Objective:To lay foundation for the functional studies of programmed cell death 5 ( PDCD5 ) and develop new technical pathway for bioinformatics analysis of human functional genes. Methods:Using PDCD5 as the target molecule, intensive bioinformatics analysis of the nucleic acid and protein sequences were conducted. Data mining and comprehensive analysis by sequence against database similarity searching, ortholog structure comparison, expression profile analysis and gene “neighbor” listing were performed. Results: Two human putative pseudogenes on chromosomes 12 and 5, and one mouse putative pseudogene on chromosome 1 were identified. The methanobacterium thermoautotrophicum ortholog was classified as the same fold as ubiquitin and ribosomal protein S13. The C. elegans ortholog, ubiquitin and IAP (inhibitor of apoptosis proteins) belonged to the same expression profile cluster. This cluster was related to biosynthesis and protein synthesis. PDCD5 orthologs in various genomes were adjacent to various ribosomal proteins on the chromosome. Conclusion: The human genome contains at least two processed pseudogenes of PDCD5 . Besides the relationship with cell apoptosis, PDCD5 is predicted to have functional relationship with ubiquitin and participate in the translation regulation.

Homo sapiens PDCD10(programmed cell death 10,alias,"TF-1 cell apoptosis related gene 15,TFAR15"),cloned by means of cDNA-representational differences analysis,had been initially identified associated with cell apoptosis.Recent research suggested mutations within the PDCD10 gene or deletion were responsible for cerebral cavernous malformations,and PDCD10 was the third CCM gene.On the other hand,other research demonstrated that PDCD10 was strictly modulated and up regulated in many kinds of tumors,which implicated that PDCD10 participated in tumorous signal transduction.The recent research confirmed that PDCD10 interacts with MST4,a member of Ste20-related kinases,and the interaction promoted cell proliferation and transformation via modulation of the ERK-MARK pathway.In conclusion,all these demonstrate that PDCD10 has many biological effects,which suggests that it is a novel player in vascular morphogenesis and/or remodeling,as well as tumorigenesis and cancer progression.

Objective　To probe into roles of hCKLF-1 in the development of systemic lupus erythematosus in BXSB mice in vivo.Methods　The recombinant eukaryotic expression plasmid for hCKLF-1 (pCDI-hCKLF-1) was transferred and expressed in lupus-prone BXSB mice with the technique of muscle-mediated transgene by electric pulse;the levels of serum IgM and IgG anti-DNA antibodies as well as serum BUN and urine protein were detected.Results　hCKLF-1 expression in vivo did not make any effects on the levels of IgM and IgG anti-DNA antibodies in female and male BXSB mice,but markedly enabled abnormal elevation of urine protein in male BXSB mice in short time,suggesting its ability for accelerating glomerulonephritis.Conclusion　hCKLF-1 may be one of inflammation mediators,playing an important role in the lupus glomerulonephritis of male BXSB mice,and its target cells may be monocyte and macrophage.

Nuclear factor κB (NF-κB) is an important cellular transcription factor. The important role of NF-κB-mediated cell signal transduction pathway in apoptosis is a hot topic at home and abroad. In order to discover new regulators in NF-κB signaling pathway, a high-throughput cell-based screening model based on dual luciferase reporters system was established, a number of genes that can activate NF-κB signal pathway were obtained by screening of 439 novel function genes. Among them, TMEM9B can obviously activate NF-κB signaling pathway. Further experiments showed that TMEM9B activated NF-κB signaling pathway in a dose-dependent pattern. Western blotting and EMSA experiments confirmed that TMEM9B can promote the degradation of IκBα (a cytoplasm inhibitor of NF-κB), and cause NF-κB shift from the cytoplasm to nucleus. At the same time, flow cytometry result demonstrated TMEM9B can induce apoptosis in HEK293T and HeLa cells. In short, a stable and effective screening system for NF-κB has been established, through which TMEM9B was identified to be able to significantly activate NF-κB signal transduction pathway and thus cause cells apoptosis.

Objective: To investigate the effects of chemokine like factor 1 (CKLF1) on proliferation and metabolism of chondrocytes. Methods: Cell culture, 3H TdR and 3H Proline incorporation methods were used. Chondrocytes were harvested from rabbit articular cartilage. First passage cells were seeded on 96 well plates. After synchronization,the medium was replaced by DMEM containing 5% FCS with various concentration of CKLF1 conditioned medium. The synthesis of mucopolysaccharide was detected by Saffraan O staining. The transcription of inducible nitricoxide synthase (iNOS) was detected by semi quantitative RT-PCR. Results: CKLF1 inhibited the DNA, collagen and mucopolysaccharide synthesis significantly, meanwhile, stimulated the transcription of iNOS. Conclusion: CKLF1 inhibits the proliferation and matrix synthesis of chondrocytes, which might be an important factor resulting in cartilage destructive lesions. CKLF1 may exert its effects on chondrocytes through iNOS pathway.

Objective: To characterize the expression of programmed cell death 5 (PDCD5) in normal and osteoarthritic human cartilage. Methods: Articular cartilage specimens were obtained from 20 patients with osteoarthritis and 10 with femoral neck (normal cartilage) at the time of arthroplasty. Expression of PDCD5 was detected by flow cytometry, immunofluorescence, RT-PCR and immunohistochemical analysis. Results: PDCD5 expression in osteoarthritis cartilage was significantly higher than in normal cartilage especially in the nucleus. PDCD5 positive chondrocytes were mainly observed in the superficial and deep zone of osteoarthritis tissue sections,and in contrast, in the superficial and middle regions of normal controls. Conclusion: Since apoptotic chondrocyte death occurs more frequently in osteoarthritis compared to normal cartilage and PDCD5 is an apoptosis related protein, the different expression patterns of PDCD5 in osteoarthritis and normal cartilage suggest that it might be involved in the pathogenesis of osteoarthritis.

Objective: To obtain monoclonal antibodies against putative protein X4 of SARS-CoV for further study of the structure and function of protein X4. Methods: Balb/c mice were immunized with recombinant Protein X4, hybridoma cell lines secreting monoclonal antibodies against protein X4 were screened by regular cell fusion and subcloning approach. The specificities of these monoclonal antibodies were determined by Western blotting and Immunofluorescecence assay. Results: Three hybridoma cell lines (8A5, 8H6 and 4A2) stable in secreting specific monoclonal antibodies were successfully obtained. They could bind specifically to protein X4 proved by Western blotting. All of them belonged to the IgG2a isotype proved by antigen mediated ELISA. Indirect Immunofluorescence assay indicated that they could specifically bind to protein X4 expressed in SARS-CoV infected Vero E6 cells. Conclusion: Monoclonal antibodies of high specificity against protein X4 have been successfully prepared, which laid the foundation for the further study of protein X4.

Objective To construct a T-bet response reporter gene, for the detection of T-bet tran-scriptional activity and application in high-throughput screening for the functional genomies. Methods The cis-acting DNA element, ThRE, based on CNS-22 T box site of IFN-γ gene, was recombined into a reporter vector pLUC-MCS. The reporter gene was transfected into HEK 293T cells to detect its response to T-bet. And the binding of T-bot to TbRE was identified with electrophoretic mobility shift assay(EMSA). Results ThBE was successfully cloned into pLUC-MCS, named as TbRE-LUC. Using a luciferase assay, expression of the reporter gene is found to be induced by T-bet in a dose dependent manner and correlate with T-bet ex-pression positively with activation up to 20 folds. Moreover, the binding specificity of T-bet to TbRE is vali-dated by EMSA. Conclusion We successfully constructed a T-bet response reporter gene, ThRE-LUC, which responds to T-bet keenly and specifically. TbRE-LUC will be a useful tool in high-throughput screen-ing for human genes associated with transcription activity of T-bet.

Objective To express human chemokine-like factor 1 (CKLF1) in Drosophila S2 cells and study its function. Methods The pMT/V5-His-CKLF1 expression plasmid was constructed and transfected into Drosophila S2 cells. The positive clones were selected through PCR and RT-PCR. The culture medium was analyzed by Western blot with anti-CKLF1 polyclonal antibody. Chemotaxis and MTT assays on human peripheral blood and C2C12 cells, respectively, were then carried out with the medium. Results CKLF1 was transcribed efficiently in S2 cells. The expressed CKLF1 protein could be detected in the culture supernatant by Western blot, which showed weak chemotactic activity on both human peripheral blood neutrophils and lymphocytes as well as enhancing effect on the proliferation of C2C12 cells. Conclusion CKLF1 was expressed successfully in Drosophila S2 cells and secreted into the culture medium. The recombinant CKLF1 expressed in Drosophila cells can chemoattract leucocytes and promote the proliferation of C2C12 cells.

Objective: In order to further investigate the structural/functional relationship of TRALL. Methods: We did homology modeling for the extracellular segment of TRAIL, which is from R117 to G281, totally 165 aa residues long. The modeling software is Insight II from MSI/Biosym and the modeling work is based on the three dimensional structure of TNF-?. Results: From the modeling result, it can be seen that the modeled structure of TRAIL contains 10 ?-sheets and homologs for all these sheets could be found in TNF-?. This just confirms with the principle that the structurally con-seived regions within molecules of the same structure family should experience relatively small sequence mutations. In addition, the credibility of the modeled structure is checked by the way of inverse folding from Profile-3D. Conclusion: The result shows modeled structure is generally correct.