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OBJECTIVE: To investigate the efficacy of novel object recognition (NOR) test in assessment of learning and memory ability in ICR mice in different experimental conditions. METHODS: One hundred and thirty male ICR mice were randomly divided into 10 groups: 4 groups for different inter-trial intervals (ITI: 10 min, 90 min, 4 h, 24 h), 4 groups for different object materials (wood-wood, plastic-plastic, plastic-wood, wood-plastic) and 2 groups for repeated test (measured once a day or every 3 days, totally three times in each group). The locomotor tracks in the open field were recorded. The amount of time spent exploring the novel and familiar objects, the discrimination ratio (DR) and the discrimination index (DI) were analyzed. RESULTS: Compared with familiar object, DR and DI of novel object were both increased at ITI of 10 min and 90 min (P<0.01). Exploring time, DR and DI were greatly influenced by different object materials. DR and DI remained stable by using identical object material. NOR test could be done repeatedly in the same batch of mice. CONCLUSION: NOR test can be used to assess the learning and memory ability in mice at shorter ITI and with identical material. It can be done repeatedly.
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Aprendizaje , Memoria , Animales , Masculino , Ratones , Ratones Endogámicos ICR , Factores de TiempoRESUMEN
Magnetic nanomaterials were functionalized with dopamine hydrochloride as the functional reagent to afford a core-shell-type Fe3O4 modified with polydopamine (Fe3O4@PDA) composite, which was used for the adsorption of cadmium ions from an aqueous solution. In addition, the effects of environmental factors on the adsorption capacity were investigated. Furthermore, the adsorption kinetics, isotherm, and thermodynamics of the adsorbents were discussed. Results revealed that the adsorption of cadmium by Fe3O4@PDA reaches equilibrium within 120 min, and kinetic fitting data are consistent with the pseudo-second-order kinetics (R2 > 0.999). The adsorption isotherm of Cd2+ on Fe3O4@PDA was in agreement with the Freundlich model, with the maximum adsorption capacity of 21.58 mg/g. The thermodynamic parameters revealed that adsorption is inherently endothermic and spontaneous. Results obtained from the adsorption-desorption cycles revealed that Fe3O4@PDA exhibits ultra-high adsorption stability and reusability. Furthermore, the adsorbents were easily separated from water under an enhanced external magnetic field after adsorption due to the introduction of an iron-based core. Hence, this study demonstrates a promising magnetic nano-adsorbent for the effective removal of cadmium from cadmium-containing wastewater.
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OBJECTIVE: To observe the effect of glucagon-like peptide-1 (GLP-1) on interleukin-1ß (IL-1ß)-induced damage in INS-1 cells and explore its possible mechanisms. METHODS: INS-1 cells were divided into normal control group, IL-1ß group, and GLP-1+IL-1ß group with corresponding treatments. The cell viability was determined by MTT assay, the expression of IKKß mRNA was detected by real-time PCR, and that of the protein p65 was detected by Western blotting. RESULTS: In comparison with the normal control group, the cells in the IL-1ß group showed a significantly decreased viability by 29% (P < 0.01); compared with those in IL-1ß group, the cells in GLP-1+IL-1ß group exhibited an significant increase in the cell viability by 30% (P < 0.01). In comparison with the normal control group, the cells in IL-1ß group showed an significantly increased expression of IKKß mRNA (1.967 ± 0.091 vs 1 ± 0, P < 0.05); GLP-1 significantly reduced IL-1ß-induced increment of IKKß mRNA expression to 1.287 ± 0.084 (P < 0.05). IL-1ß treatment significantly increased NF-κB protein expression as compared to the control level (0.814 ± 0.111 vs 0.396 ± 0.026, P < 0.01), and GLP-1 significantly inhibited such effect (0.622 ± 0.059, P < 0.05). CONCLUSIONS: GLP-1 inhibits IL-1ß-induced ß-cell damage probably by inhibiting the NF-κB pathway.
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Péptido 1 Similar al Glucagón/farmacología , Células Secretoras de Insulina/patología , Interleucina-1beta/farmacología , FN-kappa B/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Línea Celular , Supervivencia Celular , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Células Secretoras de Insulina/citología , Interleucina-1beta/antagonistas & inhibidores , Sustancias Protectoras/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
OBJECTIVE: To study of the role of nuclear transcription factor-κB (NF-κB) in high glucose-induced apoptosis in INS-1 cells. METHODS: Rat insulinoma (INS-1) cells cultured in RPMI 1640 medium were treated with 11.1 mmol/L glucose, 33.3 mmol/L glucose, or 33.3 mmol/L glucose plus 5 µmol/L NF-κB inhibitors for 48 h. The expression of NF-κB subunit P65 protein in the cell nuclei was detected by Western blotting, IKK belta mRNA level by quantitative RT-PCR, and cell apoptosis by Annexin V-PI double staining. RESULTS: Compared with the control levels, IKK belta mRNA levels of the cells significantly increased in response to 33.3 mmol/L glucose exposure (P<0.01), which also resulted in significantly increased P65 protein expression in the cell nuclei (P<0.01) and cell apoptosis rate (P<0.05). Compared with those in the high glucose group, the expression of IKK belta mRNA and P65 protein and cell apoptosis rate decreased significantly after treatment with 33.3 mmol/L glucose plus 5 µmol/L NF-κB inhibitors (P<0.05). CONCLUSION: High glucose induces NF-κB activation in INS-1 cells, and inhibition of NF-κB activation may protect INS-1 cells from high glucose-induced cell apoptosis.
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Glucosa/efectos adversos , Insulinoma/patología , Neoplasias Pancreáticas/patología , Factor de Transcripción ReIA/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Regulación de la Expresión Génica , Glucosa/metabolismo , RatasRESUMEN
Protein-disulfide isomerase (PDI), a critical enzyme responsible for oxidative protein folding in the eukaryotic endoplasmic reticulum, is composed of four thioredoxin domains a, b, b', a', and a linker x between b' and a'. Ero1-Lalpha, an oxidase for human PDI (hPDI), has been determined to have one molecular flavin adenine dinucleotide (FAD) as its prosthetic group. Oxygen consumption assays with purified recombinant Ero1-Lalpha revealed that it utilizes oxygen as a terminal electron acceptor producing one disulfide bond and one molecule of hydrogen peroxide per dioxygen molecule consumed. Exogenous FAD is not required for recombinant Ero1-Lalpha activity. By monitoring the reactivation of denatured and reduced RNase A, we reconstituted the Ero1-Lalpha/hPDI oxidative folding system in vitro and determined the enzymatic activities of hPDI in this system. Mutagenesis studies suggested that the a' domain of hPDI is much more active than the a domain in Ero1-Lalpha-mediated oxidative folding. A domain swapping study revealed that one catalytic thioredoxin domain to the C-terminal of bb'x, whether a or a', is essential in Ero1-Lalpha-mediated oxidative folding. These data, combined with a pull-down assay and isothermal titration calorimetry measurements, enabled the minimal element for binding with Ero1-Lalpha to be mapped to the b'xa' fragment of hPDI.
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Flavina-Adenina Dinucleótido/química , Peróxido de Hidrógeno/química , Glicoproteínas de Membrana/química , Oxidorreductasas/química , Oxígeno/química , Proteína Disulfuro Isomerasas/química , Pliegue de Proteína , Flavina-Adenina Dinucleótido/genética , Humanos , Glicoproteínas de Membrana/genética , Oxidación-Reducción , Oxidorreductasas/genética , Proteína Disulfuro Isomerasas/genética , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/genéticaRESUMEN
R-spondins (Rspos) potentiate Wnt/beta-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein,demonstrating for the first time specific amino acid residues in Rspos that are critical for beta-catenin signaling. The diminished activity of Rspo2/C78Y and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a beta-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor, low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important.
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Furina/química , Mutación Puntual/genética , Trombospondinas/química , Trombospondinas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Cisteína/metabolismo , Disulfuros , Genes Reporteros , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Péptidos/química , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Receptores de LDL/metabolismo , Relación Estructura-Actividad , Trombospondinas/aislamiento & purificación , beta Catenina/metabolismoRESUMEN
We presented for the first time a small angle x-ray scattering study of intact protein-disulfide isomerase (PDI) in solution. The restored model revealed that PDI is a short and roughly elliptical cylinder with a molecular mass of 69 kDa and dimensions of 105 x 65 x 40 A, and the four thioredoxin-fold domains in the order a-b-b'-a' are arranged in an annular fashion. Atomic force microscope imaging also supported the finding that PDI appears as an approximately flat elliptical cylinder. A PDI species with apparent molecular mass of 116 kDa measured by using size-exclusion chromatography, previously assumed to be a dimer, was determined to exist mainly as a monomer by using analytical ultracentrifugation. The C-terminal fragment 441-491 contributed to the anomalous molecular mass determination of PDI by size-exclusion chromatography. The annular model of PDI accounted for the cooperative properties of the four domains in both the isomerase and chaperone functions of PDI.
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Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , Cristalografía por Rayos X , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Microscopía de Fuerza Atómica , Modelos Moleculares , Estructura Terciaria de Proteína , Dispersión de Radiación , Soluciones , Rayos XRESUMEN
Protein disulfide isomerase (PDI, EC 5.3.4.1) is a chaperone and catalyzes the formation and rearrangement of disulfide bonds in proteins. Domain c-(463-491), containing 18 acidic residues, is an interesting and important C-terminal extension of PDI. In this study, the PDI mutant abb'a', in which domain c is truncated, was used to investigate the relationship between the C-terminal structure and chaperone function. Reactivation and light-scattering experiments show that both wild-type PDI and abb'a' interact with lactate dehydrogenase (LDH, EC 1.1.1.27), which tends to self-aggregate during reactivation. The interaction enhances reactivation of LDH and reduces aggregation. According to these results, it seems as if domain c might be dispensable to the chaperone function of PDI. However, abb'a' is prone to self-aggregation and causes increased aggregation of LDH during thermal denaturation. In contrast, wild-type PDI remains active as a chaperone under these conditions and prevents self-aggregation of LDH. Furthermore, measurements of intrinsic fluorescence and difference absorbance during denaturation show that abb'a' is much more labile to heat or guanidine hydrochloride denaturation than wild-type PDI. This suggests that domain c is required for the stabilization and maintenance of the chaperone function of PDI under extreme conditions.