Abstract:Conventional neuroimaging methods primarily focus on functional localization, through which specific cognitive functions are localized to specific brain regions. However, fully understanding the human brain function requires characterization of functional integration within and among the functionally specialized regions in addition to functional localization. Functional connectivity and effective connectivity analyses have been developed to investigate functional integration in human brain. Several approaches for modeling functional connectivity and effective connectivity, including the time-series correlation, psychophysiological interaction (PPI), structural equation modeling (SEM), dynamic casual modeling (DCM), and diffusion tensor imaging (DTI) are reviewed. The applications of functional connectivity analysis to the studies of object representation, motor coordinate, language, and autism are demonstrated. Functional connectivity study will highly enrich our knowledge about the dynamic integration in the human brain.

Abstract:ASICs are H+-gated novel cation ion channels, which belong to the epithelial sodium channels (NaC/DEG) superfamily. As recent studies focus, ASICs are expected to be pharmacological targets on protecting the neuron from ischemia and damage, improving the ability of memory and study, curing epilepsy and analgesia. It is not until the most recentness that the subunits of ASICs have been cloned. Now, researchers have paid more attention to the distribution, expression, function and modulation of ASICs in the organism.

Abstract:The pathological presentation of Alzheimer disease (AD), the leading cause of senile dementia, involves regionalized neuronal death and an accumulation of intraneuronal and extracellular filaments termed neurofibrillary tangles and senile plaques, respectively. One of the β-amyloid peptides (Aβ), the Aβ1-42 form, is primarily responsible for neuronal damage and cell death that is the main component in the senile plaques. Over the past twenty years, the amyloid hypothesis has been strongly supported by a wealth of evidence, including data from genetic studies of Alzheimer disease. Amyloid cascade hypothesis states that the accumulation and deposition of fibrillar Aβ is the primary driver of neurodegeneration and cognitive decline leading to dementia. AD is a clinicopathological syndrome in which different gene defects can lead——directly or indirectly——to alter APP expression or proteolytic processing as such to change Aβ stability or aggregation. These result in a chronic imbalance between Aβ production and clearance. Gradual accumulation of aggregated Aβ initiates a complex, multistep cascade that includes gliosis, inflammatory changes, neuritic/synaptic change, tangles and transmitter loss. The evidence that links Aβ to the pathogenesis of AD is substantial, but the means by which these peptides exert their toxic effects, and where in neuronal cells they act, is far from clear. The up-to-date proceeding in the molecular mechanism of β-amyloid peptides is overviewed.

Abstract:To investigate the effect of caveolin-1 on invasion and metastasis of human breast carcinoma Hs578T doxorubicin-resistanted cells, Hs578T/Dox+cav-1 was as experiment group which up-regulated caveolin-1 by introducing the pCI-neo caveolin-1 gene, and Hs578T/Dox+vector introduced the pCI-neo vector was as controlled group. It was revealed that caveolin-1 in Hs578T/Dox cells is an important factor for mediating filopodia formation, which may enhance the invasive of cells. Hs578T/Dox+cav-1 cell formed long and abundant pseudopodia and only few filopodia were detectable in Hs578T/Dox+vector cells. It was shown that adhesive capability of Hs578T/Dox cells enhanced with up-regulated expression of caveolin-1 protein(P＜0.01). Introducing the caveolin-1 gene into Hs578T/Dox cells enhanced their migration and invasive capability, as revealed by an in vitro chamber invasion assay(P＜0.01). Apoptosis index of Hs578T/Dox+cav-1 group resulting from loss of cell-matrix interactions decreased comparing Hs578T/Dox+vector group(P＜0.01). Caveolin-1 inhibited anoikis of Hs578T/Dox cells and that allowed survival of cancer cells during systemic circulation, thereby facilitating secondary tumor formation in distant organs. The efficacy in vivo of caveolin-1 was tested using cells xenografted into nude mice. Each mouse in both fifteen-mice groups were hypodermically injected with equivalent doses of 5×10⁵ cells from Hs578T/Dox+cav-1 or Hs578T/Dox+vector. Tumors in Hs578T/Dox+cav-1 group were all formed after injection followed for 4 weeks, while no one in Hs578T/Dox+vector group. Lung metastasis was found in one mouse injected Hs578T/Dox+cav-1 cells. In conclusion, up-regulated caveolin-1 level in Hs578T doxorubicin-resistanted cells markedly elevates their capacity to tumor invasion and metastasis.

Abstract:MicroRNAs (miRNAs) are a class of endogenous non-coding RNA, which regulate target gene expression via mRNA degradation and translational repression. MicroRNA has been linked to the development of plants and animals, to cell growth and apoptosis, to fat metabolism and other significant physiological processes. Misregulation of microRNA function might contribute to human diseases. Profiling microRNA expression will facilitate the study of biological functions of microRNAs. To detect microRNA expression, a high-density oligonucleotide microarray was constructed, which contains oligonucleotides corresponding to 313 human microRNAs, 261 mouse microRNAs, 196 rat microRNAs and 122 predicted microRNAs. Firstly, high molecular weight RNA was removed from total RNA by polyethylene glycol (PEG) precipitation, and low molecular weight RNA (LMW RNA) was obtained. Then LMW RNA was directly labeled based on a published method that uses T4 RNA ligase to couple LMW RNA to a fluorescence modified dinucleotide. By using a special spotting solution in combination with a swirling hybridization method, the performance of the microRNA microarray was greatly improved. The results of the microRNA microarray experiments indicated its good reproducibility, sensitivity and specificity. It was also showed that the signal of the microRNA microarray was derived from mature microRNAs, but not from their precursors. Using the microarray, microRNA expressions in human brain, heart, liver and in HeLa, HepG2, HL60 cells were profiled. The results revealed a good overall concordance with previously published data. Clustering analysis showed the tissue specificity pattern of microRNA expression, and the same tissue from different individuals was clustered together. Then the expression of miR-122a, miR-9 and miR-124a in human tissues and cell lines with RT-PCR (reverse transcription-PCR) were validated. The results which showed the liver-specific expression of miR-122a and the brain-specific expression of miR-9 and miR-124a were consistent with the microarray results.

Abstract:It had been proved by many evidences that several heat shock proteins (HSPs) expression is up-regulated in tissue-derived primary lung cancer, and HSPs may play important roles in development, in resistant to drugs and in prognosis of lung cancer. However, there have not still systemic research on which HSPs，especially HSP70 can be or not thought as a new biological target in the therapy to lung cancer. In order to address the expression and roles of heat shock protein 70 (HSP70) in lung adenocarcinoma, immunoblotting was performed to detect the expression of HSP70 in tissue specimens from lung adenocarcinoma which were diagnosed unambiguously by branch fibromicroscopy and were excised. It showed that in the normal lung tissues, the expression of HSP70 was less then that in cancer tissues. After down-regulation of HSP70 protein by HSP70 anti-sense oligonucleotides in A549 cell line, MTT assay showed that the proliferation of A549 cells was inhibited remarkably after the treatment with HSP70 antisense oligonucleotides and Act D. There had significant differential in HSP70 antisense treatment group followed by Act D treatment and Act D treatment group. Results of Hoechst33258 staining revealed that HSP70 antisense oligonucleotides could promote Act D-mediated apoptosis in A549 cells with a higher percentage of apoptotic cells (26.91±3.73)% than that of Act D-treated group (16.83±3.41)% (P < 0.01). Furthermore, the expression of HSP70 protein was increased remarkably by transfection with a full length HSP70 recombinant plasmid into A549 cells. MTT assay revealed that HSP70 over-expression could block significantly the inhibition of proliferation induced by Act D, there had statistic significance between HSP70 over-expression group and only Act D-treated group (P < 0.01). Furthermore, over-expression of HSP70 could significantly inhibit Act D-mediated apoptosis in A549 cells with a lower percentage of apoptotic cells (4.25±1.48)% in HSP70 transfection group, (12.89±2.03)% in vector transfection group and (14.37±2.56)% in Act D treatment group and disappearance of DNA laddering. These results suggests that increased levels of HSP70 in lung adenocarcinoma tissues decrease the sensitivity of lung adenocarcinoma to Act D, promote proliferation and suppress apoptosis of lung adenocarcinoma cells.

Abstract:The zinc finger antiviral protein (ZAP) specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN), but has very modest inhibitory effect on HIV. Previous studies suggest that ZAP directly binds to the viral RNA and recruit the RNA degradation machinery to degrade the target RNA. The HIV-1 Tat and Rev are regulatory proteins which bind to HIV RNA. Tat and Rev were fused with ZAP in various manners. Two fusion proteins, ZAP-Tat and ZAP-Rev were found to be able to inhibit the expression HIV-1 vector.

Abstract:Aquaporins (AQP) are integral membrane proteins that serve as channels in the transfer of water, and in some cases, small solutes across the membrane. In order to detect the expression of AQP_1～10 mRNA in the human pleural mesothelial cells, culture reproducible model of human pleural mesothelial cells was established in vitro.The value of aquaporin water channels was investigated in pleural fluid dynamics. Mesothelial cells were isolated from pleural effusion fluid of nonmalignant pleural effusion patients and cultured in vitro. The mesothelial cells were identified by morphology and streptomyces protein-peroxidase(S-P) procedure. RT-PCR was used to detect the expression of AQP_1～10 mRNA in human pleural mesothelial cells. Establishment of culture reproducible model achieved success of human pleural mesothelial cells in vitro. Immunocytochemistry revealed that cytokeratin and vimentin expressed positive, VⅢ factor associated antigen and CD₄₅ was negtive. Confluent human pleural mesothelial cells appeared multipolar and like cabblestone.Under electron microscopy numerous surface microvilli and abundant endoplasmic reticulum were observed. The cells was identified that it was mesothelial cells. The cells isolated from human pleural effusion fluid have higher purity quotient . RT-PCR studies showed that all of the AQP_1～10 mRNA expressed in mesothelial cells. AQP₁, AQP₉, AQP₁₀ mRNA have abundant expression in mesothelial cells and significant deviation was found between AQP₁, AQP₉, AQP₁₀ and AQP₂, AQP₃, AQP₇, AQP₈. Human pleural mesothelial cells expressed AQP_1～10 mRNA, all of them have relation with the function of human pleural mesothelial cells. It was confirmed that mesothelial cells have important contribution in pleural fluid dynamics. Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes.Expression of aquaporins was intervened, new treat method may be found of pleural effusion.

Abstract:The aging process of human colonic epithelium involves a slow decline in physiological vigor and an increasing susceptibility to age-related diseases, especially, colon cancer, but the molecular mechanisms of the aging and susceptibility of aged colonic epithelium to carcinogenesis is still unclear. Identification of aging related proteins in colonic epithelium will help to reveal the molecular mechanisms of colonic epithelial aging and age-related colonic diseases. Therefore, the total proteins of human normal colonic epithelial tissues from 10 young and 10 old men were separated by two-dimensional gel electrophoresis(2-DE), respectively. PDQuest software was applied to analyze 2-DE images, the differentially expressed protein spots of colonic epithelium between young and old groups were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS), and the expression levels of partial identified proteins were determined by real-time quantitative PCR and immunohistochemistry. Well-resolved, reproducible 2-DE maps of human colonic epithelial tissues from young and old men were established, 17 aging related proteins were identified by MALDI-TOF-MS, and the differential expression levels of partial identified proteins were confirmed by real-time quantitative PCR and immunohistochemistry. The results indicate that injury of mitochondrial function and decline of antioxidant capability are important reasons for the aging of human colonic epithelium, and four differential proteins (guanine nucleotide-binding protein beta subunit-like protein, stress-70 protein, 40 S ribosomal protein SA and chloride intracellular channel protein1) may be involved in susceptibility of aged colonic epithelium to carcinogenesis.

Abstract:High affinity, fully human antibody fragment Fab that specifically bind to HGF receptor, Met (a proto-oncogene product and a key molecule on tumorigenesis, invasion and metastasis), was selected from phage display antibody library. To construct antibody library, the Fab gene fragment was constructed in 3 steps. The variable region genes were amplified from VH and VL of anti-Met Fab selected from error-prone PCR mutation library. Fab gene was assembled by overlap PCR and purified and digested with SfiⅠ, and inserted into pComb3XSS. Positive phage-displayed antibody clones were selected on live cell lines and immobilized protein. The purified Fab was verified by SDS-PAGE and Western blot, which showed two bands at about 25 ku and 27 ku at the expected sizes. To analyze the immunological characters of Fab for Met binding, flow cytometry and immunoprecipitation assays were set up and carried out with S114, MKN45 and NIH3T3 cell lines. The results demonstrated Fab could bind native Met specifically on the S114 and MKN45 cell surface. In vitro cytotoxic assay showed that only Hum-ZAP/anti-Met Fab complex could inhibit the Met positive cell growth, it illuminated that anti-Met Fab bound the Met on Met positive cell surface and were internalized. For Met negative cell line NIH3T3, no significant inhibition among the different dosages of Fab and Hum-ZAP. The results showed that anti-Met Fab antibody fragments could recognize Met extracellular domain in native conformation with relatively high affinity. Importantly, these antibody fragments were able to be internalized into Met over-expressed tumor cell lines through binding to the Met receptor, and could be applied as a potential powerful reagents for clinical therapy.

Abstract:Recombinant AAV serotype 8 (rAAV8) vector is new and promising for gene delivery，which transduces muscular and hepatic cells highly efficiently. The transduction varies with the administration routes. It was observed that the rAAV8 virus injected intraperitoneally had been delivered to many different tissues through the blood circulation and resulted in extensive and prolonged gene expression. The interest gene (human coagulation factor Ⅸ) has been highly expressed via the rAAV8 vector injected intraperitoneally into mice at the dose of 5×10¹⁰ gc/ mouse. Considerable expression was detected in mouse plasma at two weeks after the administration. Expression peak up to 1 000% level compared to the UCRP occurred between 1～2 months after administration, and then the expression declined gradually but obvious expression persisted at 4 months post administration. APTT test proved the clotting activity of the recombinant hFⅨ in mouse plasma. Immunohistochemical assay showed that the human coagulation factor has been expressed significantly in several organs including liver, kidney, heart and muscles (abdominal and hindleg). These suggest that the rAAV8 virus injected intraperitoneally has been delivered via blood circulation and transduced different cells of multi-organs, and the product of the gene mediated by the rAAV8 vector is biologically active.

Abstract:It was reported that glycerol and 1,2-propanediol was converted to 1,3-propanediol and 1-propanol in Lactobacillus diolivorans(DSM14421)under the anaerobic condition，respectively. Its putative genes encoding diol dehydratase, a key enzyme in the pathway, were sequenced. However, their reactivating factor (gldG and gldH) was not completely sequenced. Based on several glycerol dehydratase and diol dehydratase reactivating factors sequence alignment, it was amplified from L. diolivorans with degenerated primers. Then it was inserted into expression vector pSE380. A recombinant plasmid pSE-gldGH was constructed and transformed into Escherichia coli BL21. Recombinant gldG and gldH protein were co-purified by metal chelating affinity chromatography and gel filtration from cell-free extracts of E. coli overexpressing the gldGH genes. They existed a putative reactivating factor, with an apparent molecular mass of 325 000. The protein complex consisted of equimolar amounts of the two subunits with M_r of 68 000 (α) and 13 000 (β), encoded by the gldG and gldH genes, respectively. Therefore, its subunit structure was most likely α₄β₄, different from the common structure α₂β₂ of the other diol or glycerol dehydratase reactivating factors. In the presence of free adenosylcobalamin, ATP, and Mg²⁺, the factor reactivated diol dehyadratase from L. diolivorans, which had been inactivated to be enzyme-cyanocobalamin complex.

Abstract:Overexpressed CKLFSF2 (chemokine-like super family member 2) can be secreted into the supernatant of cultured cells, which exhibits the effects on cell-chemotaxis. To study the structural and functional characteristics of CKLFSF2 and make the polycolonal antibody, the recombinant plasmid pGEX-4T-3-CKLFSF C51 was constructed, following the expression in E. coli with high efficiency, purification using Glutathione Sepharose 4B and Sephacyl S-200, the recombinant CKLFSF2 C51 protein (typically >95% pure) was obtained. The N-terminal animo acid sequencing of CKLFSF2 C51 was performed and relative molecular mass is 5871.48 measured by ASIMA-CFR^TM plus MALDI-TOF. ELISA assay detected the reactivity of the polycolonal antibody and Western blot show that CKLFSF2 was overexpressed in mammalian cells and the specific band was corresponding to the expectation. The recombinant CKLFSF2 C51 protein exhibits significantly chemotactic effect in PC-3 cells, which can be neutralised by anti-CKLFSF2 C51 polyantibody. The anti-CKLFSF2 C51 antibody can also be used for immunohistochemistry. Taken together, the recombinant CKLFSF2 C51 from E.coli has similar bioactivity as that from eukaryocytic expression system. The polycolonal antibodies of recombinant CKLFSF2 C51 can be used in immunohistochemistry, Western blot and to neutralize the chemotactic effect.

Abstract:In order to screen EGFR-regulated secreted proteins in human nasopharyngeal carcinoma(NPC), and to reveal the role and mechanism of epidermal growth factor receptor(EGFR) in the pathogenesis of NPC. NPC cell line CNE2 cells were cultured in serum-free medium and stimulated by transforming growth factor-α (TGF-α) for 24 h in experimental group. Control CNE2 cells were cultured at the same condition but without TGF-α stimulation. The culture medium of control and experimental cells was desalted and concentrated through ultrafiltration to prepared the total secreted proteins. Two-dimensional gel electrophoresis (2-DE) was used to separate the secreted proteins of control and experimental cells, PDQuest software was applied to analyze 2-DE images, and the differential protein spots between the control and experimental cells were identified by desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The 2-DE patterns of the secreted proteins of TGF-α stimulated and un-stimulated CNE2 cells were established, 22 differential proteins spots between the two groups of cells were found, and 8 non-redundant proteins were identified with MALDI-TOF-MS, the functions of which were involved in invasion, metastasis, apoptosis and proliferation of cancer cells. The data will be valuable for further to study the role and mechanism of EGFR in the pathogenesis of NPC.