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Efficient bifunctional catalysts for oxygen evolution and reduction reactions (OERs/ORRs) are of great importance for sustainable and renewable clean energy, especially for metal-air batteries. Herein, we investigated ß12-borophene with double-hole sites capped with 3d transition metal atoms to explore its catalyst performance for hydrogen evolution reactions (HERs), OERs and ORRs. It was found that the borophene is a good platform for diatomic site catalysts (DASCs) due to their advantage of stability over the corresponding single-atom catalysts (SACs) or clusters. The HER performance of DASCs on ß12-BM was further improved compared to the SAC case. Furthermore, the supported FeNi DASC exhibited good catalytic performance for both OERs and ORRs, the overpotentials for which were 0.43 and 0.55 V, respectively, better than those of the corresponding supported Ni or Fe SAC due to synergistic effects. We herein propose a novel descriptor involving the Bader charges of coordinated atoms explicitly, behaving much better than the d-band center and integrated crystal orbital Hamilton population (-ICOHP) for DASCs. The synergistic effect of Fe-Ni pairs balanced the too strong binding of OH and further activated OH to achieve better catalytic performance. The results of this study can provide theoretical guidance for the design of efficient bifunctional electrocatalysts.
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BACKGROUND: Cisplatin is effective against various types of cancers. However, its clinical application is limited owing to its adverse effects, especially acute kidney injury (AKI). Dihydromyricetin (DHM), a flavonoid derived from Ampelopsis grossedentata, has varied pharmacological activities. This research aimed to determine the molecular mechanism for cisplatin-induced AKI. METHODS: A murine model of cisplatin-induced AKI (22 mg/kg, I.P.) and a HK-2 cell model of cisplatin-induced damage (30 µM) were established to evaluate the protective function of DHM. Renal dysfunction markers, renal morphology and potential signaling pathways were investigated. RESULTS: DHM decreased the levels of renal function biomarkers (blood urea nitrogen and serum creatinine), mitigated renal morphological damage, and downregulated the protein levels of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. It upregulated the expression levels of antioxidant enzymes (superoxide dismutase and catalase expression), nuclear factor-erythroid-2-related factor 2 (Nrf2) and its downstream proteins, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic (GCLC) and modulatory (GCLM) subunits, thus eventually reducing cisplatin-induced reactive oxygen species (ROS) production. Moreover, DHM partially inhibited the phosphorylation of the active fragments of caspase-8 and -3 and mitogen-activated protein kinase and restored glutathione peroxidase 4 expression, which attenuated renal apoptosis and ferroptosis in cisplatin-treated animals. DHM also mitigated the activation of NLRP3 inflammasome and nuclear factor (NF)-κB, attenuating the inflammatory response. In addition, it reduced cisplatin-induced HK-2 cell apoptosis and ROS production, both of which were blocked by the Nrf2 inhibitor ML385. CONCLUSIONS: DHM suppressed cisplatin-induced oxidative stress, inflammation and ferroptosis probably through regulating of Nrf2/HO-1, MAPK and NF-κB signaling pathways.
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Lesión Renal Aguda , Ferroptosis , Animales , Ratones , Cisplatino/farmacología , Especies Reactivas de Oxígeno/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/prevención & control , Riñón , FN-kappa B/metabolismo , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/prevención & controlRESUMEN
People can infer the weather from clouds. Various weather phenomena are linked inextricably to clouds, which can be observed by meteorological satellites. Thus, cloud images obtained by meteorological satellites can be used to identify different weather phenomena to provide meteorological status and future projections. How to classify and recognize cloud images automatically, especially with deep learning, is an interesting topic. Generally speaking, large-scale training data are essential for deep learning. However, there is no such cloud images database to date. Thus, we propose a large-scale cloud image database for meteorological research (LSCIDMR). To the best of our knowledge, it is the first publicly available satellite cloud image benchmark database for meteorological research, in which weather systems are linked directly with the cloud images. LSCIDMR contains 104 390 high-resolution images, covering 11 classes with two different annotation methods: 1) single-label annotation and 2) multiple-label annotation, called LSCIDMR-S and LSCIDMR-M, respectively. The labels are annotated manually, and we obtain a total of 414 221 multiple labels and 40 625 single labels. Several representative deep learning methods are evaluated on the proposed LSCIDMR, and the results can serve as useful baselines for future research. Furthermore, experimental results demonstrate that it is possible to learn effective deep learning models from a sufficiently large image database for the cloud image classification.
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Bases de Datos Factuales , HumanosRESUMEN
Ceramides are bioactive sphingolipids that have been implicated in insect development; however, their role in insect reproduction remains poorly understood. Here, we report the pivotal role of neutral ceramidase (NCER) in the female reproduction of the brown planthopper (BPH), Nilaparvata lugens (Stål), a significant pest in rice cultivation in Asia. LC-MS/MS demonstrated that, among different developmental stages of BPH, the levels of ceramides were highest in 1st instar nymphs and lowest in adults. The transcription of NCER was negatively correlated with the levels of ceramides at different developmental stages of BPH, in that the transcript levels of NCER were the highest, whereas ceramides levels were the lowest in BPH adults. Knocking down NCER through RNA interference (RNAi) increased the levels of ceramides in BPH females and ovaries, which resulted in a delay in oocyte maturation, a reduction in oviposition and egg hatching rate, as well as the production of vulnerable offspring. Transmission electron microscopy (TEM) analysis and TdT-mediated dUTP Nick-End Labeling (TUNEL) assays showed mitochondrial deficiency and apoptosis in NCER-deficient oocytes. Taken together, these results suggest that NCER plays a crucial role in female reproduction in BPH, likely by regulating the levels of ceramides.
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Sphingolipids are ubiquitous structural components of eukaryotic cell membranes which are vital for maintaining the integrity of cells. Alkaline ceramidase is a key enzyme in sphingolipid biosynthesis pathway; however, little is known about the role of the enzyme in the male reproductive system of Drosophila melanogaster. To investigate the impact of alkaline ceramidase (Dacer) on male Drosophila, we got Dacer deficiency mutants (MUs) and found they displayed apparent defects in the testis's phenotype. To profile the molecular changes associated with this abnormal phenotype, we performed de novo transcriptome analyses of the MU and wildtype (WT) testes; and revealed 1239 upregulated genes and 1102 downregulated genes. Then, six upregulated DEGs (papilin [Ppn], croquemort [Crq], terribly reduced optic lobes [Trol], Laminin, Wunen-2, collagen type IV alpha 1 [Cg25C]) and three downregulated DEGs (mucin related 18B [Mur18B], rhomboid-7 [Rho-7], CG3168) were confirmed through quantitative real-time polymerase chain reaction in WT and MU samples. The differentially expressed genes were mainly associated with catalytic activity, oxidoreductase activity and transmembrane transporter activity, which significantly contributed to extracellular matrix-receptor interaction, fatty acids biosynthesis as well as glycine, serine, and threonine metabolism. The results highlight the importance of Dacer in the reproductive system of D. melanogaster and provide valuable resources to dig out the specific biological functions of Dacer in insect reproduction.
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Ceramidasa Alcalina/genética , Drosophila melanogaster/genética , Testículo/metabolismo , Ceramidasa Alcalina/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Genes de Insecto , Masculino , Mutación , Receptores de Superficie Celular/metabolismo , Reproducción , Esfingolípidos/metabolismo , Testículo/patologíaRESUMEN
Alkaline ceramidase (Dacer) in Drosophila melanogaster was demonstrated to be resistant to paraquat-induced oxidative stress. However, the underlying mechanism for this resistance remained unclear. Here, we showed that sphingosine feeding triggered the accumulation of hydrogen peroxide (H2O2). Dacer-deficient D. melanogaster (Dacer mutant) has higher catalase (CAT) activity and CAT transcription level, leading to higher resistance to oxidative stress induced by paraquat. By performing a quantitative proteomic analysis, we identified 79 differentially expressed proteins in comparing Dacer mutant to wild type. Three oxidoreductases, including two cytochrome P450 (CG3050, CG9438) and an oxoglutarate/iron-dependent dioxygenase (CG17807), were most significantly upregulated in Dacer mutant. We presumed that altered antioxidative activity in Dacer mutant might be responsible for increased oxidative stress resistance. Our work provides a novel insight into the oxidative antistress response in D. melanogaster.
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Ceramidasa Alcalina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Estrés Oxidativo , Esfingosina/administración & dosificación , Ceramidasa Alcalina/efectos de los fármacos , Ceramidasa Alcalina/genética , Animales , Catalasa/metabolismo , Proteínas de Drosophila/efectos de los fármacos , Proteínas de Drosophila/genética , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Peróxido de Hidrógeno/metabolismo , Paraquat , ProteomaRESUMEN
Ceramidases (CDases) are vital enzymes involved in the biosynthesis of sphingolipids, which are essential components of eukaryotic membranes. The function of these enzymes in insects, however, is poorly understood. We identified a neutral ceramidase (NlnCDase) from the brown planthopper, Nilaparvata lugens, one of the most destructive hemipteran pests of rice. The C12-ceramide was the most preferred substrate for the NlnCDase enzyme. The activity of the NlnCDase enzyme was highest in the neutral-pH range (pH 6.0). It was inhibited by EGTA, Cs+ and Fe2+, while stimulated by EDTA and Ca2+. Moreover, the NlnCDase has higher transcript level and activity in adults than in eggs and nymphs, and in the reproductive organs (ovaries and spermaries) than in other tissues (i.e. heads, thorax, legs, midguts), which suggested that the NlnCDase might be elevated to mediate developmental process. In addition, transcripts and activity of the NlnCDase were up-regulated under abiotic stresses including starvation, abnormal temperature, and insecticides, and biotic stress of resistant rice varieties. Knocking down NlnCDase by RNA interference increased female survival under starvation and temperature stresses, suggesting that NlnCDase might be involved in the stress response in N. lugens.
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Hemípteros/fisiología , Ceramidasa Neutra/genética , Estrés Fisiológico , Animales , Activación Enzimática , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hemípteros/clasificación , Informática/métodos , Ceramidasa Neutra/metabolismo , Filogenia , Transporte de Proteínas , Análisis de Secuencia de ADN , Estrés Fisiológico/genéticaRESUMEN
Temperature affects the persistence of diverse symbionts of insects. Our previous study indicates that the whitefly symbionts confined within bacteriocytes or scattered throughout the body cavity outside bacteriocytes may have differential thermal sensitivity. However, the underlying mechanisms remain largely unknown. Here, we report that following continuous heat stress, Portiera and Hamiltonella were almost completely depleted in two species of Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) of the Bemisia tabaci whitefly cryptic species complex. Meanwhile, proliferation of bacteriocytes was severely inhibited and approximately 50% of the nymphs had lost one of the two bacteriomes. While cell size of bacteriocytes was increased, cell number was severely decreased leading to reduction of total volume of bacteriocytes. Moreover, bacteriocyte organelles and associated symbionts were lysed, and huge amount of electron-dense inclusions accumulated. Eventually, Portiera and Hamiltonella failed to be transmitted to the next generation. In contrast, Rickettsia could be detected although at a reduced level, and successfully transmitted to eggs. The results suggest that the thermal sensitivity of bacteriocytes may limit thermal tolerance and vertical transmission of the associated symbionts, and consequently different patterns of distribution of symbionts may affect their capacity to tolerate unfavourable temperatures and persistence in the host.
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Fenómenos Fisiológicos Bacterianos , Gammaproteobacteria/fisiología , Hemípteros/microbiología , Calor , Espacio Intracelular/microbiología , Estrés Fisiológico , Animales , Femenino , Hemípteros/citología , Hemípteros/fisiología , Hibridación Fluorescente in Situ , Masculino , Microscopía Electrónica de Transmisión , Ninfa/microbiología , Óvulo/microbiología , SimbiosisRESUMEN
Sphingolipids and their metabolites have been implicated in viral infection and replication in mammal cells but how their metabolizing enzymes in the host are regulated by viruses remains largely unknown. Here we report the identification of 12 sphingolipid genes and their regulation by Rice stripe virus in the small brown planthopper (Laodelphax striatellus Fallén), a serious pest of rice throughout eastern Asia. According to protein sequence similarity, we identified 12 sphingolipid enzyme genes in L. striatellus. By comparing their mRNA levels in viruliferous versus nonviruliferous L. striatellus at different life stages by qPCR, we found that RSV infection upregulated six genes (LsCGT1, LsNAGA1, LsSGPP, LsSMPD4, LsSMS, and LsSPT) in most stages of L. striatellus Especially, four genes (LsCGT1, LsSMPD2, LsNAGA1, and LsSMS) and another three genes (LsNAGA1, LsSGPP, and LsSMS) were significantly upregulated in viruliferous third-instar and fourth-instar nymphs, respectively. HPLC-MS/MS results showed that RSV infection increased the levels of various ceramides, such as Cer18:0, Cer20:0, and Cer22:0 species, in third and fourth instar L. striatellus nymphs. Together, these results demonstrate that RSV infection alters the transcript levels of various sphingolipid enzymes and the contents of sphingolipids in L. striatellus, indicating that sphingolipids may be important for RSV infection or replication in L. striatellus.
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Regulación de la Expresión Génica , Hemípteros/genética , Hemípteros/virología , Proteínas de Insectos/genética , Esfingolípidos/genética , Tenuivirus/fisiología , Animales , Cromatografía Líquida de Alta Presión , Femenino , Hemípteros/enzimología , Hemípteros/metabolismo , Proteínas de Insectos/metabolismo , Masculino , Ninfa/enzimología , Ninfa/genética , Ninfa/metabolismo , Ninfa/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Esfingolípidos/metabolismo , Espectrometría de Masas en TándemRESUMEN
Natural compounds are becoming popular for the treatment of illnesses and health promotion, but the mechanisms of action and safety profiles are often unknown. Xyloketal B (XKB) is a novel marine compound isolated from the mangrove fungus Xylaria sp., with potent antioxidative, neuroprotective, and cardioprotective effects. However, its molecular targets and effects on drug-metabolizing enzymes are unknown. This study aimed to investigate the potential molecular targets of XKB using bioinformatic approaches and to examine the effect of XKB on the expression and activity of rat cytochrome P450 3a (Cyp3a) subfamily members using midazolam as a model probe. DDI-CPI, a server that can predict drug-drug interactions via the chemical-protein interactome, was employed to predict the targets of XKB, and the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to analyze the pathways of the predicted targets of XKB. Homology modeling was performed using the Discovery Studio program 3.1. The activity and expression of rat hepatic Cyp3a were examined after the rats were treated with XKB at 7 and 14 mg/kg for 8 consecutive days. Rat plasma concentrations of midazolam and its metabolite 1'-OH-midazolam were determined using a validated high-performance liquid chromatographic method. Bioinformatic analysis showed that there were over 324 functional proteins and 61 related signaling pathways that were potentially regulated by XKB. A molecular docking study showed that XKB bound to the active site of human cytochrome P450 3A4 and rat Cyp3a2 homology model via the formation of hydrogen bonds. The in vivo study showed that oral administration of XKB at 14 mg/kg to rats for 8 days significantly increased the area under the plasma concentration-time curve (AUC) of midazolam, with a concomitant decrease in the plasma clearance and AUC ratio of 1'-OH-midazolam over midazolam. Further, oral administration of 14 mg/kg XKB for 8 days markedly reduced the activity and expression of hepatic Cyp3a in rats. Taken together, the results show that XKB could regulate networks of molecular proteins and related signaling pathways and that XKB downregulated hepatic Cyp3a in rats. XKB might cause drug interactions through modulation of the activity and expression of Cyp3a members. More studies are warranted to confirm the mechanisms of action of XKB and to investigate the underlying mechanism for the regulating effect of XKB on Cyp3a subfamily members.
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Biología Computacional , Citocromo P-450 CYP3A/biosíntesis , Citocromo P-450 CYP3A/metabolismo , Piranos/farmacología , Administración Oral , Animales , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Humanos , Masculino , Midazolam/análogos & derivados , Midazolam/sangre , Simulación del Acoplamiento Molecular , Estructura Molecular , Piranos/administración & dosificación , Piranos/química , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: To study zedoary turmeric oil (ZTO) and the pharmacokinetics of its homemade compound antiviral preparation in New Zealand rabbits. METHOD: RP-HPLC was used to determinate the content of germacrone in rabbit plasma after oral administration. RESULT: After oral administration of ZTO and its homemade compound antiviral preparation, the plasma concentration-time curve of germacrone is in conformity to two-compartment open model. The pharmacokinetic parameters of ZTO: t1/2alpha, t1/2beta, Vd, CL, AUC and Ka were (1.52 +/- 0.59), (1.97 +/- 0.27) h, (47.59 +/- 2.29) L x kg(-1), (176.77 +/- 7.65) L x h(-1) x kg(-1), (5.70 +/- 0.70) mg x h x L(-1) and (0.97 +/- 0.11) h(-1), respectively, while those of compound preparation were (0.41 +/- 0.03), (1.47 +/- 0.35) h, (75.21 +/- 5.21) L x kg(-1), (287.79 +/- 6.39) L x h(-1) x kg(-1), (3.91 +/- 0.53) mg x h x L(-1) and (5.14 +/- 1.26) h(-1), respectively. There was no significant difference between the above two groups of pharmacokinetic parameters, expect that Ka of compound preparation was significantly higher than that of ZTO (P < 0.05). CONCLUSION: Hypericum perforatum in compound preparation doesn't impact the distribution and elimination of active ingredients of ZTO in New Zealand rabbits, but it improves the absorption speed, and shortens the time of drug absorption, which contributes to rapid efficacy of ZTO in rabbits.
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Antivirales/farmacocinética , Curcuma/química , Medicamentos Herbarios Chinos/farmacología , Hypericum/química , Aceites de Plantas/farmacocinética , Animales , Composición de Medicamentos , Interacciones Farmacológicas , Masculino , Conejos , Sesquiterpenos de Germacrano/farmacocinéticaRESUMEN
The HER2/neu proto-oncogene encodes a 185-kDa trans-membrane glycoprotein kinase with extensive homology to the epidermal growth factor receptor and plays a key role in the transformation and growth of malignant tumors. To date, two antibody drugs targeting HER2/neu have been developed successfully. In order to reduce the cost and the time of clinical treatment, we produced a fusion protein composed of human beta defensin 2 (hBD2) and anti-HER2/neu single-chain variable fragment (scFv 4D5), which is capable of specifically targeting, significantly inhibiting, and promptly killing HER2/neu-positive cancer cells. The recombinant protein was expressed in Escherichia coli using the small ubiquitin-related modifier (SUMO) as the molecular chaperone, and the optimal expression level reached to 40.2 % of the total supernatant protein. After purifying by Ni-NTA affinity chromatography, the fusion protein was cleaved with a SUMO-specific protease to obtain hBD2-4D5, which was further purified by Ni-NTA affinity chromatography. The purity of hBD2-4D5 was higher than 95 %, and the yield was 19 ± 2 mg/L in flask fermentation. The cell number count and flow cytometry results showed that hBD2-4D5 exerted cytotoxic and anti-proliferative effects on HER2/neu-positive breast cancer cell line, SKBR-3. The results of scanning electron microscope and transmission electron microscope observation indicated that hBD2-4D5 could induce intracellular ultrastructure changes and cell necrosis by disrupting the cell membrane. Immunofluorescence analysis showed that hBD2-4D5 could bind to SKBR-3 cells and further be internalized into the cytoplasm. Moreover, hBD2-4D5 could also mediate apoptosis of SKBR-3 cells by up-regulating the ratio of Bax to Bcl-2.
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Receptores ErbB/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , beta-Defensinas/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Cromatografía de Afinidad , Receptores ErbB/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proto-Oncogenes Mas , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , beta-Defensinas/genéticaAsunto(s)
Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Quinolonas/farmacología , Factores R/genética , beta-Lactamasas/genética , Animales , China/epidemiología , Enfermedades de los Perros/epidemiología , Enfermedades de los Perros/microbiología , Perros , Regulación Bacteriana de la Expresión Génica/fisiología , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/microbiología , beta-Lactamasas/metabolismoRESUMEN
As a novel important regulator of glucose and lipid metabolism homeostasis, human fibroblast growth factor 21 (hFGF21) has become a potential drug candidate for the treatment of metabolic diseases including obesity, and type 2 diabetes, as well as non-alcoholic fatty liver disease. To improve the production of recombinant hFGF21 to meet the increasing demand in clinical applications, an artificial gene encoding its mature peptide sequence was constructed, cloned into vector pET-3c and then expressed in Escherichia coli Origami B (DE3). Under optimal conditions in a 50-L fermentor, the average bacterial yield and the soluble expression level of recombinant hFGF21 of six batches attained 1750 ± 185 g and 32 ± 1.5%, respectively. The target protein was purified by the combination of nickel-nitrilotriacetic acid affinity chromatography and Sephadex S-100 resin. 5% (w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. The purity of rhFGF21 was higher than 97%, and the yield was 213 ± 17 mg/L. The preliminary biochemical characterization of rhFGF21 was confirmed using Western blot and peptide map finger analysis. Based on the glucose oxidase-peroxidase assay, the EC50 of glucose uptake activity of the purified rhFGF21 was 22.1 nM.
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Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Glucosa/metabolismo , Reactores Biológicos , Biotecnología/métodos , Cromatografía Liquida/métodos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Factores de Crecimiento de Fibroblastos/aislamiento & purificación , Expresión Génica , Genes Sintéticos , Vectores Genéticos , Humanos , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Cadmium (Cd) directly inhibits testosterone production in Leydig cells, but its mechanism is still unclear. To further explore the signaling pathway of Cd-mediated toxicity to Leydig cells, various concentrations of Cd were cultured with R2C cells for 24h, and two-dimensional gel electrophoresis (2DE)-based proteomics profiling was used to analyze the change of protein expressions. Cd caused a concentration-dependent inhibition of cell viability with IC(25), IC(50) and IC(75) of 2.42×10(-5)M, 4.83×10(-5)M and 7.39×10(-5)M, respectively. Cd significantly reduced progesterone production and mitochondrial membrane potential (ΔΨ(m)) in a concentration-dependent manner. 2DE-based proteomics showed 34 protein spots with altered expression by 2-folds or more, and dihydrolipoamide dehydrogenase (DLD) was the hub in the network of these altered proteins. Real-time polymerase chain reaction (PCR) and Western blotting showed that Cd downregulated the expression of DLD. Cd also decreased intracellular levels of cyclic adenosine monophosphate (cAMP). The results suggest that DLD and cAMP may be key elements related to Cd toxicity to Leydig cells.
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Cadmio/toxicidad , AMP Cíclico/metabolismo , Dihidrolipoamida Deshidrogenasa/metabolismo , Contaminantes Ambientales/toxicidad , Células Intersticiales del Testículo/efectos de los fármacos , Animales , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Progesterona/metabolismo , Mapeo de Interacción de Proteínas , Proteoma/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In vitro alternative tests aiming at replacing the traditional animal test for predicting the irritant potential of chemicals have been developed, but the assessment parameters or endpoints are still not sufficient for analysis. To discover novel endpoints for skin irritation responses, a proteomics approach was used to analyze the protein expression in human keratinocytes exposed to sodium lauryl sulfate in the present study. Among the 20 identified proteins with altered expression, small heat shock protein 27 (HSP27) and superoxide dismutase [Cu-Zn] were down-regulated while cofilin-1 was up-regulated significantly in response to the chemical challenge. Keratinocytes were exposed to acid and basic chemicals for further validation of the proteins. HSP27 displayed the most significant alteration both in mRNA and protein levels, accompanied by nuclear translocation. The irritation also induced an increased production of interleukin-1α in keratinocytes. These findings suggest that these proteins may be combinational biomarkers or additional endpoints for skin hazard assessment. Further investigation into the protein alterations would be helpful for the mechanistic understanding of skin irritation.
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Proteómica/métodos , Enfermedades de la Piel/inducido químicamente , Piel/efectos de los fármacos , Enfermedad Aguda , Biomarcadores/análisis , Células Cultivadas , Cofilina 1/genética , Cofilina 1/metabolismo , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Interleucina-1alfa/metabolismo , Irritantes/toxicidad , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Medición de Riesgo , Piel/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Regulación hacia ArribaRESUMEN
The objective of the current study is to investigate the bacterial colonization within the gut of the house fly, Musca domestica L. (Diptera: Muscidae), at the larval stage and the bacterial community of the gut of the house fly at the newly emerged adult stage. After using ethidium monoazide to inhibit recovery of nucleic acids from dead bacteria, three polymerase chain reaction (PCR)-amplified 16S rDNA libraries from wheat bran, larvae, and newly emerged adults was constructed, analyzed, and compared. In total, 24, 11, and four phylotypes in the 16S rDNA libraries of wheat bran and the gut of larvae and adults, respectively, were found and assigned to three phylogenetic phyla of the domain Bacteria: Firmicutes, Proteobacteria, and Bacteroidetes. In the wheat bran library, 76% of the total number of sequences were affiliated to the genera Pseudomonas, Halomonas, Providencia, and Ignatzschineria. The three genera Morganella (79.05%), Providencia (8.78%), and Ignatzschineria (9.46%) dominated the library of the larval gut. Compared with the wheat bran library, the relative abundance of Morganella morganii (Winslow) was significantly higher (79.05 versus 0.8%), whereas that of Ignatzschineria larvae and of Providencia spp. was similar. These results demonstrate that M. morganii, Providencia spp., and I. larvae colonized the gut of the house fly larvae. Live bacteria of M. morganii, Providencia spp., and Proteus spp. were found in the gut of newly emerged adults. Therefore, the bacteria M. morganii and Providencia spp. colonized the larval gut could survive in the gut from larval metamorphosis to adult eclosion of the house fly.
Asunto(s)
Azidas/farmacología , Moscas Domésticas/genética , Triticum/parasitología , Animales , Bacillus/efectos de los fármacos , Bacillus/genética , Bacillus/aislamiento & purificación , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/aislamiento & purificación , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Erysipelothrix/efectos de los fármacos , Erysipelothrix/genética , Erysipelothrix/aislamiento & purificación , Flavobacterium/efectos de los fármacos , Flavobacterium/genética , Flavobacterium/aislamiento & purificación , Variación Genética , Moscas Domésticas/efectos de los fármacos , Moscas Domésticas/microbiología , Filogenia , Proteobacteria/efectos de los fármacos , Proteobacteria/genética , Proteobacteria/aislamiento & purificación , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/aislamiento & purificaciónRESUMEN
The present study aimed to produce and pathophysiologically evaluate the metallothionein (MT) fusion protein. A recombinant plasmid containing DNA segment coding the pET-glutathione transferase (GST)-small ubiquitin-related modifier (SUMO)-MT fusion protein was inserted into Escherichia coli for expression. The expression level of the fusion protein was very high, reaching to 38.4% of the total supernatant proteins from the organism. Subsequent filtration through glutathione Sepharose 4B gel and Sephadex G-25 yielded an MT fusion protein with purity more than 95%. When exposed to metals, E. coli containing the GST-SUMO-MT fusion protein showed an increased accumulation of Cd(2+), Zn(2+), or Cu(2+) at approximately 4.2, 4.0, or 1.6 times higher, respectively, than those containing the control protein. Administration of GST-SUMO-MT to mice that were also treated with D-galactose to induce neuronal and hepatic damage showed a significant improvement of animal learning and memory capacity, which was depressed in mice treated by D-galactose alone. Administration of MT fusion protein also prevented D-galactose-increased malondialdehyde contents and histopathological changes in the brain and liver. Furthermore, supplement of the fusion protein significantly prevented D-galactose-increased nitric oxide contents and -decreased superoxide dismutase activity in the brain, liver, and serum. The fusion protein was also able to prevent ionizing radiation-induced DNA damage of the mouse thymus. The present study indicates that GST-SUMO-MT has a normal metal binding feature and also significantly protects the multiple tissues against oxidative damage in vivo caused by chronic exposure to D-galactose and by ionizing radiation. Therefore, GST-SUMO-MT may be a potential candidate to be developed for the clinical application.