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RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP Staufen may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS-but not with mutated endogenous NLS-potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/-), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/- is protective. stau+/- also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.
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Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Demencia Frontotemporal/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Proteínas de Unión al ARN/genética , ARN/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Arginina/genética , Proteína C9orf72/genética , Núcleo Celular/genética , Citoplasma/genética , Dipéptidos/genética , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Demencia Frontotemporal/patología , Técnicas de Silenciamiento del Gen , Humanos , Neuronas/metabolismo , Neuronas/patología , Señales de Localización Nuclear/genética , Procesamiento Postranscripcional del ARN/genéticaRESUMEN
Acrylic pressure sensitive adhesives (PSAs) were prepared by UV polymerization under varying curing conditions of both fast and slow curing, employing high- and low-intensity UV radiation, respectively. The influences of curing conditions and isobornyl acrylate (IBOA) content on PSA performance were comprehensively investigated by measurement of their rheological, thermal, and adhesive properties. In particular, rheological characterization was accomplished by several analytical methods, such as in situ UV rheology, frequency sweep, stress relaxation, and temperature ramp tests, to understand the effect of the UV curing process and IBOA content on the viscoelastic behavior of acrylic PSAs. The slow-cured samples were observed to form more tightly crosslinked networks compared to the fast-cured. On the other hand, at high loading levels of IBOA, in the case of slow curing, the sample exhibited a contrasting trend, having the shortest stress relaxation time and the highest energy dissipation; this was due to molecular chain scission occurring in the crosslinked polymer during UV polymerization. Consequently, we successfully demonstrated the influence of monomer composition of acrylic PSAs, and that of curing conditions employed in UV polymerization. This study provides valuable insights for the development of crosslinked polymer networks of acrylic PSAs for flexible display applications.
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BACKGROUND: Periodontitis is reported to be associated with preterm birth (spontaneous preterm labor and birth). Gastroesophageal reflux disease (GERD) is common during pregnancy and is expected to be related to periodontitis. However, little research has been done on the association among preterm birth, GERD and periodontitis. This study uses popular machine learning methods for analyzing preterm birth, GERD and periodontitis. METHODS: Data came from Anam Hospital in Seoul, Korea, with 731 obstetric patients during January 5, 1995 - August 28, 2018. Six machine learning methods were applied and compared for the prediction of preterm birth. Variable importance, the effect of a variable on model performance, was used for identifying major determinants of preterm birth. RESULTS: In terms of accuracy, the random forest (0.8681) was similar with logistic regression (0.8736). Based on variable importance from the random forest, major determinants of preterm birth are delivery and pregestational body mass indexes (BMI) (0.1426 and 0.1215), age (0.1211), parity (0.0868), predelivery systolic and diastolic blood pressure (0.0809 and 0.0763), twin (0.0476), education (0.0332) as well as infant sex (0.0331), prior preterm birth (0.0290), progesterone medication history (0.0279), upper gastrointestinal tract symptom (0.0274), GERD (0.0242), Helicobacter pylori (0.0151), region (0.0139), calcium-channel-blocker medication history (0.0135) and gestational diabetes mellitus (0.0130). Periodontitis ranked 22nd (0.0084). CONCLUSION: GERD is more important than periodontitis for predicting and preventing preterm birth. For preventing preterm birth, preventive measures for hypertension, GERD and diabetes mellitus would be needed alongside the promotion of effective BMI management and appropriate progesterone and calcium-channel-blocker medications.
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Reflujo Gastroesofágico , Periodontitis , Nacimiento Prematuro , Diabetes Gestacional , Femenino , Reflujo Gastroesofágico/epidemiología , Edad Gestacional , Humanos , Hipertensión Inducida en el Embarazo , Masculino , Periodontitis/epidemiología , EmbarazoRESUMEN
Amyotrophic lateral sclerosis is a devastating neurodegenerative disease with a complex genetic basis, presenting both in familial and sporadic forms. The hexanucleotide (G4C2) repeat expansion in the C9orf72 gene, which triggers distinct pathogenic mechanisms, has been identified as a major contributor to familial and sporadic Amyotrophic lateral sclerosis cases. Animal models have proven pivotal in understanding these mechanisms; however, discrepancies between models due to variable transgene sequence, expression levels, and toxicity profiles complicate the translation of findings. Herein, we provide a systematic comparison of 7 publicly available Drosophila transgenes modeling the G4C2 expansion under uniform conditions, evaluating variations in their toxicity profiles. Further, we tested 3 previously characterized disease-modifying drugs in selected lines to uncover discrepancies among the tested strains. Our study not only deepens our understanding of the C9orf72 G4C2 mutations but also presents a framework for comparing constructs with minute structural differences. This work may be used to inform experimental designs to better model disease mechanisms and help guide the development of targeted interventions for neurodegenerative diseases, thus bridging the gap between model-based research and therapeutic application.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Levamisol/análogos & derivados , Enfermedades Neurodegenerativas , Animales , Drosophila/genética , Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genéticaRESUMEN
MDC1, a mediator of DNA damage response, recruits other repair proteins on double-strand break (DSB) sites. MDC1 is necessary for activating checkpoint kinases Chk1 and Chk2. It is unclear whether Chk1 interacts with MDC1. MDC1 also comprises many discrete domains. The role of the proline-serine-threonine (PST)-repeat domain of MDC1 in the DNA damage response is unclear. Here, we showed that MDC1 directly binds Chk1 through this PST-repeat region. Phosphorylation of Chk1 by ionizing radiation (IR) also required this PST-repeat domain. Degradation of intact MDC1 was accelerated depending on the PST-repeat domain after IR exposure. In the IR damage response, the PST-repeat-deleted MDC1 levels remained elevated with slow degradation. This abnormal regulation of MDC1 was F-box- and WD40 repeat-containing 7 (FBXW7)-dependent. The mutation of lysine 1413 within the PST-repeat of MDC1 deregulated MDC1 with or without damage. K1413R mutant and PST-deleted MDC1 displayed reduced ability to repair the damaged genome post-IR exposure. These results provide that the PST domain of MDC1 is involved in Chk1 and DNA repair activation. The findings suggest new insights into how MDC1 connects the checkpoint and DNA repair in the DNA damage response.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Prolina/metabolismo , Serina/metabolismo , Treonina/metabolismo , Roturas del ADN de Doble Cadena , Humanos , Fosforilación , TransfecciónRESUMEN
Neurovascular coupling is a precise mechanism that induces increased blood flow to activated brain regions, thereby providing oxygen and glucose. In this study, we hypothesized that N-methyl-D-aspartate (NMDA) receptor signaling, the most well characterized neurotransmitter signaling system which regulates delivery of essential molecules through the blood-brain barrier (BBB). Upon application of NMDA in both in vitro and in vivo models, increased delivery of bioactive molecules that was mediated through modulation of molecules involved in molecular delivery, including clathrin and caveolin were observed. Also, NMDA activation induced structural changes in the BBB and increased transcellular permeability that showed regional heterogeneity in its responses. Moreover, NMDA receptor activation increased endosomal trafficking and facilitated inactivation of lysosomal pathways and consequently increased molecular delivery mediated by activation of calmodulin-dependent protein kinase II (CaMKII) and RhoA/protein kinase C (PKC). Subsequent in vivo experiments using mice specifically lacking NMDA receptor subunit 1 in endothelial cells showed decreased neuronal density in the brain cortex, suggesting that a deficiency in NMDA receptor signaling in brain endothelial cells induces neuronal losses. Together, these results highlight the importance of NMDA-receptor-mediated signaling in the regulation of BBB permeability that surprisingly also affected CD31 staining.
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N-Metilaspartato , Receptores de N-Metil-D-Aspartato , Animales , Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Endoteliales/metabolismo , Ratones , N-Metilaspartato/farmacología , Permeabilidad , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Among the components of the blood-brain barrier (BBB), endothelial cells (ECs) play an important role in supplying limited materials, especially glucose, to the brain. However, the mechanism by which glucose is metabolized in brain ECs is still elusive. To address this topic, we assessed the metabolic signature of glucose utilization using live-cell metabolic assays and liquid chromatography-tandem mass spectrometry metabolomic analysis. We found that brain ECs are highly dependent on aerobic glycolysis, generating lactate as its final product with minimal consumption of glucose. Glucose treatment decreased the oxygen consumption rate in a dose-dependent manner, indicating the Crabtree effect. Moreover, when glycolysis was inhibited, brain ECs showed impaired permeability to molecules utilizing transcellular pathway. In addition, we found that the blockade of glycolysis in mouse brain with 2-deoxyglucose administration resulted in decreased transcellular permeability of the BBB. In conclusion, utilizing glycolysis in brain ECs has critical roles in the maintenance and permeability of the BBB. Overall, we could conclude that brain ECs are highly glycolytic, and their energy can be used to maintain the transcellular permeability of the BBB.
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Barrera Hematoencefálica , Células Endoteliales , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Glucosa/metabolismo , Glucólisis , Ratones , PermeabilidadRESUMEN
For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, in the current study, we investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, we observed dynamic changes in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. We further showed that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons.
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Drosophila , alfa Carioferinas , Animales , Acetilación , Transporte Activo de Núcleo Celular , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Ataxina-3/genética , Ataxina-3/metabolismo , Calcio/metabolismo , Drosophila/metabolismo , Neuronas/metabolismoRESUMEN
The responses of central nervous system (CNS) cells such as neurons and glia in neurodegenerative diseases (NDs) suggest that regulation of neuronal and glial functions could be a strategy for ND prevention and/or treatment. However, attempts to develop such therapeutics for NDs have been hindered by the challenge of blood-brain barrier (BBB) permeability and continued constitutive neuronal loss. These limitations indicate the need for additional perspectives for the prevention/treatment of NDs. In particular, the disruption of the blood-brain barrier (BBB) that accompanies NDs allows brain infiltration by peripheral factors, which may stimulate innate immune responses involved in the progression of neurodegeneration. The accumulation of blood factors like thrombin, fibrinogen, c-reactive protein (CRP) and complement components in the brain has been observed in NDs and may activate the innate immune system in the CNS. Thus, strengthening the integrity of the BBB may enhance its protective role to attenuate ND progression and functional loss. In this review, we describe the innate immune system in the CNS and the contribution of blood factors to the role of the CNS immune system in neurodegeneration and neuroprotection.
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Barrera Hematoencefálica , Enfermedades Neurodegenerativas , Encéfalo , Sistema Nervioso Central , Humanos , NeuroglíaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease (COVID-19), is currently infecting millions of people worldwide and is causing drastic changes in people's lives. Recent studies have shown that neurological symptoms are a major issue for people infected with SARS-CoV-2. However, the mechanism through which the pathological effects emerge is still unclear. Brain endothelial cells (ECs), one of the components of the blood-brain barrier, are a major hurdle for the entry of pathogenic or infectious agents into the brain. They strongly express angiotensin converting enzyme 2 (ACE2) for its normal physiological function, which is also well-known to be an opportunistic receptor for SARS-CoV-2 spike protein, facilitating their entry into host cells. First, we identified rapid internalization of the receptor-binding domain (RBD) S1 domain (S1) and active trimer (Trimer) of SARS-CoV-2 spike protein through ACE2 in brain ECs. Moreover, internalized S1 increased Rab5, an early endosomal marker while Trimer decreased Rab5 in the brain ECs. Similarly, the permeability of transferrin and dextran was increased in S1 treatment but decreased in Trimer, respectively. Furthermore, S1 and Trimer both induced mitochondrial damage including functional deficits in mitochondrial respiration. Overall, this study shows that SARS-CoV-2 itself has toxic effects on the brain ECs including defective molecular delivery and metabolic function, suggesting a potential pathological mechanism to induce neurological signs in the brain.
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Barrera Hematoencefálica/metabolismo , Encéfalo/patología , COVID-19/patología , Células Endoteliales/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/virología , Células Endoteliales/virología , Humanos , Ratones , Mitocondrias/metabolismo , Dominios Proteicos , SARS-CoV-2/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Cytoplasmic accumulation of TDP-43 in motor neurons is the most prominent pathological feature in amyotrophic lateral sclerosis (ALS). A feedback cycle between nucleocytoplasmic transport (NCT) defect and TDP-43 aggregation was shown to contribute to accumulation of TDP-43 in the cytoplasm. However, little is known about cellular factors that can control the activity of NCT, thereby affecting TDP-43 accumulation in the cytoplasm. Here, we identified via FRAP and optogenetics cytosolic calcium as a key cellular factor controlling NCT of TDP-43. Dynamic and reversible changes in TDP-43 localization were observed in Drosophila sensory neurons during development. Genetic and immunohistochemical analyses identified the cytosolic calcium-Calpain-A-Importin α3 pathway as a regulatory mechanism underlying NCT of TDP-43. In C9orf72 ALS fly models, upregulation of the pathway activity by increasing cytosolic calcium reduced cytoplasmic accumulation of TDP-43 and mitigated behavioral defects. Together, these results suggest the calcium-Calpain-A-Importin α3 pathway as a potential therapeutic target of ALS.
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Calcio/metabolismo , Calpaína/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Drosophila melanogaster , Neuronas/metabolismoRESUMEN
Novel disubstituted adamantyl derivatives were synthesized and evaluated in a P-glycoprotein dependent multidrug resistance cancer cell line. The hit to lead optimization provided potent MDR reversal agents. Some potent adamantyl derivatives were more than 10-fold more potent than verapamil without considerable intrinsic cytotoxicity. The 3-trifluorophenyl derivative 14f did not affect the metabolism of CYP450 3A4, whereas most of MDR revertants had a weak inhibitory effect.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adamantano/química , Adamantano/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Adamantano/síntesis química , Línea Celular Tumoral , Citocromo P-450 CYP3A/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Sarcoma/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
We conducted a preliminary study on fiber structural development in the high-speed melt spinning of environmentally friendly polyethylene terephthalate (Ti-PET) synthesized with 25 ppm of titanium-based catalyst, which was compared with conventional PET (Sb-PET) synthesized with 260 ppm of antimony-based catalyst. Gel permeation chromatography of Ti- and Sb-PET resins of intrinsic viscosity 0.63 confirmed that both resins have similar molecular weights and distributions. However, differential scanning calorimetry revealed that the Ti-PET resin exhibited a lower melt-crystallization peak and isothermal melt-crystallization rate than the Sb-PET resin. High-speed melt spinning of the Ti- and Sb-PET was possible up to a spinning velocity of 6 km/min. Two-dimensional wide-angle X-ray diffraction analyses showed that the molecular orientation of the obtained as-spun Ti- and Sb-PET fibers increased with spinning velocity, and a highly oriented, crystalline structure by orientation-induced crystallization started to appear from 5 km/min. Notably, Ti-PET fibers showed a lower degree of crystalline structural development and lower tensile strength compared with Sb-PET fibers under the high-speed spinning conditions. Our results suggest that the catalyst in PET resins can act as nucleating agents in thermal- and orientation-induced crystallization, and that differences in catalyst content can influence PET fiber structure development under extreme conditions in high-speed melt spinning.
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: Bacterial and fungal pathogens have caused serious problems to the human health. This is particularly true for untreatable infectious diseases and clinical situations where there is no reliable treatment for infected patients. To increase the antimicrobial activity of materials, we introduce silver nanoparticle (NP) patches in which the NPs are incorporated to the surface of smooth and uniform poly(acrylic acid) (PAA) nanofibers. The PAA nanofibers were thermally crosslinked with ethylene glycol via heat treatment through a mild method. The characterization of the resulting PAA-silver NP patches was done using scanning electron microscopy (SEM), UV spectroscopy, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). To demonstrate the antimicrobial activity of PAA, we incorporated the patches containing the silver NPs into strains of fungi such as Candida albicans (C. albican) and bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA). The PAA-silver fibers achieved zones of inhibition against C. albicans and MRSA indicating their antimicrobial activity against both fungi and bacteria. We conclude that silver NP patches exhibited multiple inhibitory actions for the interruption and blockage of activity fungal and bacterial strains, which has the potential as an antimicrobial agent in infectious diseases. Moreover, the proposed material has the potential to be used in antimicrobial textile fabrics, food packaging films, and wound dressings.
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Carbonate-type macrodiols synthesized by base-catalyzed polycondensation of co-diols and dimethyl carbonate as an environmentally-friendly route were subsequently utilized for the preparation of transparent and self-healable thermoplastic polyurethanes (TPUs) containing a carbonate-type soft segment. Three types of macrodiols, obtained from mono, dual and triple diol-monomers for target molecular weights of 1 and 1.5 kg mol-1, were analyzed by ¹H NMR integration and the OH titration value. Colorless transparent macrodiols in a liquid state at a room temperature of 20 °C were obtained, except the macrodiol from mono 1,6-hexanediol. Before TPU synthesis, macrodiols require pH neutralization to prevent gelation. TPUs synthesized by a solution pre-polymer method with 4,4'-methylene(bisphenyl isocyanate) and 1,4-butanediol as a chain extender exhibited moderate molecular weights, good transparencies and robust mechanical properties. Especially, the incorporation of 3-methyl-1,5-pentanediol within carbonate-type macrodiols enhanced the transparency of the resultant TPUs by decreasing the degree of microphase separation evidenced by ATR-FTIR and DSC. Interestingly, packing density of hard segments and the degree of microphase separation determined the self-healing efficiency of TPUs, which showed good performances in the case of sourced macrodiols from triple diol-monomers.
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We conducted a genetic yeast screen to identify salt tolerance (SAT) genes in a maize kernel cDNA library. During the screening, we identified a maize clone (SAT41) that seemed to confer elevated salt tolerance in comparison to control cells. SAT41 cDNA encodes a 16-kDa protein which is 82.4% identical to the Arabidopsis Multiprotein bridging factor 1a (MBF1a) transcriptional coactivator gene. To further examine salinity tolerance in Arabidopsis, we functionally characterized the MBF1a gene and found that dehydration as well as heightened glucose (Glc) induced MBF1a expression. Constitutive expression of MBF1a in Arabidopsis led to elevated salt tolerance in transgenic lines. Interestingly, plants overexpressing MBF1a exhibited insensitivity to Glc and resistance to fungal disease. Our results suggest that MBF1a is involved in stress tolerance as well as in ethylene and Glc signaling in Arabidopsis.
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Proteínas de Arabidopsis/biosíntesis , Arabidopsis/metabolismo , Transactivadores/biosíntesis , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Etilenos/metabolismo , Glucosa/metabolismo , Datos de Secuencia Molecular , Presión Osmótica , Plantas Modificadas Genéticamente , Canales de Sodio/metabolismo , Transactivadores/genética , Transactivadores/fisiología , Zea mays/genéticaRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) induces the activation of all three types of mitogen-activated protein kinase (MAPK): c-Jun NH(2)-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). This cytokine also induces the production of several types of reactive oxygen species, including H(2)O(2). With the use both of HeLa cells expressing wild-type or dominant negative forms of the cytosolic peroxidase peroxiredoxin II and of mouse embryonic fibroblasts deficient in this protein, we evaluated the roles of H(2)O(2) in the activation of MAPKs by TNF-alpha. In vitro kinase assays as well as immunoblot analysis with antibodies specific for activated MAPKs indicated that H(2)O(2) produced in response to TNF-alpha potentiates the activation of JNK and p38 induced by this cytokine but inhibits that of ERK. Our results also suggest that cytosolic peroxiredoxins are important regulators of TNF signaling pathways.
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Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Peroxidasas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Citosol/metabolismo , Activación Enzimática/efectos de los fármacos , Células HeLa , Humanos , MAP Quinasa Quinasa 4 , Peroxirredoxinas , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Lipoxygenases (LOXs) catalyze the formation of fatty acid hydroperoxides involved in responses to stresses. This study examines the expression of a non-traditional dual positional specific maize LOX in response to wounding or methyl jasmonate (MeJA). Full-length maize LOX cDNA was expressed in Escherichia coli, and recombinant LOX was purified and characterized enzymatically. RP-HPLC and GC-MS analysis showed that the purified LOX converts alpha-linolenic acid into 13-hydroperoxylinolenic acid and 9-hydroperoxylinolenic acid in a 6:4 ratio. LOX mRNA accumulated rapidly and transiently in response to wounding reaching a peak of expression about 3 h after wounding. This increase followed an initial increase in endogenous jasmonic acid (JA) 1 h after wounding (JA burst). However, the expression of LOX induced by MeJA lasted longer than the expression induced by wounding, and the MeJA-induced expression seemed to be biphasic pattern composed of early and late phases. The expression of LOX in the presence of inhibitors of JA biosynthesis was not completely inhibited, but delayed in wound response and the expression period was shortened in MeJA response. These results suggest that wound-responsive JA burst may trigger the early phase of LOX expression which facilitates biosynthesis of endogenous JA through its 13-LOX activity, and subsequently leads to the activation of the late phase LOX expression in MeJA-treated maize seedlings. Implications of dual positional specificity of maize LOX in the observed expression kinetics are discussed.
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Acetatos/farmacología , Ciclopentanos/farmacología , Lipooxigenasa/genética , Plantones/genética , Zea mays/genética , Secuencia de Aminoácidos , Aspirina/farmacología , Cromatografía Líquida de Alta Presión/métodos , Ciclopentanos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Lipooxigenasa/química , Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , Datos de Secuencia Molecular , Oxilipinas , Filogenia , Reguladores del Crecimiento de las Plantas/farmacología , Pirazoles/farmacología , Plantones/enzimología , Homología de Secuencia de Aminoácido , Estrés Mecánico , Relación Estructura-Actividad , Especificidad por Sustrato , Factores de Tiempo , Zea mays/enzimologíaRESUMEN
In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.