Abstract:Moderate correlations between transcriptome and proteome were found in most studies. According to different data types, current studies could be divided into four categories. They are comparison on single point, comparison between two differential points, comparison among multiple time-sequence points and comparison among multiple non-time-sequence points. In addition to the experimental error and the different datasets, the post-transcription restriction and regulatory would affect the correlation very much. Different results might be got for the different genes, cells, organs, organisms and even the different developmental phases. Because of the differentia and complementary between them, parallel studies of transcriptome and proteome trended to be performed, which could get the panorama screen of gene expression and discover the genes regulated in post-transcription.

Abstract:Regulatory T cells, which play an important role in the maintenance of the immune tolerance and immune homeostasis, have become a hot research field in immunology in recent years. Researching on the regulatory T cell is not only very important to realize the complex immune system, but also has the great potential application to the therapy of autoimmune diseases, cancer, HIV infection and transplantation. The recent experimental results have demonstrated that a transcription factor Foxp3 plays a critical role in the development of regulatory T cell and is a critical regulator for the development of regulatory T cells.

Abstract:Protein crystallization is the primary bottleneck step in X-ray protein crystallography. Among many variables that affect protein crystallization, such as growth condition, temperature, protein purity and concentration, the intrinsic characteristics of the protein is the most critical factor for protein crystallization. Common strategies and examples of protein engineering in improving protein solubility, homogeneity, and crystallizability are presented.

Abstract:Several E.coli determinants contributing to invasion of brain microvascular endothelial cells (BMEC) had been previously identified in vitro, including ibeB gene. In order to further characterize the role of the invasion locus ibeB in the absence of other E.coli K1 factors, the ibeB gene was subcloned into eukaryotic expression vector pcDNA3.1/HisC and transfected into HeLa cells. As a result, the HeLa cells expressing ibeB showed dramatic morphological changes comparing to the control, such as the obvious lamellipodial protrusions and membrane ruffles at the periphery of cells due to filamentous actin rearrangement. Cell-substratum adhesion ability and cell spreading ability of ibeB-transfected Hela cells were increased. Furthermore, ibeB could promote the aggregation of vinculin, which play a key role during focal adhesion formation. This first report provides experimental proof to probe the function of ibeB in the process of E.coli K1 invasion and the mechanism of lamellipodial protrusion.

Abstract:DNA ligase from the hyperthermophilic crenarcheon Sulfolobus shibatae (Ssh ligase) was optimally active in the presence of ATP and partially active in the presence of dATP. The enzyme was adenylated by both ATP and dATP, and the adenylate moiety covalently linked to the active site of the ligase was transferable to nicked DNA. Electrophoretic gel mobility shift assays revealed that the enzyme bound a duplex DNA fragment with a nick and that without a nick with similar affinity, but displayed little affinity for single-stranded DNA. Sso ligase, a closely related homologue of Ssh ligase from Sulfolobus solfataricus, interacted with PCNA-1, one of the three PCNA homologues found in the organism, as detected by yeast two-hybrid assays. No interaction of the enzyme with the other two PCNA homologues (PCNA-like and PCNA-2) was detected. Ssh10b, a member of the highly conserved Sac10b protein family of Archaea, stimulated DNA ligation by Ssh ligase, whereas Ssh7, a major Sulfolobus chromatin protein showed no effect on the activity of the ligase.

Abstract:The purpose of the study is to clone, identify, and express the mature secreted protein MPB63 from Mycobacterium bovis(MB) and to play a foundation for diagnosis of MB, for applying MB vaccine into clinic practice, and for detecting of immunity effectiveness. The gene encoding MPB63 was amplified from M. bovis Vallee111 chromosomal DNA by using PCR technique, PCR product was approximately 400 bp. Clone vector pGEM-T-63 was successfully constructed by the PCR product that was cloned into pGEM-T vector by using T-A clone technique. pGEM-T-63 and pET28a(+) were digested by BamHⅠ and EcoRⅠ double enzymes. The porkaryotic expression vector pET28a-63 was constructed by using the purified MPB63 gene that was subcloned into the expression vector pET28a(+). Plasmid containing pET28a-63 was transformed into competence E.coli BL21 (DE3). The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. An approximately 18ku exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting and it had antigenic activity of MB. These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and DNA vaccine against bovine tuberculosis.

Abstract:SD rats were applied as experiment materials to screen differentially expressed genes of the uterus in different stage of pregnancy by suppression subtractive hybridization (SSH), in which the uterus from the 5th and the 19th day in pregnancy were respectively used as driver and tester. Seventy positive clones from forward subtractive library are selected by differential screening. Sequencing analysis and homology comparison showed that all these clones are homologous to eight known genes in GenBank by the degree from 90% to 100%. These genes are differentially expressed in the uterus from the 19th day in pregnancy, in which the expression of uroguanylin and interferon-inducible protein 16 in the uterus in pregnancy are reported for the first time. Semi-quantitative analysis showed that the expression of uroguanylin was significantly higher in the uterus of 19th day in pregnancy than that of 5th day (P < 0.001), however, there was no difference about the expression of interferon-inducible protein 16. The expression of uroguanylin increased after embryo implantation, decreased on the 15th day and reached the peak on the 19th day in pregnancy. The result suggested that uroguanylin might be concerned with parturition.

Abstract:A novel human β3-galactosyltransferase gene β3GALT7 was isolated from human lung cDNA library and characterized. β3GALT7 is mapped to chromosome 19q13.2 by browsing the UCSC genomic database. It contains an ORF with length of 1 191 bp, encoding a protein with a signal peptide sequence and galactosyl-T domain, and its molecular mass and isoelectric point are predicted to be 43.3 ku and 8.37 respectively. The molecular mass of recombinant β3GALT7 protein expressed in prokaryote was coincide with the predicted. Northern blot showed that β3GALT7 was highly expressed in lung, throat and ileum, while the expression level was low in tongue, breast, uteri, testis. In addition, it was also demonstrated that β3GALT7 is differentially transcribed in human tumor cell lines.

Abstract:Red grouper (Epinephelus akaara) females between two and four years of age were successfully reversed to functional males by feeding 17 α -methyltestosterone (17 α -MT) over 42 days. A gene, called ECaM, was cloned from sex-reversed male gonads by using the combinative methods of suppressive subtraction hybridization (SSH), SMART cDNA synthesis and RACE-PCR. The full-length cDNA of ECaM is 582 bp, containing a 450 bp open reading frame that encodes a 149 amino acid protein. It has a 5 ′ untranslated region (UTR) of 74 nt and a 3 ′ UTR of 58 nt. Virtual Northern blotting shows that ECaM is transcribed in sex-reversed male gonads but only slightly transcribed in normal female gonads. Semi-quantitative RT-PCR analyses from various tissues indicate that mRNA of ECaM can be detected in the brain, heart, liver, spleen, and kidney but slightly in the muscle. Semi-quantitative RT-PCR and Western blotting from gonads during various stages of sex reversal reveal that expression levels of ECaM in gonads increase gradually during the transformation from female to male. Calmodulin is highly conserved across species. ECaM is one of the important genes that impels grouper sex reversal.

Abstract:Human group Ⅹ phospholipase A₂ is a member of mammalian secretory phospholipase which belongs to phospholipase A₂ superfamily. It has been expressed in a form of inclusion bodies in E.coli. It could not refold efficiently as human pancreatic phospholipase A₂ (group Ⅰ B) merely by diluting the protein unfolded in 8 mol/L of urea. The refolding reaction of human group Ⅹ phospholipase A₂ in vitro was depended on temperature, pH and protein concentration. A number of additives have been tested, among which L-arginine was the most efficient effector in improving the refolding of human group Ⅹ phospholipase A₂, and its structural analogs, L-citrulline, had less effect. L-lysine and L-arginine methyl ester could not improve phospholipase A₂ refolding. In addition, L-arginine inhibited the formation of aggregates and of intramolecular disulfide bonds. These results showed that both carboxyl and guanidyl residues of L-arginine were essential for improving protein refolding.

Abstract:A new method is developed for two-photon fluorescence anisotropy imaging. Its biological applications are tested. This system is based on a two-photon laser scanning fluorescence microscope. A polarization beam splitter was inserted into the optical path to separated fluorescence into components of orthogonal polarization. By rotating the polarization of the excitation beam by 90 ° , four images were collected for each sample. These images were then processed pixel-by-pixel to generate a new fluorescence anisotropy image. The capability of this method is tested for different samples, including FITC, FITC-CD44 in solution and FITC-CD44 attached to the membranes of tumor cells. The results show that fluorescence images can distinguish fluorescence molecules of different molecular mass, or detect changes in microenvironment.

Abstract:Potassium is at least 10 000 times more permeant than Na+, remaining to be an open question of K+ channels. To elucidate the mechanism of ion selectivity at atomic level, the density functional theory was used to calculate the potential on the basis of the X-ray structure of the KcsA K⁺ channel. In the viewpoint of statistic thermodynamics, the two most readily permeant ions, K⁺ and Rb+ ions, can pass through the selectivity filter. In contrast, Na⁺ ions, which prefer staying in site 2, can not pass through the channel. The energy barrier difference between the K⁺ and Na⁺ is 27.4 kJ/mol which induced the permeant ratio of 15 000. The energy barrier difference of Rb⁺ ions is higher than that of K⁺ ions which is corresponding to the lower permeant of Rb⁺ ions. The results are qualitatively consistent with the experimental data. It is remarkable that the system, involved in 269 atoms, is much lower than the MD simulation which is about 41 000 atoms.

Abstract:STAT3 plays very important roles in cell survive, proliferation, differentiation, and transformation. Constitutively activated STAT3 was found in many cancers and tumors, including melanoma, breast cancer, head and neck cancer. In order to reveal the mechanisms of STAT3 signaling regulation, a yeast two hybrid screening using STAT3 as bait was performed in the 7days mouse cDNA library. Among the positive clones, a novel protein with 259 amino acids, named SIPAR (STAT3 interacting protein as a repressor) with a GenBank accession number AY714985, was isolated to interact with STAT3. The interaction between STAT3 and SIPAR was analyzed using yeast two hybrid experiments and the functions of the interaction in STAT3 activities as a major signaling transducer was studied. The data from ? 茁 -Gal activity colony-lift filter assay and liquid assay showed that SIPAR had a strong interaction with STAT3. The Western blot experiment showed SIPAR was expressed as a 28 ku protein and a larger protein and mainly located in nucleus in mammalian cells. In order to investigate the function of SIPAR on STAT3 signal pathway, STAT3 luciferase reporter system and SIPAR expression plasmids were co-transfected into mammalian cells. The data demonstrated that when SIPAR was overexpressed, STAT3 transcription activity was inhibited dramatically. Finally, the effect of SIPAR on development was investigated in zebrafish. When SIPAR mRNA was injected, the zebrafish development was affected seriously with shortened body axis, which was in agreement with the experiment of inhibition of STAT3. The data suggested that SIPAR was a novel negative regulator on STAT3 activities.

Abstract:For the development of new targeted drug delivery vectors and molecular imaging reagents, it is essential to find the appropriate ligand that is both safe and efficient. Ligands against EGFR are highly sought after since it is highly expressed on many kinds of tumor cells and considered as a good target in cancer therapy. A new binding site based on EGFR 3D structure was proposed and DTP-Plated small molecule database was screened using the DOCK program. The selected molecules were further evaluated for their in vitro binding capacity using the BIAcore technique. It was shown that the molecule NSC51186 may be a novel small molecule targeting ligand for EGFR. Further studies are warranted to investigate its potential in targeted drug delivery and gene delivery, as well as molecular diagnosis applications.

Abstract:Accelerated availability of new sequences, especially ESTs, calls for computational methods to link sequences with Gene Ontology (GO) terms in a batch mode. There is currently no program for such purpose except Goblet, an online tool which uses BLAST to interpret query sequence with proper GO terms, but has a restriction of upload sequence files less than 100 kilobytes in size. GoPipe is a standalone package that integrates BLAST and InterProScan results to obtain Gene Ontology annotation with built-in statistical options. GoPipe takes any number of BLAST and/or InterProScan output files simultaneously and launches jobs sequentially to perform parsing, data integration, redundancy removal, GO distributions calculation and graphic display. A very high annotation specificity of 99.1% was achieved for a test dataset when the program was run in the "intersection" mode, which intersects the BLAST and InterProScan results, outperforming the specificity (81.1%) obtained from the InterProScan only. Statistical tools are also provided to compare GO distributions between different inputs, so that GO distributions of different sets of batch sequences can be compared, and differentially represented GO terms can be easily displayed. High specificity, speed and flexibility make GoPipe an ideal tool for streamlined GO annotation for batch sequences. The package is freely available at http://gopipe.fishgenome.org/ or by contacting the authors.