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Abstract:S-nitrosation, which involves the formation of an S-nitroso function group on a protein cysteine residue, is a prototypic redox-based post-translational modification of proteins, and thereby conveys a large part of the ubiquitous influence of nitric oxide on cellular signal transduction. A purview of this modification was given mainly concerning the characteristics, the detection methods, the functional effects, the relevant diseases and the perspectives.

Abstract:Protein-protein interactions and recognition are the focal and hot themes of the life science field in the 21st century. Molecular docking approach is the effective computer modeling technology in the study of this topic. Generally, the protein-protein docking procedure is composed of four stages: searching of the binding modes of the receptor and the ligand, filtering of docked modes to eliminate the irrational docked structures, optimizing the structures, evaluating the docked modes with the refined scoring function and ranking them to obtain the near-native structures. Combining the research group's works, in terms of the international and national progress of protein-protein docking approaches, the detailed review was made about the four stages mentioned above. Additionally, the existing major questions are analyzed and the prospects of the future study are made.

Abstract:Bacterial ghost is intact bacterial envelope which is lysised by the lysis geneE of PhiX174. It can be used as vaccine directly. Foreign antigen can be targeted into outer membrane, inner membrane or the periplasmic space of bacterial, as a result, a recombinant bacterial ghost is constructed. Bacterial ghost, as a novel drug delivery system, is becoming more and more concerned, which can deliver DNA and protein vaccine or other drugs in order to have a better immune responses and therapeutic effects.

Abstract:TSH1, a novel transcription factor in rice containing conserved AP2/EREBP domain, was screened from the EST database. The specific interaction of TSH1 and DNA elements was detected by two kinds of traditional methods, electronic mobility assay and yeast one-hybrid assay, which suggested that THS1 specifically binds to GCC element in vitro and in vivo. Furthermore, It was confirmed by atom force microscopy (AFM) that TSH1 binds to GCC element (core sequence GCCGCC) not DRE element (core sequence GCCGAC), and the single molecular force between TSH1 and GCC element was quantitatively measured. The molecular force of TSH1 with GCC element is (83.9 ± 2.2) pN. This interaction can be observably blocked by the dissociate TSH1 protein in the solution. These above results proved that TSH1 recognizes and binds to GCC with specifically interaction, which also demonstrate the high sensitivity of the AFM measurements for the single base substitution of the GCC core sequence greatly reduced the binding affinity. Comparing these methods for identification the binding of transcription factor with its DNA element, it was considered that AFM is expected to be a sensitive and reliable method that offers valuable information for the characterization of transcription factors.

Abstract:The posttranscriptional modification of messenger RNA precursors by base deamination can alter profoundly the function of the encoded proteins. Adenosine deaminases that act on RNA (ADARs) are capable of catalyzing the site-specific conversion of adenosine to inosine in pre-mRNA transcripts, thereby affecting coding potential of mature mRNAs. ADAR1 is a member of ADAR1 family and is abundantly expressed in the brain and ubiquitously present in most tissues of the mammals. Alternatively spliced forms of ADAR1 cDNA were cloned from the mouse liver and the localization of these splicing forms were examined under a fluorescent microscope by fusing them to EGFP. The cDNAs of these splicing forms were cloned into a baculovirus expression vector and the recombinant protein of these splicing forms were produced in insect cells and purified with Ni-NTA resin. The double-stranded RNA editing activity of the recombinant proteins were determined and compared. The results showed four major splicing forms of ADAR1 were naturally present in mouse liver and consisted of cDNAs of 3.5 kb, 3.6 kb, 1.8 kb and 2.0 kb in length, respectively. These three splicing forms were La, Lb, Sa and Sb forms and found to have two distinct translation initiator codons. Furthermore, the four splicing forms of ADAR1 protein were localized to either the cytoplasm or nucleus of NIH 3T3 cells as shown by fluorescent microscopy. The double-stranded RNA editing activity of the four splicing forms also differ significantly, with the Lb form possessing the highest editing activity. In summary, four major mouse ADAR1splicing forms are present naturally in the mouse liver with either cytoplasmic or nuclear localizations and widely varied editing activities, which indicate they might have different substrates and functions.

Abstract:To investigate the effects of glucose regulated proteins (GRP78 and GRP94) on brain development, the expression of GRP78 and GRP94 in brain during mouse development were examined by Western blot, Northern blot and immunofluorescence. The results showed that the GRP78 and GRP94 proteins were expressed in the different spatio-temporal patterns during brain development. The level of GRP78 was higher than GRP94 in embryonic brain during the early stages of organogenesis, and gradually decreased at the later fetal stages. In contrast, the level of GRP94 was gradually increasing and exceeded the level of GRP78 at later fetal stages. Furthermore, the level of GRP78 was gradually decreasing from telencephalon to hindbrain on E16.5 but GRP94 was not. As stress proteins, the levels of GRP78 and GRP94 in brain were similar during postnatal development of mouse. In addition, the distribution of GRP78 in brain was in agreement with GRP94, which was localized within the neurons and glial cells. These results suggested that GRP78 and GRP94 played the different roles in the process of nerve cell differentiation and brain morphogenesis. They had distinct functions in the different stages during brain development of mouse.

Abstract:To explore the role of impaired brain glucose uptake/metabolism in neurodegeneration of Alzheimer's disease (AD), the relationship between an impaired brain glucose uptake/metabolism (down-regulation of tau O-GlcNAcylation) and the abnormal hyperphosphorylation of tau was investigated in fasting mice. It was found that fasting caused reduction of tau O-GlcNAcylation and a concomitant increase of tau phosphorylation at several hyperphosphorylation sites as seen in AD brain. Furthermore, hyperphosphorylation of tau induced by fasting was reversible in the brain after re-feeding. These findings provide a novel mechanism explaining how impaired brain glucose uptake/metabolism may contribute to AD-like tau hyperphosphorylation and also suggest feasibility to treat AD by reversing abnormal hyperphosphorylation of tau at early stages of the disease.

Abstract:An improved two-dimensional blue native/SDS polyacrylamide gel-electrophoresis (BN/SDS-PAGE) followed by nano-HPLC-ESI-MS/MS was used to study thylakoid membrane protein complexes between a low chlorophyll b rice mutant (Oryza sativa L. Zhenhui 249Y) and its wild type (Oryza sativa L. Zhenhui 249W). The PSⅠ-LHCⅠ super-complexes, LHCⅠ-less PSⅠ, ATP synthase, Cytochrome b6f, CP43-less PSⅡ, trimeric and monomeric LHCⅡ were resolved. 24 proteins, which belong to the subunits of four major photosynthetic apparatus were identified. Comparative study indicated that the mutant had moderately decreased amount of LHCⅡ, greatly decreased amount of LHCⅠ, and increased amount of PSⅡ reaction center and ATP synthase. Western blotting verified BN/SDS-PAGE analysis. These results provided clues about molecular foundation of higher photochemical efficiency and light stability of the mutant, and showed that the improved BN/SDS-PAGE is suitable for not only membrane protein complex separation, but also for comparative study of complex subunits in different physiological condition or between mutant and its wide type.

Abstract:With the limitations of conventional sequencing technologies, there still remains many gaps in complex genomes including the completed human genome. The closure of gaps as well as the solution of other problems caused by repeat sequences call for the development of novel techniques and strategy. Thus an ordered sequencing strategy is proposed based on the experimental advancements in positioning dissection and isolation of single DNA molecules with AFM nanomanipulation. The preliminary results of computer simulation verify its feasibility and indicate the potentialities in overcoming some intractable problems in current sequencing projects.

Abstract:A 1 270 bp full-length cDNA fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3′and 5′ ends of the incomplete expression sequence tag (EST) of hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum (SjHGPRT) were amplified by the anchored PCR with 2 pairs of primer that were designed according to the published incomplete SjHGPRT EST and the sequence of multiclone sites of library λgt11 vector. Sequence analysis indicated that this fragment, with an identity of 82% to hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni (SmHGPRT), contained a complete open reading frame(ORF). The deduced amino acid sequence showed 83% identity to that of SmHGPRT. This fragment was cloned into the prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE revealed that M of the recombinant protein was about 28 ku. Western-blot analysis showed that the recombinant protein was recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Mice vaccinated with recombinant protein revealed significant worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs of the female worms reduction percentage, compared with the controls. Taken together, the SjHGPRT full-length cDNA can be cloned and expressed in E.coli as a recombinant protein that elicited immunity against the challenge infection with Schistosoma japonicum, indicating its potential as a partial protection vaccine candidate.

Abstract:Deoxycytidylate (dCMP) deaminase is an enzyme belonged to dCMP cyt deam family. The dCMP deaminase from Streptococcus mutans UA159 was cloned and expressed in E. coli, and purified to homogeneity. The FPLC size exclusion chromatography analysis reveals that the S. mutans dCMP deaminase forms hexamer in solution. The protein was crystallized using hanging drop vapour-diffusion method and diffracted to a resolution of 3.1Å. The diffraction data were collected at BSRF beamline 3W1A. The crystals belong to P2₁3 space group, with unit cell parameters a=b=c=113.2Å, α=β=γ=90°. Assuming there are two subunits per asymmetric unit, the Matthews coefficient is 3.6ų·Da^-1. This is the first crystallization report of the wild-type deoxycytidylate deaminase.

Abstract:Conventional electrophysiological technique was used to determine the distribution of auditory-visual multisensory neurons and to study the auditory-visual information integration in rat cortex. 130 auditory-visual bimodal neurons including 65 A-V neurons, 28 v-A neurons and 37 a-A neurons were isolated. These bimodal neurons located in the boundary area between auditory and visual cortex were mainly in the dorsal bank of auditory cortex. There are divided strip trends in the distribution of different types of bimodal neurons that most of v-A neurons were recorded in the cortex close to auditory cortex, a-V neurons were mostly appeared in the part adjacent to visual cortex and a majority of A-V neurons were between of them. One point must be pointed out that bimodal neurons were presented not uniformly but in the way of patchy distribution. According to the different integration effects, all bimodal neurons were divided into three types: enhancement, modulation and depression. The temporal disparity among combinations of two stimuli was showed to be a critical factor influencing the integration of auditory and visual information. At the same time the best interstimulus interval time under which the strongest interaction can be evoked has been thoroughly studied and found that it has somewhat regular arrangement that 98 of the 130 (75%) auditory-visual bimodal neurons in this experiment have the best integration among the 30～50 ms interval time. The results suggest that the auditory-visual bimodal neurons exist in the rat cortex having the similar multisensory and auditory-visual integration characteristics of the multimodal neurons in the other animals′analogy cortex.

Abstract:A new and effective method to produce transgenic animals was established. Without a surgical incision, the recombinant plasmid containing green fluorescence protein (GFP) cDNA was repeatedly injected into male mouse testis at multi-sites. After few weeks of the final injection, the injected male was mated with normal oestrus female to produce transgenic mice. The presence of the GFP cDNA in F₁ transgenic individuals were detected by polymerase chain reaction and Southern blot hybridization, which showed that the transgenic rate of mouse F₁ offspring was 41%. The transferred gene was integrated into the host genome and could be transmitted to its offspring. When the positive F₁ individuals were mated with the wild type ICR mice, the F₂ individuals had a transgenic rate of 37%. The results indicate that the high efficiency of gene transfer and the limited number of manipulations make the method suitable for creating a large number of transgenic animals, especially, for producing domestic animals.

Abstract:Chitinases are ubiquitous chitin-fragmenting hydrolases, however until a few years ago that chitinase like proteins have been found in mammalian. A silica-induced bronchoalveolar lavage protein (iSBLP⁵⁸) with fibroblast growth promoting activity in silicotic rat, which had high sequence homology with members of the mammalian chitinase protein family has been previously purified and characterized. Bioinformatics analysis showed that several human EST clones from pooled colon, kidney or stomach matched the rat protein sequence. Thereafter clone from human kidney RNA samples with several pairs of primers was managed and a set of sequences was obtained, whose cDNA and amino acid sequences have high similarity with each other and several human chitinases in GenBank. Comparison with human genome sequence suggests that these molecules may be from variant transcripts of same a Pre-mRNA. Here the characterization of these sequences is reported.

Abstract:A cDNA (VvSUC27) of grape sucrose transporter was inserted into an expression vector pBI121 in the anti-sense orientation under the control of the cauliflower mosaic virus 35 S promoter, and subsequently transferred into tobacco(Nicotiana tobacum cv. Samsun). The plantlets of the transformed anti-sense VvSUC27 cDNA tobacco were grown normally on MS media containing 20g/L sucrose, but it was found that roots of the transformants developed slowly and the leaves have more chloroplasts when observed under slice. Sucrose content in the roots of the transformants was only 51% of the controls. The capability of sucrose uptaking and transporting into leaves among the transformed tobaccos was greatly decreased.