Abstract:Aquaporins (AQP) are a family of hydrophobic intrinsic membrane proteins that efficiently and selectively transport water. Since the discovery of first water channel (AQP1) from red blood cell membrane by Agre et al in 1992, rapid and serial progresses have been made in characterization of AQP structure and function. At least 11 homologous members (AQP0 - AQP10) have been molecularly identified in mammals. AQPs are expressed in various epithelium and endothelium involving fluid secretion and absorption, and in many cell types such as erythrocytes, white blood cells, adipocytes, and muscle fibers that have no obvious relationship with fluid transport. The extensive expression pattern of AQPs may indicate functional importance in multiorgan physiology and pathophysiology. Gene-targeting technology has been a powerful tool in defining physiological functions of specific genes. So far transgenic knockout models of AQP1, AQP3, AQP4 and AQP5, and a knock-in model introducing a point mutation (T126M) that causes autosomal recessive nephrogenic diabetes insipidus (NDI) in human have been successfully established. Significant progresses have been made in characterizing the physiological functions of these AQPs by systematic mouse phenotype studies.

Abstract:Autophagy occurs in all types of eukaryotic cells, which has a rigid connection to normal development of cells and a variety of diseases. There are many molecular control elements and multiple signaling pathways involved in regulating autophagy. Autophagic cell death is considered as the type Ⅱ programmed cell death. There is a crosstalk between autophagy and apoptosis. Both of them participate in maintaining cell homeostasis and pathogenesis of certain diseases. The roles of autophagy in development, aging, tumor, neurodegenerative diseases and infectious diseases are reviewed.

Abstract:In the field of infectious diseases there is an urgent need for global researches that can efficiently, precisely and integratively study structural and functional genomics and proteomics of microbial infection (infectomics). The combination of new (e.g. DNA and protein microarrays) and traditional approaches (e.g. cloning, PCR, gene knockin and knockout, and antisense) will help overcome the challenges we are facing today. It was assumed that the global phenotypic changes (infectomes) in microbes and their host during infections are encoded by the genomes of microbial pathogens and their hosts, expressed in certain environmental conditions devoted to specific microbe-host interactions. Global drug responses (pharmacomes) in microbes and their host can be detected by genomic and proteomic approaches. Genome-wide approaches to genotyping and phenotyping or expression profiling will eventually lead to global dissection of microbial pathogenesis, efficient and rapid diagnosis of infectious diseases, and the development of novel strategies to control infections. The key fundamental issue of infectious diseases is how to globally and integratively understand the interactions between microbial pathogens and threir hosts by using infectomics.

Abstract:Tumor angiogenesis is a mandatory step of tumor growth, invasion, and metastasis involving endothelial cell proliferation and migration from blood vessels in peritumoral tissues and formation of tubules. Water channel aquaporin-1 (AQP1) has been reported to express in microvessels of many different type of tumors, indicating the possible involvement of AQP1 in tumor angiogenesis. A melanoma-bearing model in AQP1-knockout mice was used to evaluate the role of AQP1 in tumor angiogenesis. The results demonstrated nearly 30% retarded growth of subcutaneously inoculated melanoma in AQP1 knockout mice compared to tumors in wildtype mice. Immunohistochemistry of melanoma sections revealed strong AQP1 protein labeling in endothelium of tumor blood vessels in wildtype mice and negative labeling of AQP1 in the counterpart structures in AQP1-knockout mice. On the same tumor sections stained by hematoxylin, melanoma cells form islands with microvessels in the center. It is easily seen that high-density and thin microvessels (142/mm^{2) locate in the center of tumor islands in wildtype mice, whereas sparse and enlarged vessels (47/mm2) are evident in tumor islands in AQP1 knockout mice coincident with larger necrotic area in outer layer of the islands. These results provide definitive evidence that lacking of water channel AQP1 results in defective tumor angiogenesis and retarded tumor growth that may be involved in insufficient blood supply and/or abnormal transendothelial fluid transport. The underlying molecular and cellular mechanisms are worth further investigation.}

Abstract:Microcantilever array biosensor shows peculiarities of high speed, trace detection and label-free in bioanalysis. A method to increase the detection sensitivity and to speed up analysis of microcantilever array biosensor is reported. The gold covered microcantilever array immobilized by DNA probes is applied as a microcantilever array biosensor with electrostatic field. Microcantilever arry is acted as anode in hybridization solution cell where DNA molecules are removed to and congregated around anode, so the DNA hybridization is enhanced. The data demonstrated: (1) the time of DNA hybridization on the cantilever system drived by direct electric field was no more than 3 min; (2) the detection sensitivity was improved from 30 μg/L to 300 ng/L level.

Abstract:Leukemia inhibitory factor (LIF) plays important roles in varieties of biological processes. This factor is highly conserved in mammalian animals and only one heterozygous LIF mutation was reported to cause the infertility of women. A LIF mutation was generated and the evidences were provided that the mutation of mature LIF at the 29th amino acid totally abolished its functions, including stimulation of STAT activation assayed by Luciferase reporter gene expression and EMSA experiments. In addition, the mutated LIF failed to inhibit the proliferation of M1 cells. The data indicated that the mutation of LIF did not have a dominant negative effect but lost the biological functions, suggesting that the 29th amino acid is critical for maintaining the activities of LIF.

Abstract:LRRC4 is a novel brain relatively specific gene and a member of LRR superfamily, which displayed significant down-regulation in primary brain tumor biopsies and has the potential to suppress brain tumor growth. The establishnent of LRRC4 with doxycycline (Dox) induced Tet regulating system in U251 glioblastoma cell line was reported. Firstly, Tet-on regulating plasmid was transfected into U251 cells and screened by G418 to construct the single-stable U251 Tet-on cell line. Low background and high expression clone was acquired by testing luciferase activity. Successively, pTRE-2hyg/LRRC4 plasmid was transfected into the clone and screened by hygromycin. Two positive clones were received by RT-PCR and Northern blot analysis. The positive clones showed well dose-response and time-response in expression of LRRC4 with Dox inducement. Results show that establishment of LRRC4 with doxycycline induced Tet regulating system in U251 glioblatoma cell line is successfully, which provides an ideal experimental platform for understanding the mechanism of LRRC4 in glioma tumorigenesis and development.

Abstract:In order to search for new effective Schistosoma japonicum vaccine candidate genes and study its immune protection against challenge infection in mice, S. japonicum adult worm cDNA library was screened with sera from rabbits immunized with S. japonicum female worm antigen. The novel gene (designated Sj-F1, GenBank accession number are AY261995) was cloned into the prokaryotic expression vector pTWIN1 and the eukaryotic expression vector pcDNA3 respectively. The positive recombinants were identified by PCR and restriction enzyme digestion. The plasmid pTWIN1/Sj-F1 was transformed into E.coli ER2566. The soluble recombinant fusion protein (rSj-F1/intein2) was expressed in E. coli under low IPTG concentration and low temperature, then analyzed by SDS-PAGE and Western blot. The plasmid pcDNA3/Sj-F1 was transformed into E.coli ER2502 for preparing DNA vaccine. Mice were immunized with rSj-F1 protein or/and pcDNA3/Sj-F1 DNA vaccine. Two weeks after the third vaccination, a challenge infection was carried out with S. japonicum cercariae. Worms and eggs collected from the livers of mice were counted 42 days after challenge infection. Levels of specific antibody were detected by ELISA before infection. Immunization experiment showed the recombinant Sj-F1 protein with FCA adjuvant or with chitosan adjuvant provided 28.07%, 24.69% worm reduction rates and 48.30%, 46.38% egg reduction rates in mice respectively. The naked pcDNA/Sj-F1 provided 18.47% worm reduction rate and 35.06% egg reduction rate. The worm and egg reduction rates were increased to 40.42%, 42.38% and 56.17%, 62.87% respectively when mice were immunized with the pcDNA/Sj-F1 at priming and with rSj-F1 by subcutaneously or intranasally at booster. The results suggest that both recombinant Sj-F1 protein and naked DNA can induced partial protection immunity against S. japonicum infection and the protection can be enhanced via DNA priming and rSj-F1 boosting.

Abstract:Nasopharyngeal cancer (NPC) is a type of head and neck cancer and occurs in the uppermost region of the throat situated behind the nasal cavity.98% of them are poor differential nasopharyngeal squamous carcinoma.75% of NPC were found with enlarged lymph nodes in the neck, and also usually cranial nerve dysfunction (usually Ⅱ ~ Ⅵ or Ⅸ ~ Ⅻ ). CNE2 is a kind of poor differential nasopharyngeal squamous carcinoma cell line, which existed some vital characteristic of poor differential nasopharyngeal squamous carcinoma. High malignant NPC tissues (in Ⅳ -stage, poor differential nasopharyngeal squamous carcinoma) and high malignant poor differential nasopharyngeal squamous carcinoma cell line CNE2 as the test samples were chosed and good 2-DE patterns of the 6 cases nasopharyngeal squamous carcinoma tissues and poor differential pharyngeal were established squamous carcinoma cell line CNE2 proteins with high resolution and good reproducibility using Immobilized pH gradient two-dimensional technology. Total 145 protein spots (66 spots in the tissues and 79 spots in the cell line) were analyzed by MALDI-TOF-MS, and 74 protein spots were identified. 3 proteins of the identified proteins were found to over express in poor differential pharyngeal squamous carcinoma cell line, as well as in 6 cases Ⅳ -stage nasopharyngeal squamous carcinoma tissues. Their mRNAs in cell line CNE2 and in 14 cases of human nasopharyngeal squamous carcinoma tissues were tested by RT-PCR. All mRNAs of TIM1 、 DJ1 and NM23-H1 were expressed both in cell line CNE2 and in tissues (3 cases in Ⅳ -stage, 1 cases in Ⅲ -stage and 1 cases in Ⅱ -stage, but none of 6 cases in chronic epithelium tissues), and one of 6 cases chronic epithelium tissues did expressed none of TIM1, DJ1 and NM23-H1 mRNAs. mRNAs of nm23-H1, DJ1 and TIM1 in Ⅲ , Ⅳ -stage NPC tissues were expressed simultaneity, which were compared with chronic nasopharyngitis epithelium tissues, were existed the evidently difference (x2=10.214 ， P＜ 0.005). The over expressions of nm23-H1, DJ1 and TIM1 in both the NPC tissues and the cell line may be correlated with the pathogenesis of poor differential nasopharyngeal squamous carcinoma.

Abstract:In order to evaluate the immunogenicity and protective efficacy of tetravalent combination M.tuberculosis DNA vaccine, DNA vaccines encoding Ag85B, MPT64, MPT70 and PstS-3 protein were constructed with eukaryotic expression vector pJW4303. Combination DNA vaccines were inoculated intramuscularly into C57BL/6 mice three times at 3 weeks interval. After 21 days of the third injection, the specific antibody titers against the four antigens were 1∶6 400, 1∶51 200, 1∶6 400 and 1∶6 400. Meanwhile the mixed spleen cells could produce the high antigen-specific IFN-γ level in response to the four antigen proteins, IFN-γ level of Ag85B, MPT64, MPT70 and PstS-3 reached 10 582.14 ng/L, 13 635.97 ng/L, 14 213.15 ng/L and 9 657.35 ng/L respectively. After the last injection, mice were challenged with M.tuberculosis H37Rv. When compared to negative group, the bacterial counts of lung and spleen from the mice vaccinated with tetravalent combination vaccine were reduced about 650 and 130 folds. Microphotographs showed clearly that lungs of mice vaccinated with combination vaccine were much better protected against Mycobacterium tuberculosis challenge than negative mice. The results showed that tetravalent combination M.tuberculosis DNA vaccine elicited both T-cell and humoral immune response, and protected against tuberculosis effectively.

Abstract:Double-muscling pig is good at meat yielding, but the genetic reason for this trait was not clear till now. A SSH (suppression subtractive hybridization) library was built with cDNA from LD (longissimus dorsi) muscle of double-muscling pigs as tester, and that of non-double-muscling as driver. 686 clones in this library were single insertion. And they were all sequenced. The sequences were BLAST on line with the GenBank dbase, GenBank EST dbase and also the Tigr Porcine EST dbase, 11 of them were not matched in any of the databases which might represent new genes related to porcine double-muscling trait. 3 of the functional genes, which are RYR1, CAMK2 and IGFBP 7, were chosen firstly to do quantitative PCR to confirm the expression differentiation between double-muscling LD tissue and non-double-muscling pigs LD tissue. The genes expressed in the former tissue were 1.87, 1.90, 1.85 times higher, respectively, than in the later tissue. 3 clones of the new ESTs were also detected with quantitative PCR, they were 1.48, 1.44 and 1.78 times higher in the former, respectively. These results implied that new candidate genes could be selected from the SSH library constructed in this research, and this could be a way to make the genetic base of double-muscling pig more clearly.

Abstract:Recombineering is a new developed genetic engineering technology in the past few years. A new recombineering system named pYM-Red was constructed by gap-repair, that is a technology called in vivo cloning. Linear PCR fragments that amplified from low copy plasmid pACYC184 were used as gene targeting vector. The length of subcloned DNA sequence including Red gene and a series regulatory sequences are about 6.7 kb. The biology function of Red gene in pYM-Red was tested by gene replacement (galk<>kan) of W3110 chromosome. Factors that effect recombination efficiency were precisely confirmed. When induced 10 min at 42 ℃ and using 300 ng linear DNA fragment as targeting molecules, the efficiency of pYM-Red mediated recombination can reach one positive recombination clone per four thousands electroporation survived cells, it is 5~6 folds higher than pBR322-Red and pKD46 recombination system.

Abstract:Diphtheria toxin is an exotoxin secreted by Corynebacterium diphtheriae that has been lysogenized by β bacteriophage that carries the DT gene. It blocks protein synthesis and kills the target eukaryotic cell. The R82A ， K84A ， H86A mutant of vascular endothelial growth factor (VEGF) specially binds its receptor 1 (VEGFR-1) that is expressed highly on surface of tumor blood vessel. DT genomic DNA was extracted first and then the gene that coding the T domain and C domain of DT (DT391) were amplified. The R82A ， K84A ， H86A mutant were introduced to VEGF by site-directed mutagenesis. Then a VEGFR-1 targeting fusion protein, DT391-mVEGF, was constructed by substituting the receptor binding domain of DT with the VEGF mutant which shows high affinity to a receptor expressed on tumor vascular. With DT391, a protein without the mVEGF domain of DT391-mVEGF, as a negative control in cytotoxicity assay, the hybrid protein DT391-mVEGF showed an inhibition to the growth of VEGFR-1 positive tumor cell.

Abstract:In order to detect the distribution of silica-binding proteins in rice and other graminaceous plants, specific polyclonal antibodies against a silica-binding protein from rice, namely SBP117, are successfully raised by synthesized peptides which are conjugated with Keyhole Limpet Hemocyanin and used as antigens to immunize rabbits. Western blot and the immunoblot results indicate that the antibodies not only can react with silicon-binding proteins of rice, but also can cross react with the proteins of other silicon-accumulated graminaceous plants, while it does not react with the proteins of non-silicon accumulated dicotyledonous plants (such as tomato leaves) and BSA. These findings indicate that the homologous proteins of SBP117 are widely existed in the graminaceous plants. Furthermore, tissue printing study shows that SBP117 is mainly located at the epidermis of roots, shoots and leaves as well as in the vascular bundle of the rice roots and leaves. The distribution of SBP117 in rice plants is coincided with the sites of Si accumulation in rice reported previously. Therefore it is concluded that the silicon-binding protein (SBP117) may be involved in the control of silicon deposition in rice plants.

Abstract:RNA interference (RNAi), which could silence specific gene expression post-transcriptionally, has become a powerful tool for identifying gene function in eukaryotic cells. One important approach of RNA interference is to construct a vector system which can direct the synthesis of small interfering RNAs (siRNA) in cultured mammalian cells. The H1 RNA promoter (374 bp) was cloned from HepG2 genome DNA. Two RNAi vector systems, pSL and pESL which has an EGFP gene, were constructed and used to knock down the expression of p53 gene. Then, the mRNA and protein expression of p53 gene were detected by semi-quantitative RT-PCR and Western blotting after the RNAi plasmids were transiently transfected to the HepG2 cells. It turned out that the RNAi efficiency of pSL and pESL are much higher than that of pSilencerTM 3.1-H1 hygro RNAi vetor. Therefore, the data suggested that pSL and pESL RNAi vector systems, which could suppress specific gene expression with high efficiency, are new useful tools to identify gene functions in cultured mammalian cells.