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The importance of methylation in the tumorigenic responses of nonhistone proteins, such as TP53, PTEN, RB1, AKT, and STAT3, has been emphasized in numerous studies. In parallel, the corresponding nonhistone protein methyltransferases have been acknowledged in the pathophysiology of cancer. Thus, this study aimed to explore the pathological role of a nonhistone methyltransferase in gastric cancer (GC), identify nonhistone substrate protein, and understand the underlying mechanism. Interestingly, among the 24 methyltransferases and methyltransferase family 16 (MTF16) proteins, EEF1AKMT3 (METTL21B) expression was prominently lower in GC tissues than in normal adjacent tissues and was associated with a worse prognosis. In addition, EEF1AKMT3-knockdown induced gastric tumor invasiveness and migration. Through gain and loss-of-function studies, mass spectrometry analysis, RNA-seq, and phospho-antibody array, we identified EEF1AKMT3 as a novel tumor-suppressive methyltransferase that catalyzes the monomethylation of MAP2K7 (MKK7) at K296, thereby decreasing the phosphorylation, ubiquitination, and degradation of TP53. Furthermore, EEF1AKMT3, p-MAP2K7, and TP53 protein levels were positively correlated in GC tissues. Collectively, our results delineate the tumor-suppressive function of the EEF1AKMT3/MAP2K7/TP53 signaling axis and suggest the dysregulation of the signaling axis as potential targeted therapy in GC.
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Neoplasias Gástricas , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , MAP Quinasa Quinasa 7/metabolismo , Metiltransferasas/metabolismo , Invasividad Neoplásica , Neoplasias Gástricas/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Korean Red Ginseng (KRG) is a traditional herb that has several beneficial properties including anti-aging, anti-inflammatory, and autophagy regulatory effects. However, the mechanisms of these effects are not well understood. In this report, the underlying mechanisms of anti-inflammatory and autophagy-promoting effects were investigated in aged mice treated with KRG-water extract (WE) over a long period. METHODS: The mechanisms of anti-inflammatory and autophagy-promoting activities of KRG-WE were evaluated in kidney, lung, liver, stomach, and colon of aged mice using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and western blot analysis. RESULTS: KRG-WE significantly suppressed the mRNA expression levels of inflammation-related genes such as interleukin (IL)-1ß, IL-8, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6 in kidney, lung, liver, stomach, and colon of the aged mice. Furthermore, KRG-WE downregulated the expression of transcription factors and their protein levels associated with inflammation in lung and kidney of aged mice. KRG-WE also increased the expression of autophagy-related genes and their protein levels in colon, liver, and stomach. CONCLUSION: The results suggest that KRG can suppress inflammatory responses and recover autophagy activity in aged mice.
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CONTEXT: Among the plants in the genus Barringtonia (Lecythidaceae) used as traditional medicines to treat arthralgia, chest pain, and haemorrhoids in Indonesia, Barringtonia racemosa L. and Barringtonia acutangula (L.) Gaertn. have demonstrated anti-inflammatory activity in systemic inflammatory models. OBJECTIVE: The anti-inflammatory activity of Barringtonia angusta Kurz has not been investigated. We prepared a methanol extract of the leaves and stems of B. angusta (Ba-ME) and systemically evaluated its anti-inflammatory effects in vitro and in vivo. MATERIALS AND METHODS: RAW264.7 cells stimulated with LPS or Pam3CSK4 for 24 h were treated with Ba-ME (12.5, 25, 50, 100, and 150 µg/mL), and NO production and mRNA levels of inflammatory genes were evaluated. Luciferase reporter gene assay, western blot analysis, overexpression experiments, and cellular thermal shift assay were conducted to explore the mechanism of Ba-ME. In addition, the anti-gastritis activity of Ba-ME (50 and 100 mg/kg, administered twice per day for two days) was evaluated using an HCl/EtOH-induced gastritis mouse model. RESULTS: Ba-ME dose-dependently suppressed NO production [IC50 = 123.33 µg/mL (LPS) and 46.89 µg/mL (Pam3CSK4)] without affecting cell viability. Transcriptional expression of iNOS, IL-1ß, COX-2, IL-6, and TNF-α and phosphorylation of Src, IκBα, p50/105, and p65 were inhibited by Ba-ME. The extract specifically targeted the Src protein by binding to its SH2 domain. Moreover, Ba-ME significantly ameliorated inflammatory lesions in the HCl/EtOH-induced gastritis model. DISCUSSION AND CONCLUSIONS: The anti-inflammatory activity of Ba-ME is mediated by targeting of the Src/NF-κB signalling pathway, and B. angusta has potential as an anti-inflammatory drug.
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Antiinflamatorios/administración & dosificación , Barringtonia , Sistemas de Liberación de Medicamentos/métodos , Gastritis/tratamiento farmacológico , Extractos Vegetales/administración & dosificación , Familia-src Quinasas/antagonistas & inhibidores , Animales , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/metabolismo , Relación Dosis-Respuesta a Droga , Gastritis/inducido químicamente , Gastritis/metabolismo , Células HEK293 , Humanos , Masculino , Metanol/administración & dosificación , Metanol/metabolismo , Ratones , Ratones Endogámicos ICR , FN-kappa B , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Hojas de la Planta , Tallos de la Planta , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Melicope accedens (Blume) Thomas G. Hartley is a plant included in the family Rutaceae and genus Melicope. It is a native plant from Vietnam that has been used for ethnopharmacology. In Indonesia and Malaysia, the leaves of M. accedens are applied externally to decrease fever. AIM OF THE STUDY: The molecular mechanisms of the anti-inflammatory properties of M. accedens are not yet understood. Therefore, we examined those mechanisms using a methanol extract of M. accedens (Ma-ME) and determined the target molecule in macrophages. MATERIALS AND METHODS: We evaluated the anti-inflammatory effects of Ma-ME in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and in an HCl/EtOH-triggered gastritis model in mice. To investigate the anti-inflammatory activity, we performed a nitric oxide (NO) production assay and ELISA assay for prostaglandin E2 (PGE2). RT-PCR, luciferase gene reporter assays, western blotting analyses, and a cellular thermal shift assay (CETSA) were conducted to identify the mechanism and target molecule of Ma-ME. The phytochemical composition of Ma-ME was analyzed by HPLC and LC-MS/MS. RESULTS: Ma-ME suppressed the production of NO and PGE2 and the mRNA expression of proinflammatory genes (iNOS, IL-1ß, and COX-2) in LPS-stimulated RAW264.7 cells without cytotoxicity. Ma-ME inhibited NF-κB activation by suppressing signaling molecules such as IκBα, Akt, Src, and Syk. Moreover, the CETSA assay revealed that Ma-ME binds to Syk, the most upstream molecule in the NF-κB signal pathway. Oral administration of Ma-ME not only alleviated inflammatory lesions, but also reduced the gene expression of IL-1ß and p-Syk in mice with HCl/EtOH-induced gastritis. HPLC and LC-MS/MS analyses confirmed that Ma-ME contains various anti-inflammatory flavonoids, including quercetin, daidzein, and nevadensin. CONCLUSIONS: Ma-ME exhibited anti-inflammatory activities in vitro and in vivo by targeting Syk in the NF-κB signaling pathway. Therefore, we propose that Ma-ME could be used to treat inflammatory diseases such as gastritis.
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Antiinflamatorios/farmacología , FN-kappa B/metabolismo , Extractos Vegetales/farmacología , Rutaceae/química , Quinasa Syk/metabolismo , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Ciclooxigenasa 2/genética , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Etanol/toxicidad , Gastritis/inducido químicamente , Gastritis/tratamiento farmacológico , Gastritis/patología , Células HEK293 , Humanos , Ácido Clorhídrico/toxicidad , Inflamación/genética , Interleucina-1beta/genética , Lipopolisacáridos/toxicidad , Masculino , Metanol/química , Ratones , Ratones Endogámicos ICR , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Células RAW 264.7 , Transducción de Señal/efectos de los fármacosRESUMEN
Traditional medicinal practices have used natural products such as adaptogens to treat inflammatory, autoimmune, neurodegenerative, bacterial, and viral diseases since the early days of civilization. Panax ginseng Myer is a common herb used in East Asian countries for millennia, especially in Korea, China, and Japan. Numerous studies indicate that ginseng can modulate the immune system and thereby prevent diseases. Although the human immune system comprises many different types of cells, multiple studies suggest that each type of immune cell can be controlled or stimulated by ginseng or its derivatives. Provisional lists of ginseng's potential for use against viruses, bacteria, and other microorganisms suggest it may prove to be a valuable pharmaceutical resource, particularly if higher-quality evidence can be found. Here, we reviewed the role of ginseng as an immune-modulating agent in attempt to provide a valuable starting point for future studies on the herb and the human immune system.
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CONTEXT: Sauropus brevipes Müll. Arg. (Phyllanthaceae) has been used as an effective ingredient in a decoction for the treatment of diarrhoea. However, there was no report on its modulatory role in inflammation. OBJECTIVE: This study investigates anti-inflammatory effect of S. brevipes in various inflammation models. MATERIALS AND METHODS: The aerial part of S. brevipes was extracted with 95% ethanol to produce Sb-EE. RAW264.7 cells pre-treated with Sb-EE were stimulated by lipopolysaccharide (LPS), and Griess assay and PCR were performed. High-performance liquid chromatography (HPLC) analysis, luciferase assay, Western blotting and kinase assay were employed. C57BL/6 mice (10 mice/group) were orally administered with Sb-EE (200 mg/kg) once a day for five days, and peritonitis was induced by an intraperitoneal injection of LPS (10 mg/kg). ICR mice (four mice/group) were orally administered with Sb-EE (20 or 200 mg/kg) or ranitidine (positive control) twice a day for two days, and EtOH/HCl was orally injected to induce gastritis. RESULTS: Sb-EE suppressed nitric oxide (NO) release (IC50=34 µg/mL) without cytotoxicity and contained flavonoids (quercetin, luteolin and kaempferol). Sb-EE (200 µg/mL) reduced the mRNA expression of inducible NO synthase (iNOS). Sb-EE blocked the activities of Syk and Src, while inhibiting interleukin-1 receptor associated kinases (IRAK1) by 68%. Similarly, orally administered Sb-EE (200 mg/kg) suppressed NO production by 78% and phosphorylation of Src and Syk in peritonitis mice. Sb-EE also decreased inflammatory lesions in gastritis mice. DISCUSSION AND CONCLUSIONS: This study demonstrates the inhibitory effect of Sb-EE on the inflammatory response, suggesting that Sb-EE can be developed as a potential anti-inflammatory agent.
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Antiinflamatorios/farmacología , Sistemas de Liberación de Medicamentos/métodos , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Extractos Vegetales/uso terapéutico , Quinasa Syk/antagonistas & inhibidores , Familia-src Quinasas/antagonistas & inhibidores , Animales , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/uso terapéutico , Etanol/farmacología , Etanol/uso terapéutico , Gastritis/tratamiento farmacológico , Gastritis/metabolismo , Células HEK293 , Humanos , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Peritonitis/tratamiento farmacológico , Peritonitis/metabolismo , Componentes Aéreos de las Plantas , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Células RAW 264.7 , Quinasa Syk/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Muscle atrophy is an abnormal condition characterized by loss of skeletal muscle mass and function and is primarily caused by injury, malnutrition, various diseases, and aging. Leaf of lotus (Nelumbo nucifera Gaertn), which has been used for medicinal purposes, contains various active ingredients, including polyphenols, and is reported to exert an antioxidant effect. In this study, we investigated the effect of water extract of lotus leaf (LL) on muscle atrophy and the underlying molecular mechanisms of action. Amounts of 100, 200, or 300 mg/kg/day LL were administered to dexamethasone (DEX)-induced muscle atrophy mice for 4 weeks. Micro-computed tomography (CT) analysis revealed that the intake of LL significantly increased calf muscle volume, surface area, and density in DEX-induced muscle atrophy mice. Administration of LL recovered moving distance, grip strength, ATP production, and body weight, which were decreased by DEX. In addition, muscle damage caused by DEX was also improved by LL. LL reduced the protein catabolic pathway by suppressing gene expression of muscle atrophy F-Box (MAFbx; atrogin-1), muscle RING finger 1 (MuRF1), and forkhead box O (FoxO)3a, as well as phosphorylation of AMP-activated kinase (AMPK). The AKT-mammalian target of the rapamycin (mTOR) signal pathway, which is important for muscle protein synthesis, was increased in LL-administered groups. The HPLC analysis and pharmacological test revealed that quercetin 3-O-beta-glucuronide (Q3G) is a major active component in LL. Thus, Q3G decreased the gene expression of atrogin-1 and MuRF1 and phosphorylation of AMPK. This compound also increased phosphorylation levels of mTOR and its upstream enzyme AKT in DEX-treated C2C12 cells. We identified that LL improves muscle wasting through regulation of muscle protein metabolism in DEX-induced muscle atrophy mice. Q3G is predicted to be one of the major active phenolic components in LL. Therefore, we propose LL as a supplement or therapeutic agent to prevent or treat muscle wasting, such as sarcopenia.
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Dexametasona/toxicidad , Lotus/química , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/metabolismo , Extractos Vegetales/uso terapéutico , Hojas de la Planta/química , Agua/química , Animales , Western Blotting , Línea Celular , Cromatografía Líquida de Alta Presión , Masculino , Ratones , Extractos Vegetales/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Microtomografía por Rayos XRESUMEN
The JMJD10 gene and its encoded protein MYC-induced nuclear antigen (MINA53) are associated with multiple cancers. Besides having both an oncogenic and tumor suppressor function, the intricate role of JMJD10 in cancer is complex as it depends on the cancer type. In particular, the functional role of JMJD10/MINA53 in gastric cancer has been poorly understood. In this study, we have unraveled the molecular signatures and functional roles of JMJD10/MINA53 in gastric cancer by multiple approaches, i.e., multi-omics bioinformatics study, analysis of human gastric cancer tissues, and studies in vitro using knockdown or overexpression strategies in gastric cancer cell lines. The results indicated that the JMJD10 gene and MINA53 protein are commonly overexpressed in cancer patients. JMJD10/MINA53 is involved in the regulation of proliferation and survival of gastric cancer by controlling cell cycle gene expression. These processes are highly associated with MINA53 enzymatic activity in the regulation of H3K9me3 methylation status and controlling activation of AP-1 signaling pathways. This highlights the oncogenic role of JMJD10/MINA53 in gastric cancer and opens the opportunity to develop therapeutic targeting of JMJD10/MINA53 in gastric cancer.
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Inflammation is a complex protective response of body tissues to harmful stimuli. Acute inflammation can progress to chronic inflammation, which can lead to severe disease. Therefore, this research focuses on the development of anti-inflammatory drugs, and natural extracts have been explored as potential agents. No study has yet examined the inflammation-associated pharmacological activity of Potentilla glabra Var. mandshurica (Maxim.) Hand.-Mazz ethanol extract (Pg-EE). To examine the mechanisms by which Pg-EE exerts anti-inflammatory effects, we studied its activities in lipopolysaccharide (LPS)-treated murine macrophage RAW264.7 cells and an HCl/EtOH-induced gastritis model. LPS-triggered nitric oxide (NO) release and mRNA levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1ß) in RAW264.7 cells were suppressed by Pg-EE in a dose-dependent manner. Using a luciferase assay and western blot assay, we found that the NF-κB pathway was inhibited by Pg-EE, particularly by the decreased level of phosphorylated proteins of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) subunits (p65 and p50), inhibitor of kappa B alpha (IκBα), p85, and Src. Using an overexpression strategy, cellular thermal shift assay, and immunoprecipitation analysis, we determined that the anti-inflammatory effect of Pg-EE was mediated by the inhibition of Src. Pg-EE further showed anti-inflammatory effects in vivo in the HCl/EtOH-induced gastritis mouse model. In conclusion, Pg-EE exerts anti-inflammatory activities by targeting Src in the NF-κB pathway, and these results suggest that Pg-EE could be used as an anti-inflammatory herbal medicine.
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Antiinflamatorios/farmacología , Etanol/química , FN-kappa B/antagonistas & inhibidores , Extractos Vegetales/farmacología , Potentilla/química , Familia-src Quinasas/antagonistas & inhibidores , Enfermedad Aguda , Animales , Gastritis/patología , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Modelos Biológicos , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Células RAW 264.7 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
Despite previous reports of anti-aging effects of Korean red ginseng (KRG), the underlying mechanisms remain poorly understood. Therefore, this study investigated possible mechanisms of KRG-mediated anti-aging effects in aged mice. KRG significantly inhibited thymic involution in old mice. Interestingly, KRG only increased protein expression, but not mRNA expression, of aging-related genes Lin28a, GDF-11, Sirt1, IL-2, and IL-17 in the thymocytes of old mice. KRG also modulated the population of some types of immune cells in old mice. KRG increased the population of regulatory T cells and interferon-gamma (IFN-γ)-expressing natural killer (NK) cells in the spleen of old mice, but serum levels of regulatory T cell-specific cytokines IL-10 and TGF-ß were unaffected. Finally, KRG recovered mRNA expression of Lin28a, GDF-11, and Sirt1 artificially decreased by concanavalin A (Con A) in both thymocytes and splenocytes of old mice without cytotoxicity. These results suggest that KRG exerts anti-aging effects by preventing thymic involution, as well as modulating the expression of aging-related genes and immune cell subsets.
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Envejecimiento/genética , Envejecimiento/inmunología , Regulación de la Expresión Génica , Leucocitos/inmunología , Panax/química , Animales , Concanavalina A/farmacología , Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Leucocitos/efectos de los fármacos , Ratones Endogámicos C57BL , Extractos Vegetales/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Timo/crecimiento & desarrolloRESUMEN
Dehydrotrametenolic acid (DTA) is a lanostane-type triterpene acid isolated from Poria cocos Wolf (Polyporaceae). Several studies have reported the anti-inflammatory and antidiabetic effects of DTA; however, its effects on the skin are poorly understood. In this study, we investigated the effects of DTA on skin barrier function in vitro and its regulatory mechanism in human keratinocyte cell line HaCaT cells. DTA increased the microRNA (mRNA) expression of natural moisturizing factor-related genes, such as HAS-2, HAS-3, and AQP3 in HaCaT cells. DTA also upregulated the mRNA expression of various keratinocyte differentiation markers, including TGM-1, involucrin, and caspase-14. Moreover, the protein expression of HAS-2, HAS-3, and TGM-2 were significantly increased by DTA. To examine the regulatory mechanisms of DTA, Western blotting, luciferase-reporter assays, and RT-PCR were conducted. The phosphorylation of mitogen-activated protein kinases (MAPKs) and IκBα were increased in DTA-treated HaCaT cells. In addition, AP-1 and NF-κB transcriptional factors were dose-dependently activated by DTA. Taken together, our in vitro mechanism studies indicate that the regulatory effects of DTA on skin hydration and keratinocyte differentiation are mediated by the MAPK/AP-1 and IκBα/NF-κB pathways. In addition, DTA could be a promising ingredient in cosmetics for moisturizing and increased skin barrier function.
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Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Piel/efectos de los fármacos , Piel/metabolismo , Triterpenos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Modelos Biológicos , Estructura Molecular , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo , Triterpenos/químicaRESUMEN
Methods for delivering genes into the nuclei of cancer cells are desired in various clinical and therapeutic usages of non-viral vectors. The present study describes the production of surface-modified poly-(DL)-lactic-co-glycolic acid (PLGA) nanoparticles (NPs), which facilitated the delivery of specific genes into the cancer cell nuclei. Plasmid DNA (pDNA) encoding Bak and Bak-like (BL) apoptotic genes were generated and successfully delivered into cancer cell nuclei, resulting in apoptosis of the cancer cells. The abilities of Bak and BL genes to promote cancer cell (HeLa and 293T cells) apoptosis were compared. In fluorescence-activated cell sorting (FACS) analysis, approximately 61% and 51% of BL pDNAs fused with EGFP complexed PLGA NPs were transfected into 293T cells and HeLa cells, respectively; however, a low transfection efficiency was obtained for lipofectamine-complexed BL pDNAs. In both DePsipher and cytochrome c staining analysis, larger amount of apoptotic 293T and HeLa cells were observed after cell transfection with Bak and BL pDNAs complexed with PLGA NPs as compared with lipofectamine complexed with Bak and BL pDNAs or control groups. The apoptosis of 293T and HeLa cells transfected with Bak and BL against several types of non-viral vectors was detected. Western blotting, and immunohistochemical analyses showed that the complexes of biodegradable PLGA NPs coated with polyethyleneimine (PEI; M(w) 25,000) Bak and BL can be formed, and used for efficient delivery of the apoptotic genes into the cell nuclei.
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Apoptosis/genética , Técnicas de Transferencia de Gen , Genes Transgénicos Suicidas , Ácido Láctico/química , Nanopartículas/uso terapéutico , Neoplasias/terapia , Polietileneimina/química , Ácido Poliglicólico/química , Células Cultivadas , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Portadores de Fármacos/uso terapéutico , Terapia Genética/métodos , Células HEK293 , Células HeLa , Humanos , Ácido Láctico/uso terapéutico , Nanopartículas/química , Neoplasias/genética , Neoplasias/patología , Polietileneimina/uso terapéutico , Ácido Poliglicólico/uso terapéutico , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
This study describes a single gold nanoparticle (AuNP)-based observation of biomolecular interaction using a near-field scanning microscope (NSOM) in transmission mode. To observe streptavidin molecules, a glass surface was first patterned with a micro-scale line of (3-aminopropyl)trimethoxysilane (APTMS) by micro-contact printing (microCP) with a subsequent reaction of N-hydroxysuccinimide (NHS)-biotin. The AuNP-conjugated streptavidin was then applied to the biotin-modified glass surface and NSOM was employed to detect the resulting specific interaction between streptavidin and biotin on the glass surface. Using the optical and topological images generated from the NSOM analysis, the interaction could be observed at the nanoscopic scale. This study demonstrates that the NSOM is a powerful tool for the detection of protein interactions at the nanoscopic level when the protein is conjugated with AuNPs.
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Oro/química , Nanopartículas del Metal/química , Nanopartículas/química , Estreptavidina/química , Biotina/química , Microscopía de Sonda de Barrido/métodos , Estreptavidina/análisisAsunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Compuestos Férricos , Imagen por Resonancia Magnética , Magnetismo , Nanocápsulas , Nanopartículas , Neoplasias/tratamiento farmacológico , Neoplasias de la Mama/diagnóstico , Línea Celular Tumoral , Medios de Contraste , Femenino , Oro/química , Humanos , Imagen por Resonancia Magnética/instrumentación , Imagen por Resonancia Magnética/métodos , Microscopía Electrónica de Transmisión , Modelos Biológicos , Nanopartículas/química , Nanopartículas/ultraestructura , Neoplasias/diagnóstico , Fotoquimioterapia , Plata/química , Propiedades de SuperficieRESUMEN
Protein G is an antibody binding protein, which specifically targets the Fc region of an antibody. It therefore has been widely used to immobilize different types of antibodies in numerous immunoassays. Here, we have engineered Streptococcus protein G to contain various numbers of cysteine residues at the N-terminus and therefore to form well-oriented protein G films on bare gold. SPR and SPR imaging analyses indicated that a gold surface treated with cysteine-tagged protein G possesses a superior antibody binding ability compared to one treated with tag-free protein G. AFM images indicated a higher surface coverage by antibody binding on the cysteine-tagged protein G surface than the intact protein G surface. The proper orientation of cysteine-tagged protein G on a gold surface also afforded better orientation of immobilized antibodies, resulting in enhanced antigen detection. Moreover, the protein G surfaces maintained their high antibody binding ability during multiple rounds of antibody interaction tests. The cysteine-tagged protein G constructed in this study can be a valuable link for oriented antibody immobilization in a variety of immunosensors.
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Cisteína/química , Oro/química , Proteínas del Tejido Nervioso/química , Adsorción , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo , Sitios de Unión , Humanos , Microscopía de Fuerza Atómica/métodos , Nanopartículas/química , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Sensibilidad y Especificidad , Streptococcus/química , Resonancia por Plasmón de Superficie/métodos , Propiedades de SuperficieRESUMEN
Sol-gel transition temperature-controllable Pluronic F127/F68 mixtures including mildly crosslinked alginate and nonsteroidal anti-inflammatory drug (ibuprofen) were prepared to evaluate their potential as tissue adhesion barrier gels. The sol-gel transition temperatures of the Pluronic mixtures could be controlled by adjusting F127/F68 ratio and polymer concentration. The mildly crosslinked alginate with still flow property provided the residence stability of Pluronic mixture gels in the body. Ibuprofen was loaded in Pluronic mixtures to reduce inflammatory response in the body and, thus, to prevent tissue adhesion. The gelation temperatures of the Pluronic mixtures were not affected by the alginate but lowered by the addition of ibuprofen. The in vitro drug release behavior and in vivo peritoneal tissue adhesion of the Pluronic mixtures with the sol-gel transition just below body temperatures were investigated. The drug release behavior from the ibuprofen (1 wt%)-loaded Pluronic mixture gels at 37 degrees C was examined using a membrane-less dissolution model. The drug in the mixture gels was released continuously up to about 45-65% of the total loading amount during the first 7 days. For in vivo evaluation of tissue anti-adhesion potential, the Pluronic mixtures with/without drug were coated on the peritoneal wall defects of rats and their tissue adhesion extents and tissue reactions (inflammatory response, granulation tissue formation, and toxicity in organs) were compared. It was observed that ibuprofen has a positive effect for the peritoneal tissue anti-adhesion. The Pluronic F127/F68/alginate/ibuprofen mixture gel (25 wt% of F127/F68 [7/3], 1 wt% ibuprofen) was highly effective for the prevention of peritoneal tissue adhesion and showed a relatively low inflammatory response and non-toxicity, and thus can be a good candidate material as a coatable or injectable tissue adhesion barrier gel.