Abstract:Blood plasma is a complex body fluid that contains various proteins and small molecules including peptides, salts, lipids, amino acids and sugars. Plasma proteins perform different kinds of housekeeping functions, such as immunoresponse, coagulation, anticoagulation, transportation, nutrition and regulating signaling cascades, which normally are altered both in structure and amount during pathogenesis. These characteristics of plasma proteome are critical for disease diagnosis and therapeutic monitoring. Nevertheless, only a handful of proteins are currently well known and used in routine clinical diagnosis. A comprehensive, systematic characterization of circulating proteins and peptides in health and disease will greatly facilitate development of biomarkers for many human diseases. The human proteome organization has launched the Human Plasma Proteome Project as one of the major initiatives since April 2002. In the pilot phase of the project, as a “pathfinder” of human proteomics study, the major goals are (1) to compare the advantages and limitations of many technology platforms; (2) to compare reference specimens of human plasma (EDTA, heparin, citrate-anti-coagulated) and serum prepared specifically for this project with the technology platforms; and (3) to create a knowledge base. The brief introduction about the plasma proteome project including research status, major problems faced and technical strategies been used are reviewed.

Abstract:Adipose tissue is not only an energy storage organ but also an important endocrine organ. It assists nerve system and other endocrine organs to maintain body homoeostasis. In recent years, some studies suggested a tight relationship between adipose tissue and immunity. Leptin, a hormone secreted by mature adipocyte which functions in regulating not only energy metabolism and body fat mass but also the immune effects of monocyte, macrophage, and lymphocytes. Adiponectin, another factor secreted by adipose tissue，can also regulate the immune responses of cells. In addition, immune stimulation can induce lipolysis of fat depot around lymph node. The interaction between adipocyte and immune system further demonstrates that the living body is an intergrated organic unity. The advancement in this area of study would likely provide new perspectives to therapeutic methods on related diseases.

Abstract:Riboswitches are metabolite-binding RNA structures that serve as a novel gene regulatory element for mRNAs. Located in certain mRNAs, they can directly bind relevant small metabolites but not protein factors, and subsequently allosteric rearrangements modulate associated mRNA activities. They are believed to regulate in a wide set of fundamental metabolic pathways including vitamin B₁₂ and methionine biosynthesis in bacteria. The recent discovery of natural riboswitches, especially its property that precisely controlling basic metabolic pathways via binding to certain ligands with high affinity and specificity, inspires scientists to notice its potential application in medical research. The progress on the riboswitches, its characteristics, the function mechanism as well as the thinking and research trend triggered by its finding were introduced.

Abstract:In an attempt to get insight into the biological functions of p93 in the cardiovascular system and its potential roles in the signaling pathway, a yeast two-hybrid technology was carried out to screen the adult heart cDNA library using the N-terminal fragment of p93 (101～372 aa) as bait. Peroxiredoxin 3 (PRX 3) was identified as a novel p93 binding protein. Subsequently, the interaction was confirmed by in vitro binding assay and co-immunoprecipitation and p93 was showed to co-localize in part with PRX 3 in HEK-293FT cells by immuofluorescence. Taken together, these observations suggest that p93 may participate in various pathophysiologic processes mediated by PRX3 in cardiomyocyte, such as cell growth, differentiation, development，apoptosis and oxygen stress.

Abstract:The class A scavenger receptor (SR-A) is a glycoprotein expressed on the cell surface of macrophages that mediates internalization of chemically modified lipoprotein. It was reported that the receptor internalization required the presence of internalization signal motif and the rate of receptor internalization was governed by the pattern of receptor phosphorylation induced by the ligands. However, the role of the cytoplasmic domain played in the receptor-mediated endocytosis is not fully characterized. Here the changes in internalization process of the receptor were reported when the whole cytoplasmic domain sequence (150 base pairs) was truncated. Both the full length and truncated were recombinated into PcDNA3.1/HisB vector and were then transfected to CHO cells separately. Measurement of uptake of DiI-AcLDL by transfected cells with FACS showed that the bind and uptake of the ligand in full length SR-A was higher than that of truncated receptor(1.3 fold increase).After incubated with DiI-acetyl-LDL (DiI-AcLDL), the full length SR-A-transfected cells showed a diffuse distribution of the DiI-AcLDL in cytoplasm as well as in cell membrane when monitored under laser confocal microscopy. But in the truncated SR-A-transfected CHO cells, DiI-AcLDL mostly distributes at the cell surface only. In order to elucidate the role of phosphorylation played in mediating the function of cytoplasmic domain of SR-A, transfected CHO cells were preincubated with Staurosporine for 1 h at the concentration of 0.4 μmol/L. Then the cells were refed with medium containing DiI-AcLDL at the concentration of 10 mg/L at 37℃ for 2 h, the DiI specially associated to cells was measured by spectrofluoremeter. The result indicated that staurosporine did not changed DiI-AcLDL bound and untaken by truncated receptor, which was different from the full length SR-A that increased obviously. The research here demonstrated that cytoplasmic domain regulate the receptor activity of SR-A, in which the phosphorlation or dephosphorlation of the cytoplasmic domain might play a key role,the MSR-A cytoplasmic domain may be indispensable in mediating binding and uptaking as well as internalization.

Abstract:Three cloned cattle from the same somatic cell line (TT, QQ, XW) and three transferred cloned cattle from the same gene transferred cell line (JM, LW, 8C2), together with two randomly selected cattle, one LUXI (LX) and one Holstein (HS), were genetically analyzed using 24 fluorescent microsatellite markers. There were 1～5 alleles at 24 loci, the average being 3.17. Among TT, QQ, XW, JM, LW, 8C2, the somatic cell line and the gene transferred somatic cell line, the probabilities of matching (P_M) between individuals were 1.17×10^-36 using allele frequencies obtained from public data on internet and 1.90×10^-23 by defining the allele frequencies at each locus as the reciprocal of the number of alleles at that locus. In addition, their genotypes differed from those of the randomly selected LX and HS at 23 and 20 marker loci respectively.

Abstract:To adapt to the hierarchical structural property of Gene Ontology, the standard Apriori algorithm is modified into a novel algorithm, RuleGO, which mines association rules of GO function classes and gene expression difference. The inputs of RuleGO are one set of differential expressed genes and another set of non-differential expressed genes, and the outputs of RuleGO are association rules linking GO function combinations to gene differential expression. Rules mined by RuleGO may guide insights into gene expression difference at the functional level, towards the clarification of the process of pathological changes or the mechanism of medicine. Both RuleGO and OntoExpress are applied to the datasets of colon cancer and adenocarcinoma, and RuleGO turned out to be more powerful to mine relevant function rules than OntoExpress. The experimental results also reveal that rules with both high significance and high support mostly involve more than one gene function classes, suggesting that considering the combination of multiple gene function classes may be more resonable in gene expression analysis than taking into account only a single gene function class.

Abstract:The initial barrier to the transplantation of pig organs to human is hyperacute rejection (HAR). HAR was initiated by the conjugation of human natural antibodies (XNA) with the Gal-α1,3-Gal which is thought to be the major xenoantigenic epitope present on pig tissues. Removal of antibodies directly against that structure may be critical to the success of pig to human xenotransplantation. The lectin GS-I-B₄ was used to screen phage-displayed peptide library XCX₁₅ and identified a peptide mimetic of Gal-α1,3-Gal. A phage bearing the peptide SCTALSFPSFAFLARGT has been identified to bind human natural antibody strongly. This binding reaction can be competitively inhibited by melibiose. The peptide can also inhibit the human natural antibody-mediated agglutination of pig RBCs.

Abstract:Helicobacter pylori is one of the most common infectious pathogenic agents in human being which causes gastritis, peptic ulcer disease, gastric adenocarcinoma and malignant tumour of the stomach. ure B and hsp A gene were cloned and fused into a shuttle vector pADTrack-CMV. The resultant plasmid was linarized by digesting with restriction endonuclease PmeⅠ, and subsequently were contransformed into E.coli. BJ5183 with an adenoviral backbone plasmid PAdEasy-1. The linearized recombinant plasmid was transfected into adenovirus packaging cell lines HEK-293. The recombinant adenoviruses were typically generated within 7 to 12 days. The recombinant adenoviruses titer was monitored by GFP expression. ELISA was used to test whether adenoviruses efficiently express the Ure B-Hsp A antigen protein in HEK-293. To determine the immunogenicity of the recombinant adenovirus, the delayed type serum IgG, secreted IgA, in the immunized mice were tested. The results showed that both humoral and mucusal immune response in mice were induced.

Abstract:Parkinson's disease (PD) is a neurodegenerative disease due to lack of dopamine (DA) in nigrostriatal system resulting from the degeneration and necrosis of dopaminergic neurons. No effective cure has been found. Neurturin (NTN) has been demonstrated to protect mesencephalic dopaminergic neurons specifically. Parkinson's disease was induced in rhesus monkeys by injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）. Rhesus monkeys were randomly divided into a PD model, NTN treatment and normal control groups. In the NTN treatment group, 1 mg of Pichia pastoris -derived recombinant human NTN was injected into the cerebral ventricles 48 h prior to injection of MPTP. Rhesus monkeys in the PD model group acquired PD symptoms that progressed over time, while monkeys treated with NTN had less apparent or no symptoms. Using fluorospectrophotometry, the dopamine (DA), 5-hydroxytry-ptamine (5-HT) and the 5-hydroxyindoleacetic acid (5-HIAA) in substantia nigra, putamen and caudate nucleus in monkeys from the model group were found to be significantly lower than in the normal control group. No significant differences were found between the NTN treatment and normal control groups, but the contents of DA, 5-HT and 5-HIAA in the NTN treatment group were higher than those observed in the PD model group. A dramatic loss of neurons in the substantia nigra in monkeys in the PD model group was observed by light microscopy, while no obvious loss was observed in the NTN treatment group in which the numbers of neurons were similar to those in normal controls. These results indicate that Pichia pastoris -derived recombinant human NTN can prevent PD symptoms as well as protect dopaminergic neurons and preserve DA content in midbrain substantia nigra in rhesus monkeys exposed to MPTP.

Abstract:To investigate the expression of suppressor of cytokine signaling 3（SOCS-3）in Balb/c mice brain and the association with brain lateralization, the paw preference test was used to select right-pawed, left-pawed mice. The SOCS-3 expression level was detected by RT-PCR. The results showed that,（1）The SOCS-3 expression level in right cortex was higher than that in left cortex of left-pawed mice (P＜0.05) and it was also higher in the left cortex of right-pawed mice than that in the left cortex of left-pawed mice(P＜0.05),（2）The SOCS-3 expression level in right hippocampus was higher than that in the left hippocampus of left-pawed mice (P＜0.05) and it was also higher in the left hippocampus of right-pawed mice than that in the left hippocampus of left-pawed mice(P＜0.05),（3）The SOCS-3 expression level in hypothalamus of right-pawed mice was higher than that of the left-pawed mice (P＜0.05). These results indicate that the SOCS-3 expression level in Balb/c mice brain is associated with brain lateralization.

Abstract:Fluorescence polarization template-directed dye-terminator incorporation(FP-TDI) is a simple, cheap and high throughput SNP (single nucleotide polymorphism) genotyping technology, but the quality of genotyping pictures cannot be evaluated, thus the reaction condition is very difficult to optimize. A numeric standard which can efficiently evaluate the SNP scoring picture was introduced, based on this standard, the reaction system was optimized. Using this method, randomly selected 337 SNP locus of human chromosome 3 were applied to high throughput SNP genotyping, the first time success rate reached 59.94%.

Abstract:Factor C is a serine protease enzymogen presented in the circulating blood cell of horseshoe crab. Owing to its extreme sensitivity to endotoxin, Factor C (FC) plays an important role in pyrogen-detection in pharmaceutical products. Previous study has shown that three Sushi domains (S123) in the N-terminal of FC are critical for its LPS-recognition activity. The effect of three other domains in N-terminal of FC, namely Cys-rich, EGF-like and Lectin-like, on its LPS-neutralization function was not clear. The recombinant FC and its four truncated fragments were expressed using Bac-to-Bac baculovirus expression system in Tn cells. The five recombinant peptides were then purified and tested for the LPS-binding activity and bactericidal activity in vitro. The experiments showed that the LPS-binding site of Factor C resides in the S123 region. Even though Cys-rich, EGF-like and Lectin-like domains do not harbor LPS-binding site, the presence of all three domains can improve LPS-binding activity of S123. Recombinant peptide rCES123L containing Cys-rich, EGF-like, S123, and Lectin-like domains exhibits almost the same LPS-binding activity as that of the full-length parental FC. Provided the fact that rCES123L has 4-fold higher production yield than that of recombinant FC in Tn cells, this peptide has a potential in biotechnology and pharmaceutical application for LPS-neutralization.

Abstract:Plant expression vectors, pBCry3A and pBC₃Vhb, containing cry3A gene and both cry3A and vhb genes respectively, were constructed. Potato leaf-explants were transformed via Agrobacterium harboring pBCry3A or pBC₃Vhb respectively. Results from PCR and genomic DNA Southern blotting analysis indicate that cry3A or cry3A+vhb gene have been integrated into the genome of the transformed potato lines with 1～3 copies at least for cry3A gene. The transgenes were stable and expressed even after three successive clonic propagations. Results from ELISA analysis showed that the highest expression level of Cry3A protein reached 0.1% of total leaf soluble protein among cry3A transgenic plants, while that of the cry3A+vhb transgenic plant reached 0.065%. RT-PCR analysis of vhb mRNA and waterlogging test demonstrated that the transgenic potato plants transformed with cry3A+vhb genes expressed vhb gene at least at transcription level and showed obvious higher tolerance against low-oxygen stress. These results together with that of the ELISA for Cry3A indicate that this kind of transgenic plants could be valuble in application for insect resistance and waterlogging tolerance.

Abstract:The xylulokinase gene XKS1 was cloned from Saccharomyces cerevisiae NAN-27 and ligated into plasmids pMA91 and YEp24, producing pMA-xy203 and YEpP-xy204, respectively. In both plasmids, XKS1 was under the control of PGK promoter. pMA-xy203 was transferred into the pre-constructed recombinant yeast strain H158-XR-XDH, which contains the XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase and xylitol dehydrogenase respectively, in an episomal plasmid vector. This new recombinant strain was named HSXY-251. YEpP-xy204 was transferred into the pre-constructed recombinant yeast strain H158-XI, which contains the xylA gene from Thermus thermophilus encoding xylose isomerase in an episomal plasmid vector, resulting in recombinant strain HSXY-252. The xylulokinase activities in HSXY-251 and HSXY-252 were respecfively 14 and 6.7 times higher than that in the parent strain. Glucose and xylose co-fermentation carried out with HSXY-251 under oxygen-limited conditions at 30℃ resulted in 9.4 g/L ethanol concentration with 12.4 g/L xylose consumed. Xylose consumption and ethanol production were respectively 120.9% and 36% higher than in the parent strain. Furthermore, the by-product xylitol was 0.7 g/L, a decrease of 84.9%.

Abstract:NDM-1, the cucumber mosaic virus containing a satellite RNA depresses the accumulation of helper viruses and attenuates the symptoms induced under field conditions. The kinetics of the accumulation of helper virus RNA and satRNA in infected tissues was studied in 4 different hosts. Dot blot hybridization was used in the quantitative analysis to compare the disciplinarian changing of viral RNA and satRNA at 5,10 and 40 d after inoculation. The analyses suggest that the relative loading of genomic RNA and satRNA of the CMV-DNM1 displayed a similar host- and time-effect trend but in different degree. The result in tomato plants is distinguishable from the other hosts: the loading of relative genomic RNA and satRNA ascended during the 5 d to 10 d post inoculation, and descended to the lowest at the 40 d. Also the NDM-1 in tomato tissue accumulated to the lowest levels among all the hosts.　To disclose the relationship of the sat RNA and helper virus of NDM-1, the ratios of the accumulation of satRNA and genomic RNA in 4 hosts were compared, which shows that the highest ratio is 15.8,occurred at 40 d in tomato. Analyses suggest that the mechanism of resistance to viral RNA is that the accumulation of satRNA of NDM-1 reduces the yield of the accumulated viral RNA. The changing disciplinarians of NDM-1 in other 3 hosts showed few differences.

Abstract:To investigate the in situ expression of the genes in large numbers of cultured cells, a high-throughput technique was established. A novel method of constructing the cell microarrays was devised and the cell microarrays including 100 spot arrays of mixture of the graft cells and paraffin from 20 kinds of cell lines was made successfully. Expression of P53, P21, PTEN and P16 protein in the cell microarrays was detected by immunohistochemistry. Meanwhile, expression of BRD7 and NGX6 mRNA in the cell microarrays was estimated by in situ hybridization. In situ expression profiles of P53, P21, PTEN, P16 protein and BRD7, NGX6 mRNA in different kinds of cultured cells were established. The cell microarrays provided a new high-throughput tool for biological function studies of the genes. The cell microarrays could be widely used the in situ analysis of the gene expression at the DNA, RNA, and protein level. In additional, the cell microarrays could also be applied to estimate drug target validations.

Abstract:Massively parallel signature sequencing, MPSS, is an open platform that reveals the expression level of virtually all genes expressed in a sample by counting the number of individual mRNA molecules produced from each gene.The MPSS process involves cloning each mRNA molecule onto the surface of a 5 μm bead. The DNA combitag sequence is attached to a fragment of cDNA. The cDNA library is hybridized to beads. After hybridization, each of the beads displays amplified copies of one and only one starting mRNA molecule.MPSS has a routine sensitivity of a few molecules of mRNA per cell and the results are in a digital format that simplifies data management and analysis. MPSS results will be particularly useful for generating the type of complete data sets that will help to identify the functionally important genes in the sample of interest.