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<p><b>OBJECTIVE</b>To explore the functional role of protein kinase D1 (PKD1) in the activation of nuclear factor-κB (NF-κB) signal pathway and NF-κB transcription mediated by Aspergillus fumigatus.</p><p><b>METHODS</b>A549 cells and HEK293 cells were transfected with green fluorescence protein (GFP) or GFP-PKD1 followed by treatment with 1×10(5) CFU/ml Aspergillus fumigatus conidia for different time lengths. The phosphorylation levels of PKD1, IκB and p65 (pS276) in the transfected cells were measured by Western blotting. A549 cells were transfected with GFP-PKD1 or siRNA-PKD1, and the phosphorylation of IκB and p65 (pS276) was examined. Finally, NF-κB-luc and renilla luciferase reporter pRL-SV40 were cotransfected into GFP- or GFP-PKD1-transfected A549 cells before exposure of the cells to Aspergillus fumigatus conidia for 24 h, and NF-κB transcriptional activity in the cells was determined using dual-luciferase reporter assay.</p><p><b>RESULTS</b>Overexpression of PKD1 significantly increased Aspergillus fumigatus conidia-stimulated phosphorylation of PKD1, IκB and p65 (pS276), whereas PKD1 knockdown by siRNA-PKD1 suppressed IκB and p65 (pS276) phosphorylation. Dual luciferase assay demonstrated that PKD1 overexpression markedly enhanced Aspergillus fumigatus-induced NF-κB transcription in A549 cells.</p><p><b>CONCLUSION</b>PKD1 may contribute to the activation of NF-κB signal pathway and NF-κB transcription induced by Aspergillus fumigatus.</p>
Subject(s)
Humans , Aspergillus fumigatus , Cell Line, Tumor , HEK293 Cells , I-kappa B Kinase , Metabolism , NF-kappa B , Metabolism , Phosphorylation , Protein Kinase C , Metabolism , Signal Transduction , Transcription Factor RelA , Metabolism , Transcription, Genetic , TransfectionABSTRACT
The cell culture technology is the basis for the postgraduate students to undertake researches in furure.The department of cell biology of our university carried out the teaching reformation on the cell culture course by means of writing outstanding teaching materials, making supporting DVD, arranging suitable teaching contents and plans and setting up flexible examining systems, which promoted the teaching effects and profited students to master this skill quickly.
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Objective To investigate the effects of different combinations of antioxidants VitC, VitE and GSH on the biomechanical and biochemical markers in ovariectomized rats, Methods Seventy female SD rats, aged 4 months, were divided into 2 groups (control and model) randomly. Fifty rats in the model group underwent ovariectomy, while the other 20 rats in the control group had sham operations. Three month slater, 10 rats in the model group and 10 rats in the control group were selected randomly to detect their body weight, uterus weight, BMD of the left femur and lumbar vertebra, biomechanical characteristics of the left femur, levels of serum Ca2 +, Cr, ALP, SOD, MDA and GSH-Px and serumal resistance to OH<'->. Then the rest were divided into 5 groups: A (sham), B (model), C (VitC + VitE), D (GSH), and E (VitC + VitE +GSH). VitC, VitE and GSH were given in 750 mg/kg, 250 mg/kg, and 125 mg/kg every day for 3 months,respectively. And then the biomechanical and biochemical markers were measured. Results Three months after ovariectomy, the body weight of the rats in the model group increased markedly compared with the control group, while BMD of the left femur and lumbar vertebra, biomechanical maximal load and uterus weight decreased. The serum levels of Ca<'2+>, ALP and Cr increased. Three months after antioxidant treatment,the biomechanical maximal load and elastic load of the left femur and the maximal load of the 5th lumbar vertebra, the serum levels of SOD, GSH-Px and the serumal resistance to OH<'-> in groups D and E increased markedly, while the serum level of MDA decreased in groups C and D and the level of serum ALP decreased in all the treatment groups. Conclusion GSH and combination of VitC, VitE and GSH play a positive role in treatment of osteoperesis in ovariectomized rats.
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Objective To investigate the effects of different combinations of vitamin (Vit) C, Vit E and GSH on the biochemical markers and bone mineral density (BMD) in ovariectomized rats. Methods Seventy female SD rats aged 4 months were divided into two groups, 20 rats with sham operation in control group and 50 rats with bilateral oophorectomy in model group. 3 months later, 10 rats in the model group and 10 rats in the control group were randomly selected and their body weight, uterus weight, BMD of the left femurs and lumbar spines,biomeehanical characteristics of the left femurs, serum levels of Ca2+ , creatinine (Cr), alkaline phosphatase (ALP),superoxide dismutase (SOD) , malondialdehyde (MDA) , GSH-peroxidase (GSH-Px) and serum resisting abilities to OH- were determined. Then the rest rats were divided into five groups: A (sham), B (OVX control), C (Vit C +Vit E), D (GSH) and E (Vit C +Vit E +GSH). Vit C, Vit E and GSH were given 750rng/kg, 250 mg/kg, and 125 mg/kg, respectively daily for 3 months. And then the biochemical markers and BMD were measured. Results 3 months after treatment with antioxidants, BMD of left femurs and lumbars spines was increased, while the level of serum ALP was decreased markedly in B, C and D group as compared with that in B group. The serum level of SOD, GSH-Px and serum resisting ability to OH- were increased in D and E groups and the level of MDA decreased in C and D groups as compared with that in B group. Conclusion Vit C, Vit E and GSH increased BMD, prevented the decrease of SOD and GSH-Px and elevated serum resisting ability to OH-in ovariectomized rats.
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Objective: To investigate the function and mechanism of phospholipase Cγ1 (PLCγ1) in cell-matrix adhesion in colorectal cancer. Methods: Highly metastatic colorectal cancer cell line LoVo and lowly metastatic colorectal cancer cell line SW480 were subjected to cell-matrix adhesion assay. U73122 (a specific inhibitor of PLC) and pyrrolidine dithiocarbamate (PDTC) (an inhibitor of NF-κB) were used to study the effect of PLCγ1 and NF-κB on cell-matrix adhesion. Furthermore, Western blot and gel electrophoresis mobility shift assay (EMSA) were performed to detect the mechanism of PLCγ1 in colorectal cancer cell adhesion to matrix. Results: Inhibition of PLCγ1 or NF-κB resulted in reduction of cell-matrix adhesion in a dose-dependent manner in LoVo cells(P<0.05), but had no marked effect on SW480 cells. Western blot analysis showed that epidermal growth factor (EGF) stimulated the phosphorylation of PLCγ1 in LoVo. The results of EMSA indicated that inhibition of PLCγ1 signaling pathway also down-regulated the activity of NF-κB while EGF reversed the function. Conclusion:These data suggest that PLCγ1 plays a pivotal role in the EGF-induced cell-matrix adhesion of highly metastatic colorectal cancer cells and that NF-κB is also functional in this signaling pathway.
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Changes of rats peritoneal macrophages after hyperthermia were stimulated by ConA with ACAS 570 Interactive Laser Cytometer. The results show that the influx of Ca2+ increased further and the macrophages endocytosis enhanced the lysosomal pH fluctuated quickly, which was benefit for lysosomal content excretion, thus enhanced the lysosome activity.
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Objective To investigate signal transduction mechanism of phospholipase C?1 (PLC?1) in colorectal cancer cells. Methods Gel electrophoresis mobility shift assay (EMSA), immunocytochemistry, zymography and RT-PCR were performed to investigate the function of PLC?1 on nuclear factor-Kappa B (NF-?B), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in LoVo cell. Results Compared with control group, nuclear positive rate of cells treated with epidermal growth factor (EGF) increased significantly (from 26.91%?2.84% to 40.83%?4.36%), while that of cells treated with 2.5mol/L U73122 decreased to 12.20%?1.89%. Meanwhile, pretreatment with 2.5mol/L U73122 before EGF treatment decreased nuclear positive rate of cells from 40.83%?4.36% to 18.21%?1.34%. The results of EMSA further verified that PLC?1 can regulate the activity of NF-?B. RT-PCR results showed that EGF, PLC?1 or NF-?B had no significant effect on the expression of MMP-2, TIMP-2 at mRNA level. Furthermore, zymography indicated that the activity of MMP-2 did not change dramatically by EGF, PLC?1 or NF-?B. Conclusion These data suggested that EGF-PLC?1-NF-?B signaling pathway was operative in LoVo cell, but MMP-2 and TIMP-2 may not be regulated by EGFR-PLC?1-NF-?B signaling pathway.
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Objective: To preare recombinant lentivirus stably suppressing PLC?1 in human colorectal carcinomas Lo-Vo cells, so as to establish LoVo cell lines deficient in PLC1 and to investigate the role of this gene. Methods: Recombinant lentivirus produceing PLC-?1 siRNA were prepared. After LoVo cells were transduced with lentivirust, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC?1 was examined by Western-blot and RT-PCR analysis. The effect of the lentivirus on the cell proliferation and cell adhesion was analyzed by XTT method and ctll adhesion assay, respectively. Results: PLC?1 siRNA knocked down PLC?1 expression in LoVo cells obviously. The silenced efficiency of siRNA transducted by recombinant lentivirus was very high. Adhesion of human colorectal carcinomas LoVo cell Lines was significantly decreased, while proliferation was not affected. Conclusion: Our research confirm that PLC?1 plays an important role in cell adhesion of colorectal carcinomas, and provides experimental evidences for targeting PLC?1 in gene therapy against cancer.
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To investigate dynamic characteristics of the hydrolization lipid phosphatidylinositol(4,5)-bisphosphate(PIP 2 )with the stimulation of epidermal growth factor(EGF),the mathematical model to simulate the metabolizability of PIP 2 is established based on Law of Mass Action and Law of the Quasi-steady-State Approximation.Differential equations of concentration relations between PIP 2 and EGF receptor are formulated,and the effect of the parameters on the changing trend of PIP 2 is analyzed.This mathematical model describes biology characteristics of metabolized PIP 2 and dependency relationships of concentration between key signal producuts.
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Objective: To investigate the function and mechanism of phospholipase C?1 (PLC?1) in cell-matrix adhesion in colorectal cancer. Methods: Highly metastatic colorectal cancer cell line LoVo and lowly metastatic colorectal cancer cell line SW480 were subjected to cell-matrix adhesion assay. U73122 (a specific inhibitor of PLC) and pyrrolidine dithiocarbamate (PDTC) (an inhibitor of NF-?B) were used to study the effect of PLC?1 and NF-?B on cell-matrix adhesion. Furthermore, Western blot and gel electrophoresis mobility shift assay (EMSA) were performed to detect the mechanism of PLC?1 in colorectal cancer cell adhesion to matrix. Results: Inhibition of PLC?1 or NF-?B resulted in reduction of cell-matrix adhesion in a dose-dependent manner in LoVo cells(P