Selenoprotein SELENOK Enhances the Migration and Phagocytosis of Microglial Cells by Increasing the Cytosolic Free Ca2+ Level Resulted from the Up-Regulation of IP3R.

Enhancing the migration and phagocytosis of microglial cells is of great significance for the reducing of the risk of the neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The effect of mouse selenoprotein K (mSELENOK) on the migration and phagocytosis of BV2 microglial cells and its mechanism were studied. The results showed that the over-expression of mSELENOK can increase the migratory and phagocytic abilities of the microglial cells, while the knockdown of mSELENOK can decrease the migratory and phagocytic abilities of the cells. The cytosolic free Ca2+ level and inositol trisphosphate receptor (IP3R) mRNA transcript and protein expression were also increased significantly as the consequence of the over-expression of mSELENOK in the microglial cells. On the contrary, the level of cytosolic free Ca2+ and the mRNA transcript and protein expression of IP3R in mSELENOK knockdown cells were decreased significantly. 2-aminoethoxydiphenyl borate (2-APB), an antagonist of IP3R, could prevent the increased migration, phagocytosis, and cytosolic free Ca2+ level of mSELENOK over-expressed microglial cells, and knockdown of IP3R3 could reduce the increased cytosolic Ca2+ level in mSELENOK over-expressed microglial cells. Further studies revealed that selenium supplement (Na2SeO3) can increase the expression of mSELENOK in microglial cells significantly. In summary, these data suggest that mSELENOK can increase cytosolic free Ca2+ level of microglial cells by up-regulating the expression of IP3R, thus enhancing the migration and phagocytosis of microglial cells. Our results indicated that mSELENOK is an important selenoprotein, which plays a role in trace element selenium's functions and can enhance the migration and phagocytosis of microglial cells.

[Prokaryotic expression of Drosophila selenoprotein D-SelK and preparation of polyclonal antibody to D-SelK].

AIM: To clone and encode Drosophila selenoprotein D-SelK structure gene, express it in E.coli efficiently, and after purification, to generate its antibody in rabbits. METHODS: D-SelK gene segment amplified from pGM-T-D-SelK plasmid by PCR was inserted into pGEX-6p-1 to construct recombinant plasmid pGEX-6p-1-D-SelK. The recombinant plasmid was transfected into E.coli BL21(DE3) to express the recombinant protein D-SelK in E.coli under IPTG induction. The protein was purified by denaturation and electrophoresis, and then identified by SDS-PAGE and Western blotting. Polyclonal antibody to D-SelK was obtained by immunizing rabbits with the protein. Quality and quantity of the antibody was examined. RESULTS: D-SelK gene segment was successfully inserted into pGEX-6p-1 and the positive clones of the recombinant plasmid was identified by PCR screening and restriction endonuclease analysis. The target protein was effectively expressed in E.coli by the IPTG induction. Through immunizing rabbits with the purified target protein, we obtained the specific antibodies to D-SelK, the titer of which was more than 1:51 200. The polyclonal antibody had a good specificity to D-SelK. CONCLUSION: D-SelK recombinant protein and rabbit anti-D-SelK polyclonal antibody with high specificity were obtained, which provides good tools for further research on the functional characterization of D-SelK.

[Comparison of ICP-MS and two other analytical methods for determination of selenium content in Cordyceps militaris].

Samples were digested by microwave digestion. The selenium content in selenium enriched Cordyceps militaris was determined by ICP-MS method, HPLC/fluorometric method, and 3,3-diaminobenzidine method separately. And the detection conditions, the lowest detection limit and the relative standard deviation (RSD) of the three determination methods were compared. The detection conditions of the three methods for the detection of selenium content in selenium enriched Cordyceps militaris were established. It was showed that the lowest detection limit of ICP-MS method, HPLC/fluorometric method, and 3,3-diaminobenzidine method was 0.260 7, 0.182 1 and 10.485 9 microg x L(-1) respectively, and this means that the lowest detection limit of ICP-MS method was the lowest and that of 3,3-diaminobenzidine method was the highest. For the same sample the relative standard deviation (RSD) of ICP-MS method was the lowest and the RSD of 3,3-diaminobenzidine method was the highest. It was recommended that selenium content is determined by ICP-MS and HPLC/fluorometric method when the selenium in the sample is very low and by 3,3-diaminobenzidine method when the content is rather high.

[Immunoregulation of IL-18 in mice with radiated damage].

AIM: To explore the immunoregulation of IL-18 in the mice radiated by 60Co. METHODS: 32 C57 mice were radiated by 60Co and then treated by IL-18.2 weeks later, the transformation of lymphocytes, the ability of NK cells to kill tumor cells, the subtype of T cells and the content of IgG in serum were tested. RESULTS: IL-18 increased the function of lymphocyte transformation in 60Co radiated mice, enhanced the cytotoxic activity of NK cells against tumor cells of A375, U973 and KG1, and up-regulated the amount of CD4+T cells. However, the level of IgG in the serum of the radiated mice was not regulated by IL-18. CONCLUSION: IL-18 can enhance the immune function of the mice radiated by 60Co.

[Construction of apoptin gene delivery system and its effect on apoptosis of A375 cells].

AIM: To construct the delivery nanoparticles system of apoptin gene with O-carboxymethylated chitosan(CMC) and study its effect on inducing apoptosis of human melanoma cells A375 in vitro. METHODS: CMC nanoparticles containing apoptin gene were prepared by an ultrasonic method. Restriction enzymes, DNA gel retardation assay and PCR were used to identify apoptin gene stability and to decide the best N/P ratio as well as the model effect in the progress of replication. Human melanoma cells A375 are transiently transformed by nanoparticles containing apoptin gene and apoptosis was measured by MTT assay at various time period. RESULTS: Morphology studies revealed that the particles were spherical in shape with smooth surface. The mean particle diameter ranged from 200-300 nm. The ratio of the chitosan to apoptin DNA (N/P ratio) was 5.5:1. The apoptin gene in chitosan/apoptin nanoparticles could be protected from DNase degradation and could be used as the model in the process of replication. The nanoparticles with apoptin gene could induce apoptosis of A375 cells in dose-dependent manner in vitro at 48 h after transformation. CONCLUSION: The chitosan vector and apoptin gene could be combined to be a safe gene delivery nanoparticles system, which could induce apoptosis in human melanoma cells A375.

[Mechanism of apoptin-induced apoptosis of human lymphoma cells U937].

AIM: To study the role of c-Jun N-terminal kinase (JNK) in the apoptosis induced by apoptin gene in human lymphoma cell U937. METHODS: The U937 cells were transiently transfected by pcDNAA3 plasmids containing apoptin gene. Apoptosis of U937 cells was measured by flow-cytometry. Activation of JNK signal pathway was detected by Western blot. RESULTS: Apoptin could induce apoptosis of U937 cells in vitro at 48 h after transfection. The level of phosphorylated JNK was increased at 24 h and reached the peak level at 48 h after transfection. CONCLUSION: Apoptin gene can induce apoptosis of U937 cells in which JNK signal pathway plays an important role.