Abstract:Glucocorticoid modulates multiple important physiological and pharmacol- ogical processes, in addition to the classic genomic model of glucocorticoid action. Recent evidence suggests more and more important role of nontranscriptional effects. Receptors, kinases and signal molecules may be involved in nongenomic pathway. Despite the many differences between genomic and nongenomic pathways, cross-talks between each other might occur. Therefore, the research of nongenomic mechanism will make for the reasonable clinical application of glucocorticoid.

Abstract:The Ca²⁺-ATPase of sarcoplasmic reticulum is a Ca²⁺ pump that plays a key role in regulating cytosol calcium concentration in muscle cells. It undergoes a sequential conformational transition during the transport process. According to the classical E1/E2 theory, in the E1 state the binding sites have high affinity and open to the cytoplasm, whereas in the E2 state the binding sites have low affinity and face the luminal side. Crystal structures of several states during the reaction cycle of Ca²⁺-ATPase have been solved recently, including a Ca²⁺-bound form (E1-2Ca²⁺), a Ca²⁺-unbound form stabilized by a potent inhibitor thapsigargin (TG) (E2-TG), an ATP-bound form (E1-ATP), an E1-P-ADP state, and an E2-Pi state. The details of these crystal structures and the relationship between structure and function of Ca²⁺-ATPase during reaction cycle were summarized, and the issues to be addressed in future research were raised.

Abstract:Apoptosis action is primarily exerted at the level of mitochondira, in which Bcl-2 family of proteins play an important role in its regulation. Bcl-2 family consists of anti-apoptotic and pro-apoptotic members. The anti-apoptotic members usually exist in the outer mitochondrial membrane and inhibit cell death via interaction with pro-apoptotic counterparts BH3 domain. Pro-apoptotic members are commonly localize in the cytoplasm. A series of events occured, such as typical Bax conformational change, BAD and Bik phosphorylation as well as Bid and Bim proteolysis in response to several death stimuli. As a result, these pro-apoptotic proteins directly integrate to the outer mitochondrial membranes. Finally, mitochondrial permeability transition pore is opened, by followed the release of apoptogenic factors from the mitochondrial intermembrane space, including cytochrome c, apoptosis inducing factor(AIF) and Smac, then the activation of downstream caspases and execution of cell death.

Abstract:Hemojuvelin (HJV) is a newly discovered regulating protein of iron metabolism. Recent studies demonstrated that human mutation in the HJV gene is one of the causes for Juvenile hemochromatosis (Type Ⅱ hereditory hemochormatosis). New findings also indicated that HJV might be a regulator of hepcidin expression and thus play an essential role in iron homeostasis.

Abstract:Non-proline cis peptide bond is rarely found in proteins. The recent surveys revealed that this unusual peptide configuration is by no means a curiosity, but overwhelmingly occurs at functionally important sites. However one still doubts whether it is related to crystal packing interactions, since all non-proline cis peptide bonds identified so far are from crystal structures. A toxin BmK M1 from the scorpion Buthus martensii Karsch have been crystallized as a dimer in space group P2₁2₁2₁ with unit-cell dimensions a = 76.39 Å, b=52.77 Å, c=27.12 Å. This dimeric structure was solved by molecular replacement and refined to R=0.109 for all reflections at a resolution of 1.4 Å. The extensively refined structure definitely shows that the cis peptide bond Pro9-His10 equally occurs in both molecule A and molecule B in the dimer. The observation manifested that this striking non-proline cis peptide is not related to crystal packing, but caused by certain intrinsic factors. The detailed analyses and comparison with the structure of BmK M8, which is homologous to M1 but has trans peptide bond 9-10, showed that the five-residue reverse (8～12) with a consensus sequence (-KPXNC-) may be the intrinsic structural element for the cis form of this peptide bond. A pair of well organized main-chain hydrogen bond between residues 10 in cis unit and 64 at C-terminus forms main tertiary interactions to stabilize this energetically unfavorable peptide bond.

Abstract:The modulating action and mechanism of endogenous nitric oxide (NO) on the delayed rectifier potassium currents in cultured hippocampal neurons were examined using whole-cell patch clamp techniques. L-arginine (L-Arg, 2 mmol/L), a substrate of NO synthases, significantly suppressed the delayed rectifier K+ currents in hippocampal neurons, while its isomer D-arginine (D-Arg, 2 mmol/L) exerted no effect. Moreover, pretreatment with NO synthase inhibitor L-NAME (0.5 mmol/L) completely blocked the suppressing effect by L-Arg, indicating that L-Arg exerted its modulation by producing NO but not by itself. No effect was found on the L-Arg-induced inhibition by 10 min pretreatment of 10 μmol/L ODQ (a specific inhibitor of guanylate cyclase). In contrast, thiol-alkylating agent N-ethylmaleimide (1 mmol/L) completely precluded L-Arg-induced inhibition on the whole K+ currents. The results indicate that endogenous NO modulates the delayed rectifier K+ currents in cultured hippocampal neurons mostly through S-nitrosylation.

Abstract:Cotton (Gossypium hirsutum) is one of the most important economic crops in the world. Its growth and productivity were affected by environment stresses such as drought, cold and high salinity. Thus, the enhanced stress tolerance in this plant is of great importance. As the dehydration responsive element (DRE) binding protein (DBP) plays an important role in the regulation of plant resistance to environmental stresses and is quite useful for generating transgenic plants tolerant to these stresses, isolation and functional analysis of DBPs in cotton are important to cotton production. In the previous work, a DBP gene from cotton, named as GhDBP1, was isolated and its expression patterns in cotton plants was demonstrated at the transcriptional level. Here, the expression, purification and DNA binding activity of GhDBP1 were reported. The entire coding region of the GhDBP1 gene was inserted into an expression vector, pET28a, and transformed into Escherichia coli BL21 (DE3). The fusion protein was successfully expressed under IPTG induction and the purified recombinant protein was obtained by Ni-NTA affinity chromatography. Non-radioactive electrophoretic mobility shift assay revealed that the purified GhDBP1 protein was able to form a specific complex with the previously characterized DRE element. In addition, the computer modeling of the DNA-binding domain of GhDBP1 were performed using SWISS-MODEL software. The main-chain structures and the folding patterns of the DNA-binding domain of GhDBP1 were similar to the known structure of the DNA-binding domain of the Arabidopsis thaliana GCC box-binding protein AtERF1. These results indicate that GhDBP1 is a DRE-binding transcription factor and might use the structure similar to that of AtERF1 to interact with DRE sequence.

Abstract:In order to research the molecular pathogenesis of hepatocellular carcinoma (HCC), firstly, gene expression profile data of HCC, which dealt with t-test method, was used and HCC’s characteristic genes were found, secondly, SAEM method was used to analyze these data combining HCC’s characteristic genes and human liver’s HNF family transcription factors ChIP-chip data, finally, transcriptional regulatory relations of HCC’s characteristic genes and HNF family transcription factors were got, furthermore the transcriptional regulatory motifs of HNF family transcription factors regulating HCC’s characteristic genes were found. All the results indicate that HNF family transcription factors regulate many HCC’s characteristic genes, a great number of those have very important functions, and multi-HNF family transcription factors regulating HCC’s characteristic genes can form feedforward loop motif and multi-input regulatory motifs.

Abstract:Uncoupling proteins (UCPs) are mitochondrial membrane transporters, acting as an uncoupler in oxidative phosphorylation. UCPs were also discovered associated with body mass index, resting metabolic rate and percentage of fat. Three haplotypes were detected by PCR-SSCP and then confirmed by DNA sequencing. For the first time, three single strand conformation polymorphisms(SNPs), G-42A, C-50T, and T-51C, were discovered in the promoter region of the UCP2 gene in pigs. T-C-G is close linkage and C-T-A is also linkage. The potential protein factor binding sites were predicted by transcription factor analysis tool TFSEARCH ver1.3 on line. The result clewed that three mutations, T→C(－51), C→T(－50), and G→A(－42) in the promoter region of the UCP2 may have some effect on the regulation of UCP2 gene transcription and regulation because those mutations increase a AML-1a site. There are three different haplotypes which are A , B and AB haplotype detected in Neijiang pig while other pig breeds have only haplotype A, suggesting Neijiang pig have specific property.

Abstract:Resistance to anticancer drugs is one major problem preventing effective chemotherapy of gastric cancer, but the molecular mechanisms of multidrug resistance (MDR) of gastric cancer is not completely clear. In order to find out new MDR related proteins of gastric cancer, two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of vincristine-resistant human gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901, PDQuest software was applied to analyze 2-DE images, and the differential protein spots between the two cell lines were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Then the differential expressional levels of partial identified proteins were detected by Western blot analysis and real-time RT-PCR. And the effect of HSP27 on the development of MDR of SGC7901/VCR was determined by antisense oligonucleotides (ASO) technique. The well-resolved, reproducible 2-DE patterns of SGC7901/VCR and SGC7901 were established. All the 24 differential proteins were identified, and the differential expression levels of the partial proteins between the two cell lines were confirmed by Western blot analysis and real-time RT-PCR. The suppression of HSP27 expression by HSP27 ASO could enhance vincristine chemosensitivity in SGC7901/VCR. These differential proteins such as HSP27 and sorcin may be related to MDR in SGC7901/VCR cells. The data will be valuable for further to study the mechanism of MDR in human gastric cancer.

Abstract:Soluble form of human urokinase receptor was cloned into Drosophila Schneider 2 secretion expression vector pMT/Bip/v5-his, and co-transfected with pCoHygro into S2 cells, to establish stably S2 cells line. The expressed suPAR from such procedure tends to form oligomer and is unstable, presenting difficulties for its structural studies. Here a purification procedure that yields monomeric suPAR was reported. SuPAR obtained through such procedure is monomeric and quite stable. SuPAR complex with amino terminal fragment (ATF) of uPA and an anti-suPAR antibody (ATN615) was crystallized by dialysis method into diffracting crystals (1.9 Å).

Abstract:Selectively infective phage (SIP) technology was developed for screening interacting protein-protein pairs. The in vivo SIP strategy would in principle be suitable for“library-library”selections, and the co-packaged polyphage may be a suitable approach. In order to construct the antigen expression vector which can be co-packaged into polyphage with phage displaying vectors, plasmid TG10 was chosen as the basic vector which is compatible with antibody display vector. The interval sequence of phage genome was amplified with PCR and cloned into TG10 to provide the packaging signal. It was named pTMI and it can be packaged into phage particles in 10₁₁ level. The N1N2 region of gene Ⅲ was amplified and cloned into pTMI under the control of lac promoter to give pTMIN. Promoter trc was synthesized and replaced the lac promoter to give pTTMIN which permits the fusion expression of antigen with N1N2. To test its ability for fusion expression, gene code for ten-peptide of c-myc was synthesized and inserted into pTTMIN downstream to N1N2. After induction expression, the results of ELISA and SDS-PAGE showed that it has been expressed successfully. When pTTMIN was transfected into cell carrying antibody display vector p3MHHB3, it was copackaged into phage particles in 0.3% to 55% after rescuing with helper phage VCSM13.From the results it can concluded that the antigen expression vector was constructed successfully and it can be used for library-library screening in theory.

Abstract:F1-V recombinant fusion protein was purified to immunize mice and its immunogenicity and immuno-protection are detected. Different purification strategies were used to purify F1-V recombinant fusion protein. First hydrophobic chromatography, then anion exchange chromatography, and finally size exclusion chromatography. Aluminium hydroxide gel adsorbed F1-V fusion protein was intramuscularly injected into BALB/c mice. Anti-serum titer and protection ratio were detected. The purity of F1-V recombinant fusion protein was more than 90%. The anti-serum titer was high up to 1∶(51 200 ± 800). The protein antigen was capable of inducing an effective immuno-protection against subcutaneous challenge of 400 LD50 virulent Yersinia pestis with the survival rate of 90%. The F1-V recombinant fusion protein was able to protect against subcutaneous challenge of virulent Yersinia pestis, suggesting that the F1-V substance is well suited for developmentas the active ingredient of the next plague vaccine.

Abstract:Fluorescence resonance energy transfer (FRET) is the energy transfer from an activated donor fluorophore to a receiving fluorophore. The efficiency of the energy transfer is the function of the distance between the two fluorophores, and is very sensitive to the distance. Its effective response distance is between 1～10 nm, thus it can be used to measure the distance between atoms or molecules. The feature of FRET is very useful in researches on conformational changes, interaction between macromolecules and signal transductions within live cells, and FRET has become a powerful tool in biomedical investigations. However, biological processes often involve interactions between more than two macromolecules, and FRET using two color fluorophores cannot meet the research demand. Recently, two research groups made breakthrough, establishing a novel FRET technique using three color fluorophores based on confocol microscopy and flow cytometry, respectively. The invention will significantly advance researches in biological and related fields.