ABSTRACT
LPS stimulation of macrophages production of IFN-beta plays a key role in innate immunity defending the microbial invasion. In this study, the effect of S632A3 promoting LPS-induced IFN-beta production and the underlying mechanism were investigated, mRNA level was measured by real-time PCR, cytokine production was determined by ELISA, GSK-3beta activity was investigated by kinase assay, protein phosphorylation and expression were evaluated by Western blotting. The results revealed that S632A3 significantly augmented IFN-beta production by LPS-stimulated macrophages. S632A3 inhibition of the activation of GSK-3beta, reduced the threonine 239 phosphorylation of transcription factor c-Jun but increased the total level of c-Jun in LPS-stimulated macrophages. Moreover, small interfering RNA-mediated knockdown of c-Jun level abrogated the ability of S632A3 to augment IFN-beta. The study thus demonstrates S632A3 being a new anti-inflammation lead compound and provides a molecular mechanism by which S632A3 promoted LPS-induced IFN-beta production in macrophages through inhibiting the activation of GSK-3beta.
ABSTRACT
This study is to optimize the preparation process of fusion protein Fv-LDP which was expressed in the form of inclusion body and consisted of lidamycin apoprotein LDP and single-chain Fv antibody (scFv) directed against type IV collagenase. The preparation and the dissolution of inclusion body, the immobilized metal affinity chromatography of the target protein and the renaturization by stepwise dialysis were optimized by single-factor analysis or orthogonal design. In addition, the refolded fusion protein Fv-LDP was refined by Sephadex G-75 chromatography followed by fluorescence-activated cell sorter (FACS)-based saturation binding assay to measure its antigen-binding activity. After optimization of the process, the purity of fusion protein Fv-LDP existed in the inclusion body was 63.9% and the corresponding solubility was 95.7%; Under denaturing conditions, the purity of fusion protein Fv-LDP was more than 95% after the purification process. The percentage of monomeric fusion protein Fv-LDP was 60% after the refolding process, while it was further refined to 85% which was 5.6-fold higher than that of the initial refolding condition. The refined fusion protein Fv-LDP could bind to human lung adenocarcinoma PAa cells and human hepatoma BEL-7402 cells with the dissociation constants (Kd) of 0.176 micromol x L(-1) and 0.904 micromol x L(-1), respectively. The preparation process of fusion protein Fv-LDP has been successfully optimized, which provides the experimental basis for the production and future development of fusion protein Fv-LDP, and might serve as a relatively practical system for the preparation of other scFv-based proteins expressed in the form of inclusion body.
ABSTRACT
Objective Increased morbidity of auto-immune disease in senescent individual might be related with the defects of apoptosis after stimulation or induction. Changes of apoptotic features in splenic T lymphocytes were studied in senescent mice. Methods Flow cytometry was used to study the rates of apoptosis by analysing sub-G 1 peak. DNA ladder was observed by agarose gel electrophoresis. Free calcium levels in both cells were detected by Fluo-3 loading. Bcl-2 protein levels were detected by Western blot. Results After co-stimulation with IL-2/ConA, flow cytometry showed that average apoptotic percentage of old T cells was 38.3% ?10.3%,significantly lower than that of young ones (58.6% ? 4.0%, P
ABSTRACT
AIM To study the features of cell death induced by the anticancer antibiotic lidamycin (LDM) in human hepatoma BEL-7402 cells. METHODS Chromatin condensation was observed by co-staining with fluorescent dyes, hoechst 33342 and propidium iodide. “G1 sub-peak” was detected by flow cytometry and DNA ladder was observed using agarose gel electrophoresis. The caspase-3, 6 activities were measured with kits specific for them. RESULTS Typical apoptotic chromatin condensations appeared when the BEL-7402 cells were treated with the conventional antitumor agent mitomycin C 30 μmol.L-1 for 12 h. However, an abnormal type of chromatin condensation occurred when the cells were treated with LDM 1 μmol.L-1 for 6 h, which was characterized with keeping the completeness of nuclear membrane and not forming apoptotic bodies. The DNA ladder patterns were observed using agarose gel electrophoresis. The “G1 sub-peak” occurred only in the cells treated with LDM for 24 h, though chromatin condensation was earlier detected in treatment with LDM for 6 h. The caspase-3, 6 activities were increased about 5 and 4 folds, after the cells were treated with LDM 1 μmol.L-1 for 6 h, as did mitomycin C. The time of initiating chromatin condensation was earlier than that of the high peak activities of caspase-6. CONCLUSION The characterization of cell death induced by lidamycin in the human hepatoma BEL-7402 cells differs from typical apoptosis. The results make it helpful to explain the molecular mechanism of the highly potent cytotoxicities of lidamycin toward tumor cells.
ABSTRACT
AIM To study the effect of lidamycin on the expression of genes involved in invasion regulation in HCT-8 human colon cancer cells. METHODS HCT-8 human colon cancer cells were treated with lidamycin (10 nmol*L-1) for 8 h. The effect of lidamycin on the expression of genes were detected by cDNA arrays, Northern blot and RT-PCR. RESULTS Hybridization of the entire cDNA populations to Atlas Arrays showed that lidamycin down-regulated the expression level of MMP-9 and up-regulated the expression level of TIMP-1. These changes were confirmed by Northern blot and RT-PCR. CONCLUSION The results indicate that lidamycin may exhibit its anti-invasive activity by inhibitting the production of type IV collagenase whilst enhancing the production of tissue inhibitor metalloproteinase.
ABSTRACT
Objective To explore the changes of nitric oxide (NO) and nitric oxide synthase (NOS) of the aged rat brain, and the relationship between this change and neuronal apoptosis. Methods Five month old and 30 month old rats were used in the study. NO level and NOS activity were measured by Griess reaction and high presssure liquid chromatography. Neuronal NOS(nNOS) gene and bcl 2 gene were examined by in situ hybradization; nNOS protein level and free calcium level in synaptosome were determined by immunohistochemical method and Fura 2 fluorescence probe respectively. Apoptosis was observed using terminal transferase marking method. Results NO level and nNOS activity in the brain tissue of aged rats was (2 61?0 10) ?mol/L and (398 22?21 62) fmol?mg -1 ?min -1 , respectively,being significantly higher than that of the young rats(1 54?0 15) ?mol/L and (234 38?16 24)fmol?mg -1 ?min -1 respectively. Also both the nNOS gene transcription and protein expression increased in aged rats while bcl 2 gene expression reduced with aging. Free calcium level in synaptosome of aged and young rats was (485 26?28 48) nmol/L and (372 99?19 20) nmol/L respectively. Apoptosis in brain tissue was observed in aged rats, but not in young ones. Conclusions The increase of NO level in the aged rat brain is due to the increase of nNOS activity which is at least partially determined by the increased gene expression. Abnormal enhancement of NO in the aged rats may cause damage, even death of the brain. As an anti oxidant, bcl 2 gene expression reduced with aging and resulted in the brain tissue more vulnerable to oxidative stress and thus produced more lesions. Therefore, it is a promising method to screen and develop drugs that can be anti apoptotic and anti oxidative, and reduce the formation of pathologic NO to prevent and retard brain aging.