RESUMEN

The N-degron pathway, first discovered several decades ago by Varshavsky's laboratory, controls the half-life of target proteins depending on their N-terminal residues. In vivo cell biology studies have established the physiological role of the N-degron pathway. However, in vitro studies such as biochemical assays and structural biology studies are relatively limited. The N-degron substrates cannot be obtained via simple protein expression. The N-degron residues are exposed via the proteolytic process from the translated nascent polypeptide chains. Thus, methods for the fusion expression with several cleavable tags and subsequent treatment with specific proteases to design the exposed N-degron signals have been introduced. Recently, we developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B (LC3B), a key marker protein of autophagy, to obtain a high yield of the purified target proteins with variable N-terminal residues for various biochemical studies including enzymatic and binding assays, and crystallization of N-degron complex. This chapter describes the protocols that include the vector map designed for producing LC3B fused target proteins, methods for expression and purification of an example protein, p62/SQSMT1, using different N-terminal residues, and methods to obtain the purified ATG4B protease, which is used for processing LC3B tag and exposing the required N-terminal residues of the target protein.

Asunto(s)

Autofagia , Péptido Hidrolasas , Proteolisis , Autofagia/fisiología , Péptidos

RESUMEN

OBJECTIVE: Inflammation, a vital innate immune response against infection and injury, is mediated by macrophages. Spleen tyrosine kinase (Syk) regulates inflammatory responses in macrophages; however, its role and underlying mechanisms are uncertain. MATERIALS AND METHODS: In this study, overexpression and knockout (KO) cell preparations, phagocytosis analysis, confocal microscopy, reactive oxygen species (ROS) determination, mRNA analysis, and immunoprecipitation/western blotting analyses were used to investigate the role of Syk in phagocytosis and its underlying mechanisms in macrophages during inflammatory responses. RESULTS: Syk inhibition by Syk KO, Syk-specific small interfering RNA (siSyk), and a selective Syk inhibitor (piceatannol) significantly reduced the phagocytic activity of RAW264.7 cells. Syk inhibition also decreased cytochrome c generation by inhibiting ROS-generating enzymes in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and ROS scavenging suppressed the phagocytic activity of RAW264.7 cells. LPS induced the tyrosine nitration (N-Tyr) of suppressor of cytokine signaling 1 (SOCS1) through Syk-induced ROS generation in RAW264.7 cells. On the other hand, ROS scavenging suppressed the N-Tyr of SOCS1 and phagocytosis. Moreover, SOCS1 overexpression decreased phagocytic activity, and SOCS1 inhibition increased the phagocytic activity of RAW264.7 cells. CONCLUSION: These results suggest that Syk plays a critical role in the phagocytic activity of macrophages by inducing ROS generation and suppressing SOCS1 through SOCS1 nitration during inflammatory responses.

Asunto(s)

Citocromos c , Lipopolisacáridos , Citocinas , Lipopolisacáridos/farmacología , Macrófagos , Fagocitosis , ARN Mensajero , ARN Interferente Pequeño , Especies Reactivas de Oxígeno , Quinasa Syk , Tirosina

RESUMEN

BACKGROUND: During fiberoptic-guided tracheal intubation, impingement between the distal tip of the endotracheal tube and the airway tissue can cause difficulties in tube insertion or tissue damage during the tube advancement over the bronchoscope. This randomized controlled study aimed to investigate the effects of the endotracheal tube's bevel direction on the complications associated with airway injury when performing fiberoptic-guided tracheal intubation. METHODS: The study subjects were divided into 2 groups: L (control) and D (study). When advancing the tube over the bronchoscope, the tube's bevel was facing the patients' left in Group L and the dorsal direction in Group D. According to the degree of resistance at the time of tube advancement, the insertion score was graded in 3 stages; the severity of the patients' sore throat and hoarseness was evaluated and recorded postoperatively. RESULTS: The severity of postoperative sore throat was higher in Group L than in Group D 3 hours and 24 hours after surgery. (P = .008, P = .023, respectively). The tube insertion score was comparable between the groups. The severity of postoperative hoarseness did not vary significantly between the groups. CONCLUSION: Endotracheal tube insertion with the bevel facing the dorsal direction of the patient during fiberoptic-guided tracheal intubation reduced the severity of postoperative sore throat in patients undergoing laparoscopic gynecologic surgery.

Asunto(s)

Ronquera , Faringitis , Broncoscopios/efectos adversos , Femenino , Tecnología de Fibra Óptica , Ronquera/etiología , Humanos , Intubación Intratraqueal/efectos adversos , Dolor/complicaciones , Faringitis/etiología

RESUMEN

RATIONALE: The prone position is the most commonly required position during spinal surgery. Decreasing lumbar lordosis is necessary to facilitate the accessibility of the surgical field. And this can affect the hemodynamic circulation of the patients. The Jackson spine table is one of the most preferred methods, known to have minimal effects on cardiac function. PATIENT CONCERNS: We report a case of sudden arrhythmia that developed during the prone position using a Jackson spine table. It occurred 30 minutes after the positional change. DIAGNOSES: Arrhythmia showed bizarre P and QRS waves. Ectopic P, bundle branch block, or both was suspected. INTERVENTIONS: Because it was difficult to define the exact type or cause of this sudden arrhythmia and considering that other vital signs remained stable, we decided to keep close observation during the operation rather than applying uncertain antiarrhythmic medication. OUTCOMES: Arrhythmia spontaneously developed and subsided repeatedly. And it recovered to normal sinus rhythm immediately after the positional change to the supine position. Therefore, increased intrathoracic pressure caused by the prone position was highly suspected to be the cause of this event. LESSONS: Although the Jackson spine table is known to have the least effect on cardiac function, the patient experienced arrhythmia in our case. Hence, to achieve better clinical outcomes, an understanding of physiological alterations and possible complications caused by the prone position is necessary for earlier diagnosis and management.

Asunto(s)

Posicionamiento del Paciente , Columna Vertebral , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/etiología , Arritmias Cardíacas/cirugía , Humanos , Vértebras Lumbares/cirugía , Procedimientos Neuroquirúrgicos , Posicionamiento del Paciente/efectos adversos , Posición Prona/fisiología , Columna Vertebral/cirugía

RESUMEN

We report on morphology-controlled remote epitaxy via hydrothermal growth of ZnO micro- and nanostructure crystals on graphene-coated GaN substrate. The morphology control is achieved to grow diverse morphologies of ZnO from nanowire to microdisk by changing additives of wet chemical solution at a fixed nutrient concentration. Although the growth of ZnO is carried out on poly-domain graphene-coated GaN substrate, the direction of hexagonal sidewall facet of ZnO is homogeneous over the whole ZnO-grown area on graphene/GaN because of strong remote epitaxial relation between ZnO and GaN across graphene. Atomic-resolution transmission electron microscopy corroborates the remote epitaxial relation. The non-covalent interface is applied to mechanically lift off the overlayer of ZnO crystals via a thermal release tape. The mechanism of facet-selective morphology control of ZnO is discussed in terms of electrostatic interaction between nutrient solution and facet surface passivated with functional groups derived from the chemical additives.

RESUMEN

Flexible capacitive humidity sensors are promising for low-cost, wearable, and radio frequency identification sensors, but their nonlinear response is an important issue for practical applications. Herein, the linearity of humidity response was controlled by surface water wettability and operating frequency of sensor, and the mechanism was explained in detail by surface water condensation. For a sensor with a Ag interdigitated electrode (IDE) on a poly(ethylene terephthalate) substrate, the capacitance showed a small linear increase with humidity up to 70% RH but a large nonlinear increase in the higher range. The response linearity was increased by a hydrophobic surface treatment of self-assembled monolayer coating while it was decreased by an ultraviolet/ozone irradiation for hydrophilicity. It was also increased by increasing the frequency in the range of 1-100 kHz, more prominently on a more hydrophilic surface. Based on experiment and simulation, the increase in sensor capacitance was greatly dependent on the geometric pattern (e.g., size, number, and contact angle) and electrical permittivity of surface water droplets. A larger and more nonlinear humidity response resulted from a larger increase in the number of droplets with a smaller contact angle on a sensor surface with higher water wettability and also from a higher permittivity of water at a lower frequency.

Asunto(s)

Agua , Electrodos , Humedad , Interacciones Hidrofóbicas e Hidrofílicas , Humectabilidad

RESUMEN

Preoperative laryngoscopic examination of the airway informs general anaesthesia management and planning. However, the same glottic opening view cannot always be obtained during direct laryngoscopy of anaesthetised patients. In this case report, a patient underwent preoperative rigid laryngoscopy due to medical history, and no problems were anticipated in performing tracheal intubation; however, the direct laryngoscopic view was a Grade 4 on the Cormack-Lehane Scale after anaesthesia induction. A jaw thrust manoeuvre to facilitate fibreoptic-assisted nasotracheal intubation was not feasible. In order to compensate, a modified method of jaw thrust was implemented, where both thumbs were placed on the floor of the patient's mouth, leading to a successful result. Safe airway management should be implemented with proper planning based on a careful preoperative evaluation.

Asunto(s)

Laringoscopios , Laringoscopía , Anestesia General , Tecnología de Fibra Óptica , Humanos , Intubación Intratraqueal

RESUMEN

Dipterocarpus tuberculatus Roxb. has been used traditionally as a remedy for many diseases, especially inflammation. Therefore, we analyzed and explored the mechanism of the anti-inflammatory effect of a Dipterocarpus tuberculatus Roxb. ethanol extract (Dt-EE). Dt-EE clearly and dose-dependently inhibited the expression of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1ß in lipopolysaccharide (LPS)-treated RAW264.7 cells. Also, Dt-EE suppressed the activation of the MyD88/TRIF-mediated AP-1 pathway and the AP-1 pathway related proteins JNK2, MKK4/7, and TAK1, which occurred as a result of inhibiting the kinase activity of IRAK1 and IRAK4, the most upstream factors of the AP-1 pathway. Finally, Dt-EE displayed hepatoprotective activity in a mouse model of hepatitis induced with LPS/D-galactosamine (D-GalN) through decreasing the serum levels of alanine aminotransferase and suppressing the activation of JNK and IRAK1. Therefore, our results strongly suggest that Dt-EE could be a candidate anti-inflammatory herbal medicine with IRAK1/AP-1 inhibitory and hepatoprotective properties.

Asunto(s)

Antiinflamatorios/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Extractos Vegetales/farmacología , Sustancias Protectoras/farmacología , Factor de Transcripción AP-1/metabolismo , Animales , Antiinflamatorios/química , Línea Celular , Modelos Animales de Enfermedad , Hepatitis/tratamiento farmacológico , Hepatitis/etiología , Hepatitis/metabolismo , Humanos , Masculino , Ratones , Extractos Vegetales/química , Sustancias Protectoras/química , Células RAW 264.7

RESUMEN

Background: Inflammation, a vital immune response to infection and injury, is mediated by macrophage activation. While spleen tyrosine kinase (Syk) and myeloid differentiation primary response 88 (MyD88) are reportedly involved in inflammatory responses in macrophages, their roles and underlying mechanisms are largely unknown. Methods: Here, the role of the MyD88-Syk axis and the mechanism by which Syk and MyD88 cooperate during macrophage-mediated inflammatory responses are explored using knockout conditions of these proteins and mutation strategy as well as flowcytometric and immunoblotting analyses. Results: Syk rapidly activates the nuclear factor-kappa B (NF-κB) signaling pathway in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the activation of the NF-κB signaling pathway is abolished in Syk-/- RAW264.7 cells. MyD88 activates Syk and Syk-induced activation of NF-κB signaling pathway in LPS-stimulated RAW264.7 cells but Syk-induced inflammatory responses are significantly inhibited in MyD88-/- RAW264.7 cells. MyD88 interacts with Syk through the tyrosine 58 residue (Y58) in the hemi-immunoreceptor tyrosine-based activation motif (ITAM) of MyD88, leading to Syk activation and Syk-induced activation of the NF-κB signaling pathway. Src activates MyD88 by phosphorylation at Y58 via the Src kinase domain. In addition, Ras-related C3 botulinum toxin substrate 1 (Rac1) activation and Rac1-induced formation of filamentous actin (F actin) activate Src in LPS-stimulated RAW264.7 cells. Conclusions: These results suggest that the MyD88-Syk axis is a critical player in macrophage-mediated inflammatory responses, and its function is promoted by an upstream Src kinase activated by Rac1-generated filamentous actin (F-actin).

Asunto(s)

Inflamación/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Quinasa Syk/inmunología , Animales , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Activación de Macrófagos/genética , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Células RAW 264.7 , Transducción de Señal/inmunología , Quinasa Syk/genética , Quinasa Syk/metabolismo , Tirosina/genética , Tirosina/inmunología , Tirosina/metabolismo

RESUMEN

3-Deazadenosine (3-DA) is a general methylation inhibitor that depletes S-adenosylmethionine, a methyl donor, by blocking S-adenosylhomocysteine hydrolase (SAHH). In this study, we investigated the inhibitory activity and molecular mechanisms of 3-DA in inflammatory responses. 3-DA suppressed the secretion of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide-treated RAW264.7 cells and phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells. It also reduced mRNA expression of inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, interleukin-1ß (IL-1 ß), and IL-6, indicating that 3-DA has anti-inflammatory properties in murine and human macrophages. Moreover, 3-DA strongly blocked AP-1 and NF-κB luciferase activity under PMA-, MyD88-, and TRIF-stimulated conditions and decreased the translocation of c-Jun, c-Fos, p65, and p50 into the nucleus. In addition, the p-ERK level in AP-1 signaling and the p-IκBα level in NF-kB signaling were diminished by 3-DA treatment. Interestingly, 3-DA did not alter the phosphorylation of MEK1/2, an ERK modulator, or IKKα/ß, an IκBα regulator. Instead, 3-DA prevented MEK1/2 and IKKα/ß from combining with ERK and IκBα, respectively, and directly suppressed MEK1/2 and IKKα/ß kinase activity. These results indicate that MEK1/2 and IKKα/ß are direct targets of 3-DA. In addition, suppression of SAHH by siRNA or treatment with adenosine dialdehyde, another SAHH inhibitor, showed inhibitory patterns against p-ERK and IκBα similar to those of 3-DA. Taken together, this study demonstrates that 3-DA inhibits AP-1 and NF-κB signaling by directly blocking MEK1/2 and IKKα/ß or indirectly mediating SAHH, resulting in anti-inflammatory activity.

Asunto(s)

Adenosilhomocisteinasa/antagonistas & inhibidores , Mediadores de Inflamación/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , FN-kappa B/antagonistas & inhibidores , Factor de Transcripción AP-1/antagonistas & inhibidores , Tubercidina/farmacología , Adenosilhomocisteinasa/metabolismo , Animales , Antiinflamatorios/farmacología , Células HEK293 , Humanos , Mediadores de Inflamación/metabolismo , Ratones , FN-kappa B/metabolismo , Células RAW 264.7 , Factor de Transcripción AP-1/metabolismo , Células U937

RESUMEN

Isoprenylcysteine carboxylmethyltransferase (ICMT) has been reported to regulate the inflammatory response through the Ras/MAPK/AP-1 pathway. Nevertheless, the potential of ICMT inhibitors as therapeutic agents against inflammatory diseases has not been examined. Therefore, in this study, we investigated the anti-inflammatory properties of two ICMT inhibitors, cysmethynil (CyM) and 3-methoxy-N-[2-2,2,6,6-tetramethyl-4-phenyltetrahydropyran-4-yl)ethyl]aniline (MTPA), using in vitro analyses and in vivo analyses (lipopolysaccharide (LPS)/D-GalN-triggered hepatitis and DSS-induced colitis mouse models). CyM and MTPA inhibited the production of nitric oxide (NO) and prostaglandin E (PGE)2 and the expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in LPS-induced RAW264.7 cells and peritoneal macrophages without cytotoxicity. CyM also reduced AP-1-mediated luciferase activity in LPS-stimulated RAW264.7 cells and MyD88- and TRIF-expressing HEK293 cells. In addition, CyM and MTPA suppressed the translocation of Ras to the cell membrane and ER as well as phosphorylation of Ras-dependent AP-1 signaling molecules including Raf, MEK1/2, ERK p38, and JNK. Consistent with these results, CyM diminished the expression of inflammatory genes (COX-2, TNF-α, IL-1ß, and IL-6), AP-1-Luc activity, and phosphorylation of Ras-mediated signaling enzymes in Ras-overexpressing HEK 293 cells. Moreover, CyM and MTPA ameliorated symptoms of hepatitis and colitis in mice and restrained the ICMT/Ras-dependent AP-1 pathway in inflammatory lesions of the mouse model systems. Taken together, our results indicate that CyM and MTPA alleviate the LPS-induced ICMT/Ras/AP-1 signaling pathway, thereby inhibiting the inflammatory response as promising anti-inflammatory drugs.

Asunto(s)

Antiinflamatorios/farmacología , Indoles/farmacología , Proteína Metiltransferasas/antagonistas & inhibidores , Proteína Metiltransferasas/metabolismo , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Hepatitis/tratamiento farmacológico , Hepatitis/metabolismo , Humanos , Indoles/química , Indoles/uso terapéutico , Macrófagos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Células RAW 264.7

RESUMEN

There have been rapidly increasing demands for flexible lighting apparatus, and micrometer-scale light-emitting diodes (LEDs) are regarded as one of the promising lighting sources for deformable device applications. Herein, we demonstrate a method of creating a deformable LED, based on remote heteroepitaxy of GaN microrod (MR) p-n junction arrays on c-Al2O3 wafer across graphene. The use of graphene allows the transfer of MR LED arrays onto a copper plate, and spatially separate MR arrays offer ideal device geometry suitable for deformable LED in various shapes without serious device performance degradation. Moreover, remote heteroepitaxy also allows the wafer to be reused, allowing reproducible production of MR LEDs using a single substrate without noticeable device degradation. The remote heteroepitaxial relation is determined by high-resolution scanning transmission electron microscopy, and the density functional theory simulations clarify how the remote heteroepitaxy is made possible through graphene.

RESUMEN

Our objective in this study was to investigate a sensor for volatile organic compounds based on a graphite (G)/polypropylene glycol (PPG) hybrid composite (HC) for sensing hybrid elements. The G/PPG HC sensor films for organic-matter detection were successfully fabricated on polyethylene terephthalate (PET) film with a simple blade-coating method. The sensing paste based on G/PPG (1:2) HC showed good dispersibility and stability. In addition, G/PPG HC sensor films with organic compounds showed different thickness changes as a function of the G/PPG ratio because of the swelling effect of the polymer. The observed differences in resistance of the G/PPG HC films corresponded to those of common organic compounds, suggesting that the disconnection of graphite caused by the swollen PPG matrix caused explosive resistance change. Moreover, we evaluated the sensitivity of typical hydrocarbon materials, such as benzene and toluene, in the sensor film as well as petroleum materials without moisture-induced malfunctions. This study could provoke knowledge about superior sensing with cost-effective and easily scalable materials using polymer/graphite composite-based sensors to improve the sensitivity, selectivity, and stability of chemical sensor applications.

RESUMEN

In this study, we investigated the functional role of isoprenylcysteine carboxyl methyltransferase (ICMT) and its methylatable substrate Ras in Toll-like receptor (TLR)-activated macrophages and in mouse inflammatory disease conditions. ICMT and RAS expressions were strongly increased in macrophages under the activation conditions of TLRs by lipopolysaccharide (LPS, a TLR4 ligand), pam3CSK (TLR2), or poly(I:C) (TLR3) and in the colons, stomachs, and livers of mice with colitis, gastritis, and hepatitis. The inhibition and activation of ICMT and Ras through genetic and pharmacological approaches significantly affected the activation of interleukin-1 receptor-associated kinase (IRAK)s, tumor necrosis factor receptor associated factor 6 (TRAF6), transforming growth factor-ß-activated kinase 1 (TAK1), mitogen-activated protein kinase (MAPK), and MAPK kinases (MAPKKs); translocation of the AP-1 family; and the expressions of inflammation-related genes that depend on both MyD88 and TRIF. Interestingly, the Ras/ICMT-mediated inflammatory reaction critically depends on the TIR domains of myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-ß (TRIF). Taken together, these results suggest that ICMT and its methylated Ras play important roles in the regulation of inflammatory responses through cooperation with the TIR domain of adaptor molecules.

Asunto(s)

Inflamación/enzimología , Proteína Metiltransferasas/metabolismo , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Inflamación/patología , Sistema de Señalización de MAP Quinasas , Activación de Macrófagos , Macrófagos/enzimología , Masculino , Metilación , Ratones , Modelos Biológicos , Factor 88 de Diferenciación Mieloide/metabolismo , Células RAW 264.7 , Especificidad por Sustrato , Factor de Transcripción AP-1/metabolismo , Proteínas ras/metabolismo

RESUMEN

The hydroxymethylation of cytosine bases plays a vital role in the phage DNA protection system inside the host Escherichia coli. This modification is known to be catalyzed by the dCMP hydroxymethylase from bacteriophage T4 (T4dCH); structural information on the complexes with the substrate, dCMP and the co-factor, tetrahydrofolate is currently available. However, the detailed mechanism has not been understood clearly owing to a lack of structure in the complex with a reaction intermediate. We have applied the X-ray free electron laser (XFEL) technique to determine a high-resolution structure of a T4dCH D179N active site mutant. The XFEL structure was determined at room temperature and exhibited several unique features in comparison with previously determined structures. Unexpectedly, we observed a bulky electron density at the active site of the mutant that originated from the physiological host (i.e., E. coli). Mass-spectrometric analysis and a cautious interpretation of an electron density map indicated that it was a dTMP molecule. The bound dTMP mimicked the methylene intermediate from dCMP to 5'-hydroxymethy-dCMP, and a critical water molecule for the final hydroxylation was convincingly identified. Therefore, this study provides information that contributes to the understanding of hydroxymethylation.

Asunto(s)

Bacteriófago T4/enzimología , Electrones , Transferasas de Hidroximetilo y Formilo/química , Transferasas de Hidroximetilo y Formilo/genética , Rayos Láser , Mutación , Timidina Monofosfato/metabolismo , Cristalografía por Rayos X , Transferasas de Hidroximetilo y Formilo/metabolismo , Modelos Moleculares , Conformación Proteica , Agua/química

RESUMEN

This study presents BN82002 as an anti-inflammatory drug candidate. It was found that BN82002 inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 cells and peritoneal macrophages that were activated by toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). BN82002 dose-dependently down-regulated mRNA levels of nitric oxide synthase, tumor necrosis factor-α, and cyclooxygenase-2. The nuclear translocation of nuclear factor (NF)-κB (p65 and p50) was also blocked by BN82002 in RAW265.7 cells stimulated by LPS. According to reporter gene assay performed with NF-κB construct, BN82002 clearly reduced increased level of luciferase activity mediated by transcription factor NF-κB in LPS-treated RAW264.7 cells and in MyD88- and AKT2-overexpressing HEK293 cells. However, BN82002 did not inhibit NF-κB activity in AKT1- or IKKß-overexpressing HEK293 cells. NF-κB upstream signaling events specifically targeted AKT2 but had no effect on AKT1. The specific target of BN82002 was Tyr-178 in AKT2. BN82002 bound to Tyr-178 and interrupted the kinase activity of AKT2, according to a cellular thermal shift assay analysis of the interaction of BN82002 with AKT2 and an AKT2 mutant (Tyr-178 mutated to Ala; AKT2 Y178A). These results suggest that BN82002 could reduce inflammatory pathway by controlling NF-κB pathway and specifically targeting AKT2.

Asunto(s)

Antiinflamatorios/farmacología , Etilaminas/farmacología , Nitrocompuestos/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatasas cdc25/antagonistas & inhibidores , Fosfatasas cdc25/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos/métodos , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7

RESUMEN

The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC.

Asunto(s)

Fibroblastos/efectos de los fármacos , Macrófagos/efectos de los fármacos , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Fibroblastos/enzimología , Células HEK293 , Humanos , Quinasa I-kappa B/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/enzimología , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multienzimáticos , Fosforilación , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Mycetia cauliflora Reinw. (Rubiaceae) has been used as a traditional remedy to ameliorate clinical signs of inflammatory diseases, including pain, inflammation, ulcers, and wounds. Among the Mycetia subfamilies, the molecular and cellular mechanisms of Mycetia longifolia (Rubiaceae) have been studied. However, those of Mycetia cauliflora are not clearly understood. Comprehensive investigation of this plant is necessary to evaluate its potential for ethnopharmacological use. MATERIALS: and methods: The activities of Mycetia cauliflora methanol extract (Mc-ME) on the secretion of inflammatory mediators, the mRNA expression of proinflammatory cytokines, and identification of its molecular targets were elucidated using lipopolysaccharide (LPS)-induced macrophage-like cells. Moreover, the suppressive actions of Mc-ME were examined in an LPS-induced peritonitis mouse model. RESULTS: At nontoxic concentrations, Mc-ME downregulated the release of nitric oxide (NO), the mRNA expression of inducible nitric oxide synthase (iNOS), and the mRNA expression of interleukin (IL)-1ß from LPS-activated RAW264.7 cells. This extract also inhibited the nuclear translocation of p65 and p50 and the phosphorylation of IκBα, IKK, and AKT. Western blot analysis and in vitro kinase assays confirmed that phosphoinositide-dependent kinase-1 (PDK1) is the direct immunopharmacological target of Mc-ME effect. In addition, Mc-ME significantly reduced inflammatory signs in an animal model of acute peritonitis. These effects were associated with decreased NO production and decreased AKT phosphorylation. CONCLUSION: Our results suggest that Mc-ME displays anti-inflammatory actions in LPS-treated macrophage-like cells and in an animal model of acute inflammatory disease. These actions are preferentially managed by targeting PDK1 in the nuclear factor (NF)-κB signaling pathway.

Asunto(s)

Antiinflamatorios/farmacología , Extractos Vegetales/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Rubiaceae , Animales , Antiinflamatorios/uso terapéutico , Células HEK293 , Humanos , Interleucina-1beta/genética , Lipopolisacáridos , Macrófagos Peritoneales/efectos de los fármacos , Masculino , Metanol/química , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Peritonitis/inducido químicamente , Peritonitis/tratamiento farmacológico , Peritonitis/metabolismo , Extractos Vegetales/uso terapéutico , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Solventes/química

RESUMEN

Two-dimensional atomic layered materials (2d-ALMs) are emerging candidates for use as epitaxial seed substrates for transferrable epilayers. However, the micrometer-sized domains of 2d-ALMs preclude their practical use in epitaxy because they cause crystallographically in-plane disordering of the overlayer. Ultrathin graphene can penetrate the electric dipole momentum from an underlying crystal layer to the graphene surface, which then drives it to crystallize the overlayer during the initial growth stage, thus resulting in substantial energy saving. This study demonstrates the remote homoepitaxy of ZnO microrods (MRs) on ZnO substrates across graphene layers via a hydrothermal method. Despite the presence of poly-domain graphene in between the ZnO substrate and ZnO MRs, the MRs were epitaxially grown on a- and c-plane ZnO substrates, whose in-plane alignments were homogeneous within the wafer's size. Transmission electron microscopy revealed a homoepitaxial relationship between the overlayer MRs and the substrate. Density-functional theory calculations suggested that the charge redistribution occurring near graphene induces the electric dipole formation, so the attracted adatoms led to the formation of the remote homoepitaxial overlayer. Due to a strong potential field caused by long-range charge transfer given from the substrate, even the use of bi-layer and tri-layer graphene resulted in remote homoepitaxial ZnO MRs. The effects of substrate crystal planes were also theoretically and empirically investigated. The ability of graphene, which can be released from the mother substrate without covalent bonds, was utilized to transfer the overlayer MR arrays. This method opens a way for producing well aligned, transferrable epitaxial nano/microstructure arrays while regenerating the substrate for cost-saving device manufacturing.

RESUMEN

The preparation of crystalline materials on incommensurate substrates has been a key topic of epitaxy. van der Waals (vdW) epitaxy on two-dimensional (2D) materials opened novel opportunities of epitaxial growth overcoming the materials compatibility issue. Therefore, vdW epitaxy has been considered as a promising approach for the preparation of building blocks of flexible devices and thin film-based devices at the nano/microscale. However, an understanding of vdW epitaxy has not been thoroughly established. Especially, controlling nucleation during vdW epitaxy has not been achieved although nucleation in vdW epitaxy is suppressed due to the absence of surface dangling bonds on 2D materials. Here we show an enhancement of nucleation probability of germanium on graphene via introducing an out-of-plane dipole moment without any change in the chemical nature of graphene. A graphene/hexagonal boron nitride stack and transferred graphene on a polarized ferroelectric thin film were employed to demonstrate the significant enhancement of Ge nucleation on graphene. Theoretical calculations and chemical vapor deposition were employed to elucidate the effect of the out-of-plane dipole moment on nucleation in vdW epitaxy.