Abstract:Introduction of new concept may promote science development. In recent years, the concept of redundancy has gradually infiltrated into life science. Functional redundancy of cytokines and transcriptional factors has attracted much attention. However, cellular redundancy remains ill-characterized yet. Recent development in study of adult stem cell plasticity and professional and amateur phagocytes arouse exploration of cellular redundancy. Relevant information concerning cellular redundancy and antiredundancy are summarized, the function and significance of cellular redundancy are discussed from the angle of biomedicine, and active investigation in cellular redundancy is suggested.

Abstract:Accumulating evidence suggests that cyclooxygenase (COX) family members are associated with a proliferative potential and apoptosis resistance of many cancers. COX-2 is an important molecular target for both therapy and prevention of malignancies. In vitro studies have revealed that treatment of various human epithelial cancer cell lines with specific COX-2 inhibitors induce apoptotic cell death. However, little is known about tumor cell lines of hematopoietic origin. The effects of COX-2 specitic inhibitor-celecoxib on proliferation and apoptosis on K562 cells are sought to be determined. K562 cells were exposed to increasing concentration of celecoxib (0, 10, 20, 40, 80, 160 μmol/L). The growth inhibition was evaluated by trypan blue dye exclusion, MTT assay and colony- formation inhibiting test. Apoptosis was determined by DNA ladder in agarose gel. The morphology apoptotic cells was identified by AO/EB staining combined with fluorescence microscopy observation, and by caspase-3 assay. The percentage of apoptotic cells was determined by flow cytometry. A blocking experiment for caspase-3 was carried out by incubating K562 cells together with DEVD-fmk. In addition, whether there is an expression of COX-2 mRNA or protein in K562 cells were investigated by RT-PCR and Western blot. Celecoxib(40～160 μmol/L) can effectively inhibit the proliferation and induce apoptosis of K562 cells in a dose-dependent manner. IC₅₀ of celecoxib for inhibiting cells proliferation was 46 μmol/L. The regulation of apoptosis induction was related to an upregulated caspase-3 expression and activation. When caspase-3 activity was blocked by DEVD-fmk, the effect of celecoxib on apoptosis induction was partly abolished. Importantly, It is first demonstrated that K562 cells indeed exists in COX-2 expression both mRNA and protein (IL-1β inducement assay and RT-PCR/Western blot confirmation), celecoxib (80～160 μmol/L) could significantly down-regulate the expression of COX-2 mRNA and protein. In conclusion, these data indicated that a selective COX-2 inhibitor celelcoxib inhibits cell proliferation, induces apoptosis in human K562 cells, thus suggesting that COX-2 inhibitors may be a promising strategy in treatment of CML.

Abstract:To accelerate study of gene function, a home-made high-density cDNA microarray was developed, using the cDNA clones from different types of tissues. In order to cover as far as possible the whole genome, cDNA clones that essentially represented genes with low abundance were introduced from Resgene Company. Totally 384 quality control DNA and 12 630 cDNA fragments, which includes 12 508 Unigenes and 122 ESTs, were spotted on the chip, after optimization. The home-made cDNA microarray was evaluated with Microarray ScoreCard and proved to be representative and reliable. These microarrays were then used to study the differential gene expression profiles of visceral adipose tissues between obese patients and normal subjects. The results showed that genes involved in immune-regulation, apoptosis and metabolism of energy were up-regulated in obese patients, while genes involved in lipid synthesis were down-regulated in obese patients. Further exploration of these genes will greatly improve the understanding of the pathogenesis of obesity.

Abstract:RNA interference(RNAi) is the process of sequence-specific posttranscriptional gene silencing initiated by double-stranded RNA that is homologous to the silenced gene. To investigate the inhibitory effect of endogenous siRNA on target gene, a DNA-based vector was constructed for intracellular transcription of a hTERT specific short hairpin RNA(shRNA) and evaluate the silencing effect for hTERT gene expression. hTERT cDNA sequences from nucleotides 3565～3583 followed by 9 bp to form a loop and the corresponding antisense hTERT nucleotides followed by five thymines were cloned to the downstream of the U6 promoter in pPUR/U6 vector, resulting in the RNA interference vector pPUR/U6/hTERT. The constructed vector pPUR/U6/hTERT and the vancant vector pPUR/U6 were transferred into HepG2 cells respectively by Liposome-mediated transfection. The antibiotic-resistant transfected cells were selected and enriched by applying puromycin in culture medium. The surviving cells were seeded in 12-well plate for assaying cell growth rate by cell counting or harvested for evaluation of hTERT gene expression by RT-PCR and Western-blot assay. Telomerase activity was determined by TRAP(Telomerase Repeat Amplification Protocol) and p53 expression level was assayed by Western-blot. Compared to the cells transfected with pPURU6 control vector, pPUR/U6/hTERT caused efficient down-regulation of hTERT gene expression and telomerase activity. It also significantly increased p53 expression level and inhibited cell growth. These results suggested that the endogenous hairpin siRNA producted by DNA plasmid is capable of mediating robust hTERT gene inhibition. It is promised to be a potential tool for gene function analysis.

Abstract:Lung cancer is one of the malignant tumors. The death rate and incidence are very high through the whole world. Aberrant methylation of p16 gene promoter is thought to be an early event in lung cancer development. To improve the sensitivity and specificity, the microfluidic chip method was established to detect the aberrant methylation of p16 gene. The plasma samples of lung cancer patients were detected. The p16 gene methylation can perhaps become a biomarker for early lung cancer. It may become a new and reliable method for early diagnosis of lung cancer.

Abstract:This is a research of the relationship between the characteristics of local degeneracy on mutational hotspots regains on genes of human genome. There includes the introduction of related research background and a draft of the calculation methods of local degeneracy and clustering. SNP spots are regarded as the foundation to decide mutational hotspots. 2 831 genes of human genome are selected and analyzed by their characteristics of local degeneracy on hotspots regains. Furthermore, genes whose hotspots are aggregated in relatively high local degeneracy regains and their low counterpart are analyzed and classified by their biological function. The selected genes are clustered by their parameters of local degeneracy. Some function classes whose selected genes are clustered together through clustering are found. For certain function classes, all selected genes of each class have similar parameters of local degeneracy. It appears that the local degeneracy of amino acid residues that contain SNPs of certain gene depends on the function of the proteins these genes produce. The fundamental reason for this phenomenon may be that by their nature, SNPs connect local degeneracy in mutational hotspot regions and biological function. Depending on whether they are degenerate SNPs affect the functions of proteins, and each no degenerate SNP may be a potential source of phenotypic diversity. Human genes appear to select high local degeneracy at the amino acid residues that contain SNPs. This may be the result of natural selection. This result shows that their trait of local degeneracy also like each other, which is a good clue to predict gene function by their local degeneracy.

Abstract:To elucidate the role of cellular repressor of E1A-stimulated genes (CREG) in the phenotype modulation of vascular smooth muscle cells (VSMCs), human CREG (hCREG) was overexpressed in primary rat VSMCs was using a mammalian expression vector pRC/CMV-hCREG. Transient transfection of VSMCs with hCREG inhibited cell proliferation and DNA synthesis as evidenced by decrease in BrdU incorporation. Moreover, CREG overexpression increased SM α-actin protein level in VSMCs. Co-transfection of VSMCs with SM α-actin promoter reporter gene with hCREG enhanced SM α-actin promoter activity, suggesting that elevated SM α-actin protein production resulted from increased gene transcription. The mechanism by which CREG regulates VSMC phenotype conversion was further investigated by analysis of interaction of CREG with serum response factor (SRF), an important transcriptional factor involved in VSMC differentiation. CREG co-immunoprecipitated with SRF and CREG overexpression increased the SRF bound to CREG. In addition, gel mobility shift assay and supershift assay showed that CREG bound together with SRF to the CArG site of SM α-actin promoter. These results suggest that CREG acts as a transcriptional factor and interacts with SRF to promote VSMC differentiation.

Abstract:In order to reveal proteins differentially expressed in the process of rat pancreas development, two-dimensional gel electrophoresis and mass spectrometry were performed on rat pancreas at different stages. After rat pancreas microseperated, protein extracted from of four stages: 15.5 embryonic days (E15.5), E18.5, newborn and adult were separated by immobilized pH gradient two-dimensional gel electrophoresis. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS)and ESI-MS were performed with the proteins which were found to be significantly different presented in these four maps, 8 of which were down-expressed and 1 of which up-expressed at the adult stage, 4 of which specially expressed at adult stage while 6 of which specially expressed in E18.5 stage. All together 18 protein spots were identified. 7 spots were identified as alpha fetoprotein (AFP). 5 spots were pancreatic lipase related protein 1 precursor. 1 spot was tublin beta 5.2 spots were protein disulfide isomerase. The other 3 spots were similar to FLN29 gene product, Trypsin V-A precursor, and Peroxiredoxin 4.Among them, AFP specially expressed in new born and E18.5 stage. The development of pancreas gave rise to the changes of the protein expression patterns. This helps to understand the molecular mechanisms of rat embryonic pancreatic development.

Abstract:The Arabidopsis thaliana AtNHX2 gene is one member of the Arabidopsis NHX Na⁺/H⁺ antiporter gene family and play an important role in salt tolerance. A sequence-analyzed 2.8 kb DNA fragment on upstream of ATG start codon of AtNHX2 gene was cloned into pCAMBIA1301-1. The promoter activity was detected by transient expression in onion epidermis. The reconstructed vector pCAMBIA1301-1/ AtNHX2 promoter was transformed into Arabidopsis thaliana by Floral Dip method. AtNHX2 promoter-GUS analysis in transgenic Arabidopsis showed that AtNHX2 was expressed in all tissues. Strong GUS expression was detected in guard cells suggesting the possibility that it is involved in stomatal regulation. AtNHX2 promoter activity was decreased by NaCl and up-regulated by KCl, demonstrating that NaCl and KCl regulation to AtNHX2 expression occurs at the transcriptional level. GUS activity in old leaves was higher than in new leaves, which reveals a role of AtNHX2 in salt tolerance. Strong GUS activity in root hair cells was observed. It suggests that AtNHX2 may play an important role in partitioning of Na⁺ into the enlarged vacuoles of root hair cells.

Abstract:A family of synapse-associated protein(SAPs) has recently emerged as a central player in the molecular organization of synapses. This work is to analyze the epitope of a novel Homo sapiens synapse associated-protein(FRG4) and synthesize polyclonal antibody, then research FRG4 protein expression in smooth muscle cells and endothelial cells. FRG4 full-length sequence was obtained by PCR from human fetal liver library. Its epitope, motifs and the second structure of amino acids encoded were detected by bioinformatics technique. By solid-phase peptide synthesis method to synthesize FRG4 peptides, then peptides were immunized to rabbits; by immunohistochemistry to detect the expression of FRG4 in vascular smooth muscle cells and endothelial cells. The 13-peptides PKLVKEEVFWRNY was selected by bioinformatic analysis to synthesize rabbit anti-human FRG4 polyclonal antibody. Antibody purity was 82.79% and antibody dilution was 1∶16 000 detected by ELISA. The antibody has a good reaction and speciality in Western blot, it was mainly expressed in the cytoplasm of smooth muscle cells and endothelial cells by immunohistochemistry. The results suggested a novel Homo sapiens synapse associated protein (FRG4) antibody was synthesized successfully. FRG4 expressed mainly in the cytoplasm of vascular smooth muscle cells and endothelial cells.

Abstract:Two recombinants of haemadin which contain RGD amino acids sequence in its forth loop was cloned, expressed and identificated on the basis of the analysis of structure and function of the haemadin and RGD sequence. The chimera genes coding for mature haemadin with inserted RGD sequence were obtained by overlapping PCR and cloned into the shuttle expression vector pPICZαA. This process was identified by restriction endonuclease digestion and DNA sequence analysis. The recombinant plasmids were electroporated into Pichia pastoris strain GS115. The recombinant proteins, HRGD1 and HRGD2, were expressed and induced by methyl alcohol, secreted into media leading by signal peptides and purificated by combination of ultrafiltration, cation exchange chromatography and gel filtration chromatography. The biological activity assays approved the chimera proteins show activity not only in antithrombin but also in antiplatete aggregation as what have been predicted.