Abstract:After the full sequence map of human genome has been completed, the great challenges for the life scientists is how to transform the genomic sequence into gene function information, to elucidate the molecular mechanism of biological process, to improve human health and supply impetus powers to the progression of biotechnology. Among the series of novel functional genomic technologies, high-throughput and high-content cell-based screen platforms have showed enormous potential and will play more and more important roles in human functional genomics research. By overexpression or knock-down of genes in cultured mammalian cells in vitro and analysing the consequent changes in signal transduction pathways and/or cell phenotypes, gene functions can be identified directly. With the recent technique progresses, the cell-based screening assays can possess the characteristics of miniaturization, automation, high efficiency, high-throughput and feasibility, and have become one of the pivotal methods in functional genomics. In the last 2~3 years, the successful applications of cell-based screen technology in large-scale functional genomics research have been reported in the literatures. In China, investigation in this field has been set out, and it will have profound influences on boosting the R & D of Chinese biotechnology.

Abstract:Autophagy (eating oneself) is the lysosomal degradation of cytosolic components, usually the long-lived proteins and organelles, and recycle the digested food for cellular metabolism during starvation. Hence, autophagy is functionally involved in cell development, immunity, tissue remodeling and cell adaptation to the adversary circumstances. Recently it suggests that autophagic machinery plays a critical role in protecting eukaryotes from infection of microbial infections, the process called xenophagy. The genetic basis of the intracellular digestioin was highlighted, and physiological and pathophysiological regulation of autophagy is discussed.

Abstract:Nobel Prize in Physiology or Medicine 2005 goes to Barry Marshall and Robin Warren, who with tenacity and a prepared mind challenge prevailing dogmas. They made the remarkable and unexpected discovery that inflammation in the stomach (gastritis) as well as ulceration of the stomach or duodenum (peptic ulcer disease) is the result of an infection of the stomach caused by the bacterium Helicobacter pylori.

Abstract:pf40 is obtained from millet unmature seeds cDNA library. It has high similarity with mental transporter family by blasting in DDBJ, GENBANK, EMBL. It is predicted that PF40 is included in ZIP family. Software analysis indicates that PF40 is a membrane protein. But which membrane system it localized in is unclear. A full PF40 cDNA fused at the 5′ end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco by agrobacterium-mediated transformation. The subcellular localization of GFP was analyzed by fluorescence microscope and laser confocal microscope. The fluorescence of PF40-GFP fusion protein was detected mainly in the ER, demonstrating that PF40 was mainly localized in the ER. 4 truncated pf40 fragments were fused with gfp. The truncated fusion genes also were introduced and stably expressed in tobacco. N-terminal amino acid region can locate the protein in the ER.

Abstract:Data analysis poses a significant challenge to the large-scale proteomics studies. Based on the structured and controlled vocabularies-Gene Ontology (GO), and the GO annotation from related databases, a strategy composed of several programs and local databases is developed to identify the functional distribution and the significantly enriched functional categories of the proteomic expression profile. It would be helpful for understanding the overall functions of these identified proteins and supply the fundamental information for further bioinformatics exploration. This strategy has been successfully used in the Human Fetal Liver (HFL) proteomic research, which is available online at http://www.hupo.org.cn/GOfact/.

Abstract:Severe acute respiratory syndrome (SARS) is a newly emerged human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. Previously study demonstrated that amino acids 318 to 510 is the receptor binding domain of SARS-CoV spike protein. The receptor-binding domain of the spike protein was expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that this fragment could be recognized by SARS convalescent sera and spike protein specific monoclonal antibody. To map the antigenic epitope of this region, a set of 23 partially overlapping fragments spanning the fragment were fused with GST and expressed. Then Western blot and ELISA reactivity of these short peptide fused protein to immunized sera and monoclonal antibody D3D1 were surveyed. Two linear antigenic epitopes SRBD3 (334~349) and D3D1 (447~455) were identified. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for severe acute respiratory syndrome.

Abstract:In order to understand the mechanism of growth and development of Schistosoma japonicum, the differentially expressed gene cDNA libraries of Schistosoma japonicum cercariae, egg and adult worm were constructed separately for the first time by SSH (suppressed subtractive hybridization) technique, which can be used to isolate and analyze differentially expressed genes on the whole genome scale. The results of subtractive efficiency and identification of stage difference among above three subtracted cDNA libraries indicated the quality were high. The 257 clones which inserted fragments more than 500 bp from above three cDNA libraries were sequenced. BLASTn results showed that these 257 ESTs represented 182 schistosome genes, including 22 schistosome known genes, 128 schistosome ESTs and 32 no match genes which may represent schistosome novel gene. Results of these ESTs function prediction indicated that differentially expressed genes from cercariae subtracted cDNA library were mainly involved in movement, energy metabolism, transcriptional regulation and pathogenesis; differentially expressed genes from egg were mainly involved in signal transduction, cell adhesion, protein and carbohydrate metabolism and response to oxidative stress; differentially expressed genes from adult worm were mainly involved in protein biosynthesis, transport and body movement. To isolate and analyze differentially expressed gene on large scale must provide the valuable information for understanding the mechanism of parasite growth and development on the molecular level, for screening highly effective antigen candidates, target of drug and diagnostic antigen.

Abstract:Anoikis is a type of apoptosis that results from cell detachment from the extracellular matrix. Resistance to anoikis may allow survival of cancer cells during metastasis. It has been found that PI3K-PKB/Akt and MAPK pathways confer anoikis resistance to cancer cells. However, the tyrosine kinase pathways upstream of PI3K-PKB/Akt and MAPK have not been adequately explored. In an attempt to identify specific phosphotyrosine-containing proteins and potential tyrosine kinase pathways involved in anoikis resistance, a functional screening method based on the specific interaction between src homologue 2 (SH2) domains and phosphotyrosine (p-Tyr) containing proteins was designed. Cell detachment rendered normal MDCK cells to undergo anoikis. However, the survival and proliferation of tumor cells was anchorage-independent. Consistent with this phenomenon, cell detachment induced rapid decrease in SH2s-binding tyrosine phosphorylated proteins in MDCK cells, while tyrosine phosphorylation of SH2-binding proteins in tumor cells was anchorage-independent. It was also found that tyrosine phosphorylation levels of Abl SH2-associated proteins decreased in detached MDCK cells. However, the tyrosine phosphorylation levels of Abl SH2-associated proteins in H460 lung tumor cells increased after a transient decrease, and the levels increased in H1792 lung tumor cells upon detachment. Using this functional screening method, some of the Fyn SH2 and Crk SH2-binding proteins were identified as FAK and p130Cas respectively, which are critical in mediating cell-matrix interactions. The present data suggest that multiple phosphotyrosine-containing proteins and potential tyrosine kinase pathways may act to support the anoikis resistance of tumor cells. The SH2-domain screening method may be an efficient way to explore anoikis-resistance-related phosphotyrosine-containing proteins and potential tyrosine kinase pathways in tumor cells.

Abstract:Frequency and intensity are two of the important parameters of sounds. Many researchers use spike rate of auditory neurons to represent the two parameters generally. Some studies showed that response latency may represent frequency and intensity too. However, it is not clear where the two parameters are encoded by response latency in the auditory pathway. GABA and glycine are two ubiquitous inhibitory neurotransmitters. Iontophoretically bicuculline (antagonist to GABA-A) or strychnine (antagonist to glycine) was injected to observe the change of response latency. If the relationship between response latency and intensity or frequency has changed, sound may be encoded locally. If the relationship keeps unchanged, this information may be transferred from the inferior nervous centres. The auditory neurons in the inferior colliculus and A1 area of auditory cortex of BALB/c mouse were studied. The results show that the relationship between response latency and intensity or frequency didn't change after the application of bicuculline or strychnine. It suggests that the encoding relationship between response latency and intensity or frequency didn't be completed in the inferior colliculus or A1 area of auditory cortex, but it is probably transferred from the inferior nervous centres.

Abstract:In order to construct a fusion gene encoding hVEGF165 and fused hirudin(FH), which can not only accelerate endothelial cell proliferation but also inhibit thrombosis, the eukaryotic expression vector hVEGF165-FH/pcDNA3.0 was constructed which contained the hVEGF165 and fused hirudin(FH). Then, the constructed vector was transfected into endothelial cell strain(ECV304). The hVEGF165-FH fusion gene transcription and protein expression were examined by RT-PCR and Western blot. The activities of fusion hVEGF165-FH were identified by serials of endothelial cell proliferation tests and antithrombotic tests in vitro. The results showed that the fusion protein was successfully expressed and it had biological activities of both hVEGF165 and fused hirudin. This new fusion gene with multi-function might be a new weapon to prevent restenosis after percutaneous coronary intervention(PCI).

Abstract:MTT reduction assay was used for the quantitative detection of the inhibitory activity of GIF in the rat hippocampal nerve call culture system. Evaluation of the influence of presence of Rab3a on the inhibitory activity of GIF indicated that Rab3a is enough to support the inhibitory activity of GIF instead of AD brain extract. Subsequently, the influence of antibody of GIF or Rab3a on the interaction between GIF and Rab3a was examined. The results indicated that Rab3a was most important to the inhibitory activity of GIF, and the interaction between GIF and Rab3a deeply depend on the spatial conditions. The mechanism and significance of the interaction between GIF and Rab3a are discussed.

Abstract:By measurement of intracellular free Ca2+ concentration using micro-fluorometric assay, whole cell calcium current and capacitance, regulative effects of extracelluar ATP on insulin secretion in primary rat β cells were studied. ATP elevated intracellular free Ca2+ concentration by mobilizing intercellular IP3 sensitive calcium pool, therefore it induced a strong secretion in resting β cells. Meanwhile, ATP attenuated depolarization-evoked Ca2+ influx and inhibited secretion induced by excitation of cell. The results demonstrated that ATP had dual regulative effects on insulin secretion in rat β cell. It is suggested extracellular ATP signal maybe have different effects on the two phases of insulin secretion in organisms: a negative feedback effect on the first phase, and a positive feedback effect on the second. Although, there are open questions to be resolved, the data support cue to the further study of mechanism underlying ATP regulation of insulin secretion.

Abstract:Using in vitro selection method to isolate nucleic acids, peptides and proteins has been studied intensively in recent years. In vitro mRNA display is a new and effective technique for peptides selection, and the rationale of this technique is that a synthetic mRNA with puromycin could covalently link with the protein that it encodes, thus an mRNA-protein fusion is formed. This approach has been used in identification of many functional peptides. The peptides binding with thymidylate synthase RNA were isolated using mRNA display technique from a large peptide library (>1013 different sequences) . The selection scheme was constructed, and the experimental conditions, including library synthesis, formation of RNA-peptide fusion and RNA immobilization were optimized. Eight cycles have been processed and the results confirmed that the selected peptides could bind with thymidylate synthase mRNA specifically. Compared the amino acid sequences of the selected peptides with those from the initial random library, the basic and aromatic residues in selected peptides were enriched significantly, suggesting these peptide regions may be important in the peptide-TS mRNA interaction. As a novel in vitro selection approach, mRNA display technique would be developed as a powerful tool for isolation of functional peptides and proteins that could interact with immobilized targets with high affinity and specificity.

Abstract:Mammalian visual system develops after birth. In rats, postnatal 3 to 5 weeks are critical for their development (namely, critical period), during which the excitatory and inhibitory synapses mature to form functional circuits in visual cortex. Extracelluar single-unit recording techniques were used in vivo to investigate the difference of response characteristics of between neurons in young and adult rats' primary visual cortexes. It was found that: (1) The adaptation to sustaining flashing stimuli is significantly higher in neurons of young rats; (2) The evoked response rate of young visual cortex neurons is significantly lower than that of adult rats; (3) The spontaneous rate (without flashing stimuli) is significantly higher in young instead of adult rats; (4) Signal to noise ratio (SNR) is significantly lower in young visual cortex. These results indicate that the response capability to continued stimuli and the detectability is lower in developing visual cortex. It might result from the sequential development of excitatory and inhibitory synapses in postnatal rat neocortex.

Abstract:For a long time, it has been an intriguing but unresolved question whether the immune system, especially thymus derived T lymphocytes, can recognize and eliminate randomly occurring neoplastic cells, known as “Immunosurveillance”. With the development of biology and transgenic technology, scientists have in recent years found that deficiency of either IFN-γ or T cells in knockout mice increases their sensitivity to methylcholanthrene-induced tumorigenesis. This has been hailed as the “ Renaissance” of cancer immunosurveillance in the field of immunology. Nevertheless, different views have also been raised claiming that there is still no new evidence for the specific immune response-mediated surveillance against cancer cells. A brief review of the different opinions on this topic is given.