Abstract:Avian influenza is a severe disease in poultry and human all over the world, which become more important after SARS broken. Because of the variability of the avian influenza virus in its antigenicity and pathogenicity, the control strategies for avian influenza should include early detection, the identification of the virus using advanced molecular biological technique, immunization and the establishment of national or international network for the detection of avian influenza.

Abstract:Notch is an evolutionarily conserved single-span transmembrane protein family that is used to regulate cell fate determinations during metazoan development. Notch signalling is mediated by a well-established mechanism that relies on a transmembrane ligand-induced release of the Notch intracellular domain (NICD) and the interaction of this fragment with the CSL (CBF1, Suppressor of Hairless, Lag-1) family of transcription factors within the nucleus. Level, duration and spatial distribution of Notch activity can be regulated through combination with intrinsic or extrinsic inducing factors at multiple levels including Notch-ligand interactions, trafficking and ubiquitination of Notch and ligands. The molecular components that participate in Notch signalling, central events of Notch signalling, multiple levels of Notch signal regulation and the relation with some human diseases are reviewed.

Abstract:Gene expression is regulated at different levels. RNA interference is the mechanism through which double-stranded short RNAs silence cognate genes. Recent research work demonstrates that double short RNAs can not only repress the gene expression at post-transcriptional level, but also have a close relationship with chromatin silencing by directed H3 Lys9 methylation, DNA methylation and combining with other heterochromatin proteins. The mechanism existed in RNA interference, short RNAs, chromatin modification and chromatin silencing has been disclosed.

Abstract:Fatty acids and prostaglandins, function not only through their cell membrane receptors but also through nuclear receptors to regulate gene expression. PGI₂ can interact with G protein-coupled receptor IP on the surface of cell membrane and nuclear receptor PPARs. Recently it was found that PGE₂ receptors localized not only on the cell membrane but also on the envelope of the nucleus. There exists difference in the signaling pathway and in regulation mechanism between nuclear receptor and membrane receptors.

Abstract:Hepatitis C virus (HCV) core protein is considered an important viral structural protein and has been known to regulate proliferation and apoptosis of the cells, thus, it is belived to be related to cirrhosis of liver and hepatocarcinogenesis resulting from HCV infection. To further study the relationship between HCV core protein and hepatocarcinogenesis. The eukaryote expressing recombinant plasmid vector were constructed, then transferred it into human hepatocyte-derived Chang-liver cell line by lipofectin and established the cell model expressing HCV core protein. HCV core mRNA in the Chang-liver cells was detected by RT-PCR. Expression and distribution of the HCV core protein in the Chang-liver cells were identified by Western blot and immunocytochemistry (ICC). Phenotype of the cells expressing HCV core protein altered and displayed long fusiform shape under light microscope cultured more than 20 passages after transfection, Compared with control group cells, the cells showed a markedly increased proliferation rate, and a higher variation index of DNA content. 6/6 of BALB/c-nu/nu nude mice generated tumors until 20 days after subcutaneous inoculation of the cells expressing HCV core protein. Furthermore, constitution structure of the tumors coincided with that of hepatocarcinoma. Control group nude mice did not generated tumors. Above results indicated that HCV core protein promoted the malignant transformation of the Chang-liver cells, which implied that HCV core protein was directly related to hepatocarcinogesis result from HCV infection.

Abstract:In human cells, the heterogeneous nuclear ribonucleoproteins (hnRNP) are represented by a group of polypeptides, with various molecular properties, comprizing the most abundant constituents of the cell nucleus. Autoantibodies to hnRNPs have been reported in patients suffering from different rheumatic dieseases since 1980s. Experimental evidence indicates that hnRNP complexes undergo substantial structural changes during mRNA formation and export. However, how this contributes to disease development still has to be elucidated. Here some preliminary physicochemical features of RNA-protein folding and stability patterns of newly characterized hnRNP A3 with further functional implications in development of systemic human autoimmune states are reported.

Abstract:Human MOB is a novel gene first cloned in lung tissue and lymphoma tissue. It is proposed to express predominately in brain, and be a five-pass transmembrane protein. A new expressed sequence tag (EST) (GenBank accession number: BI740300) matched completely with human MOB gene was obtained. The 2 230 bp sequence of human MOB cDNA, which contains a 1 242 bp (nt 415～1 656) open reading frame, encoding a protein of 413 amino acid residues, was successfully cloned. In addition, rat and chicken MOB cDNA sequences, which also contain a 1 242 bp open reading frame, were cloned in silico by aligning dozens of overlapping rat and chicken ESTs and cDNA sequences identified from GenBank. Human MOB was mapped on chromosome 10q11.1～11.2. Homology searches with the deduced 413 amino acid residues revealed human MOB shares 97% similarity with murine MOB, 97% with rat MOB, 91% with chicken MOB and 45%～73% with lots of hypothetical proteins of various origin. The predicted protein contains SAM domain, which has been suggested to be an evolutionarily conserved protein-binding domain that is involved in the regulation of numerous developmental processes in diverse eukaryotes and can potentially function as a protein interaction module through its ability to homo- and heterooligomerise with other SAM domains. Homology searches and domain query indicate that human MOB is a member of potential phylogenetically conserved MOB family. RT-PCR revealed that human MOB was almost expressed in all kinds of tissues and cells, which is completely consistent with the in silico expression pattern identified by bioinformatics analysis. In contrast to bioinformatics analysis and previous study, however, human MOB has widespread subcelluar expression, primarily in nuclei. Further study in HeLa cells shows that overexpression of human MOB seems not to influence cell cycle and apoptosis.Taken together, human MOB is a novel phylogenetically conserved gene, which is proposed to express in almost all tissues and cells, and has widespread subcelluar expression, primarily in nuclei. It may play a role in signal transduction and thus be involved in development regulation, but seems not to influence cell cycle and apoptosis. Further investigation remains needed.

Abstract:Using bioinformatics analysis and 5′RACE technology, a new gene Mip1 which is up-regulated during rat myocardial ishemia-reperfusion had been cloned. The full length of Mip1 was proved by RT-PCR sequencing and multiple tissue Northern blot. Bioinformatics analysis indicate that Mip1 is located in chromosome 1q12, including 5 exons and 4 introns. The full-length cDNA has an open reading frame of 1 827 bp, which encodes 608 amino acids. There is a KRAB domain at the N terminal of the hypothetical protein and 14 successive C2H2 type zinc finger domain at the C terminal of the protein. There is a bipartite nuclear targeting sequence from amino acid 277 to 193. Mip1 is expressed abundantly in brain and heart and seldom in other tissues. It is worthwhile to further study the biological functions of Mip1.

Abstract:In order to understand the characterization and genetic mutations of the NS1 gene of the H9N2 and H5N1 subtype avian influenza viruses(AIV),the nucleotide sequences of NS1 regions of 12 strains of AIV from 2000～2003 in southern China were tested. The NS1 genes were amplified by RT-PCR and inserted into the pMD 18-T vector, then the recombinant plasmids were transformed into competent DH5α. The sequence analysis demonstrated that the NS1 genes of H9N2 subtype contained 654 bp and encoded 217 amino acids, and the NS1 genes of H5N1 subtype contained 678 bp and encoded 225 amino acids. The homologies of amino acid sequences of NS1 protein between H9N2 were 97.7%～94.9％, and the homologies of amino acid sequences of NS1 protein between H5N1 were 99.1%～97.8％. The H9N2 and H5N1 subtype isolates were closer to some AIV strains prevalent in southern China and Hong Kong in recent years. The results require further studying the role of the NS1 protein in the evasion of the host innate defense and potential contribution to pandemic influenza.

Abstract:The transgenic expression vector was constructed by using 9.5kb α-lactalbumin genomic fragment cloned from human cosmid library. Eight transgenic mice （4♂，4♀）were detected by PCR and Southern blot analysis from 68 F₀ mice produced through microinjection. The copy number ranges from 1 to 8, and the integration rate is 11.7%. Human α-lactalbumin have been detected in milk samples of the four founder female transgenic mice by SDS-PAGE gel electrophoresis and Western blotting, and the concentrations of the human α-lactalbumin are quantified to be 0.62g/L, 0.48g/L, 0.56g/L, 3.21g/L respectively by radioimmunoassay (RIA) method. Meanwhile, one offspring of the No.50 male transgenic mice has also expressed the human α-lactalbumin, and the concentration is 1.03 g/L in the milk. The transgenic construct herewith has the characterization of a high-level expression and stable inheritance. A foundation has been provided for improving the quality of cow milk thorough transgenesis of human α-lactalbumin.

Abstract:In order to develop an assay for rapid, specific, quantitative detection of SARS-associated coronavirus, the primers and quantitative probes were designed and applied to detect coronavirus, based on the principle of complex probes quantitative assay. Sensitivity, specificity, reproducibility and range of quantitation of this method were determined. The quantitative RT-PCR for coronavirus with complex probes and an easy to handle and high efficiency method for isolation RNA from sample were established. The detect limit is 5 copies RNA per reaction and no negative samples or other RNA/DNA were detected with this method. It allows for a high sample throughput. It shows a very good precision, the coefficient of variation of threshold cycle was less than 5%. 42 clinical SARS samples were detected with this quantitative RT-PCR, the rate of SARS samples can be detected was 79%. It can be concluded that the method established for quantitaion of SARS-CoV is highly sensitive,rapid and easy to handle and shows a very good reproducibility,it can be applied to clinical diagnosis.

Abstract:By the means of overlapping peptides expressed in Escherichia coli in combination with Western blotting, two linear epitopes were identified on non-structural protein 3ABC of foot-and-mouth disease virus(FMDV), which covering amino acid residues 106～155 and 156～190 on 3ABC. The epitopes gave positive reaction with sera from pigs or guinea pigs infected with different serotypes of FMDV, but not with sera from vaccinated or naive animals. The method based on expressed peptide for mapping epitope on viral protein was reliable and applicable.

Abstract:In order to search for the sequence that affects the entering of PC-1 into cell nucleus，different part of PC-1 cDNA were fused to that of EGFP. Through the observation of the distribution of green fluorescence in the cell，the region between 112～190 amino acids was identified as independent functional domain which excluded PC-1 from cell nucleus and there appeared many dotted structure in the cell cytoplasm. The other part of PC-1 could accumulated in the nucleus when this region was deleted. Simultaneously，PC-1 was also found to attach to the cell membrane by the yeast two-hybrid system based on the SOS recovery sytem，what is more，the region between 112～190 amino acids was identified as independent domain responsible for it is cell membrane anchoring.

Abstract:Calreticulin is an calcium binding protein existed in all animal cells. It was found that calreticulin and its N terminal 1～180 amino acid inhibit the proliferation of endothelial cells and angiogenesis. In order to search for efficient and small molecular angiogenesis inhibitor, the DNA sequence coding for the N terminal 122～180 amino acid was amplified by PCR and cloned into the prokaryotic vector pET-3c. The recombinant plasmid pET-N58 was transformed into E.coli BL21(DE3). The recombinant protein was expressed efficiently in E.coli BL21(DE3,pET-N58) as inclusion body with a yield of about 35.4% of the bacterial total protein. The partial purified recombinant protein inhibits the proliferation of human umbelical vein endothelial cells and the CAM angiogenesis. The recombinant protein also suppresses the growth of primary B16 murine melanoma in C57BL/6 mice.

Abstract:The C-phycocyanin and the R-phycoerythrin were purified from the blue-green alga Spirulina platensis and red alga Polysiphonia urceolata respectively. Both sodium periodate and glutaraldehyde are effective coupling agents being capable of constructing the R-phycoerythrin-C-phycocyanin conjugate, which was also called phycobiliproteins energy transfer model. The two artificial conjugates constructed with different methods were purified by Sephadex G-200 chromatography respectively. Spectra analysis indicated that energy transfer occurred in the two conjugates. The conjugate with sodium periodate had the higher efficiency of energy transfer than that with glutaraldehyde conjugate.

Abstract:In order to study hypoglycemic effect after direct injection of a doxycyline-controlled insulin expression plasmid to the skeletal muscle of streptozotocin-induced diabetic mice, Balb/C mice were induced to diabetes. 300 μg of prTA-tet4-rhINS was injected into the muscles of mouse hind legs bilaterally and fed with drinking water containing different concentrations of doxycycline right after the injection. Tail blood glucose (random glucose level) was measured everyday with glucose oxidase method. Total RNA were isolated from muscles and liver tissues and RT-PCR was used to analyze the rhINS mRNA level and mouse β-actin mRNA was used as an internal control. After injection of the plasmid prTA-tet4-rhINS, the blood glucose in diabetes mice decreased markedly for an average of 10 mmol/L and 5 mmol/L at doxycycline concentration of 2 g/L and 0.5 g/L in the drinking water respectively. prTA-tet4-rhIN injected diabetic mice gained mass, and urine volume decreased. This hypoglycemic effect lasted for nearly two weeks. Furthermore, the hypoglycemic effect was closely related to the concentration of doxycycline in the drinking water in a dose-dependent manner. RT-PCR proved the expression of human proinsulin mRNA in the total muscle RNA. It can be concluded that a single tetracycline controlled recombinant pro-insulin gene expression plasmid could decrease the glucose levels of streptozotocin-induced diabetic mice dose-dependently by doxycycline in the drinking water. Muscle can be used as good target tissue for foreign insulin gene transfer and expression.

Abstract:A heat stable protein, named GFAP, was isolated and purified from the fruiting bodies of edible fungus Grifola frondosa by a procedure of (NH₄)₂SO₄ precipitation, and ion-exchange chromatography on DEAE-Sepharose column and gel filtration on Sepharose-6B column. According to PAGE and IEF,GFAP had a single band with pI 3.67. And it consisted of two subunits of 34 ku and 40 ku when encounted by SDS-PAGE. GFAP is identified as a glycoprotein containing 2% sugar. The N-terminal amino acid sequence of the 40 ku subunit is ACCVPSVTEFENAINSDPVM,which has no homology with other sequences in GenBank. GFAP possesses the inhibitory activity against the infection of Tobacco mosaic virus. When mixed with TMV(10 mg/L) and inoculated in local lesion host,Nicotiana glutinosa, GFAP was able to completely inhibit the infection of TMV,and with TMV(40 mg/L),it still had an infection effect of higher than 60% at the concentration of 4 mg/L.