Abstract:PRDM9(PR domain containing 9) is one kind of histone trimethylase which catalyzes the H3K4 trimethylation, and possesses the activity of transcription factor. PRDM9 transcripts were only expressed in germ cells entering meiotic prophase in female fetal gonads and in postnatal testis, whose deficiency results in sterility. The zinc finger motif of mammalian PRDM9 rapidly evolves, which is respond to the sequences of recombination hotspots. Several researches indicated that PRDM9 was involved in the binding to recombination hotspots and initiation of recombination. These progresses are important for understanding of species evolution and the mechanism of genetic recombination.

Abstract:Screening small molecules having anti-aging effect and researching the mechanism are of great practical significance for discovering new effective targets for treatment of age-related diseases and tumors, developing new drugs and promoting human health. What is more important is that these small molecules can be used as breakthrough points for the specific molecular mechanisms of aging, cancer and other biological phenomena via in-depth study. And this has an important role in promoting the development of molecular biology and other related life sciences. This article summarizes some representative anti-aging small molecules of the past decade or two and focuses on their molecular mechanisms.

Abstract:Chromosome conformation is thought to play an important role in gene expression regulation. Chromosome conformation capture (3C), circular chromosome conformation capture (4C), 3C-carbon copy (5C), ChIP-loop assay, ChIA-PET and Hi-C method were developed to study spatial organization of long genomic regions in living cells. All steps of 3C, those 3C-based methods and key procedures in experiment were also discussed. The aim of this paper is to introduce some new methods to study gene regulation in 3D.

Abstract:Dendritic cells (DCs) are the most potent professional APCs and play an important role in cancer immune response. With the rapid development of DC-based cancer vaccines, tumor-DC hybrids are the most effective ones —— they could present antigens and keep on producing endogenous antigens. The tumor-DC hybrids are currently being evaluated in many animal and clinical trials, available published data showed that hybrids could induce tumor specific T lymphocyte response and be well-tolerated in patients. The progress of tumor-DC hybrids, as well as some issues was discussed.

Abstract:Metabolic diseases including obesity and diabetes are emerging as major health threats over the world. Cidea, a member of CIDE family proteins, plays an important role in energy homeostasis in brown adipose tissue by regulating lipid storage and AMPK stability. Cidea is tightly regulated at both transcriptional and posttranslational levels, but little is known about its translational regulation. Here, a novel N-terminal truncated isoform of Cidea (mCidea-22) was identified in mouse brown adipose tissue. Using mutational analysis, it was demonstrated that this isoform was generated by alternative translation. Ectopic expression of mCidea cDNA in various cell lines showed that expression of mCidea-22 was cell type specific with its highest expression in preadipocyte 3T3-L1 cells. In addition, it was observed that mCidea-22 had a short half-life and was localized to ER and lipid droplets. Taken together, the data provide strong evidence that translational control played an important role in the regulation of Cidea expression.

Abstract:Somatic cell nuclear transfer (SCNT, Somatic cloning) has been succeeded in some species, but the low cloning efficiency limits its application in many areas, including medicine industry, therapeutic cloning and the propagation of rare animals. In somatic cell nuclear transfer process, the donor nucleus requires epigenetic reprogramming to a totipotent ground state. Although molecular events within hours of nuclear transfer determine the fate of cloned embryos, it is disappointing that so little is known about these events during the early development of cloned embryos. Genomic imprinting is the differential expression of a gene based upon parental inheritance. DNA methylation is a known regulator of major genomic imprinting, and many imprinted genes are associated with DMRs that play a role in regulating their allele specific expression. Mash2 is an imprinted gene that plays important role in embryo development and organogenesis. Aberrations were often observed in the lungs of most deceased cloned calves died around birth. In an effort to determine whether the DNA methylation reprogramming of Mash2 is efficient in somatic cloning animals, the DNA methylation status of Mash2 was analyzed in lungs of deceased somatic cloning bovines that died within 48 h of birth using bisulfite sequencing analysis. The results demonstrated that cloned bovines showed significantly lower DNA methylation of Mash2 than controls (P < 0.05), and both showed low methylation (20.04% and 5.55%)，and the percentage of overall mCpG in 9C5 was only 0.4%；the types of methylation patterns were five in 9N3 and one in 9N4，while only one type were found in cloned cow．The abnormal DNA methylation of Mash2 may contribute to the lung development defects and the low efficiency of somatic cloning.

Abstract:The carboxylesterase gene of Geobacillus stearothermophilus CICC 20156 was cloned by bioinformatics technology and then was inserted into the expression vectors pPIC9K and pYG1.2. The recombined plasmids were transformed into yeast Pichia pastoris GS115 and Aspergillus niger M54 respectively. It was revealed by SDS-PAGE and Western blot that the exogenous protein of about 29 ku with a His-tag each were secreted by the transformed hosts into the external mediums. The concentration of recombinant carboxylesterase in medium excreted by Pichia pastoris and Aspergillus niger was 30.7 mg/L and 15.3 mg/L respectively. Bioactivity assay indicated that the carboxylesterase activity was 22 671 U/mg for the enzyme expressed by Pichia pastoris and 21 438 U/mg for that expressed by Aspergillus niger. The research results also showed that both enzymes expressed by the two kinds of hosts possess similar characteristic. The recombinant enzyme exhibited thermostability with optimum temperature at 60℃ and remained 76.7%～67.6% activity even after incubation at 70℃ for 30 min. The enzyme also showed a broader pH tolerance and the optimum pH for the enzyme activity was at 8.0. This is the first report that the thermostable carboxylesterase of Geobacillus stearothermophilus was highly secretively expressed in Aspergillus niger and Pichia pastoris. Though the amount of carboxylesterase excreted by recombined Aspergillus niger was lower than that excreted by recombined Pichia. pastoris, the engineering Aspergillus niger may still show a better perspective for potential applications in biotechnological industries, for no inducer is needed to get crude carboxylesterases in the culture supernatants of it.

Abstract:The C-terminal four residues of influenza A virus non-structural protein 1 (NS1) comprise the binding site for PDZ (the domain of PSD95, Dig and ZO-1) and is named PL domain (PDZ ligand domain). Previous study showed that NS1 proteins from different subtypes/strains of influenza A virus varied in their amino acids sequence of PL domain, which may affect the interaction between NS1 and cellular proteins and is closely associated with the pathogenicity of influenza A virus. To further explore the role of PL domain in the biological properties of NS1 protein, four wild type NS1-expressing plasmids harboring the NS1 encoding sequence from different influenza A virus subtypes (H1N1, H3N2, H5N1 and H9N2) were constructed firstly, based on pEGFP-c1 vector. The mutant NS1 (H3N2)-expressing plasmids were subsequently generated by either deletion or replacement of PL domain from other influenza A virus subtypes. Comparative analysis of the localization of these NS1 proteins in HeLa cells showed that wild type NS1 protein from H3N2 virus mainly localized in the nucleoli, whereas wild type NS1 proteins from H1N1, H5N1 and H9N2 viruses or mutant NS1 proteins from H3N2 virus mainly localized in the nuclei (but not nucleoli). In MDCK cells, none of the above NS1 proteins located in the nucleoli. The results indicated that PL domain can significantly influence the nuclear localizing pattern of NS1 protein in HeLa cells, which may be responsible for the variation of NS1 protein in its biological function; the distributed pattern of NS1 protein is closely associated with the origin of host cells.

Abstract:Parkinson disease (PD) is the common movement disorder. Along with the development of molecular genetics, genes related to the familial forms of Parkinson's disease such as Parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1——PINK1 (PARK6) have been identified. GST pull-down assays were performed to identify which domain of PINK1 interacted with Parkin, and the result showed that Parkin directly interacted with PINK1, and the PINK1 kinase domain interacted with Parkin. Based on these results, then the co-immunoprecipitation technique was used to investigate the possible interaction between Parkin and PINK1 in 293A cells. The results indicated that Parkin stabilized PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway, and PINK1 decreased level of Parkin by promoting its degradation via the ubiquitin-mediated proteasomal pathway. The results confirmed that Parkin directly interacted with PINK1, and they regulated each other via the ubiquitin-mediated proteasomal pathway.

Abstract:In plants, chloroplast is the key organelle for the photosynthesis, the knowledge about biological processes in chloroplast has been accumulated. However, limited information exists on the expression of chloroplast proteins. To investigate the expression profiling of rice chloroplast proteins in different growth and developmental stages and provide a pilot experiment for rice antibody-based proteomics. To address this questions, ten rice chloroplast genes were chosen and antibodies were generated using proteins expressed in E. coli or epitope peptides synthesized in vitro as immunogen, protein expression profiling were investigated by Western blotting for root, stem, leaf and panicles at five developmental stages. The results indicated that all chloroplast proteins tested were expressed in leaf, but not detectable in root. The photosynthesis primary reaction protein CAB1 and CAB2, the electron transport protein OEE1, and the ROS scavenging-related proteins 2-CysP and Trx were expressed in stem, but four carbon fixation proteins RCA, GAPDH, FBPA and SBPase, which involved in Calvin cycle, were not detected in stem. In panicle, the chloroplast proteins showed different expression patterns, CAB2 and 2-CysP were expressed at all stages during panicle growth and development, CAB1 and OEE1 were expressed at late stage, and the four proteins involved in Calvin cycle were expressed only in the middle stage. Interestingly, four proteins in Calvin cycle showed the same expression patterns, supporting their cohesive relationship. In addition, the data revealed possible clues of post-translational modification, dimer and different forms of transcripts. Comparison analysis between the profiling of gene transcription and translation revealed parallel phenomena; however, they are quite different at least in some instances. Taking together, this experiment revealed the expression patterns of rice chloroplast proteins in a direct and relative quantitative way, provided helpful information for better understanding their function and also provided a preliminary proof for the concept of a rice antibody-based proteomics strategy.

Abstract:Uncovering the underlying regulatory mechanism has become a major research in bioinformatics studies. The availability of various kinds of high-throughput biological data makes the reconstruction of regulatory networks on a genomic scale possible. Since each single data source provides only partial and noisy information of the regulatory relationships, methods combining diverse data sources are expected to get more reliable networks. Here a method was presented to infer the regulatory networks by combining ChIP-chip, TF (transcription factor) knock out and expression data. Since ChIP-chip and TF knock out data provide direct physical binding and functional evidences of relations between TF and target genes, combining these two data is expected to obtain high prediction accuracy. However，the overlap of these two data is low. Based on the assumption that co-regulated genes often have high expression similarity, the method reduced the effect of the low overlap of these two data to some extent. The results show that most inferred regulatory relations are validated by YEASTRACT, high quality ChIP-chip data and literatures, which demonstrate our method is powerful and reliable. Moreover, the comparison between our method and others also shows that it has better performance.

Abstract:Intron as a kind of non-coding DNA is rich in eukaryote genomes. The functions and involution mechanisms are not very clear besides the splicing. It was thought that introns play a very important role in maintaining and regulating the functional mRNA structure after splicing in the process of mRNA export and translation elongation, etc. Moreover, intron sequence and its corresponding coding sequence are existed interaction or co-evolution relations. The relations between intron sequences and its corresponding coding sequences were studied. For the C. elegans ribosomal protein genes, 85 genes were selected from RPG (http://www.cbi.pku.edu.cn/chinese/mirrors.html). The intron sequences were divided into first introns, second introns, other introns, short introns, and long introns and the corresponding coding sequences were divided into exons and all protein coding sequences (CDS), then the matching local alignment between introns and the corresponding coding sequences were done with Smith-Waterman local alignment software. The results show that there are really the interaction regions in introns when it is aligned with coding sequences. When intron sequences are aligned with CDSs, the significant interaction regions for the first intron and the other intron are located in about 15%～55% of intron length and it is located in about 30%～80% of intron length for the second intron. The distribution of interaction regions for short introns is similar to the distribution of the first introns. For long introns, there are two significant interaction regions. The first peak region is located about 15%～30% of intron sequence and the second peak region is located about 54%～78% of intron sequence. When long introns are aligned with exons, there is only one peak region. It is located in about 5%～20% of intron upstream region. When CDS are aligned with every kind of introns, it was found that there are many interaction regions and forbidden regions in CDSs. It was also found that there are two common forbidden regions in the CDSs, they are located at the 10% and 80% of coding sequence. The distribution of interaction regions for the first introns is different from the second introns. When compared the distributions of long introns aligned with CDS and aligned with exons, it can be concluded that the segment of the first peak region are acted on the inner exon segment, the segment of the second peak region are acted mainly on the exon-exon junction regions. Furthermore, there are many peak regions and forbidden regions which are distributed in protein coding sequences. It is speculated that the forbidden regions may be the combined regions of protein complex. In a word, all of the intron sequences besides the 5' end and 3' end correlate closely with their corresponding coding sequences or the two kinds of sequence segments are existed co-evolution relation.

Abstract:The objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) with specific monoclonal antibodies for quick detection and screening of a broad spectrum of O,O-diethyl organophosphorus pesticides. Diethylphosphono acetic acid was used as a hapten and conjugated with bovine sera albumin (BSA) or ovalbumin (OVA) to prepare immunogen and coating antigen, respectively. Two hybridoma cell lines that produce monoclonal antibodies against organophosphorus pesticides have been obtained by fusing mouse myeloma cells SP2/0 and splenocytes from Balb/c mice immunized with the immunogen.The immunoglobulin class was analyzed by double immunodiffusion and the affinity and specificity of McAbs were determinated with indirect enzyme-linked immunosorbent assay (ELISA). Results indicated that the immunoglobulin class of McAb was IgG1, McAbs have a high affinity for diethylphosphono acetic acid (1.4×10⁷ L/mol) and are able to cross-react to Chlorpyifos, Parathion, Profenofos, Omethoate, Dichlofenthion, Diazinon, Bromophos, Phoxim. This immunoassay is therefore capable of detecting 8 different organophosphorus pesticides. It is concluded that this assay is a qualified screening tool for quantitative or semi-quantitative detection of the 8 organophosphorous pesticides.

Abstract:Proteomics has become a powerful technology being successfully used in plant science research to investigate different biological processes, including plant genetics, development, physiological ecology and responses to climate change. Two-dimensional electrophoresis (2-DE) is a potential analytical tool in proteomics to identify qualitative and quantitative changes of proteins and reveal the changes of protein expression under different growth conditions. However, protein sample preparation is the key fundamental steps of 2-DE analysis, and it is also a prerequisite for proteomics analysis. L638-y is a chlorophyll-deficient mutant naturally generated from the wild type L638-g in Brassica juncea L., which is typical characterized with yellow leaves and no other different traits from the wild type except leaves color. In order to efficiently analyze the differential expression proteins between the mutant L638-y and its wild type L638-g, two protein extraction methods for proteomic analysis, TCA/acetone extraction method and an improved polyethylene glycol ( PEG) fractionation method, were compared. Total proteins were extracted from leaves of the mutant L638-y and its wild type L638-g at five leaves period with the two protocols, then the samples were analyzed by 2-DE, pH of IPG strip and concentration of SDS-PAGE being optimized. The results showed that when using 17 cm linear IPG strips pH ranged from 4 to 7, 11% SDS-PAGE, loading 180 μg/350 μl, proteins were better separated. Much more protein spots had been observed in 2-DE maps of the protein samples prepared by the PEG fractionation than TCA/acetone precipitation. In 2-DE profiles of the protein sample from mutant L638-y prepared by the PEG fractionation, as much as 1 235±6 protein spots were identified, 330 more spots than those by TCA/acetone precipitation method. By using the PEG fractionation, 190 differential protein spots were identified between the 2-DE maps of the mutant L638-y and wild type L638-g, 100 more spots than those by TCA/acetone precipitation. Thus, the improved PEG fractionation is more efficient and simple for proteomic analysis of chlorophyll-deficient mutants in Brassica Juncea L.