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Objective: To investigate whether grain-sized moxibustion at Xinshu (BL15) and Shenshu (BL23) can alleviate cognitive decline and other pathologic features in early-stage Alzheimer disease (AD) using transgenic mice with 5 familial AD mutations (5XFAD). Methods: The genotype of transgenic mice was detected by polymerase chain reaction. A total of 40 transgenic mice (1.5 months old) were randomly and equally allocated to an AD model group (5XFAD group) or a grain-sized moxibustion group (5XFAD + GM group), with 20 wild-type (WT) mice (C57BL/6J) serving as the normal control group (WT group). Mice in the 5XFAD + GM group were treated by grain-sized moxibustion at bilateral Xinshu (BL15) and Shenshu (BL23). Mice in the WT group and 5XFAD group received no treatment but were restrained to ensure exposure to a similar experimental condition. Cognitive function and memory were assessed with the Morris water maze and Y-maze tests. The amyloid β 40 (Aβ40) and amyloid β 42 (Aβ42) levels in the brain were evaluated by enzyme-linked immunosorbent assay; amyloid plaque deposition in brain tissue sections was detected by thioflavin-S staining; the expression of glial fibrillary acidic protein (GFAP), cluster of differentiation 11b (CD11b), brain-derived neurotrophic factor (BDNF), and choline acetyltransferase (ChAT) in the hippocampus and prefrontal cortex was analyzed by immunohistochemistry. Results: In the Morris water maze test, compared with the 5XFAD group, mice in the 5XFAD + GM group had a shorter escape latency and more target area crossings and spent more time in the target quadrant (P<0.05). In the Y-maze test, compared with the 5XFAD group, the number of training times of the 5XFAD + GM group was significantly decreased (P<0.05), together with more correct responses (P<0.05). Compared with the 5XFAD group, the levels of Aβ40 and Aβ42 in the brain tissue of the 5XFAD + GM group were significantly lower (P<0.05); in the hippocampus and prefrontal cortex, the total number of amyloid β plaque deposition were significantly lower (P<0.05); the expression levels of GFAP and CD11b were significantly reduced (P<0.05); and the expression levels of ChAT and BDNF were significantly increased (P<0.05).Conclusion: Grain-sized moxibustion at Xinshu (BL15) and Shenshu (BL23) greatly improves learning and memory functions, decreases the levels of Aβ40 and Aβ42, inhibits amyloid β plaque deposition, decreases the expression of GFAP and CD11b, and increases the expression of ChAT and BDNF in AD mice to inhibit the progression of AD.
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AIM:To investigate the effects of peroxiredoxin 4 ( Prdx4) protein expression levels on the migra-tion and invasion of human cervical cancer HeLa cells.METHODS:The plasmid pcDNA3.0-HA-Prdx4 was transfected into HeLa cells.The HeLa cells were infected with LV-Prdx4 RNAi vector to establish stable Prdx4 shRNA HeLa cells. The change in the expression of Prdx4 protein was validated by Western blotting.The wound-healing assay, and Transwell migration and invasion assays were performed to detect the migration and invasion of HeLa cells, respectively.RESULTS:The expression of Prdx4 protein was up-regulated in the HeLa cells after transfection with pcDNA3.0-HA-Prdx4 plasmid ( P<0.05), whereas it was down-regulated in the Prdx4 shRNA HeLa cells (P<0.05).The abilities of migration and inva-sion were significantly increased in Prdx4-overexpressing HeLa cells compared with non-transfected and mock plasmid trans-fected control groups ( P<0.01) .When Prdx4 was knocked down by shRNA, the migration and invasion of the HeLa cells were remarkably repressed compared with blank control group and negative control group ( P<0.01 ) .CONCLUSION:The up-regulation of Prdx4 expression facilitates the migration and invasion of HeLa cells, and the down-regulation of Prdx4 expression inhibits the migration and invasion of HeLa cells, indicating that Prdx4 may be a potential molecular target for cervical cancer therapy.
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<p><b>OBJECTIVE</b>To establish a new method for studying the mechanism of nuclear localization signal (NLS)-mediated nuclear translocation in living cells.</p><p><b>METHODS</b>The cells were treated with 67 mg/L 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), followed by incubation with 1 g/L wheat germ agglutinin (WGA), and their effects on interferon- γ (IFN-γ)-induced nuclear translocation of signal transducer and activator of transcription 1 (STAT1) were observed.</p><p><b>RESULTS</b>Treatment with CHAPS alone had no effect on IFN-γ-induced nuclear translocation of STAT1, while this process was blocked by further WGA incubation.</p><p><b>CONCLUSION</b>We established a new, simple but effective method for studying the mechanism of NLS-mediated nuclear translocation in living cells by perforating the cell membrane with CHAPS treatment.</p>
Subject(s)
Humans , Active Transport, Cell Nucleus , Cell Nucleus , Metabolism , Cholic Acids , Cytological Techniques , HeLa Cells , Interferon-gamma , Metabolism , Nuclear Localization Signals , Metabolism , STAT1 Transcription Factor , Metabolism , Signal TransductionABSTRACT
In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both ≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.
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The ompA gene of Chlamyia psittaci in cows was amplified by PCR with primers designed based on those reported in GenBank.The amplified ompA gene was inserted into the bacterial plasmid vector pGEX-4T-1 and then transformed into E.coli BL21(DE3) with IPTG induction. The gene was derived from plasmid pMD18-T vector and then sequenced.It was demonstrated that this recombinant fusion protein of approximately 68kD in molecular mass was highly expressed in inclusion body and more pure proteins would be produced after purification.The fusion protein specifically reacted with positive sera of bovine Chlamydia as demonstrated by Western blotting. These results indicate that this recombinant fusion protein shows good reactivity and could be used to develop the diagnostic kit for bovine Chlamydia and genetic engineering vaccine.
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Mitogen-activated protein kinase (MAPK) signaling pathways are involved in multiple important cellular responses. To activate their downstream protein kinases by phosphorylation is a crucial manner for MAPK family members to fulfill their physiological functions. Downstream of MAPKs, there exist three structurally related MAPK-activated protein kinases (MAPKAPKs or MKs), i.e., MK2, MK3 and MK5. Once upon activated by MAPKs, MKs signal to different cellular targets, to regulate gene expression at the levels of transcription and translation, control cytoskeleton remodelling and cell cycle, and mediate cell migration and embryonic development. Recently, based on the gene knockout studies, the function divisions among different MK subfamily members are gradually clear, leading to tremendous advancements of our knowledge on MKs.
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There are some characteristics in the teaching of amateurish adult medical students.As a bridge connecting basic medicine and clinical medicine,pathophysiology is very important in medical education.Aiming at the specialties of amateurish adult medical students,it has very realistic significance for us to explore and reform the teaching methods of pathophysiology,in order to improve the teaching quality and effect.
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Objective To construct pSUPER-ARPC2 vector and to obtain its expression in eukaryotic cell, in order to down-regulate the expression of ARPC2. Methods Oligonucleotide sequences specific for ARPC2 were designed for RNAi, and cloned into pSUPER.basic vector after annealing by restriction enzyme digestion and ligation. After identification by restriction enzyme digestion and sequencing, the plasmid of positive clones was transfected into HEK 293 cells for three times. The expression of ARPC2 was detected by RT-PCR and Western blot. Results It was found that the expression of ARPC2 was definitely knocked down by transfection with pSUPER-ARPC2. Conclusions The RNAi expression vector of ARPC2 was constructed successfully, which was showed to possess the ability to knockdown the expression of ARPC2 by different methods. So, pSUPER-ARPC2 should be a powerful tool in the study of function of ARPC2.
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AIM:To investigate the role of p38 MAPK signaling pathway in the receptor-mediated endocytosis.METHODS:The effects of p38 MAPK on the receptor-mediated endocytosis were observed by using Alexa 594-conjugated Transferrin,in the presence of p38 specific inhibitor SB203580 or ERK pathway specific inhibitor PD98059,or using p38 knockout techniques.RESULTS:In the process of receptor-mediated endocytosis,p38 was activated by phosphorylation.Furthermore,the receptor-mediated endocytosis was inhibited by pretreatment with SB203580 or p38 knockout,while pretreatment with PD98059 had no effect on this process.CONCLUSION:p38 MAPK signaling pathway plays a role in the regulation of receptor-mediated endocytosis.
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AIM: To construct the plasmid vectors containing different regions of human eNOS promoter coupled to a red fluorescent protein reporter gene, which may express in mammalian cells. METHODS: Different regions of human eNOS promoter were subcloned respectively into a red fluorescent protein vector, pDsRed1-1. These recombinant vectors, pDsF1033Red, pDsF494Red and pDsF166Red, were then transfected into NIH3T3 cell lines, followed by the observation under a fluorescent microscope. RESULTS: After identified to be right by double restriction enzyme digestion, PCR and sequencing, the vectors might be effectively expressed in NIH3T3 cells. 95 % of the red fluorescent emitted by a red fluorescent protein dispersed all over the cells, appearing at 48-60 h after transfection, reaching peak at 96-144 h, becoming the strongest in light at 144 h, gradually disappearing after 168 h and remaining little red fluorescent in 21 days. The quantity and intensity in expressions of red fluorescent protein drived by different regions of human eNOS promoter were clearly lower than by a strong promoter, p CMVIE . CONCLUSION: The red fluorescent protein reporter gene vectors containing different regions of human eNOS promoter are successfully constructed and may efficaciously express in mammalian cells, appearing not strong transcriptional activities, which provide practical and feasible tools to study functions of different regions of human eNOS promoter and roles of cis-elements in it. [