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BACKGROUND: Walnut kernels are high in polyphenols (PPs), which cause low protein solubility, limiting the use of walnut protein in the food industry. To obtain the best technical parameters of the dephenolization treatment, the defatted walnut powder was dephenolized using ultrasound-assisted ethanol extraction (UAE), and the response surface optimization was performed on the basis of single factor. On this basis, the effects of dephenolization on the solubility, emulsifying properties and foaming properties of walnut protein isolates (WPIs) were compared to those of defatted walnut powder without dephenolization. RESULTS: The results showed that PP extraction in the UAE could significantly increase PP yield. The optimal process parameters were as follows: 51% (v/v) ethanol concentration, 140 W ultrasound power, 10 min extraction time, 30 °C ultrasound temperature, and a material-liquid ratio of 1:30 (w/v). The results revealed that the UAE dephenolization treatment significantly improved the functionality of WPI and that the functionality of the dephenolized WPI by UAE was superior to that of the protein without dephenolization, and that the functionality of both walnut proteins was the worst at pH 5, with solubility of 5.31% and 4.86%, emulsifying activity index (EAI) of 24.95 and 19.91 m2 /g, and foaming capacity (FC) of 3.66% and 2.94%, respectively; and the best at pH 11, with solubility of 82.35% and 73.55%, EAI of 46.35 and 37.28 m2 /g, and FC of 35.85% and 18.87%, respectively. CONCLUSION: The study found that dephenolization by UAE can significantly improve the functionality of WPI, and this method should be promoted and used in walnut and walnut protein processing industries. © 2023 Society of Chemical Industry.
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Juglans , Polifenoles , Polifenoles/química , Juglans/química , Polvos/análisis , Etanol/análisis , Nueces/químicaRESUMEN
BACKGROUND: N6-methyladenosine (m6A) has emerged as a significant regulator of the progress of various cancers. However, its role in lung adenocarcinoma (LUAD) remains unclear. Here, we explored the biological function and underlying mechanism of methyltransferase-like 3 (METTL3), the main catalyst of m6A, in LUAD progression. METHODS: The expression of m6A, METTL3, YTHDF1 and SLC7A11 were detected by immunochemistry or/and online datasets in LUAD patients. The effects of METTL3 on LUAD cell proliferation, apoptosis and ferroptosis were assessed through in vitro loss-and gain-of-function experiments. The in vivo effect on tumorigenesis of METTL3 was evaluated using the LUAD cell xenograft mouse model. MeRIP-seq, RNA immunoprecipitation and RNA stability assay were conducted to explore the molecular mechanism of METTL3 in LUAD. RESULTS: The results showed that the m6A level, as well as the methylase METTL3 were both significantly elevated in LUAD patients and lung cancer cells. Functionally, we found that METTL3 could promote proliferation and inhibit ferroptosis in different LUAD cell models, while METTL3 knockdown suppressed LUAD growth in cell-derived xenografts. Mechanistically, solute carrier 7A11 (SLC7A11), the subunit of system Xc-, was identified as the direct target of METTL3 by mRNA-seq and MeRIP-seq. METTL3-mediated m6A modification could stabilize SLC7A11 mRNA and promote its translation, thus promoting LUAD cell proliferation and inhibiting cell ferroptosis, a novel form of programmed cell death. Additionally, we demonstrated that YTHDF1, a m6A reader, was recruited by METTL3 to enhance SLC7A11 m6A modification. Moreover, the expression of YTHDF1 and SLC7A11 were positively correlated with METTL3 and m6A in LUAD tissues. CONCLUSIONS: These findings reinforced the oncogenic role of METTL3 in LUAD progression and revealed its underlying correlation with cancer cell ferroptosis; these findings also indicate that METTL3 is a promising novel target in LUAD diagnosis and therapy.
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Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are common lung disorders characterized by alveolar-capillary barrier disruption and dyspnea, which can cause substantial morbidity and mortality. Currently, a cluster of acute respiratory illnesses, known as novel coronavirus (2019-nCoV)-infected pneumonia (NCIP), which allegedly originally occurred in Wuhan, China, has increased rapidly worldwide. The critically ill patients with ARDS have high mortality in subjects with comorbidities. Previously, the excessive recruitment and activation of neutrophils (polymorphonuclear leukocytes [PMNs]), accompanied by neutrophil extracellular traps (NETs) formation were reported being implicated in the pathogenesis of ALI/ARDS. However, the direct visualization of lung epithelial injuries caused by NETs, and the qualitative and quantitative evaluations of this damage are still lacking. Additionally, those already reported methods are limited for their neglect of the pathological role exerted by NETs and focusing only on the morphological features of NETosis. Therefore, we established a cell-based assay for detecting NETs during lung epithelial cells-neutrophils co-culture using the xCELLigence system, a recognized real-time, dynamic, label-free, sensitive, and high-throughput apparatus. Our results demonstrated that lung epithelial injuries, reflected by declines in cell index (CI) values, could be induced by lipopolysaccharide (LPS)-activated PMNs, or NETs in a time and dose-dependent manner. NETs generation was verified to be the major contributor to the cytotoxicity of activated PMNs; protein components of NETs were the prevailing cytotoxic mediators. Moreover, this cell-based assay identified that PMNs from severe pneumonia patients had a high NETs formative potential. Additionally, acetylsalicylic acid (ASA) and acetaminophen (APAP) were discovered alleviating NETs formation. Thus, this study not only presents a new methodology for detecting the pathophysiologic role of NETs but also lays down a foundation for exploring therapeutic interventions in an effort to cure ALI/ARDS in the clinical setting of severe pneumonia, including the emerging of NCIP.
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Lesión Pulmonar Aguda/sangre , Infecciones por Coronavirus/sangre , Trampas Extracelulares/diagnóstico por imagen , Neutrófilos/metabolismo , Neumonía Viral/sangre , Síndrome de Dificultad Respiratoria/sangre , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/diagnóstico por imagen , Lesión Pulmonar Aguda/virología , Animales , Betacoronavirus/patogenicidad , COVID-19 , Infecciones por Coronavirus/diagnóstico por imagen , Infecciones por Coronavirus/virología , Células Epiteliales/patología , Células Epiteliales/virología , Trampas Extracelulares/virología , Humanos , Lipopolisacáridos/toxicidad , Pulmón/diagnóstico por imagen , Pulmón/virología , Masculino , Neutrófilos/virología , Pandemias , Neumonía/sangre , Neumonía/diagnóstico por imagen , Neumonía/virología , Neumonía Viral/diagnóstico por imagen , Neumonía Viral/virología , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/virología , SARS-CoV-2RESUMEN
Membrane proteins play important functions not only as receptors and transporters, but also in many other important intracellular functions such as photosynthetic and respiratory electron transport. Identification of membrane proteins is a necessary step to understand their functions. Membrane proteins are generally highly hydrophobic and difficult to be resolved by aqueous solutions, and large-scale proteomic identification of membrane proteins has been a great technical challenge. Significant efforts have been invested in the field to improve the solubility of membrane proteins in aqueous solutions that are compatible for mass spectrometry analysis. This review summarizes the main technological achievements in the field of membrane proteomics particularly for the improvement of membrane protein identification, and uses the photosynthetic model cyanobacterium Synechocystis sp. PCC6803 as an example to illustrate how technology advances push forward the field in terms of the increased coverage of membrane proteome identification.
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Proteoma , Proteómica/tendencias , Synechocystis/genética , Proteínas Bacterianas/genética , Espectrometría de MasasRESUMEN
Whelks Neptunea arthritica cumingi Crosse and Neverita didyma were processed by hot air drying and changes of thei lipids and the mechanism involved were evaluated by analyzing peroxide value, thiobarbituric acid-reactive substances, total oxidation value, fatty acid composition, activities of lipases and lipoxygenase (LOX), as well as contents of triacylglycerol (TAG), free fatty acid (FFA), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The processing significantly decreased the contents of PC, PE and TAG but increased the content of FFA. The presence of acid lipase and phospholipase in whelk tissues and their activity preservation during processing suggest that the enzymes may help hydrolyze lipids. By contrast, the reduction of PC, PE and TAG was more pronounced than the increase in FFA in whelk tissues upon processing, indicating the oxidative degradation of FFA. LOX may play a role in lipid oxidation due to the stability of the starting components during processing.
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OBJECTIVES: The hypothalamus regulates metabolism and feeding behavior by perceiving the levels of peripheral insulin. However, little is known about the hypothalamic changes after aberrant metabolism. In this study, we investigated the changes of insulin and autophagy relevant signals of hypothalamus under diabetes mellitus. METHODS: C57B/L mice were injected with low-dose streptozotocin (STZ) and fed with high-fat diet to induce type 2 diabetes mellitus. In vitro, PC12 cells were treated with oleic acid to mimic lipotoxicity. RESULTS: Results showed that the cholesterol level in the hypothalamus of the diabetic mice was higher than that of the normal mice. The expression of insulin receptors and insulin receptor substrate-1 were downregulated and the number of Fluoro-Jade C positive cells significantly increased in the hypothalamic arcuate nucleus of the diabetic mice. Furthermore, Upregulation of mammalian target of rapamycin (mTOR) and downregulation of LC 3II were obvious in the hypothalamus of the diabetic mice. In vitro, results showed that high-lipid caused PC12 cell damage and upregulated LC3 II expression. Pretreatment of cells with 3-methyladenine evidently downregulated LC3 II expression and aggravated PC12 cell death under high lipid conditions. By contrast, pretreatment of cells with rapamycin upregulated LC3 II expression and ameliorated PC12 cell death caused by lipotoxicity. CONCLUSION: These results demonstrate that autophagy activation confers protection to neurons under aberrant metabolism and that autophagy dysfunction in the hypothalamus occurs in the chronic metabolic disorder such as T2DM.
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Autofagia , Encefalopatías/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hipotálamo/metabolismo , Neuronas/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Núcleo Arqueado del Hipotálamo/metabolismo , Núcleo Arqueado del Hipotálamo/ultraestructura , Autofagia/efectos de los fármacos , Western Blotting , Colesterol/metabolismo , Dieta Alta en Grasa , Regulación hacia Abajo , Prueba de Tolerancia a la Glucosa , Hipotálamo/efectos de los fármacos , Hipotálamo/ultraestructura , Inmunosupresores/farmacología , Técnicas In Vitro , Insulina , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Ácido Oléico/farmacología , Células PC12 , Ratas , Receptor de Insulina/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Regulación hacia Arriba , Núcleo Hipotalámico Ventromedial/efectos de los fármacos , Núcleo Hipotalámico Ventromedial/metabolismo , Núcleo Hipotalámico Ventromedial/ultraestructuraRESUMEN
Tumor-associated thrombus (TAT) accounts for a high proportion of venous thromboembolism. Traditional thrombolysis and anticoagulation methods are not effective due to various complications and contraindications, which can easily lead to patients dying from TAT rather than the tumor itself. These clinical issues demonstrate the need to research diverse pathways for adjuvant thrombolysis in antitumor therapy. Previously, the phenotypic and functional transformation of monocytes/macrophages is widely reported to be involved in intratribal collagen regulation. This study finds that myeloid deficiency of the oncogene SHP2 sensitizes Ly6Clow monocyte/macrophage differentiation and can alleviate thrombus organization by increasing thrombolytic Matrix metalloproteinase (MMP) 2/9 activities. Moreover, pharmacologic inhibition by SHP099, examined in mouse lung metastatic tumor models, reduces tumor and thrombi burden in tumor metastatic lung tissues. Furthermore, SHP099 increases intrathrombus Ly6Clow monocyte/macrophage infiltration and exhibits thrombolytic function at high concentrations. To improve the thrombolytic effect of SHP099, NanoSHP099 is constructed to achieve the specific delivery of SHP099. NanoSHP099 is identified to be simultaneously enriched in tumor and thrombus foci, exerting dual tumor-suppression and thrombolysis effects. NanoSHP099 presents a superior thrombus dissolution effect than that of the same dosage of SHP099 because of the higher Ly6Clow monocyte/macrophage proportion and MMP2/MMP9 collagenolytic activities in organized thrombi.
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Monocitos , Trombosis , Animales , Ratones , Leucocitos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Terapia Trombolítica/métodos , Trombosis/metabolismo , Piperidinas/farmacología , Pirimidinas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidoresRESUMEN
SIPRα on macrophages binds with CD47 to resist proengulfment signals, but how the downstream signal of SIPRα controls tumor-infiltrating macrophages (TIMs) is still poorly clarified. Here, we report that the CD47/signal regulatory protein α (SIRPα) axis requires the deneddylation of tyrosine phosphatase SHP2. Mechanistically, Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) was constitutively neddylated on K358 and K364 sites; thus, its autoinhibited conformation was maintained. In response to CD47-liganded SIRPα, SHP2 was deneddylated by sentrin-specific protease 8 (SENP8), which led to the dephosphorylation of relevant substrates at the phagocytic cup and subsequent inhibition of macrophage phagocytosis. Furthermore, neddylation inactivated myeloid-SHP2 and greatly boosted the efficacy of colorectal cancer (CRC) immunotherapy. Importantly, we observed that supplementation with SHP2 allosteric inhibitors sensitized immune treatment-resistant CRC to immunotherapy. Our results emphasize that the CRC subtype that is unresponsive to immunotherapy relies on SIRPαhiSHP2hiNEDD8lo TIMs and highlight the need to further explore the strategy of SHP2 targeting in CRC therapy.
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Antígeno CD47 , Neoplasias del Colon , Humanos , Antígenos de Diferenciación/genética , Antígeno CD47/genética , Antígeno CD47/metabolismo , Neoplasias del Colon/genética , Endopeptidasas , Terapia de Inmunosupresión , Inmunoterapia/métodos , Fagocitosis , Receptores InmunológicosRESUMEN
The urokinase-type plasminogen activator receptor (uPAR) is an essential regulator for cell signaling in tumor cell proliferation, adhesion, and metastasis. The ubiquitous nature of uPAR in many aggressive cancer types makes uPAR an attractive target for immunotherapy. Here, we present a rapid and successful workflow for developing cross-reactive anti-uPAR recombinant antibodies (rAbs) using high-throughput optofluidic screening of single B-cells from human uPAR-immunized mice. A total of 80 human and cynomolgus uPAR cross-reactive plasma cells were identified, and selected mouse VH/VL domains were linked to the trastuzumab (Herceptin®) constant domains for the expression of mouse-human chimeric antibodies. The resulting rAbs were characterized by their tumor-cell recognition, binding activity, and cell adhesion inhibition on triple-negative breast cancer cells. In addition, the rAbs were shown to enact antibody-dependent cellular cytotoxicity (ADCC) in the presence of either human natural killer cells or peripheral blood mononuclear cells, and were evaluated for the potential use of uPAR-targeting antibody-drug conjugates (ADCs). Three lead antibodies (11857, 8163, and 3159) were evaluated for their therapeutic efficacy in vivo and were shown to suppress tumor growth. Finally, the binding epitopes of the lead antibodies were characterized, providing information on their unique binding modes to uPAR. Altogether, the strategy identified unique cross-reactive antibodies with ADCC, ADC, and functional inhibitory effects by targeting cell-surface uPAR, that can be tested in safety studies and serve as potential immunotherapeutics.
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Leucocitos Mononucleares , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Humanos , Animales , Ratones , Anticuerpos , Transducción de Señal , Linfocitos BRESUMEN
We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-ß-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry.
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Expresión Génica , Vectores Genéticos/genética , Trichoderma/genética , Trichoderma/metabolismo , Agrobacterium/genética , Agrobacterium/metabolismo , Celulasa/genética , Celulasa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/genética , Clonación Molecular , ADN Bacteriano/genética , Recombinación Homóloga , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transformación Genética , Proteína Fluorescente RojaRESUMEN
OBJECTIVE: To determine the optimum microwave extraction process of flavonoids in peanut skin. METHODS: The effects of extraction methods, microwave irradiation time, microwave power, ethanol concentration, material-liquid ratio and extraction times on the extraction of total flavonoids in peanut skin was investigated. RESULTS: Based on the single factor experiments, the extraction conditions were optimized by orthogonal experiment. Microwave extraction method had higher extraction rate, and was selected for the extraction of total flavonoids in peanut skin. The optimum extraction conditions were as follows: the microwave power was 690 W, the extraction time was 40 s, the concentration of ethanol was 55%, the material-liquid ratio was 1: 20. Under the optimum conditions, the yield of total flavonoids was 3.18%. CONCLUSION: The microwave extraction method is simple, and can be applied to the fast extraction of total flavonoids in peanut skin.
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Arachis/química , Flavonoides/aislamiento & purificación , Microondas , Semillas/química , Análisis de Varianza , Medicamentos Herbarios Chinos/aislamiento & purificación , Etanol/química , Solventes/química , Espectrofotometría Infrarroja , Tecnología Farmacéutica/métodos , Factores de TiempoRESUMEN
Prolyl hydroxylase domain proteins (PHDs) hydroxylate HIFα in the presence of oxygen, leading to HIFα proteasomal destruction. The PHDs family comprises PHD1, 2, and 3. Recent studies indicate that, in addition to HIFα, PHDs have other substrates. Paired box (Pax) 2, a transcription factor, was found aberrantly expressed in a variety of cancers. However, the underlying mechanisms remain unknown. Here we demonstrate that PHD3 is a negative regulator of expression of Pax2. We found that PHD3 bound to Pax2 and mediated Pax2 destruction directly. Inhibition of PHD3 hydroxylase activity led to upregulation of Pax2 protein but not mRNA level. We found that Pax2 protein was increased and PHD3 protein was decreased in colorectal cancer, and the increased Pax2 was associated with decreased PHD3. Our results suggest that PHD3 targets Pax2 for destruction. The findings may disclose a mechanism for the regulation of Pax2 expression in cancer cells.
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Neoplasias Colorrectales/metabolismo , Dioxigenasas/metabolismo , Factor de Transcripción PAX2/metabolismo , Línea Celular Tumoral , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Factor de Transcripción PAX2/antagonistas & inhibidores , Factor de Transcripción PAX2/genética , Transcripción GenéticaRESUMEN
Activating transcription factor 3 (ATF3) is a member of the ATF/CREB (CAMP responsive element binding protein) family of transcription factors. The expression and the function of ATF3 in vascular smooth muscle cells (VSMCs) remain unknown. The aim of this work is to determine the expression and possible function of ATF3 in VSMCs. We found that VSMCs expressed ATF3, and expression of ATF3 in VSMCs was induced by a variety of stimuli including serum, angiotensin II, and H(2)O(2). Knockdown of ATF3 induced apoptosis of VSMCs, caspase-3 cleavage, and cytochrome c release. The results suggest that ATF3 regulates survivability of VSMCs. Moreover, we found that overexpression of ATF3 promoted migration of VSMCs and induced expression of matrix metalloproteinase 1, 3, and 13. These results suggest that ATF3 plays a role in regulating migration of VSMCs. In addition, we found that the expression of ATF3 was upregulated in smooth muscle cells in the injured mouse femoral arteries compared with the uninjured control group. These results suggest that ATF3 is relevant to disease physiology.
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Factor de Transcripción Activador 3/fisiología , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Músculo Liso Vascular/citología , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Cartilla de ADN , Masculino , Ratones , Ratones Endogámicos C57BL , Plásmidos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mussel (Mytilus edulis) is an economic shellfish with a high nutritional value. Due to the high amount of protein and fat, fresh mussels are susceptible to spoilage during storage. In the present study, how a combination of pullulan, acidic electrolyzed water (AEW), and stable chlorine dioxide (ClO2) ice-glazing treatments affect the quality of mussels was investigated during 90 days of frozen storage. The results indicate that the combined glazing treatment effectively maintained the mussel muscle quality during storage mainly due to its air barrier actions. Mussel samples coated with AEW and ClO2 showed lower aerobic plate counts than other groups, resulting from the strong antibacterial action of AEW and ClO2. After 90 days of frozen storage, the mussel glazed with a combination of AEW, ClO2, and pullulan solutions showed better texture properties, higher content of myofibrillar proteins, higher Ca2+-ATPase activity, and more SH groups than the other glazing treatments. The water-holding capacity and SEM observations showed that the pullulan glazing efficiently inhibited the physical damage caused by the frozen and long-term storage, which mainly contributed to the high amount of hydrophilic hydroxyl groups in the muscle tissues. The present study supports the use of a combination of cryoprotectants for extending the shelf-life of frozen mussel products during long-term storage.
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OBJECTIVE: This article aims to investigate the effect of miRNA-200b on the proliferation and apoptosis of cervical cancer cells by targeting RhoA. METHODS: HeLa cells of cervical cancer were divided into five groups: blank control group, negative control group (miRNA-200b mimic NC), miRNA-200b mimic group, RhoA-negative control group, and RhoA overexpression group. Cells were collected 48 h after transfection. The expression levels of miRNA-200b were detected by RT-PCR. Target relationship between miRNA-200b and RhoA was verified by the dual-luciferase reporter assay. RhoA mRNA and protein expression were detected by western blot and RT-PCR methods. Flow cytometry was used to detect the apoptosis of cells in each group, and the CCK8 method was used to detect the proliferation of cells in each group. The mRNA and protein expression of Bax and cyclin D1 were detected by RT-PCR and western blot. RESULTS: The results of the dual luciferase reporter assay showed that RhoA was the target gene of microRNA 200b. Compared with the blank control group and the miRNA-200b mimic-NC group, the proportion of apoptotic cells increased significantly in the miRNA-200b mimic group, and the proliferation of cells was inhibited (P < 0.05). After overexpression of RhoA, the percentage of apoptotic cells decreased and the ability of cell proliferation increased significantly (P < 0.05). CONCLUSION: miRNA-200b can inhibit the proliferation and promote the apoptosis of cervical cancer cells by targeting the RhoA gene.
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Benzo[a]pyrene (B[a]P) has been shown to accelerate atherosclerosis development in animal models. However, the mechanisms that B[a]P induces atherogenesis are unclear. Abnormal migration and invasion of vascular smooth muscle cells (VSMCs) is a major contributor to the development of atherosclerotic lesions. In this article, we demonstrated that B[a]P promoted the migration and invasion of rat VSMCs. B[a]P increased the mRNA levels of matrix metalloproteinase (MMP) 1, 2, 3, and 9. The MMPs inhibitor GM6001 inhibited B[a]P-induced invasion of VSMCs. Among the MMPs mentioned above, MMP-3 had the maximal induction. Mechanistic studies indicate that B[a]P-induced transcriptional activation of MMP-3 is not mediated by AP-1, NF-kappaB. B[a]P-induced expression of MMPs was attenuated by alpha-naphthoflavone, the aryl hydrocarbon receptor antagonist. In addition, alpha-naphthoflavone inhibited B[a]P-induced migration and invasion of VSMCs. These results suggest that the aryl hydrocarbon receptor plays an important role in B[a]P-induced expression of MMPs and migration and invasion of VSMC. Our findings may reveal a novel role of B[a]P in inducing atherogenesis.
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Benzo(a)pireno/farmacología , Movimiento Celular/efectos de los fármacos , Metaloproteinasas de la Matriz/metabolismo , Músculo Liso Vascular/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Masculino , Músculo Liso Vascular/enzimología , Ratas , Ratas Sprague-DawleyRESUMEN
AIMS: We showed previously that insulin-like growth factor-I (IGF-I)-induced vascular smooth muscle cells (VSMCs) proliferation through the production of reactive oxygen species (ROS). However, how IGF-I-induced ROS was unknown. The aim of this study is to investigate the mechanisms by which IGF-I induces ROS production in VSMCs. Methods results Reverse transcription-PCR, real-time PCR, immunoblotting, and confocal microscopic image analysis were employed to determine protein expression, small Rho-GTPase Rac1 activation, and ROS production. Inhibition of NADPH oxidase 4 (Nox4) or Rac1 was performed by means of siRNA technology. Inhibition of Rac1 activity was accomplished using dominant-negative form of Rac1 (N17Rac1) plasmid. VSMCs from Sprague-Dawley rat thoracic aortas were used in this work.IGF-I enhanced ROS production in rat VSMCs. IGF-I increased the protein level of Nox4 but had little effect on its mRNA level. IGF-I induced the activation of Rac1. Either knockdown of Nox4 or inactivation of Rac1 impaired IGF-I-induced ROS. Over-expression of Nox4 increased NADPH oxidase activity, which was not influenced by inactivation of Rac1. Neither over-expression nor knockdown of Rac1 influenced Nox4 expression. Knockdown of Nox4 did not affect IGF-I-induced activation of Rac1. IGF-I increased matrix metalloproteinase (MMP)-2 and 9 activity and promoted VSMC migration, which was inhibited by knockdown of Nox4 and inactivation of Rac1. CONCLUSION: Our results suggest that Nox4 and Rac1 mediate IGF-I-induced ROS production and cell migration in VSMCs and that Nox4 is not regulated by Rac1.
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Movimiento Celular , Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Células Cultivadas , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Proteína de Unión al GTP rac1/genéticaRESUMEN
The present study investigated the aptness of assessing the levels of progastrin-releasing peptide (Pro-GRP) in addition to the T lymphocyte subpopulation in lung cancer patients prior to and after therapy for determining immune function. A total of 45 patients with lung cancer were recruited and stratified in to a non-small cell lung cancer (NSCLC) and an SCLC group. Prior to and after treatment by combined biological therapy comprising chemotherapy or chemoradiotherapy followed by three cycles of retransformation of autologous dendritic cells-cytokine-induced killer cells (DC-CIK), the peripheral blood was assessed for populations of CD3+, CD4+, CD8+ and regulatory T cells (Treg) by flow cytometry, and for the levels of pro-GRP, carcinoembryonic antigen, neuron-specific enolase and Cyfra 21-1. The results revealed that in NSCLC patients, CD8+ T lymphocytes and Treg populations were decreased, and that CD3+ and CD4+ T lymphocytes as well as the CD4+/CD8+ ratio were increased after therapy; in SCLC patients, CD3+, CD4+ and CD8+ T lymphocytes were increased, while Treg cells were decreased after treatment compared with those at baseline. In each group, Pro-GRP was decreased compared with that prior to treatment, and in the SCLC group only, an obvious negative correlation was identified between Pro-GRP and the T lymphocyte subpopulation. Furthermore, a significant correlation between Pro-GRP and Tregs was identified in each group. In conclusion, the present study revealed that the immune function of the patients was improved after biological therapy. The results suggested a significant correlation between Pro-GRP and the T lymphocyte subpopulation in SCLC patients. Detection of Pro-GRP may assist the early clinical diagnosis of SCLC and may also be used to assess the immune regulatory function of patients along with the T lymphocyte subpopulation. Biological therapy with retransformed autologous DC-CIK was indicated to enhance the specific elimination of tumor cells and improve the immune surveillance function in cancer patients, and also restrained the immune evasion of the tumor, leading to decreased Pro-GRP levels.
RESUMEN
Neuroligins (NLGs) are postsynaptic adhesion molecules known to play essential roles in synapse development and maturation, but their effects on synaptic plasticity at mature synapses remain unclear. In this study, we investigate the involvement of NLG1 in hippocampal long-term depression (LTD), a key form of long lasting synaptic plasticity, critical for memory formation and brain disorders, by using mice deficient in the expression of NLG1. We find that although NLG1 homozygous (NLG1-/-) mice show no impairments in either NMDA receptor- (NMDAR-LTD) or metabotropic glutamate receptor-dependent LTD (mGluR-LTD), the heterozygous (NLG1+/-) mice are significantly altered in both forms of LTD characterized by the absence of NMDAR-LTD but enhanced mGluR-LTD. Accordingly, the NLG1+/-, but not the NLG1-/- mice are altered in synaptic proteins, including PSD95, GluA2 and phosphorylated GluA1 at serine 845, all of which are involved in the expression of LTD. The NLG1+/- mice also exhibit autistic-like behaviors including increased grooming and impaired recognition memory. We further show that the expression of NLG3, a close family member of NLG1, is elevated in the NLG1-/-, but not in NLG1+/- mice, suggesting that the lack of LTD deficits in the NLG1-/- mice might be due to the increased NLG3. Our results reveal a gene dosage dependent role for NLG1 in the regulation of LTD and suggest that moderate changes in NLG1 protein level may be sufficient to cause synaptic and behavior deficits in brain disorders where copy number variants and hemizygosity of gene mutations are common.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Hipocampo/metabolismo , Depresión Sináptica a Largo Plazo/fisiología , Memoria/fisiología , Animales , Trastorno del Espectro Autista/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Células Cultivadas , Homólogo 4 de la Proteína Discs Large/metabolismo , Miedo/fisiología , Masculino , Proteínas de la Membrana/metabolismo , Trastornos de la Memoria/metabolismo , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Conducta Social , Transmisión Sináptica/fisiología , Técnicas de Cultivo de TejidosRESUMEN
Platycodin D is one of the most important monomers of the Qinbaiqingfei pellet (Qinbai), which has already been approved as the first effective new Traditional Chinese Medicine used to fight against Mycoplasma pneumoniae (M. pneumoniae) in clinic in China. In previous studies, pharmacodynamics experiment has proved that Platycodin D has anti-M. pneumoniae effect and the minimum inhibitory concentration (MIC) is 16mµg/ml. This paper further clarified that the mechanism underlying the anti-M. pneumoniae effect of Platycodin D might be due to M. pneumoniae adhesion proteins P1 and P30. P1 and P30 expression levels in M. pneumoniae strain, M. pneumoniae-infected BALB/c mice, and M. pneumoniae-infected A549 cells were determined by reverse transcription PCR. Platycodin D strongly inhibited P1 and P30 expression in M. pneumonia and high dosage of Platycodin D exhibited a greater effect on reducing P1 and P30 expression than low dose Platycodin D. Platycodin D prevented M. pneumoniae infection through inhibiting the expression of adhesion proteins, which might be one of the mechanisms for the anti-M. pneumoniae properties of Qinbai. These results provide a foundation to further explore the mechanisms of action of Qinbai in future studies.