Death of nerve cells after cerebral ischemia have a variety of forms,including cell necrosis occurs immediately in ischemic core area and the subsequent apoptosis and autophagy induced by oxidative stress and inflammatory response in the course of reperfusion.After cerebral ischemia,a variety of different molecular mechanisms eventually lead to cell death,and the process involves several signaling pathways.Intervention of different forms and mechanisms of cell death may alleviate cell death after cerebral ischemia.

In order to characterize the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface protein GapC, gapC gene of S. aureus was amplified from strain BMSA/855/23-1 by PCR, and was inserted into pQE-30 vector subsequently. The recombinant plasmid, designated as pQE/gapC, was transformed into E. coli strain M15 (pREP4). The recombinant GapC fusion proein was successfully expressed in E. coli M15 induced with IPTG and its GAPDH activity was confirmed by GAPDH activity assay. Then, the recombinant GapC protein, inactivated S. aureus whole cell and placebo (PBS) were administrated to healthy rabbits respectively. The IgG antibody titers, concentration of IFN-gamma and IL-4 cytokines in immunized rabbit sera were measured with Enzyme-Linked Immunosorbnent Assay (ELISA). Finally, immunized rabbits were challenged with S. aureus strain Wood46 to evaluate the immunoprotection. The IgG antibody titers against GapC and whole cell in rabbit sera reached their peaks at day 28 after boost immunization (1:64,000). The concentration of IL-4 and IFN-gamma in GapC groups rabbit sera increased significantly (P<0.05) at day 14 after boost immunization, while the concentration of those in whole cell group did not increase (P>0.05) compared with the placebo group. 4 rabbits in 5 of the protein immunized group were protected against challenge with 1 x 10(8) CFU S. aureus. The results above indicate that the expressed recombinant GapC protein have high GAPDH activity and immunogenicity, can also protect against S. aureus challenge to some extent. S. aureus GapC protein could be an attractive target for further genetic engineering vaccine.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glyceraldehyde-3-Phosphate Dehydrogenases ; biosynthesis ; genetics ; immunology ; Immunization ; Male ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Staphylococcal Vaccines ; immunology ; Staphylococcus aureus ; enzymology ; genetics ; immunology ; Vaccines, Synthetic ; immunology

In order to characterize the immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface Isdb, we amplified Isdb gene from S. aureus Wood46 strain. The isdb gene was subsequently inserted into pET32a(+) vector and the recombinant plasmid was transformed into E. coli strain BL21. The recombinant Isdb was expressed and purified. Then, we immunized mice with the purified recombinant protein. The antibody level was measured by enzyme-linked immunosorbent assay. Finally, immunized mice were challenged with S. aureus strains Wood46 and HLJ23-1. These results showed that isdb gene sequences were highly conserved, and the recombinant Isdb was successfully expressed. The antibody titer in the immunized groups was increased significantly (P < 0.05) compared with the control, the protective rate of Isdb protein inducted by challenge with the two S. aureus stains Wood46 and HLJ23-1 was 62.5% and 75%, respectively. These results showed that the Isdb protein had high immunogenicity and immunoprotective capacity.
Animals ; Antibodies, Bacterial ; blood ; Cation Transport Proteins ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Vectors ; genetics ; Immunization ; Male ; Mice ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Staphylococcal Infections ; immunology ; prevention & control

In order to characterize the immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface protein Clumping factor A (ClfA), we amplified clfa genes from S. aureus Newman strain, Wood46 strain and HLJ23-1. The clfa gene from Newman strain was subsequently inserted into pQE-30 vector and the recombinant plasmid was transformed into Escherichia coli strain M15 (pREP4). The recombinant ClfA protein was expressed and purified. Then, we immunized mice with the purified recombinant protein. The antibody level and the concentration of cytokines were measured by enzyme-linked immunosorbent assay. Finally, immunized mice were challenged with S. aureus Newman, Wood46 and HLJ23-1. These results suggested that clfa gene sequences were highly conserved, and the recombinant ClfA was expressed correctly with good antigenicity. The antibody titer and the concentration of cytokines in the immunized groups increased significantly (P < 0.05) compared with control, and the mice in the immunized groups were protected against the challenge strains to some extent. These results showed that the ClfA had high immunogenicity and immunoprotective potential.
Animals ; Coagulase ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Immunization ; Mice ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Staphylococcus aureus ; metabolism ; pathogenicity

Objective:Super-enhancer-associated genes may be closely related to the progression of osteosarcoma,curcumin exhibits a certain inhibitory effect on tumors such as osteosarcoma.This study aims to investigate the effects of curcumin on osteosarcoma in vitro and in vivo,and to determine whether curcumin can inhibit the progression of osteosarcoma by suppressing the expression of super-enhancer-associated genes LIM and senescent cell antigen-like-containing domain 1(LIMS1),secreted protein acidic and rich in cysteine(SPARC),and sterile alpha motif domain containing 4A(SAMD4A). Methods:Human osteosarcoma cell lines(MG63 cells or U2OS cells)were treated with 5 to 50 μmol/L curcumin for 24,48,and 72 hours,followed by the methyl thiazolyl tetrazolium(MTT)assay to detect cell viability.Cells were incubated with dimethyl sulfoxide(DMSO)or curcumin(2.5,5.0 μmol/L)for 7 days,and a colony formation assay was used to measure in vitro cell proliferation.After treatment with DMSO or curcumin(10,15 μmol/L),a scratch healing assay and a transwell migration assay were performed to evaluate cell migration ability.Real-time reverse transcription polymerase chain reaction(real-time RT-PCR)and Western blotting were used to detect mRNA and protein expression levels of LIMS1,SPARC,and SAMD4A in the cells.An osteosarcoma-bearing nude mouse model was established,and curcumin was administered via gavage for 14 days to assess the impact of curcumin on tumor volume and weight in vivo.Real-time RT-PCR was used to measure mRNA expression levels of LIMS1,SPARC,and SAMD4A in the cancer and adjacent tissues from 12 osteosarcoma patients. Results:After treating cells with different concentrations of curcumin for 24,48,and 72 hours,cell viability were all significantly decreased.Compared with the DMSO group,the colony formation rates in the 2.5 μmol/L and 5.0 μmol/L curcumin groups significantly declined(both P＜0.01).The scratch healing assay showed that,compared with the DMSO group,the migration rates of cells in the 10 μmol/L and 15 μmol/L curcumin groups were significantly reduced.The exception was the 10 μmol/L curcumin group at 24 h,where the migration rate of U2OS cells did not show a statistically significant difference(P＞0.05),while all other differences were statistically significant(P＜0.01 or P＜0.001).The transwell migration assay results showed that the number of migrating cells in the 10 μmol/L and 15 μmol/L curcumin groups was significantly lower than that in the DMSO group(both P＜0.001).In the in vivo tumor-bearing mouse experiment,the curcumin group showed a reduction in tumor mass(P＜0.01)and a significant reduction in tumor volume(P＜0.001)compared with the control group.Compared with the DMSO group,the mRNA expression levels of LIMS1,SPARC,and SAMD4A in the 10 μmol/L and 15 μmol/L curcumin groups were significantly down-regulated(all P＜0.05).Additionally,the protein expression level of LIMS1 in U2OS cells in the 10 μmol/L curcumin group was significantly lower than that in the DMSO group(P＜0.05).Compared with adjacent tissues,the mRNA expression level of SPARC in osteosarcoma tissues was significantly increased(P＜0.00l),while the mRNA expression levels of LIMS1 and SAMD4A did not show statistically significant differences(both P＞0.05). Conclusion:Curcumin inhibits the proliferation and migration of osteosarcoma both in vitro and in vivo,which may be associated with the inactivation of super-enhancer-associated gene LIMS1.