arsenic but low levels of the essential micronutrient selenium. Anthropogenic arsenic contamination of paddy soils exacerbates arsenic toxicity in rice crops resulting in substantial yield losses. Here, we report the identification of the gain-of-function arsenite tolerant 1 (astol1) mutant of rice that benefits from enhanced sulfur and selenium assimilation, arsenic tolerance, and decreased arsenic accumulation in grains. The astol1 mutation promotes the physical interaction of the chloroplast-localized O-acetylserine (thiol) lyase protein with its interaction partner serine-acetyltransferase in the cysteine synthase complex. Activation of the serine-acetyltransferase in this complex promotes the uptake of sulfate and selenium and enhances the production of cysteine, glutathione, and phytochelatins, resulting in increased tolerance and decreased translocation of arsenic to grains. Our findings uncover the pivotal sensing-function of the cysteine synthase complex in plastids for optimizing stress resilience and grain quality by regulating a fundamental macronutrient assimilation pathway.


Asunto(s)
Arsénico/metabolismo , Oryza/metabolismo , Semillas/metabolismo , Selenio/metabolismo , Azufre/metabolismo , Alelos , Cloroplastos/metabolismo , Cisteína Sintasa/metabolismo , Redes y Vías Metabólicas , Modelos Biológicos , Mutación/genética , Fenotipo , Fitoquelatinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Serina/metabolismo , Fracciones Subcelulares/metabolismo
6.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-33669913

RESUMEN

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.


Asunto(s)
Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/efectos de los fármacos , Fluconazol/farmacología , Fructosa/farmacología , Proteínas Fúngicas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Candida albicans/citología , Carbono/farmacología , Proliferación Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Fructosa/sangre , Proteínas Fluorescentes Verdes/metabolismo , Fracciones Subcelulares/metabolismo
7.
Nat Commun ; 12(1): 1049, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33594064

RESUMEN

Eukaryotic phytoplankton have a small global biomass but play major roles in primary production and climate. Despite improved understanding of phytoplankton diversity and evolution, we largely ignore the cellular bases of their environmental plasticity. By comparative 3D morphometric analysis across seven distant phytoplankton taxa, we observe constant volume occupancy by the main organelles and preserved volumetric ratios between plastids and mitochondria. We hypothesise that phytoplankton subcellular topology is modulated by energy-management constraints. Consistent with this, shifting the diatom Phaeodactylum from low to high light enhances photosynthesis and respiration, increases cell-volume occupancy by mitochondria and the plastid CO2-fixing pyrenoid, and boosts plastid-mitochondria contacts. Changes in organelle architectures and interactions also accompany Nannochloropsis acclimation to different trophic lifestyles, along with respiratory and photosynthetic responses. By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales.


Asunto(s)
Metabolismo Energético , Imagenología Tridimensional , Fitoplancton/citología , Fitoplancton/fisiología , Aclimatación/efectos de la radiación , Metabolismo Energético/efectos de la radiación , Luz , Microalgas/metabolismo , Microalgas/efectos de la radiación , Microalgas/ultraestructura , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Mitocondrias/ultraestructura , Fitoplancton/efectos de la radiación , Fitoplancton/ultraestructura , Plastidios/metabolismo , Fracciones Subcelulares/metabolismo
8.
J Vis Exp ; (167)2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33522506

RESUMEN

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to. Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres. This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.


Asunto(s)
Drosophila melanogaster/citología , Imagenología Tridimensional , Microscopía Fluorescente/métodos , Animales , Técnicas de Cultivo de Célula , Supervivencia Celular , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Microtúbulos/metabolismo , Células Madre/citología , Fracciones Subcelulares/metabolismo , Testículo/citología , Imagen de Lapso de Tiempo , Tubulina (Proteína)/metabolismo
9.
Plant Mol Biol ; 106(1-2): 49-65, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33625643

RESUMEN

KEY MESSAGE: Three novel transcription factors were successfully identified and shown to interact with the trichome-specific THCAS promoter regulatory region. Cannabinoids are important secondary metabolites present in Cannabis sativa L. (cannabis). One cannabinoid that has received considerable attention, 9-tetrahydrocannabinol (THC), is derived from Delta-9-Tetrahydrocannabinolic acid (THCA) and responsible for the mood-altering and pain-relieving effects of cannabis. A detailed understanding of transcriptional control of THCA synthase (THCAS) is currently lacking. The primary site of cannabinoid biosynthesis is the glandular trichomes that form on female flowers. Transcription factors (TFs) have been shown to play an important role in secondary-metabolite biosynthesis and glandular trichome formation in Artemisia annua, Solanum lycopersicum and Humulus lupulus. However, analogous information is not available for cannabis. Here, we characterize a 548 bp fragment of the THCAS promoter and regulatory region that drives trichome-specific expression. Using this promoter fragment in a yeast-one-hybrid screen, we identified 3 novel TFs (CsAP2L1, CsWRKY1 and CsMYB1) and provided evidence that these 3 TFs regulate the THCAS promoter in planta. The O-Box element within the proximal region of the THCAS promoter is necessary for CsAP2L1-induced transcriptional activation of THCAS promoter. Similar to THCAS, the genes for all three TFs have trichome-specific expression, and subcellular localization of the TFs indicates that all three proteins are in the nucleus. CsAP2L1 and THCAS exhibit a similar temporal, spatial and strain-specific gene expression profiles, while those expression patterns of CsWRKY1 and CsMYB1 are opposite from THCAS. Our results identify CsAP2L1 playing a positive role in the regulation of THCAS expression, while CsWRKY1 and CsMYB1 may serve as negative regulators of THCAS expression.


Asunto(s)
Vías Biosintéticas , Cannabinoides/biosíntesis , Cannabis/metabolismo , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Cannabis/genética , Flores/genética , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Filogenia , Proteínas de Plantas/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Represoras/metabolismo , Elementos de Respuesta/genética , Fracciones Subcelulares/metabolismo , Factores de Transcripción/genética , Transcripción Genética
10.
Nat Commun ; 12(1): 71, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397984

RESUMEN

Signaling complexes are often organized in a spatiotemporal manner and on a minute timescale. Proximity labeling based on engineered ascorbate peroxidase APEX2 pioneered in situ capture of spatiotemporal membrane protein complexes in living cells, but its application to cytosolic proteins remains limited due to the high labeling background. Here, we develop proximity labeling probes with increased labeling selectivity. These probes, in combination with label-free quantitative proteomics, allow exploring cytosolic protein assemblies such as phosphotyrosine-mediated protein complexes formed in response to minute-scale EGF stimulation. As proof-of-concept, we systematically profile the spatiotemporal interactome of the EGFR signaling component STS1. For STS1 core complexes, our proximity proteomics approach shows comparable performance to affinity purification-mass spectrometry-based temporal interactome profiling, while also capturing additional-especially endosomally-located-protein complexes. In summary, we provide a generic approach for exploring the interactome of mobile cytosolic proteins in living cells at a temporal resolution of minutes.


Asunto(s)
Citosol/metabolismo , Proteómica , Transducción de Señal , Biotina/metabolismo , Supervivencia Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Células HeLa , Humanos , Fenoles , Mapeo de Interacción de Proteínas , Transducción de Señal/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Factores de Tiempo
11.
Methods Mol Biol ; 2213: 49-58, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33270192

RESUMEN

New biologically active compounds are regularly discovered through screening procedures using microorganisms. This very cheap procedure is followed by drug discovery that is usually seen as a highly focused approach, testing new compounds on animals or cell lines. In vivo assays of candidate drugs in mammals are expensive and sometimes not affordable at the preliminary stages of drug development. Early screening approaches in transgenic plants would allow chemotherapeutic drug candidates further selection before their characterization in expensive biological models. The proposed screening approach is based on cell subcellular architecture observations in transgenic plants within a short time of treatment, which is better than observing the effects of compounds on growth.


Asunto(s)
Arabidopsis/metabolismo , Evaluación Preclínica de Medicamentos , Células Vegetales/metabolismo , Arabidopsis/genética , Microscopía , Plantas Modificadas Genéticamente , Fracciones Subcelulares/metabolismo
12.
Nucleic Acids Res ; 49(D1): D803-D808, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33313828

RESUMEN

Protein subcellular localization (SCL) is important for understanding protein function, genome annotation, and aids identification of potential cell surface diagnostic markers, drug targets, or vaccine components. PSORTdb comprises ePSORTdb, a manually curated database of experimentally verified protein SCLs, and cPSORTdb, a pre-computed database of PSORTb-predicted SCLs for NCBI's RefSeq deduced bacterial and archaeal proteomes. We now report PSORTdb 4.0 (http://db.psort.org/). It features a website refresh, in particular a more user-friendly database search. It also addresses the need to uniquely identify proteins from NCBI genomes now that GI numbers have been retired. It further expands both ePSORTdb and cPSORTdb, including additional data about novel secondary localizations, such as proteins found in bacterial outer membrane vesicles. Protein predictions in cPSORTdb have increased along with the number of available microbial genomes, from approximately 13 million when PSORTdb 3.0 was released, to over 66 million currently. Now, analyses of both complete and draft genomes are included. This expanded database will be of wide use to researchers developing SCL predictors or studying diverse microbes, including medically, agriculturally and industrially important species that have both classic or atypical cell envelope structures or vesicles.


Asunto(s)
Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Bases de Datos de Proteínas , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Bacterianas/química , Pared Celular/química , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Interfaz Usuario-Computador
13.
Methods Mol Biol ; 2181: 69-81, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32729075

RESUMEN

APOBEC1 is a member of the AID/APOBECs, a group of deaminases responsible for the editing of C>U in both DNA and RNA. APOBEC1 is physiologically involved in C>U RNA editing: while hundreds of targets have been discovered in mice, in humans the only well-characterized target of APOBEC1 is the apolipoprotein B (ApoB) transcript. APOBEC1 edits a CAA codon into a stop codon, which causes the translation of a truncated form of ApoB. A number of assays have been developed to investigate this process. Early assays, poisoned primer extension and Sanger sequencing, have focused on accuracy and sensitivity but rely on extraction of the RNA from tissues and cells. More recently, the need to visualize the RNA editing process directly in live cells have led to the development of fluorescence-based tools. These assays detect RNA editing through reporters whose editing causes a change in cellular localization or a change in fluorescent properties. Here we review the available assays to quantify RNA editing, and we present the protocol for cytofluorimetric analysis using a double-fluorescent reporter.


Asunto(s)
Desaminasas APOBEC-1/genética , Biología Computacional/métodos , Citidina/genética , Edición de ARN/genética , ARN Mensajero/genética , Fracciones Subcelulares/metabolismo , Uridina/genética , Desaminasas APOBEC-1/metabolismo , Citidina/química , Genes Reporteros , Células HEK293 , Células Hep G2 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN Mensajero/metabolismo , Uridina/química
14.
Int J Mol Sci ; 21(24)2020 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-33352693

RESUMEN

Drought is the largest stress affecting agricultural crops, resulting in substantial reductions in yield. Plant adaptation to water stress is a complex trait involving changes in hormone signaling, physiology, and morphology. Sorghum (Sorghum bicolor (L.) Moench) is a C4 cereal grass; it is an agricultural staple, and it is particularly drought-tolerant. To better understand drought adaptation strategies, we compared the cytosolic- and organelle-enriched protein profiles of leaves from two Sorghum bicolor genotypes, RTx430 and BTx642, with differing preflowering drought tolerances after 8 weeks of growth under water limitation in the field. In agreement with previous findings, we observed significant drought-induced changes in the abundance of multiple heat shock proteins and dehydrins in both genotypes. Interestingly, our data suggest a larger genotype-specific drought response in protein profiles of organelles, while cytosolic responses are largely similar between genotypes. Organelle-enriched proteins whose abundance significantly changed exclusively in the preflowering drought-tolerant genotype RTx430 upon drought stress suggest multiple mechanisms of drought tolerance. These include an RTx430-specific change in proteins associated with ABA metabolism and signal transduction, Rubisco activation, reactive oxygen species scavenging, flowering time regulation, and epicuticular wax production. We discuss the current understanding of these processes in relation to drought tolerance and their potential implications.


Asunto(s)
Sequías , Flores/fisiología , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Sorghum/fisiología , Estrés Fisiológico , Fracciones Subcelulares/metabolismo , Flores/genética , Regulación de la Expresión Génica de las Plantas , Genotipo , Proteoma/análisis , Sorghum/genética
15.
Anticancer Res ; 40(11): 6327-6335, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-33109570

RESUMEN

BACKGROUND/AIM: Despite numerous studies, the etiology of chronic lymphocytic leukemia (CLL) remains unknown. A hypothesis of autoantigen stimulation in leukemic clone selection might explain 'stereotypy' of B-cell receptors. In healthy cells, cofilin-1 (CFL1) has multiple functions. Its role was described in several malignancies. The aim of this study was characterization of the role of CFL1 in CLL. Materialas and Methods: Cells from peripheral blood of 180 patients and 42 healthy volunteers (HVs) were isolated. Gene expression was assessed with reverse transcription polymerase chain reaction (RT-qPCR); western blot was performed for determination of protein level and activity. After silencing of CFL1 gene, cell ability for migration and chemotaxis was investigated with Transwell method. Post-silencing, apoptosis and cell cycle was determined by flow cytometry. RESULTS: In RT-qPCR, we observed significantly higher expression of CFL1. Higher activity of protein in CLL cells when compared to HVs was detected. Knock-down of CFL1 led to decreased chemotaxis and migration of CLL cells versus cells from HVs. Apoptosis was increased amongst cells with silenced CFL1 and correlated with higher proportion of cells in the G2/M phase. CONCLUSION: Significantly higher expression of CFL1 mRNA in CLL and higher protein activity might indicate high utilization of CFL1 in malignant cells, maintaining their viability, as its inhibition affected viability, cell-cycle progression and motility of leukemia cells.


Asunto(s)
Cofilina 1/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Transducción de Señal , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular , Quimiotaxis/genética , Cofilina 1/genética , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/metabolismo
16.
PLoS Comput Biol ; 16(9): e1007728, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32970668

RESUMEN

Calcium oscillations and waves induce depolarization in cardiac cells which are believed to cause life-threathening arrhythimas. In this work, we study the conditions for the appearance of calcium oscillations in both a detailed subcellular model of calcium dynamics and a minimal model that takes into account just the minimal ingredients of the calcium toolkit. To avoid the effects of homeostatic changes and the interaction with the action potential we consider the somewhat artificial condition of a cell without pacing and with no calcium exchange with the extracellular medium. Both the full subcellular model and the minimal model present the same scenarios depending on the calcium load: two stationary states, one with closed ryanodine receptors (RyR) and most calcium in the cell stored in the sarcoplasmic reticulum (SR), and another, with open RyRs and a depleted SR. In between, calcium oscillations may appear. The robustness of these oscillations is determined by the amount of calsequestrin (CSQ). The lack of this buffer in the SR enhances the appearance of oscillations. The minimal model allows us to relate the stability of the oscillating state to the nullcline structure of the system, and find that its range of existence is bounded by a homoclinic and a Hopf bifurcation, resulting in a sudden transition to the oscillatory regime as the cell calcium load is increased. Adding a small amount of noise to the RyR behavior increases the parameter region where oscillations appear and provides a gradual transition from the resting state to the oscillatory regime, as observed in the subcellular model and experimentally.


Asunto(s)
Calcio/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Calsecuestrina/metabolismo , Modelos Biológicos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Procesos Estocásticos , Fracciones Subcelulares/metabolismo
17.
Chem Biol Interact ; 330: 109247, 2020 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-32866466

RESUMEN

This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat livers. Benoxacor was incubated for ≤30 min with microsomes or cytosol, and its enantioselective depletion was measured using gas chromatographic methods. Benoxacor was depleted in incubations with active microsomes in the presence and absence of NADPH, suggesting its metabolism by hepatic cytochrome P450 enzymes (CYPs) and microsomal carboxylesterases (CESs). Benoxacor was depleted in cytosolic incubations in the presence of glutathione, consistent with its metabolism by glutathione S-transferases (GSTs). The depletion of benoxacor was faster in incubations with cytosol from male than female rats, whereas no statistically significant sex differences were observed in microsomal incubations. The consumption of benoxacor was inhibited by the CYP inhibitor 1-aminobenzotriazole, the CES inhibitor benzil, and the GST inhibitor ethacrynic acid. Estimates of the intrinsic clearance of benoxacor suggest that CYPs are the primary metabolic enzyme responsible for benoxacor metabolism in rats. Microsomal incubations showed an enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). A greater enrichment occurred in incubations with microsomes from female (EF = 0.67 ± 0.01) than male rats (EF = 0.60 ± 0.01). Cytosolic incubations from female rats resulted in enrichment of E1-benoxacor (EF = 0.54 ± 0.01), while cytosolic incubations from male rats displayed enrichment of the second eluting enantiomer (E2-benoxacor; EF = 0.43 ± 0.01). Sex-dependent differences in the metabolism of benoxacor in rats could significantly impact ecological risks and mammalian toxicity. Moreover, changes in the enantiomeric enrichment of benoxacor may be a powerful tool for environmental fate and transport studies.


Asunto(s)
Hígado/metabolismo , Oxazinas/metabolismo , Fracciones Subcelulares/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Citosol/enzimología , Citosol/metabolismo , Femenino , Herbicidas/química , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Ratas , Factores Sexuales , Estereoisomerismo
18.
PLoS One ; 15(8): e0237930, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32841274

RESUMEN

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Asunto(s)
Microsomas/metabolismo , Proteómica , Animales , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Centrifugación , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Ontología de Genes , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Microsomas/ultraestructura , Mitocondrias/ultraestructura , Proteoma/metabolismo , Programas Informáticos , Fracciones Subcelulares/metabolismo
19.
Life Sci ; 259: 118203, 2020 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-32781069

RESUMEN

High mobility group box 1 (HMGB1) is a highly conserved nucleoprotein involving in numerous biological processes, and well known to trigger immune responses as the damage-associated molecular pattern (DAMP) in the extracellular environment. The role of HMGB1 is distinct due to its multiple functions in different subcellular location. In the nucleus, HMGB1 acts as a chaperone to regulate DNA events including DNA replication, repair and nucleosome stability. While in the cytoplasm, it is engaged in regulating autophagy and apoptosis. A great deal of research has explored its function in the pathogenesis of renal diseases. This review mainly focuses on the role of HMGB1 and summarizes the pathway and treatment targeting HMGB1 in the various renal diseases which may open the windows of opportunities for the development of desirable therapeutic ends in these pathological conditions.


Asunto(s)
Proteína HMGB1/metabolismo , Enfermedades Renales/metabolismo , Animales , Apoptosis/fisiología , Autofagia/fisiología , Humanos , Enfermedades Renales/patología , Transducción de Señal , Fracciones Subcelulares/metabolismo
20.
Chem Biol Interact ; 328: 109192, 2020 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-32712081

RESUMEN

Many natural products are prodrugs which are biotransformed and activated after oral administration. The investigation of gastrointestinal and hepatic biotransformation can be facilitated by in vitro screening methods. This study compares two widely used in vitro models for hepatic biotransformation: 1) human S9 fractions and 2) human liver microsomes and cytosolic fractions in a two-step sequence, with the purpose of identifying differences in the biotransformation of medicagenic acid, the putative precursor of active metabolites, responsible for the medicinal effects of the herb Herniaria hirsuta. The combination of liquid chromatography coupled to high-resolution mass spectrometry with subsequent suspect and non-target data analysis allowed the identification of thirteen biotransformation products, four of which are reported here for the first time. Eight biotransformation products resulting from oxidative Phase I reactions were identified. Phase II conjugation reactions resulted in the formation of three glucuronidated and two sulfated biotransformation products. No major differences could be observed between incubations with human liver S9 or when utilizing human microsomal and cytosolic fractions. Apart from two metabolites, both methods rendered the same qualitative metabolic profile, with minor quantitative differences. As a result, both protocols applied in this study can be used to study in vitro human liver biotransformation reactions.


Asunto(s)
Microsomas Hepáticos/metabolismo , Triterpenos/metabolismo , Biotransformación , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Masculino , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Triterpenos/química
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