Abstract:Insulin is one of the most important hormonal regulators of metabolism. Since the diabetes patients increase dramatically, the chemical properties, biological and physiological effects of insulin had been extensively studied. In last decade the development of NMR technique allowed us to determine the solution structures of insulin and its variety mutants in various conditions, so that the knowledge of folding, binding and stability of insulin in solution have been largely increased. The solution structure of insulin monomers is essentially identical to those of insulin monomers within the dimer and hexamer as determined by X-ray diffraction. The studies of insulin mutants at the putative residues for receptor binding explored the possible conformational change and fitting between insulin and its receptor. The systematical studies of disulfide paring coupled insulin folding intermediates revealed that in spite of the conformational variety of the intermediates, one structural feature is always remained: a “native-like B chain super-secondary structure”, which consists of B9-B19 helix with adjoining B23-B26 segment folded back against the central segment of B chain, an internal cystine A20-B19 disulfide bridge and a short α-helix at C-terminal of A chain linked. The “super-secondary structure” might be the “folding nucleus” in insulin folding mechanism. Cystine A20-B19 is the most important one among three disulfides to stabilize the nascent polypeptide in early stage of the folding. The NMR structure of C.elegans insulin-like peptide resembles that of human insulin and the peptide interacts with human insulin receptor. Other members of insulin super-family adopt the “insulin fold” mostly. The structural study of insulin-insulin receptor complex, that of C.elegans and other invertebrate insulin-like peptide, insulin fibril study and protein disulfide isomerase (PDI) assistant proinsulin folding study will be new topics in future to get insight into folding, binding, stability, evolution and fibrillation of insulin in detail.

Abstract:Synthetic double-stranded oligonucleotides with high affinity for AP-1 transcrption factor can be introduced into cells as decoy cis-elements to bind the factor and alter gene expression by competition with AP-1 element.This decoy oligonucleotide restrained tumor cell proliferation in vitro and in vivo, and has important cancer gene therapeutic potential.

Abstract:Latent signal transducers and activators of transcription(STATs) reside in the nucleus following cytokine stimulation and regulate gene expression upon activation of cytokine or growth factor receptors. While this translocation event is essential for gene regulation by STATs, their mechanism of transport through the cytoplasm to the nucleus has remained elusive. Nuclear shuttling molecules often own a mono or bipartite basic amino acid stretch, but STAT family do not own the classic nuclear localization sequence(NLS). Now, it is reported that STAT3 accumulate nucleus after IL-6 stimulation 20 min. With IL-6 stimulation, STAT3 molecule conformation changed and the NLS in DNA binding domain exposure to make it gain nuclear targeting function. Deleted a stretch(403～426aa) of basic amino acid in STAT3 DNA binding domain which destroys the nuclear importing function even with cytokine stimulation. It indicated that this stretch of basic amino acid has critical role for STAT3 nuclear import, which has nuclear localization sequence(NLS)function.

Abstract:In order to find agonists for melanocortin 4 receptor (MC4R), an assay in vitro was established for high throughout screening. The MC4R receptor plasmid (MC4R/pCDNA3.1) and reporter gene plasmid (3×CRE/3×MRE/SRE-LUC) were cotransfected HEK293 to establish a cell line for agonist screening. To identify and optimize the assay condition, the effects of some factors were examined by using α-MSH, such as cell number per well, incubation time, final concentration of DMSO and luciferase's substrate concentration on the assay. A steady cell line and a reliable method were established for MC4R agonist screening, the Z'-factor value was near to 0.7 on the condition in each well that the cell number were 4×10⁴ , the agonist incubation time was 8 h, the final concentration of DMSO was less than 1%, and the final concentration of α-MSH was 1 μmol/L. This technology can be applied to identify agonist for MC4R receptor.

Abstract:A micro-polymerase chain reaction chip fabricated by MEMS technology, was successfully used to amplify the DNA molecule of GUS gene. Kinds of micro-chamber PCR chip were designed and fabricated. The surface of the chamber was washed and prepared using a special method. Sample amplification was performed with thermal cycling system. The GUS gene amplified in chip was analyzed by slab gel electrophoresis with separation of DNA markers in parallel. The polymerase chain reaction was successfully performed in chip. The rapid PCR was completed in 37 min for 24 cycles. The thermal cycle time in chip was reduced to 1/2 with a commercial PCR instrument.

Abstract:Kidney gene expression profile of db/db diabetic nephropathy mice was analyzed by using Affymetrix oligonucleotide genechip. Mice diabetic nephropathy related genes (and ESTs) were found out. Then, a 1.4 kb cDNA (mouse diabetic nephropathy related cDNA No.411, mdnr411, GenBank accession number AY256858) was amplified and cloned by using RACE method, based on the sequence of EST. Homologous analysis of the sequence was carried out. And the result shows that it is a new cDNA, which is highly homologous with mouse “RIKEN cDNA 5830436L09 gene (5830436L09Rik) mRNA”. The ORF of mdnr411 was predicted by using DNASIS v 2.5 Demo program. The result shows that the peptide coded by mdnr411 mRNA has 90 amino acids. And the result of homology screening did not find any obviously homologous protein, except Archaeoglobus fulgidus Na⁺/H⁺ antiporter (napA-2), which is partially homologous with the peptide coded by mdnr411 mRNA. All these results show that a cDNA of a new DNA related gene which function is fully unknown have screened and cloned. The foundation for further study of the function of the new gene, and its possible role in the pathogenesis of mice diabetic nephropathy has been laid, which is valuable to further understand the mechanism of diabetic nephropathy.

Abstract:The mammalian oocytes are important materials for study of meiotic cell cycle in developmental biology and cell biology. Confocal microscopy is a powerful technique in detecting the spindle-associated proteins in oocytes. However, in order to obtain the ideal results, some technological modifications should be made according to the characteristics of mammalian oocytes. Among them, the oocyte preparation, fixation, membrane permeabilization, fat extraction, and antibody incubation are the key steps. Furthermore, the differences among oocytes from various mammalian species should be considered in the actual manipulation of confocal microscopy.

Abstract:［B10Glu,B25-Glu-B26］human proinsulin gene was obtained by asymmetry PCR and cloned into the expression vector pBV220 and expressed in Escherichia coli DH5α in the form of inclusion body.The target protein was refolded in vitro and purified. After lysis with trypsin and carboxypeptidase B, an insulin analogue was purified by DEAE ion exchange and RP-HPLC.The product was characterized by MALDI-TOF MS.Circular dichroism spectrum was studied. The results implied the changes of the secondary structure and the association.The association of the analogue was further studied by size exclusive chromatography with a strong monomeric property. The biology activities including RIA, RBA were assayed, exhibiting 63.5% and 114.4% those of native insulin respectively. The result of mouse convulsion test indicated the insulin analogue had in vivo activity nearly the same as native insulin.

Abstract:In order to investigate the treatment efficacy and the potential mechanism of the attenuated Salmonella typhimurium strain expressing conservative region(AB) of Helicobacter pylori(Hp) adhesion X4072(pYA248-AB) in mouse model infected with Hp, the feasibility of X4072(pYA248-AB) in Hp vaccination was determined. Hp infected mouse model was established and applied in oral vaccination. The density of bacterial colonization was determined by the semi-quantitative bacterial culture assay. The T cell subsets of spleenocytes were assayed by flow cytometry (FCM) after immunization. IL-2 and IL-4 were detected through MTT. Mice sera and intestinal fluid were separatively tested for AB-specific IgG and IgA by ELISA. The results showed that after immune treatment, the eradicate rate of vaccine treatment group was 53.3%, which was significantly higher than that of other two control groups (X4072(pYA248-AB) vs X4072(pYA248) and PBS, P＜0.01). The Hp colonized density of vaccination group with Hp infection was significantly lower than that of other two groups (X4072(pYA248-AB) vs X4072(pYA248) and PBS, P＜0.01). Comparing the ratio of spleen CD4⁺/CD8⁺ T lymphocytic cells of the immuned mice by flow cytometer, it was found that the ratio of vaccine treatment group and mice typhoid fever germ control group were significantly higher than that of negative control group (X4072(pYA248-AB) and X4072(pYA248) vs PBS, P＜0.05) respectively. In addition, the ratio of vaccine treatment was significantly higher than that of mice typhoid fever germ control group (X4072(pYA248-AB) vs X4072(pYA248), P＜0.05). The further analysis of cytokine it was found that IL-2 and IL-4 of vaccine treatment group and mice typhoid fever germ control group were significantly higher than those of negative control group (X4072(pYA248-AB) and X4072(pYA248) and PBS, P＜0.05) respectively, meanwhile, the IL-4 of vaccine treatment group was significantly higher than that of mice typhoid fever germ control group (X4072(pYA248) vs PBS, P＜0.05). Humoral immunity analysis showed that IgA was only detected in the immune treatment group, and the IgG antibody titer of immune treatment group increased significantly than that of the other two group, and the titer reached 1∶10 000 two weeks after the second intensive immunization. By the animal experiments it is suggested that X4072（pYA248-AB）can eradicate or significantly reduce the Hp colonization on the mice stomach. The potential immune therapeutic mechanism included the increase of CD4⁺/CD8⁺ ratio, induction of Th1 and Th2 response, and production of the specific antibodies.

Abstract:Small prolin-rich proteins (Sprrs) participate in the construction of cornified cell envelop in stratified squamous epithelial cell. Their functions in the simple epithelium are not clear. Northern blots and semi-quantitative RT-PCR were used to investigate the expression pattern of Sprr2 in mice uteri during the estrous cycle and pregnancy as well as their hormonal regulation. The results showed the up-regulation of Sprr2 in the proestrous and estrous uteri during the estrous cycle. The expression of Sprr2 was rapidly down-regulated at the early stage of pregnancy until the perinatal stage when it was re-induced and reached the high level after labor. Due to its unique expression pattern in the uterus at different reproductive stages and its protective function in stratified squamous epithelial cell, its role in uteri against reproductive stress such as copulation and labor was suggested.

Abstract:The conservation of nucleotides at splicing sites and the characteristics of base composition and base correlation in the adjacent segment sequences have been investigated by use of the method of diversity measure combined with quadratic discriminant analysis. About 4 000 genes in five model genomes have been studied. The splicing sites and the exon/intron boundaries are recognized and predicted. The preliminary calculation shows that, through this simple and unified approach the prediction accuracy on the nucleotide basis is from 92.5% to 97.1% for C.elegans, A.thaliana, D.melanogaster and human. The prediction sensitivity and specificity on the exon basis are 83.7%～94.5% and 87.8%～97.1% respectively for these genomes. Non-canonical splicing has also been analyzed. The prediction capacity of the present method is comparable with GeneSplicer and other current splice site detectors.

Abstract:Second harmonic generation (SHG) imaging needs no fluorescent labels and brings no photobleaching or toxicity. It's a promising tool for high-resolution, three-dimensional studies in biomedicine fields. Backward SHG imaging system is established base on a converted laser-scanning two-photon fluorescence microscope. This epifluorescence configuration is especially advantageous to thick tissue imaging and in situ detection. Various tissues were tested to find out those which give rise to strong backward SHG. Then backward SHG imaging was applied in the studies of human skins. The SHG intensity from gangrenosis skins was much lower than that from normal skins. The skins of diabetic patients, which showed no macroscopic abnormality, also displayed a distinct decrease in SHG intensity. The result indicated that collagen in these skins may have suffered from micro-structural disorder or other changes. SHG imaging may serve as a new diagnosis method for dermopathy or other diseases.

Abstract:Neurochip is a microelectromechanical device made by silicon micromachine technology, on which the neuron cells of living mammalian can be cultured. These neurons can be detected readily under the control of applied circuits, and their responses to different stimulations can also be investigated. The overall process is selective, on-line, continually. The recent progress of neurochip technology is reviewed, and its future scene is anticipated.