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BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.
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Ascomicetos , Verticillium , Melaninas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Verticillium/genética , Dedos de Zinc , Enfermedades de las Plantas/microbiologíaRESUMEN
(2S, 3R)-4-methylsulfonylphenylserine [(2S, 3R)-MPS], a key chiral precursor for antibiotics florfenicol and thiamphenicol, could be asymmetrically synthesized by l-threonine transaldolase (LTTA) coupled with an acetaldehyde elimination system. The low efficiency of acetaldehyde elimination system blocked further accumulation of (2S, 3R)-MPS. To address this issue, strengthening acetaldehyde elimination system and enzyme self-assembly strategy were combined to accelerate biosynthesis of (2S, 3R)-MPS. The new multi-enzyme cascade with intensified acetaldehyde elimination system BL21 (PsLTTAD2/ScADH/BtGDH) could produce (2S, 3R)-MPS with a titer of 157.6 mM, 1.7-folds than that produced by the original system BL21 (PsLTTAD2/ApADH/CbFDH). Moreover, self-assembly of PsLTTAD2 and ScADH by respective fusion of SpyTag and SpyCatcher were carried out to develop a self-assembled multi-enzyme cascade BL21 (ST-PsLTTAD2/SC-ScADH/BtGDH). As a result, the yield of (2S, 3R)-MPS was up to 248.1 mM with 95% de. As far as we knew, that represented the highest yield of (2S, 3R)-MPS by enzymatic synthesis, and therefore was a promising and green route for industrial production of this valuable compound.
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Acetaldehído/química , Diseño de Fármacos , Catálisis , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Ingeniería Genética , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
Inosine monophosphate dehydrogenase (IMPDH) of human is an attractive target for immunosuppressive agents. Currently, small-molecule inhibitors do not show good selectivity for different IMPDH isoforms (IMPDH1 and IMPDH2), resulting in some adverse effects, which limit their use. Herein, we used a small-molecule probe specifically targeting IMPDH2 and identified Cysteine residue 140 (Cys140) as a selective druggable site. On covalently binding to Cys140, the probe exerts an allosteric regulation to block the catalytic pocket of IMPDH2 and further induces IMPDH2 inactivation, leading to an effective suppression of neuroinflammatory responses. However, the probe does not covalently bind to IMPDH1. Taken together, our study shows Cys140 as a druggable site for selectively inhibiting IMPDH2, which provides great potential for development of therapy agents for autoimmune and neuroinflammatory diseases with less unfavorable tolerability profile.
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Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , IMP Deshidrogenasa/metabolismo , Inflamación/tratamiento farmacológico , Isoflavonas/farmacología , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/farmacología , Sitios de Unión , Dominio Catalítico , Línea Celular , Cisteína/metabolismo , Humanos , IMP Deshidrogenasa/química , IMP Deshidrogenasa/genética , Inflamación/metabolismo , Isoflavonas/química , Ratones Endogámicos BALB C , Microglía/efectos de los fármacos , Microglía/patología , Terapia Molecular Dirigida/métodos , Relación Estructura-ActividadRESUMEN
To investigate the inhibitory effects and mechanism of Cistanche tubulosa ethanol extract( CTEE) against oxygen-glucose deprivation/reperfusion( OGD/R)-induced PC12 cells neuronal injury. In this study,OGD/R-induced PC12 cells were used to explore the neuroprotective effects of CTEE( 12. 5,25,50 mg·L-1) by detecting cell viability with MTT assay,apoptosis with AO/EB and Hoechst 33258,mitochondrial membrane potential changes with JC-1 staining,mitochondrial oxidative stress with MitoSOX staining,as well as the apoptosis-related protein expression( PARP,cleaved PARP,caspase-3,cleaved caspase-3,Bax,Bcl-2) with Western blot. RESULTS:: showed that CTEE effectively protected OGD/R-induced neuronal injury and increased the survival rate of PC12 cells.AO/EB and Hoechst 33258 staining showed that CTEE could effectively inhibit apoptosis. Moreover,JC-1 and MitoSOX staining results showed that CTEE decreased mitochondrial stress and mitochondrial membrane potential imbalance in PC12 cells in a concentration-dependent manner. Meanwhile,CTEE could obviously suppress the activation of key proteins in mitochondrial apoptosis pathway such as caspase-3 and PARP,and significantly inhibit the rise of Bax and down-regulation of Bcl-2. In conclusion,CTEE has obvious protective effects on OGD/R-induced PC12 cells neuronal injury,potentially via inhibiting mitochondrial oxidative stress and apoptosis-related signaling pathway.
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Apoptosis , Cistanche/química , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Animales , Caspasa 3/metabolismo , Etanol , Glucosa , Estrés Oxidativo , Oxígeno , Células PC12 , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Proteína X Asociada a bcl-2/metabolismoRESUMEN
To investigate the immune activation effect and mechanism of low molecular weight saccharides from Cistanche deserticola(LMSC) on mouse peritoneal macrophages, RAW264.7 cells. The RAW264.7 cells were divided into the normal control group, LPS positive control group, and LMSC treatment groups. The RAW264.7 cells were treated with various concentrations of LMSC from 3.91 to 62.5 gâ¢L ⻹. The neutral red assay was employed to detect the phagocytic activity of macrophages. NO release was detected by using NO kit, and macrophage activation associated protein expression levels (TNF-α, IL-6, IKKß, p-IKKß, IκBα, p-IκBα, NF-κB, and p-NF-κB) were detected by Western blot. Results showed that LMSC had an activation effect on macrophages; it can significantly increase the release of NO in RAW264.7 cells and promote the expression of cytokines TNF-α and IL-6. Moreover, LMSC significantly increased the phosphorylation of IKKß, IκBα, and NF-κB p65. Furthermore, mannitol's one of the main constituents in LMSC significantly enhanced the phagocytic activity of macrophages. These results showed that LMSC could activate macrophages by up-regulating the NF-κB signaling pathway, and mannitol may be one of the main active components in LMSC.
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Carbohidratos/farmacología , Cistanche/química , Activación de Macrófagos/efectos de los fármacos , Manitol/farmacología , Fitoquímicos/farmacología , Animales , Macrófagos/efectos de los fármacos , Ratones , Peso Molecular , Células RAW 264.7 , Transducción de Señal , Factor de Necrosis Tumoral alfaRESUMEN
This study aims to investigate the targets and targets-involved mechanism for the macrophage activation of low molecular weight saccharides from Cistanche deserticola (LMSC). The phagocytic activity and NO release of RAW264.7 cells were detected, and results showed that LMSC exerts immune activation effect by significantly increasing the phagocytic activity and NO release. LMSC-conjugated epoxy-activated sepharose beads were prepared as affinity reagent to capture the target proteins. Twenty-four proteins such as Eef2 were identified by LC-MS/MS analysis. Pathway enrichment analysis showed LMSC activated RAW264.7 cells by regulating Fcgamma receptor dependent phagocytosis, TNF-alpha NF-κB signaling pathway, glycolysis/gluconeogenesis and the citric acid cycle and respiratory electron transport pathway.
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Carbohidratos/farmacología , Cistanche/química , Activación de Macrófagos/efectos de los fármacos , Animales , Carbohidratos/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía Liquida , Ratones , Peso Molecular , Óxido Nítrico/metabolismo , Fagocitosis , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Células RAW 264.7 , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
AIM: To describe the surgical procedure of fusiform penetrating keratoplasty (FPK) using multiple trephines of different sizes for treating patients with severe infectious keratitis. METHODS: Fourteen eyes underwent FPK, and 15 eyes received conventional penetrating keratoplasty (PK) were included in the study. The best-corrected visual acuity (BCVA), refractive outcomes, endothelial cell density, and postoperative complications were recorded. RESULTS: The FPK group was followed for an average of 15.3±2.1mo, whereas the PK group was followed for 16.1±1.9mo. The corneal ulcers were elliptical-shaped in all 14 eyes in the FPK group. The mean BCVA (logMAR, 0.26±0.13) showed no statistically significant differences from that in the PK group (logMAR, 0.21±0.12, P>0.05) at 1y after surgery. But the mean curvature, mean astigmatism, and mean spherical equivalent in the FPK group were lower than those in the PK group (P<0.05). Peripheral anterior synechia was observed in one patient in the FPK group, whereas 6 patients in the PK group. Suture loosening and neovascularization were observed in 4 and 5 eyes in the PK group, respectively. No graft immune rejection or elevation of intraocular pressure was observed in the two groups. CONCLUSION: For patients with elliptical-shaped corneas or corneal ulcers, FPK can avoid disrupting of corneal limbus, reduce the risk of postoperative complications, and can result in satisfactory visual quality.
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OBJECTIVE: We retrospectively studied the efficacy of bevacizumab as salvage therapy for recurrent malignant glioma with a focus on the overall survival (OS). METHODS: Patients who received a therapy other than surgery for recurrent malignant glioma were included. Efficacy was evaluated using MRI. Neurological function was evaluated using the Response Assessment in Neuro-Oncology (RANO). The survival rate was calculated using the Kaplan-Meier method. RESULTS: Fifty-one patients with recurrent glioma (31 grade III, 20 grade IV) were included. Among them, 22 subjects (43.1%) received bevacizumab. The median OS was 10.2 months (range, 1 to 27 months). Patients receiving bevacizumab had comparable OS (a median of 9.9 vs. 10.0 months) and similar 6-month survival rate (43% vs. 34%) to those who did not receive bevacizumab. A subgroup analysis failed to notice any significant difference in grade III glioma patients receiving bevacizumab vs. those who did not. The median survival was significantly longer at 8.9 months (range, 4 to 13 months) in grade IV glioma patients receiving bevacizumab than in those who did not (5.6 months, range, 2 to 7 months, P=0.042). The 6-month survival rate was higher (83%) in those who received bevacizumab than in those who did not (47%, P=0.046). No grade 3/4 adverse events were observed in any patient. CONCLUSIONS: Bevacizumab, as a rescue therapy, provides a survival benefit for recurrent grade IV glioma.
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Suzuki-Miyaura cross-couplings of arenediazonium salts with arylboronic acids catalyzed by highly active aluminium hydroxide-supported palladium nanoparticles catalyst have been investigated for the first time. The reactions are performed at 25 °C in MeOH without any base and ligand to afford biaryls in good to excellent yields under non-anhydrous and non-degassed conditions.
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Hidróxido de Aluminio/química , Compuestos Azo/química , Boratos/química , Ácidos Borónicos/química , Nanopartículas del Metal/química , Paladio/química , Sales (Química)/química , Catálisis , TemperaturaRESUMEN
Mitochondrial fusion/fission dynamics plays a fundamental role in neuroprotection; however, there is still a severe lack of therapeutic targets for this biological process. Here, we found that the naturally derived small molecule echinacoside (ECH) significantly promotes mitochondrial fusion progression. ECH selectively binds to the previously uncharacterized casein kinase 2 (CK2) α' subunit (CK2α') as a direct cellular target, and genetic knockdown of CK2α' abolishes ECH-mediated mitochondrial fusion. Mechanistically, ECH allosterically regulates CK2α' conformation to recruit basic transcription factor 3 (BTF3) to form a binary protein complex. Then, the CK2α'/BTF3 complex facilitates ß-catenin nuclear translocation to activate TCF/LEF transcription factors and stimulate transcription of the mitochondrial fusion gene Mfn2. Strikingly, in a mouse middle cerebral artery occlusion (MCAO) model, ECH administration was found to significantly improve cerebral injuries and behavioral deficits by enhancing Mfn2 expression in wild-type but not CK2α'+/- mice. Taken together, our findings reveal, for the first time, that CK2 is essential for promoting mitochondrial fusion in a Wnt/ß-catenin-dependent manner and suggest that pharmacologically targeting CK2 is a promising therapeutic strategy for ischemic stroke.
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Quinasa de la Caseína II/genética , GTP Fosfohidrolasas/genética , Glicósidos/farmacología , Accidente Cerebrovascular Isquémico/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Infarto de la Arteria Cerebral Media , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/patología , Factor de Unión 1 al Potenciador Linfoide/genética , Ratones , Dinámicas Mitocondriales/genética , Complejos Multiproteicos/genética , Neuroprotección/genética , Factor 1 de Transcripción de Linfocitos T/genética , Transcripción Genética/efectos de los fármacos , beta Catenina/genéticaRESUMEN
L-threonine transaldolase(PsLTTA) could asymmetric synthesize ß-hydroxy-α-amino acids (HAAs) with excellentstereoselectivity, while the poor yield limited its further application. Here we provided a combinatorial strategy to improve HAAs production, by directed evolution of PsLTTA towards enhanced activity and introducing an acetaldehyde elimination system to avoid acetaldehyde over-accumulation. A novel high throughput screening (HTS) method for evaluating PsLTTA activity was developed andapplied for directed evolution of PsLTTA. Subsequently, we co-expressedalcohol dehydrogenase andformate dehydrogenase to construct an acetaldehyde elimination system toremove acetaldehyde inhibition.Moreover, the above positive strategies were integrated. As a result,the (2S,3R)-p-methylsulfonyl phenylserine yield reached 154.0 mM andwith 94.6% devalue, the highest productivity and stereoselectivity of (2S,3R)-HAAs reported by enzymatic synthesis so far. Taken together, our studies provided an efficient and green route for chiral synthesis of (2S,3R)-HAAs, which might contribute to the industrialization production of these useful building blocks.
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Treonina , Transaldolasa , Acetaldehído , AminoácidosRESUMEN
It is an effective approach to control invasive plants based on the allelopathic effect of native plants with higher economic values, from the perspective of allelopathy. The aim of this study was to test the allelopathic effect of a local crop, sweet potato (Ipomoea batatas), on the invasive plant Alternanthera philoxeroides. Water extracts from different organs of sweet potato (roots, stems, leaves) with three concentrations (0.025, 0.05, 0.1 g·mL-1) were used in the study. To test the effect of sweet potato on rhizome growth of A. philoxeroides, the morphological index (ramet number, node number, leaf number, leaf area, plant height, total dry weight and root number of new ramets), allelopathic response index, trait values (succulent degree, root-shoot ratio, specific leaf area, leaf mass ratio, stem mass ratio, root mass ratio), CAT, POD, MDA and SOD in young leaves were measured. The results showed that 1) Water extracts from different organs of sweet potato with different concentrations differed in their effects on the growth of A. philoxeroides. Water extract from root with 0.1 g·mL-1 significantly inhibited all morphological indices. Except total dry weight and root number, other morphological indices decreased significantly with increasing concentration of water extract from all organs. 2) The synthetic allelopathic response index (RI) was negative under all treatments, indicating that water extract of sweet potato had negative effects on all indices of A. philoxeroides and thus inhibited their growth. Among all the treatments, water extract from root with a concentration of 0.1 g·mL-1 had the strongest allelopathic inhibition (RI=-0.73), followed by that from stem with a concentration of 0.1 g·mL-1(RI=-0.44) and from root with a concentration of 0.05 g·mL-1(RI=-0.44). 3) Water extract of sweet potato had significant inhibitory effects on the degree of succulence, root-shoot ratio, specific leaf area, and leaf mass ratio, but did not affect stem mass ratio and root mass ratio. 4) Water extract of sweet potato significantly increased the contents of MDA and SOD in the fresh leaves of A. philoxeroides, while had no effect on the contents of CAT and POD. All these results indicates that water extract of sweet potato significantly suppress the ramet growth of A. philoxeroides.
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Amaranthaceae , Ipomoea batatas , Alelopatía , Especies Introducidas , AguaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Aglaia odorata Lour. is a traditional Chinese medicinal plant possessing properties of improving blood circulation, and it is widely used in the treatment of dizziness, traumatic injuries and bruises. AIM OF STUDY: In this study, we are aimed to investigate the cerebral protection effect of the extracts from leaves of Aglaia odorata Lour. (ELA) and the potential mechanism in vivo and in vitro. MATERIALS AND METHODS: The therapeutic effect of ELA on ischemic cerebral stroke was measured on a middle cerebral artery occlusion (MCAO) rat model. Protective effect of ELA on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12â¯cells was measured by MTT assay. The apoptotic cells were observed by Hoechst 33258 staining and acridine orange/ethidium bromide double staining assay. Mitochondria were observed by Mitotracker staining assay. The mitochondrial membrane potential was determined by JC-1 staining assay. Western blot was used to investigate the effects of ELA on apoptosis-related proteins. RESULTS: We showed that ELA was an effective neuroprotective agent. In vivo experiments, ELA exerted significant protective effect on MCAO model. TTC staining showed that ELA could reduce cerebral infarction area against MCAO insult. HE and Nissl's staining indicated that ELA could reverse the damage of cortex and hippocampus caused by MCAO. In vitro experiments, ELA showed significant protective effect on OGD/R-induced PC12â¯cells by reducing the number of apoptotic cells, increasing mitochondrial membrane potential, and reducing superoxide aggregation, further suppressing mitochondrial caspase-9/3 apoptosis pathway. Moreover, protective effect of ELA on mitochondrial function may be exerted by inhibiting p53/Puma signal pathway. CONCLUSION: Our results suggest that ELA exerts a marked neuroprotective effect against cerebral ischemia potentially via suppressing p53/Puma-mediated mitochondrial caspase-9/3 apoptosis pathway.
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Aglaia , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Infarto de la Arteria Cerebral Media/prevención & control , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Aglaia/química , Animales , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/aislamiento & purificación , Células PC12 , Extractos Vegetales/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Histone post-translational modifications (PTMs) have been shown to be highly associated with inflammation response, suggesting a therapeutic significance of pharmacologically editing histone PTMs. Currently reported anti-inflammation small-molecules mainly target histone PTMs writers or erasers for methylation, phosphorylation, and acetylation. Although histone chaperones also appear to be involved in inflammation signaling cascades, whether small-molecules could target histone chaperones to show anti-inflammation effects has still been rarely discovered. In this study, natural product artone was found to show obvious inhibitory effects on microglia-mediated neuroinflammation by directly targeting ASF1a, which is a histone-remodeling chaperone. Mechanism study revealed that artone modulated histone H3 PTMs profile by down-regulating acetylation and trimethylation modification levels at sites K4, K9, K18 and K27. Artone-dependent regulations on PTMs further caused an effective inhibition on transcription factor NF-κB assembling to promoters of pro-inflammatory cytokine genes including Tnf-α, Il-6 and Rgs3, indicating a distinctive anti-neuroinflammation mechanism. Collectively, we reported artone as the first small-molecule targeting histone-remodeling chaperone ASF1a for anti-neuroinflammation. Moreover, these findings broaden our knowledge of histone chaperone as a druggable target protein for neuroinflammation inhibition, and open a new avenue to novel therapy strategy for inflammation-associated neurological disorders.
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Artemisia/química , Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Lipopolisacáridos/farmacología , Microglía/citología , Microglía/efectos de los fármacos , Chaperonas Moleculares/metabolismo , Extractos Vegetales/farmacología , Sustancias Protectoras/farmacología , Acetilación/efectos de los fármacos , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular , Supervivencia Celular/efectos de los fármacos , Inflamación/metabolismo , Metilación/efectos de los fármacos , Ratones , Microglía/metabolismo , Chaperonas Moleculares/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Background: Histone post-translational modifications (PTMs) are involved in various biological processes such as transcriptional activation, chromosome packaging, and DNA repair. Previous studies mainly focused on PTMs by directly targeting histone-modifying enzymes such as HDACs and HATs. Methods and Results: In this study, we discovered a previously unexplored regulation mechanism for histone PTMs by targeting transcription regulation factor 14-3-3ζ. Mechanistic studies revealed 14-3-3ζ dimerization as a key prerequisite, which could be dynamically induced via an allosteric effect. The selective inhibition of 14-3-3ζ dimer interaction with histone H3 modulated histone H3 PTMs by exposing specific modification sites including acetylation, trimethylation, and phosphorylation, and reprogrammed gene transcription profiles for autophagy-lysosome function and endoplasmic reticulum stress. Conclusion: Our findings demonstrate the feasibility of editing histone PTM patterns by targeting transcription regulation factor 14-3-3ζ, and provide a distinctive PTM editing strategy which differs from current histone modification approaches.
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Proteínas 14-3-3/antagonistas & inhibidores , Autofagia , Regulación de la Expresión Génica , Histonas/metabolismo , Fenoles/farmacología , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Acetilación , Regulación Alostérica , Animales , Línea Celular , Histonas/química , Humanos , Masculino , Metilación , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Modelos Animales , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
Background: Caesalpinia sappan L. (C. sappan) is a traditional Chinese medicinal plant. The dried heartwood of C. sappan (also known as Sappan wood) has been widely used for the folkloric medical treatment of ischemic cerebral stroke in China. However, the detailed underlying pharmacological mechanism still remains largely unexplored. Methods: In this study, a middle cerebral artery occlusion (MCAO) rat model was employed to elucidate the mechanism of the anti-cerebral ischemic effects of C. sappan ethanolic extract (CEE). Moreover, systemic multi-target identification coupled with gene ontology biological process (GO BP) and reactome pathway analysis was used to investigate the potential neuroprotective mechanism. Furthermore, the presumed mechanism was confirmed through biological analysis by determining the effects of CEE on the identified signaling pathways in PC12 cells model-induced by oxygen-glucose deprivation/reperfusion (OGD/R). Results: Our study demonstrates that CEE (both through in vivo administration at a dosage of 300 mg/kg and through in vitro incubation at a dosage of 2.4 µg/mL) is a neuroprotective agent that can effectively inhibit neuronal damage, promote synaptic generation, and suppress the activation of neutrophils, microglia, and astrocytes. Moreover, the neuroprotective mechanism of CEE is mediated via regulating 150 potential target proteins, which are associated with 6 biological processes and 10 pathways, including JAK-STAT, HSP90 and DNA damage/telomere stress. Conclusion: CEE can exert neuroprotective effect through multi-target pharmacological mechanisms to prevent ischemia/reperfusion-induced cerebral injury.
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Drastic macrophages activation triggered by exogenous infection or endogenous stresses is thought to be implicated in the pathogenesis of various inflammatory diseases. Carnosic acid (CA), a natural phenolic diterpene extracted from Salvia officinalis plant, has been reported to possess anti-inflammatory activity. However, its role in macrophages activation as well as potential molecular mechanism is largely unexplored. In the current study, we sought to elucidate the anti-inflammatory property of CA using an integrated approach based on unbiased proteomics and bioinformatics analysis. CA significantly inhibited the robust increase of nitric oxide and TNF-α, downregulated COX2 protein expression, and lowered the transcriptional level of inflammatory genes including Nos2, Tnfα, Cox2, and Mcp1 in LPS-stimulated RAW264.7 cells, a murine model of peritoneal macrophage cell line. The LC-MS/MS-based shotgun proteomics analysis showed CA negatively regulated 217 LPS-elicited proteins which were involved in multiple inflammatory processes including MAPK, nuclear factor (NF)-κB, and FoxO signaling pathways. A further molecular biology analysis revealed that CA effectually inactivated IKKß/IκB-α/NF-κB, ERK/JNK/p38 MAPKs, and FoxO1/3 signaling pathways. Collectively, our findings demonstrated the role of CA in regulating inflammation response and provide some insights into the proteomics-guided pharmacological mechanism study of natural products.
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ETHNOPHARMACOLOGICAL RELEVANCE: Albiziae Cortex (AC) is a widely used traditional medicine in China. It is possess various properties to treat insomnia, traumatic injuries, diuresis, sthenia, and confusion. Total saponins of Albiziae Cortex (TSAC) are the most abundant bioactive components of AC, which were reported to show significant anti-tumor effects in vivo and in vitro. But the underlying mechanism of TSAC remained to be revealed. AIM OF STUDY: In this study, we investigated the anti-hepatoma carcinoma effects and the potential mechanism of TSAC in vivo and in vitro. MATERIALS AND METHODS: We first purified TSAC from crude extracts and characterized the major bioactive compounds by high performance liquid chromatography (HPLC). Effects of TSAC on viability of various hepatoma carcinoma cell lines were measured by MTT. Inhibition on cell proliferation was analysed using colony formation assay. Cell cycle distribution was revealed by flow cytometry. The apoptotic cells were observed by Hoechst 33258 staining and acridine orange (AO)/ethidium bromide (EB) double staining. Microstructures of apoptotic cells were examined by Transmission electron microscopy (TEM). The mitochondrial membrane potential were determined by JC-1 staining. Western blot was used to investigate the effects of TSAC on apoptosis-related proteins, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax), and S-phase related protein cyclin A, cyclin E and cyclin-dependent kinases 2 (CDK2). Effects on tumor growth was assessed by H22-bearing ICR mice. RESULTS: TSAC significantly decreased the hepatoma carcinoma cell viability and inhibited HepG2 cell colony formation in a concentration-dependent manner. We also found that TSAC inhibited HepG2 cell growth via induction of S phase arrest. Further study showed that TSAC significantly down-regulated the expressions of cyclin A, cyclin E and CDK2 in HepG2 cells. Meanwhile, TSAC could effectively induce mitochondria-dependent caspase apoptosis pathway activation. Furthermore, TSAC increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2. In vivo assay showed that the anti-tumor effects of TSAC were significantly augmented without increasing toxicity in H22-bearing ICR mice. CONCLUSION: TSAC could inhibit cell proliferation through inducing S phase arrest and activate cell apoptosis via mitochondria-dependent apoptosis pathway. Therefore, TSAC could be a promising agent in clinical trials for anti-hepatoma carcinoma treatment.
Asunto(s)
Albizzia , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Saponinas/uso terapéutico , Animales , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Humanos , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos ICR , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Fitoterapia , Extractos Vegetales/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Fase S/efectos de los fármacos , Saponinas/farmacología , Carga TumoralRESUMEN
Microglial activation and resultant neuroinflammatory response are implicated in various brain diseases including Alzheimer's disease and Parkinson's disease. Treatment with anti-neuroinflammatory agents could provide therapeutic benefits for such disorders. Protosappanin A (PTA) is a major bioactive ingredient isolated from Caesalpinia sappan L.. In this work, the anti-neuroinflammatory effects of PTA on LPS-stimulated BV2 cells were investigated and the underlying mechanisms were explored. Results showed that PTA significantly inhibited the production of TNF-α and IL-1ß in LPS-activated BV2 microglia. Moreover, the mRNA expressions of IL-6, IL-1ß, and MCP-1 were reduced by PTA in a dose-dependent manner. Furthermore, PTA suppressed JAK2/STAT3-dependent inflammation pathway through down-regulating the phosphorylation of JAK2 and STAT3, as well as STAT3 nuclear translocation against LPS treatment. These observations suggested a novel role for PTA in regulating LPS-induced neuroinflammatory injuries.
Asunto(s)
Antiinflamatorios/farmacología , Inflamación/inmunología , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Fenoles/farmacología , Factor de Transcripción STAT3/inmunología , Animales , Humanos , Inflamación/tratamiento farmacológico , Inflamación/genética , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Ratones , Microglía/inmunología , Óxido Nítrico/genética , Óxido Nítrico/inmunología , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Heat shock protein 70 (Hsp70) is widely involved in immune disorders, making it as an attractive drug target for inflammation diseases. Nonselective induction of Hsp70 upregulation for inflammation therapy could cause extensive interference in inflammation-unrelated protein functions, potentially resulting in side effects. Nevertheless, direct pharmacological activation of Hsp70 via targeting specific functional amino acid residue may provide an insight into precise Hsp70 function regulation and a more satisfactory treatment effect for inflammation, which has not been extensively focused. Here we show a cysteine residue (Cys306) for selective Hsp70 activation using natural small-molecule handelin. Covalent modification of Cys306 significantly elevates Hsp70 activity and shows more satisfactory anti-neuroinflammation effects. Mechanism study reveals Cys306 modification by handelin induces an allosteric regulation to facilitate adenosine triphosphate hydrolysis capacity of Hsp70, which leads to the effective blockage of subsequent inflammation signaling pathway. Collectively, our study offers some insights into direct pharmacological activation of Hsp70 by specially targeting functional cysteine residue, thus providing a powerful tool for accurately modulating neuroinflammation pathogenesis in human with fewer undesirable adverse effects.