Abstract:NEDD8 is a member of the ubiquitin-like proteins. The overall structure of NEDD8 is quite similar to ubiquitin. Covalent conjugation of NEDD8 to proteins at the post-translational level is called Neddylation. Neddylation occurs similarly to ubiquitination and need enzyme cascades involving E1, E2 and E3. Neddylation has been demonstrated to be essential to maintain the ubiquitin ligase activity of Cullin-Roc based E3 ligases. Compared with the ubiquitination which was widely studied in the past two decades, few substrates were identified for Neddylation and the physiological functions of Neddylation need further investigations. The current progress of function and regulation of protein Neddylation will be reviewed.

Abstract:MicroRNAs (miRNAs) are small non-coding RNAs which are essential for posttranscriptional gene regulation and have important roles in physiology and pathology. The researches on function of miRNA will be a focus in the future. Several animal models have been built by transgenic technology, making important contributions to our understanding of gene function at the whole scale. Recently, the number of transgenic animal models for microRNAs and construction strategies have been increasing and diversifying. Some roles of miRNA in tumor and cardiovascular disease have been revealed by transgenic animals. Transgenic animals are becoming a kind of powerful tool in microRNA researches.

Abstract:Osteoclastic bone resorption and osteoblastic bone formation are coordinated as a coupled mechanism to effect the development of bone and to maintain bone homeostasis. Recently reported Eph/ephrin bidirectional signaling between osteoclasts and osteoblasts plays a pivol role in bone homeostasis and casts new light on coupling of bone resorption and bone formation, which is gaining more and more attention in researches of bone biology and bone diseases. The present article aims to address the researches on the Eph/ephrin bidirectional signaling between osteoblasts and osteoclasts with molecular constitution, mechanism of the signal transduction, biological significance and so on.

Abstract:Photodynamic therapy (PDT) relies on the interaction of light, photosensitizer, and the presence of oxygen, leading to cytotoxicity to the tissue containing the photosensitizer. The tissue specificity of photosensitizer is thus one of the key factors in photodynamic therapy. The limited targeting capability of current photosensitizers that are in the clinical applications has led to the active development of the new generation of photosensitizer. Another approach-peptide conjugation-to enhance the tissue specificity of photosensitizers were reviewed. The cell penetrating peptides, vasculature targeting peptides, and peptides targeting cell surface receptors were reviewed. The current research indicates that the effectiveness of peptide conjugation to confer the tissue specificity to photosensitizers.

Abstract:Norcantharidin ( NCTD ) is an effective anti-tumor drug developed by China independently. It has been widely used for clinical therapy especially in digestive tract cancers. It was found that NCTD can induce M arrest and apoptosis in a dose- and time-dependent manner in BGC-823 cell line. In order to reveal the molecular mechanism by which NCTD actions systemically, a comparative analysis of proteomic profiling was conducted between control cells and NCTD treated cells by 2-D and mass spectrum. The results indicated that mitochondrial heat shock protein CH60, ATP synthase d subunit , ER glucose-regulated protein GRP78, mitochondrial Hsp70 related factor GRPE1, SH3 domain-binding glutamic acid-rich-like protein SH3L3 and Histone-binding protein RBBP4 may involve in the antitumor function of NCTD. The result suggested that NCTD might induce caspase-3 dependent apoptosis through promoting the expression of mitochondrial heat shock protein and p53. NCTD can promote the apoptosis by inhibiting the activity of ERK after inducing ER stress. The combinational treatment of BGC-823 cells with oligomycin A, an inhibitor of mitochondrial ATP synthase, and NCTD inhibited the growth of BGC-823 cells more evidently compared with single drug treatment. This result confirmed that NCTD can suppress the growth of BGC-823 by inhibiting the activity of mitochondrial ATP synthase.

Abstract:The mechanism of how stroma plays an important role in tumor carcinogenesis is now a hotspot. To delineate the features of stromal protein between nasopharyngeal carcinoma (NPC) and normal nasopharyngeal mucosa(NNM), laser capture microdissection (LCM) was performed to purify stromal cells from the NPC and NNM, respectively. The protein expressed profiles of the stroma of NPC and NNM were compared using fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) and 34 differential protein spots between tumor stroma and normal stroma were chosen to be identified by mass spectrometry (MS). A total of 20 differential proteins were identified, and three differential proteins (CapG, L-plastin and S100A9) were selectively further validated by Western blotting and immunohistochemical analysis to confirm the results of 2D-DIGE. 2D-DIGE patterns of the stroma of NPC and NNM were established for the first time, the results suggested that differentially expressed proteins in the stroma of NPC and NNM may be useful for understanding the relationship between NPC cells and their surrounding microenvironment. Further studying of these proteins will be helpful to elucidate the mechanisms of NPC carcinogenesis and provide new thoughts on therapy of NPC through stroma.

Abstract:The 3′untranslated region of CCAAT/enhancer binding protein β(C/EBPβ) is a regulation element with tumor suppression activity found in previous study. Here, it is reported that deletion of 3 short sequences in this 3′UTR reduces the tumor suppression activity of it, as demonstrated by slowed-down cell growth, reduced colony formation ability in soft agar and in ordinary culture conditions, as well as the decreased tumorigenicity in nude mice. The cDNA array and real-time RT-PCR analysis showed that the loss of tumor suppression activity for the mutated 3′UTR was due to the change of the gene expression profile of the transfected cells, i.e. the up-regulation of several genes related with malignant phenotype and the down-regulation of some genes related with tumor suppression, compared with the revertant control cells. These results indicate that those short sequences are simultaneous necessary for the tumor suppression activity of the C/EBPβ 3′UTR.

Abstract:Individuals with CCR5Delta32 mutant genotype can naturally resist human immunodeficiency virus type 1(HIV-1) infection. The mechanism is mainly due to CCR5Delta32 mutant protein expressed in the individual peripheral blood mononuclear cells (PBMCs), through trans-dominant negative (TDN) effect, which can inhibit two types of HIV-1 coreceptor (CCR5 and CXCR4) on the cell surface from producing. The recombinant lentiviruses, namely Lenti-CCR5Delta32, were generated and used to infect human PBMCs. HIV-1 infection of the PBMCs transduced with Lenti-CCR5Delta32 showed that CCR5Delta32 protein expressed in human PBMCs was able to inhibit R5, X4, and R5X4 HIV-1 infection. The result is expected to be used for the gene therapy on AIDS, which deserves further study.

Abstract:Wild type (WT) and F185K mutant type of HIV-1 integrase catalytic domain (IN_C) were expressed and purified, and their solubility and activity were compared. The experiment results show that the solubility of F185K mutated IN_C was dramatically increased, whereas the activity was reduced to some extent. Subsequently, 1 800 ps molecular dynamics (MD) simulations for the WT and F185K type of IN_C in water were performed. The MD simulation results demonstrate that the flexibility of the catalytic loop region and the total mobility of F185K IN_C was reduced, which causes the decrease of activity. After the F185K mutation, changes of the salt bridge network drove the conformational change of IN_C, resulted in the burying of some hydrophobic residues and exposure of some other hydrophilic residues on the protein surface. Therefore, the relative hydrophilic solvent accessible surface of IN_C was increased. Moreover, the F185K mutation increased the hydrogen number between the IN_C protein and water molecule, as a consequence, the protein-water interaction was enhanced. These above changes contribute to the solubility increase of IN_C. It is found that the results obtained from MD simulation are in good agreement with the experiment data. The above mentioned results provides valuable insight for the understanding of protein solubility and will be helpful in protein engineering for increasing the solubility of proteins.

Abstract:53 new microRNAs which have p53-DNA binding sites and regulated the p53 upstream transcription factor and downstream target genes were screened from 676 human microRNAs. The microRNAs and p53 protein interaction networks was constructed through mined the known interaction of p53 and p53-microRNAs. Remarkably, FAS was found, which is a key factor in the apoptotic pathway regulated by a number of microRNAs. The interaction of FAS-microRNAs maybe play a key role in the apoptotic pathway. A presumptive p53-microRNA regulatory mechanism was proposed: p53 as a transcription factor regulated the target genes and direct regulatory microRNAs, whereas it also is regulated by upstream transcription factor and microRNAs. So, a balance which p53 located in the center would be formed by the factors of p53 pathway, the disease would be caused when this balance is broken. A total of 15 500 genes were predicted as targets of these 53 microRNAs and they are classed by 27 clusters according to the frequency of gene in all the 15 500 genes. Function annotation analysis of genes frequency more than 10 revealed a novel p53 functions, including cell adhesion and migration which suggested that p53 can suppress metastasis through direct transcriptional regulation of this new category of molecular targets. 30 microRNAs which involved in cell cycle, apoptosis and cell proliferation were explored through gene functional enrichment analysis, noticeably, 9/30 microRNAs (hsa-mir-181a-1, hsa-mir-181b-1, hsa-mir-181c, hsa-mir-181d, hsa-mir-195, hsa-mir-497, hsa-mir-495, hsa-mir-543 and hsa-mir-548c) regulated all three biological process, which implies that these 9 microRNAs maybe play a key role in the regulation of p53 signaling pathway and feedback loops through interaction of microRNAs. Finally, the homology and conservation of 30 microRNAs were analyzed in the 36 species and 10 new highly conserved microRNAs (hsa-mir-497, hsa-mir-495, hsa-mir-543, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-200b, hsa-mir-448, hsa-mir-28, hsa-mir-455 and hsa-mir-590) which have not included in the current microRNA database yet were found.

Abstract:It is known that (-)-Epigallocatechin-3-gallate (EGCG) is the inhibitor of TLR4 signaling pathway activation. To investigate a possible role of TLR4 signaling pathway in the development of atherosclerosis, the effects of EGCG on the development of atherosclerosis, the expression of TLR4 and inflammatory cytokine production in apoE^-/- mice was investigated. Fifty male apoE^-/- mice (5wk old) were divided into four groups: basic diet group (control group), high-fat diet group (control group), EGCG+ basic diet group (EGCG group), and high-fat diet + EGCG group (EGCG group). EGCG (10 mg/kg) was injected intraperitoneally every day. Areas of aortic plaque areas were measured by oil red O staining. Western blot was used to detect the expression of TLR4 and CD14 in mouse aorta. The expression of TLR4 mRNA and CD14 mRNA were detected by Real-time PCR. Serum concentrations of MCP-1 and TNF-α were determined with ELISA. Compared with high-fat diet group, EGCG groups showed marked decreases in aortic atherosclerosis(P < 0.05), concomitantly with significant decreases in levels of the expression of TLR4, TNF-α and MCP-1. The mean lesion area was (2.37 ± 0.08) mm² in the high-fat diet + EGCG group, whereas the atherosclerotic lesion was only (1.05 ± 0.13) mm² in the high-fat diet + EGCG group. The TLR4 expression was obviously higher in high-fat diet group than that in other groups (P < 0.05). Compared with high-diet group, the serum concentrations of MCP-1 and TNF-α were significantly decreased in EGCG groups (P < 0.05). These results suggest that TLR4 signaling pathway may play an important role in the development of atherosclerosis in apoE^-/- mice induced by high-fat diet.

Abstract:Cisplatin, an efficient anticancer agent, can trigger multiple apoptotic pathways in cancer cell. However, the signal transduction pathways in response to cisplatin-based chemotherapy are complicated, and the mechanism is not fully understood. Using fluorescence resonance energy transfer (FRET) technique, the molecular mechanism of cisplatin-induced apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) were investigated. After cisplatin treatment, the recombinant pFRET-Bid and pSCAT-3 probes were used to determine the kinetics of Bid cleavage and Caspase-3 activation, respectively. The fluorescence probes Bid-CFP and DsRed-Mit were also used to detect the spatial and temporal changes of Bid in real-time in sub-cell level. The results showed that a cleavage of the Bid-FRET probe occurring at about 4～5 h after treated with 20 μmol/L cisplatin. Cleavage of the Bid-FRET probe coincided with a translocation of tBid from the cytosolic to the mitochondria, and the translocation lasted about 1.5 h. At the anaphase of cell apoptosis, Caspase-3 was activated obviously as detected by FRET and Western blotting techniques. Using real-time single-cell analysis, it was observed the kinetics of Bid and Caspase-3 activation for the first time in living cells during cisplatin-induced apoptosis.

Abstract:The transcription factor Oct-4 is expressed specifically in mammalian preimplantation embryos and its function is related to the maintenance of embryonic stem cell pluripotency. The functional role of the heterogenous expression of Oct-4 remains unclear however. A GFP reporter construct, pOct-4(p)-GFP was generated, containing the upstream regulatory regions of bovine Oct-4 gene and its expression pattern was evaluated in the developing embryos of mouse, pig and rabbit following intracytoplasmic sperm injection. GFP fluorescence was visible early at the 2-cell stage and then became stronger in the blastocysts of all three species. However, the distribution of the GFP signals was restricted to the cells of inner cell mass and no fluorescence was detectable in trophectoderm cells. These results suggest that the bovine Oct-4 promoter is functional and that its embryonic expression activity is similar in different mammalian species.

Abstract:Transgenesis in rabbit has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock by the combination of transgenic technology. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. 24-well cell culture plates were used to isolate cell lineages obtained from a single fibroblast clone transfected with the pRNAT-U6.1/Neo plasmid. Since single fibroblast clone does not grow well in fresh medium, the use of conditioned medium was evaluated. Meanwhile, the effect of initial cell density and linear-plasimid on transgenic fibroblast colony growth were investigated. The increasing initial cell density and lining plasmid could improve colony growth and expansion. There was a significant difference in the conditioned medium or initial cell density compared to the control group. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. When the transgenic fibroblasts were used as donor cells of nuclear transfer, the blastocyst rate were 23.5%. There was not a significant difference in the transgenic fibroblasts compared to the normal group. PCR or Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal β-actin DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production; further improvements in related technologies will facilitate the use of this method for the generation of the genetic engineering of rabbits.

Abstract:Thymosin-β4 (thymosin-β4, Tb4) is an important G-actin sequestering factor in cells, which could regulate the activity of G-actin in various microfilaments events. Previously, many researches have reported that Tb4 has broad cellular and physiological functions, but until now, there is lack of systematic research on mammal oocytes and early embryonic development. Tb4 is a small hydrophilic peptide, initially, which separated and purified from bovine thymus component Ⅴ, with a molecular mass of 5 ku, and compose of 43 amino acid residues. Tb4 molecule is highly conservative, which has been founded in a variety of tissues and organs in vertebrates, and also exists in part invertebrates. This peptide has extensive biological functions. Recent studies have shown that it closely relate with many physiological and pathological activities, such as immune regulation, neurological development, wound healing , inflammatory response, angiogenesis, apoptosis, tumor occurrence and migration. Dynamic events of microfilaments in cells always accompany with G-actin activity, while Tb4 could bind G-actin with a ratio of 1∶1 to inhibit G-actin to polymerize into microfilaments. In cells, nearly half of G-actin are bound with various of sequestering factors, which effects on both G-actin activity regulation and its reservation, while Tb4 is the chief executer of G-actin sequestering factor family. The ovaries, oocytes and early embryos of Kunming mouse was used to investigate the expression and distribution of Tb4 in oocytes and eary embryos. During the experiment, the immuno-histochemistry, immuno-fluorescence-histochemistry and RT-PCR technique were utilized as chief study methods. The discipline of Tb4 expression and distribution in oocytes maturation and early embryonic development were studied both in vivo and vitro. The results showed that Tb4 were different in expression and localization during mouse oocytes maturation and early embryonic development. Immuno-histochemistry results illustrated that, accompany with follicle growing, the expression amount of Tb4 peptide in vitro oocytes is gradually increased. There are different distribution characteristics of Tb4 in GV stage oocytes, which featured by chromatin configuration, such as divided the configuration into NSN, pNSN and SN type. In other stages of oocytes maturation and early embryonic development, Tb4 could locate both in nuclear and cytoplasm, and the expression amount always changing regularly, which are closely related with cells growing status and the cell cycle. AIOD value of Tb4 was not significantly different in the process of oocytes maturation in vivo, but the fluorescence intensity rapidly increased form 8-cell embryos to blastula stages in the process of embryonic development. The tendency of Tb4 mRNA transcription are similar to the peptide expression in those stages. Specifically, in early implantation blastula, Tb4 peptide highly expresses in implanted lateral cells of blastula, indicating that Tb4 can regulate the microfilaments depolymerization in embryos implantation, so Tb4 plays an important role in the process of embryos implantation. The research concluded that, Tb4 could regulate microfilaments events (such as polymerization and depolymerization) and proliferation of cells in the developmental processes through the changes of its expression and localization. Overall, Tb4 plays an important role for mouse oocytes maturation, early embryonic development and embryos implantation.

Abstract:TAK1 and TAK2 are both nuclear-encoded kinases. TAK1 may be involved in the phosphorylation of LHCⅡ in state transitions. After the analysis of conservative activity regions on TAK1 and TAK2 sequences, a segment of hydrophilic polypeptide consisting of 12 amino acid residues was devised and linked to bovine serum albumin (BSA) before injection to the rabbit. The polyclonal antibody against TAK1 raised was examined by agar gel immunodiffusion (AGID) test and Western blot analysis. then it was used to study the expression of tak1 and tak2 influenced by the change of light and temperature in RT-PCR with specific primers and the possible regulation relationship between TAK1 and LHCⅡ phosphorylation especially by using TAK1 serum and P-Thr antibody. The result indicated that light and temperature regulated the phosphorylation of LHCⅡ and took effect on the transcription and translation level of tak1 and tak2, but the response of LHCⅡ phosphorylation to light did not coincided with the quantity of TAK1 protein. Furthermore, low light density could enhance the expression of tak2, but temperature impacted tak2 little. The different regulation pattern of tak1 and tak2 may derive from the different elements of promoter.

Abstract:The U-Box domain proteins, with similar configuration to RING finger proteins, are highly conserved among eukaryotic organisms and most of them belong to the ubiquitin/proteasome system as E3 ubiquitin protein ligases, U-Box containing proteins play a key role in the recognition and selection of abnormal proteins targeted for ubiquitination and subsequent degradation, a process for the maintenances and quality control of proteins exist in living cells. There are 77 U-Box genes in rice genome and the systematic investigation of their expression will provide basic information for the functional analysis. The specific antibodies against rice U-Box proteins was prepared to investigate the expression profile of U-Box proteins at different developmental stages and accumulate basic information for functional studies. Four rice U-Box genes were chosen as their U-Box domains are located at the N-terminal and posses ARM repeats at C-terminal. Epitopes prediction were carried out by computer software and the target protein fragments were expressed and purified in E. coli. system. Polyclonal antibodies were generated by rabbit immunization. Western blotting analysis were carried out for rice material collected at different developmental stages including shoot and root at seedling stage, root and stem at tilling stage, flag leaf and young panicle at heading stage, flag leaf and panicle at flowering stage, flag leaf and seed at filling stage. Comparison analysis was carried out with EST sequencing data. Specific antibodies were obtained by rabbit immunization of recombinant proteins expressed in E. coli. One major band were observed for Western blotting detection of rice U-Box proteins, the apparent molecular mass of two U-Box proteins (Os06g01304 and Os12g38210) were consistent with predicted size, while the other two proteins (Os01g66130 and Os08g01900) with apparent molecular mass smaller than that of predicted. Western blotting results indicated that the U-Box proteins were constitutively expressed with close abundance in tested tissues. EST analysis based on 1 million ESTs derived from 274 libraries from NCBI EST database revealed closed numbers of U-Box gene transcription, which is parallel with the results of constitutively expression of proteins. However, compared with the expression of ATPase, HSP81-3, EGF-1 alpha and RuBisCo, the number of ESTs for U-Box proteins were much lower, suggest the low abundance transcription of U-Box genes. Four rice U-Box proteins were chosen for the generation of specific antibodies via the expression of predicted epitope fragments, demonstrated the feasibility of the process. Western blotting analysis indicated that four U-Box proteins were constitutively expressed among tested rice tissues at different developmental stages, which is parallel with EST sequencing data. The antibodies will provide resources for functional studies, such as co-immunoprecipitation, ChIP-on-chip, Pull-down and stress response etc.

Abstract:Light, auxin and brassinosteroid play important roles in plant growth and development. Genetic analysis has demonstrated complex interactions between their signaling pathways, but the gene regulatory mechanisms connecting these pathways are poorly understood. CYP72B1 and AUR3 are two important genes responsive to light, auxin and brassinosteroid at transcription level. To understand the regulation mechanism of the two genes, a new tool called OCMMat was developed for identifying cis-elements, OCMMat combines both the over-representation property of regulatory elements in co-expressed genes and the conservation property in orthologous genes, for the latter, it was estimated by an enrichment score of regulatory element in orthologous promoter sequences. Using this tool, 3 regulatory motifs shared by genes CYP72B1 and AUR3 were reported, motif GAGACA which is the same as a known cis-element AuxRE, motif AAGAAAAA containing the sequence of GT element and the third ATCATG which is a new one named EDIB element. The space and the order of AAGAAAAA and EDIB show the same pattern in promoters of both the co-expressed genes and the orthologs of CYP72B1. Based on the sequence analysis and the literature knowledge to date, a model was proposed for describing the transcriptional regulatory mechanism of CYP72B1 and AUR3 in response to light, auxin and brassinosteroid. The model presents how the signaling pathways of light, auxin and brassinosteroid are interplaying at gene transcription level. In response to light, the transcription factors GT factor and an unknown protein repress the expression of CYP72B1 and AUR3, the hormone pathways are not interfered and thus work in their own way. While in the absence of light stimulation, CYP72B1 and AUR3 are expressed and the products, in turn, inhibit both the auxin pathway and the brassinosteroid pathways. On the other side, at high hormone level, gene expression is up-regulated through ARF binding, the gene products inhibit the hormone pathways in a feedback manner, and meanwhile, rescues the light signal through the photoreceptor phyB.

Abstract:As a classical problem of computational molecular biology, the multiple sequences alignment is also important foundational process. RNA is one of biological polymer, and is different from protein and DNA that the secondary structure of RNA is more conservative than its primary sequence. Therefore, RNA multiple sequences alignment require not only information of sequences, but also information of secondary structures which those sequences will form. Here, a program——QEA-MRNA, which based on quantum evolutionary algorithm(QEA) to align RNA sequences, is proposed. The program introduce a full crossover operator and a fitness function which considering the information of RNA premary sequence and secondary structure, and improving on prematurity controling and the convergent speed. The effectiveness and performance of QEA-MRNA are demonstrated by testing cases in BRAliBase.