Abstract:piRNA is a novel class of small single strand RNA that were recently isolated from testes of the mammals. These RNAs are bigger (26～31 nt) than most previously described small RNAs (21～23 nt) and are associated with Piwi-subfamily members of the Argonaute protein family.

Abstract:The recent report “The Consensus Coding Sequences of Human Breast and Colorectal Cancers” published in Science represents the first unbiased systematic mutational analysis of human genome at any disease states after the completion of The Human Genome Project. It was tentatively discussed how to take advantage of the candidate cancer genes found in this study in discovery of novel targets of therapeutic drugs. In addition, the penitential values of the candidate cancer genes in identification of patients at high risk of cancer, and in renewing classification of tumors based on underlying molecular mechanisms are introduced.

Abstract:Protein phosphorylation is one of the most important post-translational modifications. Reversible protein phosphorylation plays an essential role in regulation of cellular actions. Recently, phosphoproteomics is becoming a hotspot. Quantitative phosphoproteomics provides the possibilities to study temporal-spatial dynamics of protein phosphorylation and to better understand the regulatory networks of key processes in cells. As a part of proteomics, the quantitative phosphoproteomics faces more severe challenges since protein phosphorylation occupies high dynamics and extreme complexity. Advantages and disadvantages of several newly developed methods and technology were discussed, and the progress in quantitative phosphoproteome research realm was summarized.

Abstract:The epidermal growth factor receptor (EGFR) provides a rational target for cancer therapy as it is commonly overexpressed in a variety of solid tumors, and its deregulation is correlated with resistance to chemotherapy and radiotherapy and a poor prognosis. Cetuximab (C255), a specific monoclonal antibody directed against EGFR, is synergistic with chemotherapy and radiotherapy and has been licensed for the treatment of irinotecan refractory colorectal cancer (CRC) and squamous cell cancer of the head and neck (SCCHN), which express EGFR. In addition, the clinical trials about cetuximab for the treatment of non-small cell lung cancer (NSCLC), breast and pancreas carcinoma are ongoing, and cetuximab has been proven to a novel strategy for the treatment of cancer with the overexpression of EGFR.

Abstract:Among the tetrodotoxin-resistant(TTX-R) α-subunits, Nav1.5 mRNA showed the strongest expression in the heart, according with its consideration as the cardiac Na⁺ channel. Compared to its mRNA transcription in the heart, the Nav1.5/SCN5A gene was also expressed in high levels in the brain, especially limbic regions of the brain. Exon6A of Nav1.5/SCN5A gene was first found in cloning of Nav1.5 channel in human neuroblastoma cells. It was found that it is exon6A that encodes Nav1.5 channels in human and rat brain while it is exon6 that encodes Nav1.5 channels in other tissues in the cloning of Nav1.5 channel in these tissues, Both exon6A and exon6 have 92 base pairs, which encodes 30 amino acid residues, and are located in human chromosome 3. But these two resulting products differ by 7 amino acid residues. There is only one different base pair between the enon6As encoding Nav1.5 channels in human and rat brain, but their encoding amino acid residues are identical. The expressions of exon6A in different part of the brain are different and the similar result was found in the expressions of exon6 among different tissues of rat when detected by using RT-PCR method. The study is useful for making further investigation in the functional analysis of Nav1.5 channels in different tissues.

Abstract:Bone turnover is regulated by local concentrations of cytokines such as osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL). To explore the in vivo biological function of Opg and the mechanism of osteoporosis due to deficiency of Opg, Opg knockout mice have been generated through homologous recombination. Opg^－/－ mice exhibit a sharply decrease in bone density and strength as expected. The number of osteoclasts in Opg^－/－ mice significantly increases. Morphologically, osteoclasts appear more cuboidal in shape in Opg^－/－ mice than those of wt mice, suggesting that active osteoclastogenesis occurs in the absence of Opg. In consistent with this finding, an increase of osteoblast activity was also observed with accelerated mineral accumulation rate by histomorphometric measurement and elevated serum alkaline phosphatase activity (ALP) in Opg^－/－ mice. Interestingly, more than 50% of 2-month-old Opg^－/－ mice manifest medial calcification of aorta with comparable serum concentrations of calcium and phosphorus to wt mice. In conclusion, Opg^－/－ mice have a high-bone-turnover type osteoporosis. The aortic calcification in Opg^－/－ mice is not due to abnormality of calcium and phosphorus metabolism. The mechanism underlying aortic calcification in Opg^－/－ mice needs to be further investigated.

Abstract:IGFBP-7 plays an important role in the process of human pregnancy. However, it is not found in rats. In previous study, suppression subtractive hybridization (SSH) was utilized to analyse differentially expressed gene between pre-implantation period and implantation period and IGFBP-7 was identified as a gene differentially expressed. Temporo-spatial expression pattern of partial IGFBP-7 cDNA sequence(CDs 531～928 nt, IGFBP-7′) in early pregnancy was detected by Northern blot and in situ hybridization. RT-PCR was used to analyse IGFBP-7′ mRNA expression in multiple tissue and uterus of pseudopregnancy, artificial decidualization and delayed-implantation. Results indicated that IGFBP-7′ mRNA expression level began to increase in the day 5 of pregnancy, the expression level during day 5.5 and 6 is higher than pre-implantation period. IGFBP-7′ mRNA was mainly located in luminal epithelium and gland epithelium. The expression of IGFBP-7′ mRNA have not tissue-specific and was detected in hypothalamus, pituitary, uterus, ovary, heart, liver, spleen, lung, kidney and so on. The expression of IGFBP-7′ mRNA was not significantly different in uterus of pseudopregnancy and artificial decidualization, and significantly increased in the uterus of delayed-implantation. Collectively, these results suggest that IGFBP-7′ expression increase is mainly induced by blastocyst, not decidualization during the window of implantation, which may be beneficial to embryo implantation in the early of pregnancy in rats.

Abstract:In order to identify whether functional L-type calcium channels are expressed in neural stem cells(NSCs) from rat embryonic hippocampus, and whether L-type calcium channels participate in the modulation of proliferation and differentiation of NSCs, the rat embryonic hippocampal tissue was dispersed into a single cell suspension, and the dissociated cells were cultured in serum-free DMEM/F12 medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), N-2 and B27 supplement. Immunofluorescent labeling showed an expression of nestin-positive cells. Following five-days culture in differentiation medium, neuron-like and astrocyte-like cells were observed, which expressed β-tubulin Ⅲ(Tuj1) and Glial fibrillary acidic protein (GFAP), respectively. Western blot analysis showed an expression of Cav1.2α₁C subunits in NSCs, but no Cav1.3α₁D subunits. Moreover, L-type calcium channel currents were recorded in those cells by using whole-cell patch clamp techniques. It was found that activation of L-type calcium channels promotes proliferation and differentiation to neuronal type of NSCs. The results indicated that rat embryonic hippocampal NSCs express functional L-type calcium channels, L-type calcium channels modulate proliferation and differentiation of NSCs.

Abstract:Two-dimensional (2D) gel electrophoresis followed by mass spectrometry was utilized to compare the protein expression between male worms of single-sex infection and male worms of bisexual infection in Schistosoma japonicum. To observe the in vitro expression and subcellular localization of recombinant eukaryotic expression plasmid pEGFP-C1/ SJCHGC in COS-7 cells and analyze the antigenicity of the products expressed. 9 pairing-related proteins were identified and further validated by semi-quantitative RT-PCR analysis. Most of the candidate proteins have been evident to be somehow associated with various aspects of S. japonicum such as cell growth, development, motility, reproduction, nutrition and signal transmission. The SJCHGC gene was expressed in the transfected COS-7 cells. The expression and subcellular localization of the fusion protein were analyzed by fluorescence microscopy. The proteins expressed show the antigenicity of Schistosoma japonicum. These results provide the basis for elucidating the Schistosome mechanism of development and reproduction, and may permit the identification of protein candidates for the development of novel vaccine or new targets for drug development against schistosomiasis.

Abstract:RNA silencing is increasingly employed as an experimental strategy to probe for gene function in several organisms. The purpose of the present study was to test the effect of gene silencing by dsRNA in budding yeast Saccharomyces cerevisiae. GRE3 gene encoding an NADPH-dependent aldose reductase was chosen as an example. A recombinant plasmid psiLENT-GRE3 was constructed based on the pESC-LEU backbone and used to transform S. cerevisiae YPH499. The down regulation of GRE3 gene expression by inducing 1 kb RNA duplex and a 136 bp loop was investigated using reverse transcription - PCR. The results showed that double-stranded RNA mediated gene silencing could be used as a functional tool to decrease the expression level of a specific gene in S. cerevisiae, which would contribute to the understanding of RNA interference in budding yeast.

Abstract:As a basic work of illustrating the relationship between the structure and function of a kind of protein purified from Se-enriched Ganoderma lucidum (Se-GL-P), the primary biochemical characters of Se-GL-P were characterized. Molecular mass was determined by two methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis and size exclusion chromatogram. Amino acids composition, isoelectric point, peptide mass fingerprint, and the sequence of N terminal amino acids were determined by high performance liquid chromatography, capillary isoelectric focusing method, in-gel digestion, and Edman digestion method respectively. Results showed that, the Mr of Se-GL-P was 36 600, pI = 4.01, and the sequence of N-terminal amino acids was DINGGGATLPQKLYLTPDVL. A conclusion was drawn that Se-GL-P was a kind of new selenium-containing protein, and it was one member of DING protein family.

Abstract:The supercondensed DNA, a special kind of topological structure of plasmid DNA, was firstly found in E. coli SD108(topA⁺ gyrB225). Now, this structure is also found in E. coli DM800(topA^－ gyrB225). The result indicates that the formation of supercondensed DNA is related with decrease of the activity of gyrase in vivo. Topoisomerase Ⅳ was proved to relax the supercondensed DNA completely in vitro, which suggested that the supercondensed DNA and the supercoiled DNA could transform to each other in cells. The supercondensed DNA samples were analyzed by atomic force microscopy and compared to supercoiled DNA. The results showed that the length of supercondensed DNA decreased about 30% and the width and height of double-strand increased about 60%, which indicates that the structure of double-strand of supercondensed DNA is much more similar to A-DNA than B-DNA. The results also showed that chloroquine intercalation did not change the supercoiling level of supercondensed DNA, but made it knot and compact.

Abstract:The strong absorption of gold nanoparticles in the visible spectral range allows the localized generation of heat in a volume of only a few tens of nanometer. The efficient conversion of strongly absorbed light by plasmonic gold nanoparticles to heat energy and their easy bioconjugation suggest that the gold nanoparticles can be used as selective photothermal agents in molecular cell targeting. The selective destruction of alkaline phosphatase, the permeabilization of the cell membrane and the selective killing of cells by laser irradiating gold nanoparticles were demonstrated. The potential of using this selective technique in molecularly targeted photothermal therapy and transfection is discussed.

Abstract:The emergence of drug-resistant protease(PR) has seriously affected the anti AIDS therapy. Using mutated PR to screen phage library displaying randomized HIV PR target sites, phages susceptible to mutated PR can be obtained ,and used in drug screening of protease inhibitor(PI) against to drug-resistant HIV PR. In order to validate the feasibility of this suppose, a designed LD3-CAP2NC peptide composing of HIV CAP2NC with P2/NC target site and LD3 peptide locating in the NH2 terminus of the CAP2NC which could immobilise the phage to the plate through binding to human IgG was displayed on the surface of phage. This phage was fixed on plate, and suffered to the cleaving by HIV SF2 PR. The uncleaved phage leaving on the plate was measured using HRP/Anti-M13 conjugate, which reflects the cleavage efficiency. The results showed the phage can be cleaved effectively in a dose-dependent manner to PR concentration, with the most cleavage effect reached to more than 80%.Comparing with the control, the ELISA value of the cleaved phage decreased obviously. Furthermore, this cleavage was specifically inhibited by HIV protease inhibitor durg Indinavire. The data proved that a novel HIV protease cleavage model of the phage displaying Gag CAP2NC was firstly successfully established, which can not only be used as a new research platform to investigate the relationship between PR drug-resistant mutation and PR target sequence adapted mutation, but also lay a foundation to establish the new phage screening model for PIs especially against to drug-resistant PR.

Abstract:Gene expression and protein synthesization of osteoprotegerin (OPG) and osteoclast differentiation factor (ODF) were studied in shear stress cultured osteoblasts applying parallel-plate flow chamber system. Shear stress of 0.5, 1.0, 1.5 N/m² in 24 h, and step-wise increased shear stress from 0.5 to 1.0 and then to 1.5 N/m² and each stress for 8 h, and stepwise decreased shear stress from 1.5 to 1.0 and then to 0.5 N/m² and each stress for 8 h were applied. The expression of OPG and ODF mRNA was detected by RT-PCR, and synthesization of their protein was detected by Western blot. The results showed that the shear stress of 1.0 and 1.5 N/m² induced more significant changes in OPG mRNA and ODF mRNA than 0.5 N/m² and there was no significant difference between 1.0 and 1.5 N/m2. No marked differences were observed between the effects of step-wise increased shear stress and 1.5 N/m² after 24 h, and the same was between the effects of step-wise decreased shear stress and 0.5 N/m² after 24 h. The shear stress of 1.0 N/m² increased OPG expression and inhibited ODF expression in 24 h both in mRNA and protein level. In all, the ratio of OPG and ODF was increased by shear stress, which reminds that bone resorption was inhibited by the fluid flow, and shear stimuli regulated the new balance of bone resorption and bone formation throught OPG/ODF route.