Abstract:Cell-cell recognition is the key for multicellular organisms to survive. This recognition critically depends on protein-protein interactions from opposing cell surfaces. Recent structural investigations reveal unique features of these cell surface receptors and how they interact. These interactions are specific, but usually relatively weak, with more hydrophilic forces involved in binding. The receptors appear to have specialized ways to present their key interacting elements for ligand-binding from the cell surface. Cell-cell contacts are multivalent. A large group of cell surface molecules are engaged in interactions. Characteristic weak interactions make possible for each individual molecule pair within the group to constantly associate-dissociate-reassociate, such that the cell-cell recognition becomes a dynamic process. The immunological synapse is a good example for immune receptors to be orchestrated in performing immunological function in a collective fashion.

Abstract:Tissue acidosis can directly induce pain sensation which is mediated by ion channels in peripheral sensory neurons. To date, several ion channel superfamilies are identified to be involved in the pain from tissue acidosis. (1) ASICs can be gated by proton and directly mediate the acidosis pain; (2) VR1 can be sensitized by acidosis as well as activated by pH＜6.0; (3) P2X2 receptor is up-modulated whereas P2X3 is down-modulated by acidosis; (4) TASK is one of the tandem pore and the inwardly rectifying K⁺ channel which can be closed by acidosis.

Abstract:The molecular basis of the processes of cholesterol absorption and plasma plant sterol levels is only partially understood. A breakthrough in understanding the molecular basis of sterol absorption has recently achieved by the discovery of ABCG5 and ABCG8. The genes involved in cholesterol absorption and plant sterols metabolism are characterized by a spectrum of specificity. Studies on a recently developed novel cholesterol absorption-blocking agent, ezetimibe, provide additional insight into the genetics of cholesterol absorption and plasma plant sterol levels. Focuses on the evidence for genetic control of cholesterol absorption and plasma plant sterol levels are reviewed. The commonalities and difference in the regulation of these two traits are examined and the recent developments and future perspectives in this field are discussed.

Abstract:To explore the effect of acute hypoxia on tau phosphorylation, rat brain slices were incubated at 37℃ for 30 min or 120 min with or without oxygen supply. Then tau phosphorylation was detected by Western blots. The results showed that significant dephosphorylation of tau at Ser-396/Ser-404, Ser-422 and Ser-199/Ser-202 was induced by acute hypoxia. The activity of protein phosphatase-2A (PP-2A) and the expression of PP-2A catalytic subunit were simultaneously increased. The results suggest that acute hypoxia induces dephosphorylation of tau and the up-regulation of PP-2A may be at least one of the underlying mechanisms.

Abstract:In order to study the effect of microfilament cytoskeleton on SHEEC esophageal cancer cells and the biological activity variation of cancer cells caused by antisense blocking of NGAL gene expression, making use of antisense expressing vector of different length NGAL gene segments and antisense oligonucleotide segments of thio-modification to transfer the SHEEC cancer cells of esophagus, a series of subcellular clones aiming at the blocking of NGAL gene expression of SHEEC esophageal cancer cells were established by means of G418 selection. On the basis of the fluorescent dual-labeling of F-actin and DNA within cells and after antisense blocking of NGAL gene expression, the content of F-actin and DNA, the F-actin cytoskeleton structure in SHEEC cell and the variational characteristics of biological action on the tumor cell were tested by use of flow cytometry and confocal scanning laser microscope (CSLM). The results showed that F-actin level in the SHEEC cells was obviously reduced after antisense blocking and it was similar to that in SHEE cells, but no obvious change of cellular division index was found. This indicates that the blocking of NGAL gene expression has an obvious effect on the microfilament cytoskeleton of SHEEC cells, but no obvious influence on SHEEC cell proliferation. With the technique of CSLM, it is revealed that the blocking of NGAL gene expression could make the cellular F-actin cytoskeleton of SHEEC cell uniformly distributed, F-actin body evidently decreased, the joint structure of the cell reestablished, the cellular structure more compact, and the main structure characteristics of SHEEC cell and those of F-actin cytoskeleton structures of SHEE cell consistently approached. The result suggests that SHEEC cellular F-actin cytoskeleton of the esophagus cancer could be obviously affected by antisense blocking of NGAL gene expression, and it is conjectured that microfilament cytoskeleton F-actin in cancer cell could be a function key of NGAL gene that plays a role in SHEEC cancer cell.

Abstract:The gene PC-1 was identified as a novel gene that expressed higher in the much more malignant human prostate cancer cell lines and its translation product meets some points of transcription factors. In order to study the transcriptional activation activity of PC-1，firstly, the yeast two-hybrid system was used and the whole length as well as various regions of PC-1cDNA were cloned into the expression vector pAS2-1 and transfected into the yeast cell line CG-1945 respectively, the result of the reporter genes lacZ and His3 activation assay showed that PC-1 had transcriptional activation activity and this activity was mapped within its N terminal 46 amino acids. Then，the various regions of PC-1 cDNA were cloned into the expression vector pZHO1 and cotransfected with the pTRE-luc plasmid of the reporter gene into COS7 and C4-2 mammalian cells, the 46 amino acids in the N terminal of PC-1 were found to have transcriptional activation activity by the firefly relative luciferase activity assay.

Abstract:In order to study the effects of mechanical strain on distribution of integrins in normal human pulmonary epithelial cell line H727, a cyclic strain unit in vitro was set up, and the redistribution of α₃,α₅,β₁ integrin in H727 cells subjected to 24 h cyclic strain was analyzed. Human pulmonary epithelial H727 cells seeded on either fibronectin or collagen Ⅳ surfaces were subjected to 15% elongation at a frequency of 40 cycles/min. Confocal microscopy revealed that α₃,α₅,β₁ integrin in H727 cells were concentrated and brighter,and cells created a large fusion of focal adhesion or adhesion plaques after 24 h exposure to strain. Under static condition, α₃,α₅,β₁ integrin in H727 cells maintained a diffuse pattern with an occasional speckle concentration in some regions. Furthermore α₃,α₅,β₁ integrin in H727 cells transferred from the apical layer to the basal layer and created adhesion plaques, and affinity between integrins and extracellular matrices(ECM) was enhanced. It is concluded that the formation of effective focal contacts is the concerted interaction between receptors recruitment and ligand occupy. This interaction plays an important role in transducing mechanical stimuli into intracellular signals.

Abstract:In order to identify whether EB virus latent membrane protein 1 (LMP1) can regulate vascular epithelial growth factor (VEGF) expression via STAT3 in nasopharyngeal carcinoma cell line, Western blotting was used to detect VEGF expression in HNE2, HNE2-LMP1 and HNE2-LMP1 transient transfected with STAT3 dominant negative plasmid STAT3β. VEGF is up-regulated by LMP1 and STAT3 dominant negative STAT3β can down-regulate VEGF expression. In tet-on-HNE2-LMP1 cell line, in which LMP1 can be tightly regulated, VEGF is expressed in dose and time dependent fashion. After cotransfected LMP1 with VEGF wild promoter luciferase reporter and 848 mutated luciferase reporter respectively, luciferase assay results indicated that LMP1 can up-regulate wild promoter luciferase activity and not that of 848 mutated luciferase reporter. Electrophoretic mobility shift assay results in HNE2 and HNE2-LMP1 cell line indicated that LMP1 enhances STAT3 binding ability in VEGF promoter. In conclusion, latent membrane protein 1 encoded by EB virus can enhance VEGF transcription and expression in nasopharyngeal carcinoma cell line via activating STAT3 binding ability in VEGF 848 promoter binding site.

Abstract:Ultrastructure of nucleolar DNA is most important to elucidate transcription site of rRNA. Many judgments were made in this respect but all of them were based on direct experimental observations. A new technology for the quantitative analysis of the location of nucleolar DNA in situ from EM data of the ultrastructure of the nucleolus in allium sativum cells and from the modified NAMA-Ur DNA specific staining method was developed. For this purpose, a new methodology of multi-scale morphological gradient operator and automatic analysis software named “HEREN.CELL” were created and adopted. By using this method, superfine contour of the nucleolar ultrastruture was obtained and showed the location and location of nucleolar DNA in situ. From the results, a “petaline envelope” model was proposed, which indicated that nucleolar DNA fibrils radiate from center to periphery of the nucleolus. The study will contribute to the development of the EM image processing technology and the study of nucleolar ultrastructure of the nucleolus.

Abstract:The immunocompetent tandem fragment FB of the foot-and-mouth disease virus(FMDV) genome was inserted into the expression vehicle pBAD/TOP, to yield an identified recombinant plasmid pBAD-FB, which was used to transform the host bacteria TOP10 and, after induction with different concentrations of the inductant arabinose for varied duration, samples of the expression product were subjected to SDS-PAGE and Western blot analysis. Results revealed that using a final concentration of 0.002% arabinose for induction, expression peaked at 4 h, yielding a product approximately 26 ku in size. Software scanning demonstrated that the FB fusion protein expressed accounted for 28.9% of total bacterial protein, could react specifically with FMDV antibody, and occurred both in the form of inclusion bodies and soluble protein. The soluble fraction of the fusion protein was purified with 50% Ni-NTA resin affinity chromatography, and the fusion protein inclusion bodies extracted. After washing both fractions were separately used to prepare oil-emulsion vaccines, with which guinea pigs were immunized subcutaneously. Neutralization test in suckling mice was employed to assess the neutralization index of the guinea pig serum, and foot-and-mouth disease virus was used to challenge the guinea pigs. The result proved that when the purified soluble product and inclusion bodies were used to immunize guinea pigs, they could induce a high titer of neutralizing antibodies, and provide 100% immunoprotection against virus challenge.

Abstract:SNF2 family includes proteins essential to genome replication, repair, and expression. The cDNA cloning and initial characterization of a novel mouse member of this protein family was reported, and the new member was designated as Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like) by MGNC. The cDNA of Ercc6l was 4 002 bp and firstly cloned in silico and then identified by RT-PCR in mouse embryo heart. This gene was composed of 2 extrons and mapped to mouse chromosome X. The longest ORF encoded a putative protein of 1240 amino acids. Eight highly conserved sequence regions of SNF2 protein were present in the deduced protein. Alignment against members of SNF2 family initially placed Ercc6l into ERCC6 subfamily of SNF2 family. EGFP-tagged Ercc6l protein was localized in cytoplasm of HeLa, NIH3T3 and B16 cells. BLAST search in EST database retrieved 69 homologous ESTs of Ercc6l, most of which were from mouse embryonic and tumorous tissues. RT-PCR performed on a panel of different mouse tissues demonstrated that Ercc6l strongly expressed in mouse embryo but significantly downregulated in neonate and adult normal tissue. These results suggest that Ercc6l is a novel member of SNF2 family and may be crucial for development of embryo and tumor.

Abstract:A comparative analysis of the occurrence frequency of oligonucleotides in two sets of yeast genes with higher and lower transcription frequencies respectively has shown that the sequence structures of the two sets of introns are different. There are more potential binding sites in the introns of genes with higher transcription frequencies. After observing regulatory sites obtained by experimental analysis, many transcriptional regulatory sites in yeast consist of a pair of highly conserved oligonucleotides, spaced by a non-conserved region of fixed width (dyad). Therefore, dyad-like transcriptional regulatory sites are analyzed. Some dyads are extracted by statistical comparative analysis of the occurrence frequencies, whose occurrence frequencies in the set of introns with higher transcription frequencies are higher significantly than those in the set of introns with lower transcription frequencies. Analyzing the distribution of the extracted dyads in two sets of introns, and comparing with the regulatory sites revealed by experiments, these dyads are probably related to positive transcriptional regulation.

Abstract:In order to improve exon level sensitivity and specificity of recent gene-finding programs, strong “search by signal” components are needed to identify splice sites, translation start and other biological signal sites. A new model for the identification of 3′ splice sites (acceptors) using Hidden Semi-Markov Model (HSMM) was introduced. This model is proved to be particularly suitable for modeling the biological structure of acceptors. When tested in Burset/Guigo dataset, this new method demonstrated an improved accuracy compared with existing method. The success of this model gives a deep understanding of the structure of acceptors and the biological process of splicing.

Abstract:Two major β-glucan binding proteins (β-GBPs), 75.9 ku and 83.2 ku, were extracted from the hemolymph of Plutella xylostella larvae by using β-1,3-glucan affinity precipitation method. The proteins were present in plasma but absent in haemocytes. The β-GBPs had a specific affinity to β-1,3-glucan, a prophenoloxidase (ProPO) trigger in P.xylostella hemolymph. The phenoloxidase (PO) activity induced by the coexistence of both β-GBPs and β-1,3-glucan was significantly higher than that by the β-GBPs or β-1,3-glucan alone. Moreover, the PO activities induced by mycelial lysates of four Zoophthora radicans isolates at the presence of β-GBPs were much higher than that triggered by their protoplast lysates. The results indicate that the β-GBPs may trigger the ProPO activity in P.xylostella hemolymph only if they specifically recognized β-1,3-glucan from Z.radicans cell walls. This is a proof for a hypothesis that entomophthoralean protoplasts may evade from insect host immune defense. The protoplast or mycelial lysates of different Z.radicans isolates differed in ability to trigger the PO activities in the following order: ARSEF1342＞ARSEF2699＞F99101＞ARSEF1100. This trend was in accordance with that of their virulence to P.xylostella larvae, suggesting a possible relationship between host immune defense and fungal virulence.

Abstract:NPC-related ESTs were selected from UniGene bank. According to nr and EST database BLAST results, BG231197 was found to be a sequence that represents a new gene. The EST contig does not have complete ORF. NAP1 gene cDNA was got from normal nasopharynx tissue cDNA by PCR through searching Human Genome Draft. The Northern blot analysis revealed one transcript (0.6 kb) in lymph node and trachea. GenBank Accession number of the gene is AY190326. The length of NAP1 gene cDNA is 573 bp, ORF is from 113 bp to 370 bp. NAP1 encodes a 9 700 u protein composed of 85 amino acids. The protein resembles to FDC-SP secreted by FDC (AF435080). Bioinformatics analysis suggested that the protein is a secreted protein. NAP1 gene is located at chromosome 4q13. Genome spans 9 179 bp, containing 5 exons and 4 introns. The difference of NAP1 expression between nasopharyngeal carcinoma (NPC) and nasopharynx(NP) tissues of 40 cases was tested by differential RT-PCR. Low expression occurred in 17 NPC cases. High expression occurred in 6 NPC cases. Expression difference did not occur in 17 NPC cases. In situ hybridization (ISH) and immunohistochemistry (IH) suggested that it express in dentritic cells of NP and NPC tissues. It is meaningful to do further studies on the reason of low expression in NPC tissue and the function of NAP1 gene.

Abstract:Granzyme B (GrB) is an important serine protease involved in granule-mediated killing in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In order to study whether ectopic expression of GrB in tumor cells can induce cell death, expression vectors encoding active GrB (GrBa) and mutant GrBa (mGrBa) gene in which serine in catalytic triad was replaced with cysteine were constructed, and transiently transfected into HeLa cells with lipofectamine. It was shown by GFP coexpression, indirect immunofluorescence, cell counting and MTT analyses that ectopic expression of GrBa genes caused increased tumor cell size and multinucleation, and the growth of cells that expressed GrBa proteins was inhibited. Retarded growth was further observed in these morphologically abnormal cells isolated by 40% percoll, which directly contributed to growth inhibitory effect on GrBa-transfectants. There was cytoskeletal breakdown and abnormal mitosis characteristic of multiple spindle poles, largely resulting in accumulation of giant multinuclear cells. These results suggest that GrBa may serve as a good candidate in tumor gene therapy.