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1.
PLoS One ; 15(7): e0236185, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32730344

RESUMEN

Fluorescent markers are a powerful tool and have been widely applied in biology for different purposes. The genome sequence of Xanthomonas citri subsp. citri (X. citri) revealed that approximately 30% of the genes encoded hypothetical proteins, some of which could play an important role in the success of plant-pathogen interaction and disease triggering. Therefore, revealing their functions is an important strategy to understand the bacterium pathways and mechanisms involved in plant-host interaction. The elucidation of protein function is not a trivial task, but the identification of the subcellular localization of a protein is key to understanding its function. We have constructed an integrative vector, pMAJIIc, under the control of the arabinose promoter, which allows the inducible expression of red fluorescent protein (mCherry) fusions in X. citri, suitable for subcellular localization of target proteins. Fluorescence microscopy was used to track the localization of VrpA protein, which was visualized surrounding the bacterial outer membrane, and the GyrB protein, which showed a diffused cytoplasmic localization, sometimes with dots accumulated near the cellular poles. The integration of the vector into the amy locus of X. citri did not affect bacterial virulence. The vector could be stably maintained in X. citri, and the disruption of the α-amylase gene provided an ease screening method for the selection of the transformant colonies. The results demonstrate that the mCherry-containing vector here described is a powerful tool for bacterial protein localization in cytoplasmic and periplasmic environments.


Asunto(s)
Proteínas Bacterianas/metabolismo , Citoplasma/metabolismo , Periplasma/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Xanthomonas/metabolismo , Arabinosa/farmacología , Cromosomas Bacterianos/genética , Vectores Genéticos/metabolismo , Viabilidad Microbiana/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Almidón/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Xanthomonas/patogenicidad
2.
Biochem Biophys Res Commun ; 521(4): 957-963, 2020 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-31718798

RESUMEN

The signaling elicited by the cytokine interleukin-17A (IL-17) is important for antimicrobial defense responses, whereas excessive IL-17 production leads to autoimmune diseases such as psoriasis and multiple sclerosis. IL-17-induced stabilization of mRNAs has been recognized as a unique and important feature of IL-17 signaling. Previously, we demonstrated that IL-17 signaling protein ACT1 is required to counteract constitutive inhibitor of nuclear factor kappa B zeta (IκB-ζ) mRNA degradation by the ribonuclease Regnase-1. However, information about the mechanism of mRNA stabilization in IL-17-stimulated cells remains insufficient. In the present study, we aimed to clarify the mechanism in more detail and identify an agent that can inhibit IL-17-induced mRNA stabilization. Experiments using small interfering RNA and an inhibitor of TANK-binding kinase 1 (TBK1) revealed that TBK1 was required for IκB-ζ mRNA stabilization through Regnase-1 phosphorylation. Intriguingly, this TBK1-mediated phosphorylation of Regnase-1 was suppressed by the addition of dimethyl fumarate (DMF), an electrophilic small molecule that has been used to treat IL-17-related autoimmune diseases. Confocal microscopic observation of the cellular localization of ACT1 revealed that DMF treatment resulted in the disappearance of ACT1 nuclear dots and perinuclear accumulation of ACT1. These results suggested that DMF is a small molecule that compromises IL-17-induced activation of the ACT1-TBK1 pathway, thereby inhibiting IL-17-induced mRNA stabilization.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Dimetilfumarato/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-17/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleasas/metabolismo , Línea Celular , Humanos , Fosforilación/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo
3.
Ecotoxicol Environ Saf ; 185: 109692, 2019 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-31585391

RESUMEN

Canna indica L. is a promising species for heavy metal phytoremediation due to its fast growth rate and large biomass. However, few studies have investigated cadmium (Cd) tolerance mechanisms. In the present study, Canna plants were cultivated under hydroponic conditions with increasing Cd concentrations (0, 5, 10, 15 mg/L). We found that the plants performed well under 5 mg/L Cd2+ stress, but damage was observed under higher Cd exposure, such as leaf chlorosis, growth inhibition, a decreased chlorophyll content, and destruction of the ultrastructure of leaf cells. Additionally, Canna alleviated Cd toxicity to a certain extent. After Canna was exposed to 5, 10 and 15 mg/L Cd2+ for 45 d, the highest Cd concentration was exhibited in roots, which was almost 17-47 times the Cd concentration in leaves and 8-20 times that in stems. At the subcellular level, cellular debris and heat-stable proteins (HSPs) were the main binding sites for Cd, and the proportion of Cd in the two subcellular fractions accounted for 71.4-94.2% of the total Cd. Furthermore, we found that granules could participate in the detoxification process when Cd stress was enhanced. Our results indicated that Canna indica L. can tolerate Cd toxicity by sequestering heavy metals in root tissues, fencing out by cell wall, and binding with biologically detoxified fractions (granules and HSPs).


Asunto(s)
Cadmio/toxicidad , Contaminantes del Suelo/toxicidad , Fracciones Subcelulares/efectos de los fármacos , Zingiberales/efectos de los fármacos , Biodegradación Ambiental , Biomasa , Cadmio/metabolismo , Relación Dosis-Respuesta en la Radiación , Tolerancia a Medicamentos , Inactivación Metabólica , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Hojas de la Planta/ultraestructura , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Raíces de Plantas/ultraestructura , Contaminantes del Suelo/metabolismo , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , Zingiberales/metabolismo , Zingiberales/ultraestructura
4.
Yale J Biol Med ; 92(3): 413-422, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31543705

RESUMEN

The search for conditions that maximize the outcome of Photodynamic Therapy (PDT) continues. Recent data indicate that PDT-induced cell death depends more on the specific intracellular location of the photosensitizer (PS) than on any other parameter. Indeed, knowledge of the PS intracellular location allows the establishment of clear relationships between the mechanism of cell death and the PDT efficacy. In order to determine the intracellular localization sites of a given PS, classical co-localization protocols, which are based in the comparison of the emissive profiles of organelle-specific probes to those of the PS, are usually performed. Since PSs are usually not efficient fluorophores, co-localization protocols require relatively high PS concentrations (micromolar range), distorting the whole proposal of the experiment, as high PS concentration means accumulation in many low-affinity sites. To overcome this difficulty, herein we describe a method that identifies PS intracellular localization by recognizing and quantifying the photodamage at intracellular organelles. We propose that irradiation protocols and characterization of major sites of photodamage results from many cycles of photosensitized oxidations, furnishing an integrated picture of the PS location. By comparing the results of protocols based in either method, we showed that the analysis of the damaged organelles can be conducted at optimal conditions (low PS concentrations), providing clear correlations with cell death mechanisms, which is not the case for the results obtained with co-localization protocols. Experiments using PSs that target either mitochondria or lysosomes were described and investigated in detail, showing that evaluating organelle damage is as simple as performing co-localization protocols.


Asunto(s)
Orgánulos/patología , Fármacos Fotosensibilizantes/farmacología , Células HeLa , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/patología , Microscopía Fluorescente , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Orgánulos/efectos de los fármacos , Oxidación-Reducción , Porfirinas/farmacología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo
5.
Small ; 15(42): e1901642, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31461215

RESUMEN

Nanocellulose is increasingly considered for applications; however, the fibrillar nature, crystalline phase, and surface reactivity of these high aspect ratio nanomaterials need to be considered for safe biomedical use. Here a comprehensive analysis of the impact of cellulose nanofibrils (CNF) and nanocrystals (CNC) is performed using materials provided by the Nanomaterial Health Implications Research Consortium of the National Institute of Environmental Health Sciences. An intermediary length of nanocrystals is also derived by acid hydrolysis. While all CNFs and CNCs are devoid of cytotoxicity, 210 and 280 nm fluorescein isothiocyanate (FITC)-labeled CNCs show higher cellular uptake than longer and shorter CNCs or CNFs. Moreover, CNCs in the 200-300 nm length scale are more likely to induce lysosomal damage, NLRP3 inflammasome activation, and IL-1ß production than CNFs. The pro-inflammatory effects of CNCs are correlated with higher crystallinity index, surface hydroxyl density, and reactive oxygen species generation. In addition, CNFs and CNCs can induce maturation of bone marrow-derived dendritic cells and CNCs (and to a lesser extent CNFs) are found to exert adjuvant effects in ovalbumin (OVA)-injected mice, particularly for 210 and 280 nm CNCs. All considered, the data demonstrate the importance of length scale, crystallinity, and surface reactivity in shaping the innate immune response to nanocellulose.


Asunto(s)
Adyuvantes Inmunológicos/farmacología , Celulosa/farmacología , Inflamación/patología , Nanoestructuras/química , Animales , Supervivencia Celular/efectos de los fármacos , Celulosa/ultraestructura , Cristalización , Células Dendríticas/metabolismo , Glutatión/metabolismo , Humanos , Hidrodinámica , Inmunidad Humoral/efectos de los fármacos , Inmunoglobulina G/biosíntesis , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Nanopartículas/química , Nanopartículas/ultraestructura , Nanoestructuras/ultraestructura , Ovalbúmina/inmunología , Estrés Oxidativo/efectos de los fármacos , Tamaño de la Partícula , Especies Reactivas de Oxígeno/metabolismo , Electricidad Estática , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Células THP-1
6.
Mol Cell ; 73(1): 166-182.e7, 2019 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-30609389

RESUMEN

Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org.


Asunto(s)
Cromatografía Liquida , Espectrometría de Masas , Proteínas/metabolismo , Proteoma , Proteómica/métodos , Fracciones Subcelulares/metabolismo , Biomarcadores/metabolismo , Fraccionamiento Celular , Biología Computacional , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Gefitinib/farmacología , Humanos , Focalización Isoeléctrica , Células MCF-7 , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas , Proteínas/antagonistas & inhibidores , Proteínas/clasificación , Proteínas/genética , Reproducibilidad de los Resultados , Fracciones Subcelulares/clasificación , Fracciones Subcelulares/efectos de los fármacos
7.
Chemosphere ; 219: 740-747, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30557731

RESUMEN

Cadmium (Cd) and benzo [a]pyrene (BaP) often co-occur in the environment, and the critical body residue of organisms is used as an indicator of the toxic effects of contaminants. However, little is known about their distributions and toxicities when pollution of Cd and BaP are combined. Semi-static solution culture experiment was used to study the impacts of BaP on the subcellular distribution of the toxic metal Cd in the earthworm Eisenia fetida. We explored the mechanisms by which this organism responds to combined exposure to these pollutants by measuring the protein content of each of three subcellular fractions, as well as acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities. The subcellular partitioning of Cd was heterogeneous and Cd mainly accumulated in the cytosolic fraction (Fraction C), which was previously reported to be involved in metal immobilization. In Fraction C, Cd accumulation was correlated with the external concentration to which the earthworm had been exposed; however, in the presence of BaP, Cd accumulation was inhibited and plateaued at high external Cd concentrations. A principal component analysis revealed that this decreased Cd accumulation might be caused by increases in GST activity, which likely increased the excretion of Cd. BaP was also found to stimulate protein biosynthesis and upregulate AChE and GST activities in the debris fraction (Fraction E), indicating other potential detoxification mechanisms in this fraction. Granule fraction (Fraction D) had a lower protein content, AChE and GST activities than the other subcellular fractions, supporting previous findings that Fraction D is largely inert.


Asunto(s)
Benzo(a)pireno/farmacología , Cadmio/toxicidad , Oligoquetos/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Animales , Benzo(a)pireno/análisis , Cadmio/análisis , Antagonismo de Drogas , Glutatión Transferasa/metabolismo , Oligoquetos/metabolismo , Análisis de Componente Principal , Biosíntesis de Proteínas/efectos de los fármacos , Contaminantes del Suelo/análisis , Fracciones Subcelulares/efectos de los fármacos
8.
Glia ; 67(4): 703-717, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30485542

RESUMEN

Clostridium botulinum C3 transferase (C3bot) ADP-ribosylates rho proteins to change cellular functions in a variety of cell types including astrocytes and neurons. The intermediate filament protein vimentin as well as transmembrane integrins are involved in internalization of C3bot into cells. The exact contribution, however, of these proteins to binding of C3bot to the cell surface and subsequent cellular uptake remains to be unraveled. By comparing primary astrocyte cultures derived from wild-type with Vim-/- mice, we demonstrate that astrocytes lacking vimentin exhibited a delayed ADP-ribosylation of rhoA concurrent with a blunted morphological response. This functional impairment was rescued by the extracellular excess of recombinant vimentin. Binding assays using C3bot harboring a mutated integrin-binding RGD motif (C3bot-G89I) revealed the involvement of integrins in astrocyte binding of C3bot. Axonotrophic effects of C3bot are vimentin dependent and postulate an underlying mechanism entertaining a molecular cross-talk between astrocytes and neurons. We present functional evidence for astrocytic release of vimentin by exosomes using an in vitro scratch wound model. Exosomal vimentin+ particles released from wild-type astrocytes promote the interaction of C3bot with neuronal membranes. This effect vanished when culturing Vim-/- astrocytes. Specificity of these findings was confirmed by recombinant vimentin propagating enhanced binding of C3bot to synaptosomes from rat spinal cord and mouse brain. We hypothesize that vimentin+ exosomes released by reactive astrocytes provide a novel molecular mechanism constituting axonotrophic (neuroprotective) and plasticity augmenting effects of C3bot after spinal cord injury.


Asunto(s)
ADP Ribosa Transferasas/farmacología , Astrocitos/metabolismo , Toxinas Botulínicas/farmacología , Vesículas Extracelulares/fisiología , Neuronas/metabolismo , Vimentina/metabolismo , ADP Ribosa Transferasas/metabolismo , Animales , Astrocitos/ultraestructura , Toxinas Botulínicas/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Vesículas Extracelulares/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Inmunoelectrónica , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Ratas , Ratas Endogámicas Lew , Médula Espinal/citología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , Factores de Tiempo , Vimentina/genética
9.
CNS Neurosci Ther ; 25(2): 200-214, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-29962076

RESUMEN

BACKGROUND: Treatments immediately after spinal cord injury (SCI) are anticipated to decrease neuronal death, disruption of neuronal connections, demyelination, and inflammation, and to improve repair and functional recovery. Currently, little can be done to modify the acute phase, which extends to the first 48 hours post-injury. Efforts to intervene have focused on the subsequent phases - secondary (days to weeks) and chronic (months to years) - to both promote healing, prevent further damage, and support patients suffering from SCI. METHODS: We used a contusion model of SCI in female mice, and delivered a small molecule reagent during the early phase of injury. Histological and behavioral outcomes were assessed and compared. RESULTS: We find that the reagent Pifithrin-µ (PFT-µ) acts early and directly on microglia in vitro, attenuating their activation. When administered during the acute phase of SCI, PFT-µ resulted in reduced lesion size during the initial inflammatory phase, and reduced the numbers of pro-inflammatory microglia and macrophages. Treatment with PFT-µ during the early stage of injury maintained a stable anti-inflammatory environment. CONCLUSIONS: Our results indicate that a small molecule reagent PFT-µ has sustained immunomodulatory effects following a single dose after injury.


Asunto(s)
Activación de Macrófagos/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Traumatismos de la Médula Espinal/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Animales , Animales Recién Nacidos , Conducta Animal , Contusiones/tratamiento farmacológico , Femenino , Inflamación/tratamiento farmacológico , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Fagocitosis/efectos de los fármacos , Cultivo Primario de Células , Recuperación de la Función , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Sulfonamidas/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos
10.
Toxicol Lett ; 299: 47-55, 2018 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-30240590

RESUMEN

Environmental exposure to the highly persistent chlorinated pesticides including dieldrin and lindane is postulated to be a risk factor to the development of Parkinson's disease, a devastating movement disorder. We have previously reported that the combined treatment with dieldrin and lindane induces a cooperative toxicity in the rat N27 dopaminergic neuronal cells through increased oxidative stress and mitochondrial dysfunction. In this study, we investigated the involvement of NADPH oxidase (NOX) proteins in the combined treatment with dieldrin and lindane-induced dopaminergic neurotoxicity. Immunoblot analysis demonstrated the presence of NADPH Oxidase 1 (Nox1) isoform and p67phox in N27 neurons. Furthermore, treatment with dieldrin and lindane upregulated the cellular expression of Nox1 but not p67phox protein. Functionally, dieldrin and lindane-induced ROS production was attenuated, in a dose-dependent manner, by Nox inhibitors diphenylene iodonium and apocynin. Subcellular localization analysis of Nox1 and p67phox proteins indicated colocalization of both subunits with mitochondria in untreated cells. Treatment with dieldrin and lindane further increased mitochondrial colocalization of Nox1 protein, suggesting a potentially prominent role for mitochondrial Nox1 protein in dieldrin and lindane-induced ROS generation in dopaminergic neurons and its contribution to the combined organochlorinated pesticide-induced neurotoxicity.


Asunto(s)
Dieldrín/toxicidad , Neuronas Dopaminérgicas/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Hexaclorociclohexano/toxicidad , NADPH Oxidasa 1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular , Neuronas Dopaminérgicas/metabolismo , Sinergismo Farmacológico , Ratas , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología
11.
Exp Neurol ; 309: 160-168, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30102916

RESUMEN

Severe early life stressors increase the probability of developing psychiatric disorders later in life through modifications in neuronal circuits controlling brain monoaminergic signaling. Our previous work demonstrated that 24 h maternal deprivation (MD) in male Sprague Dawley rats modifies dopamine (DA) signaling from the ventral tegmental area (VTA) through changes at GABAergic synapses that were reversible by in vitro histone deacetylase (HDAC) inhibition which led to restoration of the scaffold A-kinase anchoring protein (AKAP150) signaling and subsequently recovered GABAergic plasticity (Authement et al., 2015). Using a combination of in situ hybridization, Western blots and immunohistochemistry, we confirmed that MD-induced epigenetic modifications at the level of histone acetylation were associated with an upregulation of HDAC2. MD also increased Akap5 mRNA levels in the VTA. Western blot analysis of AKAP150 protein expression showed an increase in synaptic levels of AKAP150 protein in the VTA with an accompanying decrease in synaptic levels of protein kinase A (PKA). Moreover, the abundance of mature brain-derived neurotrophic factor (BDNF) protein of VTA tissues from MD rats was significantly lower than in control groups. In vivo systemic injection with a selective class I HDAC inhibitor (CI-994) was sufficient to reverse MD-induced histone hypoacetylation in the VTA for 24 h after the injection. Furthermore, HDAC inhibition normalized the levels of mBDNF and AKAP150 proteins at 24 h. Our data suggest that HDAC-mediated targeting of BDNF and AKAP-dependent local signaling within VTA could provide novel therapeutics for prevention of later-life psychopathology.


Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Privación Materna , Área Tegmental Ventral/metabolismo , Acetilación/efectos de los fármacos , Animales , Dopamina/metabolismo , Inhibidores Enzimáticos/farmacología , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Técnicas In Vitro , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Área Tegmental Ventral/efectos de los fármacos
12.
Plant Mol Biol ; 98(1-2): 19-32, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-30117035

RESUMEN

Key message The OsPLS3 locus was isolated by map-based cloning that encodes a DUF266-containing protein. OsPLS3 regulates the onset of leaf senescence in rice. Glycosyltransferases (GTs) are one of the most important enzyme groups required for the modification of plant secondary metabolites and play a crucial role in plant growth and development, however the biological functions of most GTs remain elusive. We reported here the identification and characterization of a novel Oryza sativa premature leaf senescence mutant (ospls3). Through map-based cloning strategy, we determined that 22-bp deletion in the OsPLS3 gene encoding a domain of unknown function 266 (DUF266)-containing protein, a member of GT14-like, underlies the premature leaf senescence phenotype in the ospls3 mutant. The OsPLS3 mRNA levels progressively declined with the age-dependent leaf senescence in wild-type rice, implying a negative role of OsPLS3 in regulating leaf senescence. Physiological analysis, and histochemical staining and transmission electron microscopy assays indicated that the ospls3 mutant accumulated higher levels of ethylene and reactive oxygen species than its wild type. Furthermore, the ospls3 mutant showed hypersensitivity to exogenous 1-aminocyclopropane-1-carboxylic acid, H2O2 and high level of cytokinins. Our results indicated that the DUF266-containing gene OsPLS3 plays an important role in the onset of leaf senescence, in part through cytokinin and ethylene signaling in rice.


Asunto(s)
Emparejamiento Base , Genes de Plantas , Oryza/crecimiento & desarrollo , Oryza/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/genética , Proteínas de Plantas/genética , Eliminación de Secuencia/genética , Secuencia de Bases , Citocininas/farmacología , Etilenos/biosíntesis , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Fenotipo , Hojas de la Planta/efectos de los fármacos , Proteínas de Plantas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Estallido Respiratorio/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo
13.
J Neurochem ; 146(5): 585-597, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29779213

RESUMEN

TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP-43 exists as a full-length protein and as two shorter forms of 25 and 35 kDa. Full-length mutant TDP-43s found in amyotrophic lateral sclerosis patients re-localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP-43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kDa truncated form of TDP-43 is restricted to the intermembrane space, while the full-length forms also localize in the mitochondrial matrix in cultured neuronal NSC-34 cells. Interestingly, the full-length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial-transcribed mRNAs, while the 35 kDa form does not. In the light of the known differential contribution of the full-length and short isoforms to generate toxic aggregates, we propose that the presence of full-length TDP-43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP-43 forms play a major role.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismo , Neuronas , Oligonucleótidos/toxicidad , Isoformas de Proteínas/metabolismo , Línea Celular Transformada , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Chaperonina 60/genética , Chaperonina 60/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Citosol/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Inmunoprecipitación , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mutación/efectos de los fármacos , Mutación/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/ultraestructura , Consumo de Oxígeno/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Transfección
14.
Biochem Biophys Res Commun ; 501(4): 858-862, 2018 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-29752941

RESUMEN

We discovered that SU11274, a class I c-Met inhibitor, fluoresces when excited by 488 nm laser light and showed rapid specific accumulation in distinct subcellular compartments. Given that SU11274 reduces cancer cell viability, we exploited these newly identified spectral properties to determine SU11274 intracellular distribution and accumulation in human pancreatic cancer cells. The aim of the studies reported here was to identify organelle(s) to which SU11274 is trafficked. We conclude that SU11274 rapidly and predominantly accumulates in the endoplasmic reticulum.


Asunto(s)
Retículo Endoplásmico/metabolismo , Indoles/metabolismo , Indoles/farmacología , Piperazinas/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Fluorescencia , Humanos , Indoles/química , Piperazinas/química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-met/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Sulfonamidas/química
15.
Biomed Pharmacother ; 103: 118-126, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29649626

RESUMEN

Cervical carcinoma remains the second most common malignancy with a high mortality rate among women worldwide. TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) are novel compounds synthesized on the chemical structure of taspine derivatives, and show an effective suppression for HeLa cells. Our study aims to confirm the potential targets of F2 and F5, and investigate the underlying mechanism of the inhibitory effect on HeLa cells. In this study, Real Time Cell Analysis and crystal violet staining assay were conducted to investigate the effect of F2 and F5 on HeLa cells proliferation. And the analytical methods of surface plasmon resonance and quartz crystal microbalance were established and employed to study the interaction between F2 and F5 and potential target protein JAK2, suggesting that both compounds have strong interaction with the JAK2 protein. Western blot analysis, immunofluorescence staining study and PCR was conducted to investigate the molecules of JAK/Stat signaling pathway. Interestingly, F2 and F5 showed diverse regulation for signaling molecules because of their different chemical structure. F2 increased the expression of JAK2 and downregulated the level of P-JAK1 and P-JAK2, and decreased P-Stat3 (Ser727). While F5 could increase the expression of JAK2 and naturally decrease the phosphorylation of JAK1 and Tyk2, and decreased the expression of P-Stat6. Moreover, F2 and F5 showed the same downregulation on the P-Stat3 (Tyr705). Therefore, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, which suggested that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway.


Asunto(s)
Alcaloides/farmacología , Quinasas Janus/metabolismo , Quinazolinas/farmacología , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Alcaloides/química , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Células HeLa , Humanos , Modelos Biológicos , Fosforilación/efectos de los fármacos , Quinazolinas/química , Transducción de Señal/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo
16.
Neurotox Res ; 34(2): 198-219, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29532444

RESUMEN

Piracetam, a nootropic drug, has been clinically used for decades; however, its mechanism of action still remains enigmatic. The present study was undertaken to evaluate the role of mitochondrion-specific factors of caspase-independent pathway like apoptotic-inducing factor (AIF) and endonuclease-G (endo-G) in piracetam-induced neuroprotection. N2A cells treated with lipopolysaccharide (LPS) exhibited significant cytotoxicity, impaired mitochondrial activity, and reactive oxygen species generation which was significantly attenuated with piracetam co-treatment. Cells co-treated with LPS and piracetam exhibited significant uptake of piracetam in comparison to only piracetam-treated cells as estimated by liquid chromatography-mass spectrometry (LC-MSMS). LPS treatment caused significant translocation of AIF and endonuclease-G in neuronal N2A cells which were significantly attenuated with piracetam co-treatment. Significant over-expression of proinflammatory cytokines was also observed after treatment of LPS to cells which was inhibited with piracetam co-treatment demonstrating its anti-inflammatory property. LPS-treated cells exhibited significant oxidative DNA fragmentation and poly [ADP-ribose] polymerase-1 (PARP-1) up-regulation in nucleus, both of which were attenuated with piracetam treatment. Antioxidant melatonin but not z-VAD offered the inhibited LPS-induced DNA fragmentation indicating the involvement of oxidative DNA fragmentation. Further, we did not observe the altered caspase-3 level after LPS treatment initially while at a later time point, significantly augmented level of caspase-3 was observed which was not inhibited with piracetam treatment. In total, our findings indicate the interference of piracetam in mitochondrion-mediated caspase-independent pathway, as well as its anti-inflammatory and antioxidative properties. Graphical Abstract Graphical abstract indicating the novel interference of metabolic enhancer piracetam (P) in neuronal death mechanisms.


Asunto(s)
Caspasas/metabolismo , Fragmentación del ADN/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Piracetam/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Animales , Anexina A5/metabolismo , Factor Inductor de la Apoptosis/metabolismo , Caspasas/genética , Línea Celular Tumoral , Tamaño del Núcleo Celular/efectos de los fármacos , Ensayo Cometa , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Endodesoxirribonucleasas/metabolismo , Expresión Génica/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Lipopolisacáridos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Neuroblastoma/patología , Poli(ADP-Ribosa) Polimerasas/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estadísticas no Paramétricas , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo
17.
Aquat Toxicol ; 198: 231-239, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29558708

RESUMEN

Increasing concern has been focused on the potential risks associated with the trophic transfer to aquatic organisms of ambient contaminants in the presence of titanium dioxide nanoparticles (nano-TiO2). This study investigated the influence of nano-TiO2 on the trophic transfer of arsenic (As) from the microalgae Nannochloropsis maritima to the brine shrimp Artemia salina nauplii. We found that nano-TiO2 could significantly facilitate As sorption on N. maritima within an exposure period of 24 h, and this sorption subsequently led to higher As trophic transfer from the algae to A. salina according to trophic transfer factors (TTFAs+nano-TiO2 > TTFAs). However, after 48 h of depuration, the retention of As in A. salina fed As-nano-TiO2-contaminated algae was even lower than that in A. salina fed As-contaminated algae at the same exposure concentrations. This result indicates that the increased food chain transfer of As in the presence of nano-TiO2 can be explained by adsorption of As onto nano-TiO2 in contaminated food (algae), but the bioavailability of As in A. salina is reduced after the introduction of nanoparticles. Although the stress enzyme activities of superoxide dismutase (SOD) and acetylcholinesterase (AChE) in A. salina at a lower As concentration treatment in the presence of nano-TiO2 were not significantly changed, they increased with higher exposure concentrations of As with or without nano-TiO2. Our study highlighted the complex role of nanomaterials in the transfer of ambient contaminants via trophic chains and the potential of nano-TiO2 to reduce the bioavailability of As via trophic transfer to saltwater zooplankton.


Asunto(s)
Arsénico/metabolismo , Artemia/metabolismo , Cadena Alimentaria , Nanopartículas/toxicidad , Estramenopilos/metabolismo , Titanio/toxicidad , Acetilcolinesterasa/metabolismo , Adsorción , Animales , Artemia/efectos de los fármacos , Catalasa/metabolismo , Dieta , Cinética , Microalgas/efectos de los fármacos , Microalgas/metabolismo , Nanopartículas/ultraestructura , Estramenopilos/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Contaminantes Químicos del Agua/toxicidad
18.
Plant Cell Physiol ; 59(5): 916-929, 2018 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-29432551

RESUMEN

OsLEA5 acts as a co-regulator of a transcriptional fact ZFP36 to enhance the expression and the activity of ascorbate peroxidase OsAPX1 to regulate seed germination in rice, but it it unknown whether OsLEA5 is also crucial in plant seedlings under stress conditions. To determine this, we generated OsLEA5 overexpression and knockdown rice plants. We found that overexpression of OsLEA5 in rice plants enhanced the tolerance to drought and salt stress; in contrast, an RNA interference (RNAi) mutant of OsLEA5 rice plants was more sensitive to drought and salinity. Further investigation found that various stimuli and ABA could induce OsLEA5 expression, and OsLEA5 acted downstream of ZFP36 to be involved in ABA-induced generation of hydrogen peroxide (H2O2), and the regulation of the expression and the activities of antioxidant defense enzymes in plants leaves, and OsLEA5 contributed to stabilize ZFP36. Additionally, OsLEA5 participates in the accumulation of ABA by up-regulating ABA biosynthesis genes and down-regulating ABA metabolism genes. Moreover, we found that two homologs of OsLEA5 (5C700, short for Os05g0526700; and 5C300, short for Os05g0584300) which were induced by ABA also interacted with ZFP36 separately; interestingly, the nuclear-located 5C700 could also act as a co-activator of ZFP36 to modulate OsAPX1, while 5C300 which was down-regulated by ABA induction acted as an ABA-induced inhibitor of ZFP36 to regulate OsAPX1. Hence, our conclusion is that OsLEA5 participates in the ABA-mediated antioxidant defense to function in drought and salt stress response in rice, and the 5C subgroup of LEAs contribute by acting as co-regulators of the transcription factor ZFP36.


Asunto(s)
Ácido Abscísico/farmacología , Antioxidantes/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Adaptación Fisiológica/efectos de los fármacos , Sequías , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Genes de Plantas , Homeostasis , Peróxido de Hidrógeno/metabolismo , Modelos Biológicos , Oryza/efectos de los fármacos , Oryza/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Transporte de Proteínas/efectos de los fármacos , Salinidad , Plantones/efectos de los fármacos , Plantones/fisiología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo
19.
Life Sci ; 196: 156-161, 2018 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-29459024

RESUMEN

AIMS: Although baicalin could attenuate obesity-induced insulin resistance, the detailed mechanism of baicalin on glucose uptake has not been sufficiently explored as yet. The aim of this study was to survey if baicalin might facilitate glucose uptake and to explore its signal mechanisms in L6 myotubes. MATERIALS AND METHODS: L6 myotubes were treated with 100, 200, 400 µM baicalin for 6 h, 12 h and 24 h in this study. Then 2-NBDG and insulin signal protein levels in myotubes of L6 cells were examined. KEY FINDINGS: We discovered that administration of baicalin enhanced GLUT4, PGC-1α, pP38MAPK, pAKT and pAS160 contents, as well as GLUT4 mRNA and PGC-1α mRNA levels in L6 myotubes. The beneficial metabolic changes elicited by baicalin were abrogated in myotubes of L6 by P38MAPK or AKT inhibitors. SIGNIFICANCE: These results suggest that baicalin promoted glucose uptake in myotubes by differential regulation on P38MAPK and AKT activity. In conclusion, these data provide insight that baicalin is a powerful and promising agent for the treament of hyperglycemia via AKT/AS160/GLUT4 and P38MAPK/PGC1α/GLUT4 pathway.


Asunto(s)
Flavonoides/farmacología , Transportador de Glucosa de Tipo 4/biosíntesis , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Fibras Musculares Esqueléticas/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animales , Células Cultivadas , Desoxiglucosa/análogos & derivados , Desoxiglucosa/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Proteína Oncogénica v-akt/efectos de los fármacos , Proteína Oncogénica v-akt/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/efectos de los fármacos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
20.
Plant Cell Environ ; 41(1): 50-63, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28152567

RESUMEN

High sensitivity of seeds to water loss is a widespread phenomenon in the world's plant species. The molecular basis of this trait is poorly understood but thought to be associated with critical changes in membrane function. We profiled membrane lipids of seeds in eight species with varying levels of desiccation tolerance and found a close association between reducing seed viability and increasing phosphatidic acid (PA). We applied hydration-dehydration cycles to Arabidopsis seeds, which are normally desiccation tolerant, to mimic the onset of desiccation sensitivity with progression towards germination and examined the role of phospholipase D (PLD) in desiccation stress-induced production of PA. We found that PLDα1 became more abundant and migrated from the cytosol to the membrane during desiccation, whereas PLDδ did not change, and that all desiccation-induced PA was derived from PLDα1 hydrolysis. When PLDα1 was suppressed, the germination level after each hydration-dehydration cycle improved significantly. We further demonstrated that PLDα1-mediated PA formation modulates desiccation sensitivity as applying its inhibitor improved seed desiccation tolerance and its suppression in protoplasts enhanced survival under dehydration. The insights provided by comparative lipidomics enable us to propose a new membrane-based model for seed desiccation stress and survival.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Desecación , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Semillas/enzimología , Semillas/fisiología , Supervivencia Tisular/fisiología , Adaptación Fisiológica/efectos de los fármacos , Arabidopsis/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cotiledón/metabolismo , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Inhibidores Enzimáticos/farmacología , Germinación/efectos de los fármacos , Lípidos/análisis , Transporte de Proteínas/efectos de los fármacos , Protoplastos/efectos de los fármacos , Protoplastos/metabolismo , Semillas/efectos de los fármacos , Especificidad de la Especie , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Supervivencia Tisular/efectos de los fármacos
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