Abstract:The robust nuclear-cytoplasmic transport is vital for eukaryotes’ life cycle. Not only proteins and RNAs are transferred to their destination by this system, the important cellular activities such as mitosis are also regulated by the transport pathways via adjusting the local concentrations of some critical cargoes. tRNA is one of the most important biomacromolecules in the cell, synthesized in nucleus, and taking part in protein synthesis in cytoplasm. It is a long-held belief that tRNA functions only as a key player in protein synthesis, and tRNA nuclear-cytoplasmic transport is a unidirectional movement from nucleus to cytoplasm. However, the recent discoveries are overturning the traditional opinions. Nuclear-born tRNA can be exported out of nucleus, while cytoplasmic-residing tRNA can also be retrogradely transport into nucleus. The new concept tRNA nuclear-cytoplasmic dynamics was coined in 2008 to describe the tRNA communication between nucleus and cytoplasm, which was found able to regulate protein synthesis and cell cycle in S. cerevisiae. The latest results in this field are going to change the landscape of eukaryotic tRNA biology.

Abstract:It is well believed that learning and memory is one of the functions of sleep. Not only does the sleep after learning aid memory consolidation, but enough sleep before learning is necessary for memory formation. Due to the net increase in synaptic strength, waking plasticity has a cost in terms of energy requirements, space requirements, and progressively saturates the capacity to learning. The review will focus on the role of sleep which is to downscale synaptic strength to a baseline level that is beneficial for learning and memory.

Abstract:Cyclic AMP responsive element-binding protein (CREB) is a transcription factor coactivated in response to a simultaneous increase in intracellular cAMP and Ca²⁺. The target gene products of CREB involve broad physiological processes such as cell proliferation and survival, carbohydrate and lipid metabolism, steroid synthesis, and learning and memory. The newly-identified transducer of regulated CREB activity (TORC) controls transcription and expression of many CREB target genes by undergoing nucleo-cytoplasmic shuttling. Salt-inducible kinase (SIK) is a group of serine/threonine protein kinase including SIK1, SIK2 and SIK3. These kinases indirectly modulate CREB target gene transcription and expression by affecting TORC phosphorylation and their subsequent distribution between nucleus and cytoplasm. In certain organs and tissues, SIK (SIK1) is also one of the target genes for CREB. Thus, a complete negative feedback regulatory circuit may be formed between SIK and TORC-CREB complex. To date, the latter has been identified in several organs and tissues such as pancreatic β-cell, the liver, adrenal cortex and skeletal muscle where they regulate β-cell survival, hepatic glyconeogenesis, steroid synthesis, and mitochondrial biogenesis and fatty acid oxidation in the skeletal muscle. The feedback regulation of TORC-CREB complex by SIK and its implications in pathogenesis of hypertension and diabetes mellitus are focused on.

Abstract:Domains are evolutionarily conserved sequence units and they are structural and functional building blocks of proteins. Interaction between two proteins typically involves binding between specific domains, and identifying interacting domain pairs is an important step towards thoroughly understanding protein function and evolution, constructing protein-protein interaction (PPI) networks, and analyzing pathway at the domain level. A number of interacting and/or functionally linked domain pairs have been identified and the information was organized and hosted in many domain-domain interactions (DDI) databases with the help of further mining experimental data and computational predictions from various input data. First, the 8 computational predicting methods used to acquire DDI data will be introduced. Then the introduction of DDI public databases, including 3DID, iPfam, InterDom, DIMA and DOMINE will be given. And finally, some examples are described about applications of DDI in computational predicting interacting protein pairs, assessment of the reliability for PPI, protein domain annotation, and in pathway study.

Abstract:It has been found that Ezrin is aberrantly expressed in some carcinomas and there is a relationship between the high level of Ezrin expression and metastatic potential of carcinomas. However, the molecular mechanisms underlying the regulation of the human ezrin gene transcription are not well understood. Transcriptional regulation regions of the human ezrin gene were examined by linking 5′-deletion mutants and site-directed mutagenesis of the 5′-flanking region to a luciferase reporter gene, and the basal promoter region and key DNA sequence elements (an Sp1 binding site, -75/-69; and an AP-1 binding site, -64/-58) involved in the expression of the human ezrin gene in lung cancer A549 cells were explored. Furthermore, electrophoretic mobility shift assay (EMSA) showed that DIG-ddUTP labeled DNA sequences containing ezrin key elements could bind nuclear extracts from lung cancer cells to form DNA-protein complex, and the bindings to Sp1 and AP-1 sites by rhSp1 and rhAP-1, respectively, were specific. Finally, the analysis of transient transfection of A549 cells by transcription factor expression vectors indicated that Sp1 and AP-1 (c-Jun/c-Fos heterodimer) could increase the human ezrin basal transcriptional activity significantly through the Sp1 and AP-1 binding sites, respectively. In addition, over-expression of Sp1, c-Jun or c-Fos could up-regulate Ezrin expression. The data suggested that the Sp1 and AP-1 binding sites were two key elements regulating ezrin gene basal transcriptional activity in lung cancer cells, and the corresponding transcription factors Sp1 and AP-1 were crucial for the transactivation.

Abstract:To investigate function of collapsin response mediator protein-1(CRMP-1) on neurite outgrowth, the CRMP-1-phrGFPⅡ-N eukaryotic expression vector was constructed and transfected into PC12 cells induced by nerve growth factor. The lapse-time imaging and neurite extraction, as well as immunoblotting were utilized. The results demonstrated that PC12 cells were induced to be typical neurons by nerve growth factor. Lipofectamine could effectually transfect CRMP-1 into PC12 cells induced by nerve growth factor. Overexpression of CRMP-1 inhibited neurite outgrowth and collapsined the tiny neurite, then the longer neurite. Length of neurites demonstrated a tendency to be gradually shorten accompanying expression of CRMP-1 protein. Neurite extraction showed that cells overexpressing CRMP-1 possesed more and longer neurite(P < 0.01), compared with cells only induced by nerve growth factor and transfected by expression vector without CRMP-1. These data suggested that CRMP-1 play an important role in inhibiting neurite outgrowth.

Abstract:As an effective method of studying soluble protein-protein interactions, yeast display system is now widely used for affinity maturation of single-chain antibodies. Due to the strong homology recombination machinery of yeast and the high-throughput nature of FACS detection, a rapid scan for interaction between antigen-antibody pairs could be easily achieved. Based on this system, a novel and reliable method for determining conformational epitopes was developed. Different fragments of macrophage migration inhibitory factor (MIF) and several point mutations of MIF were displayed on yeast cell surface using homologous recombination technology. Three MIF monoclonal antibodies, 10C3, 2A12 and 4E10, were screened for their binding affinity to each displayed peptide. Utilizing this technology, the key amino acids of MIF that bind to the MIF monoclonal antibodies were easily identified.

Abstract:A high accuracy method for quantitative expression proteomics was developed for the identification of putative markers in gastric adenocarcinoma. Firstly, gastric adenocarcinoma and nonmalignant epithelial cells were obtained through laser capture microdissection (LCM), which were proteolysis and then prefractionated by 1D SDS-PAGE. The post-digestion ¹⁸O/¹⁶O labeling method followed by Nano-HPLC-MS/MS were conducted to identify the differentially expressed proteins between gastric adenocaicinoma and nonmalignant gastric mucosa. A total of 78 differential proteins were identified, among these proteins, 42 proteins are over-expressed in gastric adenocarcinoma tissues and 36 proteins are down-expressed. Some of these differentially expressed proteins (moesin, periostin, annexin A2, annexin A4) were further confirmed by Western blot analysis. The quantitative proteomic protocol of ¹⁸O stable isotope labeling coupled with LCM was an effectively alternate technique for complicated proteins such as gastric cancer.

Abstract:In order to study the effect of pcsk9 siRNA on THP-1 derived macrophages apoptosis induced by oxLDL，THP-1 derived macrophages were induced to differentiate into macrophages by PMA treatment for 24 h. The experiments were designed as follows: cells were incubated with oxLDL with a concentration of 0, 25, 50, 75, 100 mg/L for 48 h respectively. The apoptosis of THP-1 derived macrophages was observed by staining with Hoechst33258. The expression of pcsk9 was analyzed by reverse transcription polymerase chain reaction and Western blot. The siRNAs for pcsk9 gene were designed and synthesized，then transfected into THP-1 derived macrophages by positive ion liposome Lipofectamine 2000. Transfection efficiency was assessed by fluorescence microscope assay. RT-PCR and Western blot were conducted to detect the expression of pcsk9 after 24 h, 48 h respectively. The most efficient siRNA was selected to transfect into THP-1 derived macrophages. 24 h after transfection, cells were treated with oxLDL for 48 h, and then Hoechst 33258 staining. The results showed that the number of cells with nuclear condensation induced by 75 mg/L oxLDL increased significantly. In THP-1 derived macrophages, pcsk9 was upregulated with increasing concentration of oxLDL, while 75 mg/L oxLDL increased significantly. The RNA interference experiment showed that siRNA was successfully transfected into cells and 80 nmol/L as most effective dose of siRNA was selected by RT-PCR and Western blot. Compared with control, the suppression of apoptosis in THP-1 cells transfected with 80 nmol/L of siRNA for 24 h and incubated with 75 mg/L of oxLDL for another 48 h was detected by Hoechst 33258 staining and flow cytometer. Together, these results reveal the expression of pcsk9 mRNA and protein were increased by oxLDL in a concentration-dependent manner. Expression of pcsk9 gene could be effectively suppressed by siRNA. The apoptosis of THP-1 derived macrophages induced by oxLDL could be effectively suppressed by pcsk9 siRNA.

Abstract:APOBEC (apolipoprotein B mRNA-editing enzyme catalytic-polypeptide) family members were reported as innate immune molecules with anti-viral activity for many viruses, such as HIV and HBV. In order to understand the function of APOBEC, the APOBEC-3F and -3G were cloned, expressed, and the sub-cellular localization of them was detected. The genes of APBEC-3F and -3G were cloned from PHA-stimulated PMBC and expressed in the MDCK cell by transfection. The sub-cellular localization of APOBEC-3F and -3G were detected by immunofluorescence. APOBEC-3F and -3G were cloned by RT-PCR and confirmed by DNA sequencing. The immunofluorescence indicated APOBEC-3F and -3G were located in the cytosal. APOBEC-3F and -3G could inhibit HBV replication effectively in HepG2.2.15 cell. APOBEC-3F and -3G could not be trans-located into nuclear by nuclear location signal (NLS) or bi-NLS (B-NLS). These results will help the future research on the function of APOBEC.

Abstract:To characterize the promoter region and upstream regulation region of human and mouse SCN3A gene, 5′-Full RACE was performed to identify that the nucleotide “A” was identified as the transcription start site, which locate 27 kb upstream of the translation start site of human SCN3A and 31 kb upstream of the translation start site of mouse SCN3A. Two 5′ -untranslated exons of human SCN3A and one 5′-untranslated exon of mouse SCN3A were found by sequence blast. The core promoter region (-80 ～ +70) of human SCN3A showed 96% nucleotide homology with that of mouse. Two core promoter elements, BRE and TATA, were predicted in the region of -80～+70 from both human and mouse. The transcriptional factors PHR1, GATA-1, FOXN2, NF-1 and AP-4 predicted in the region of -400 to +200 of human SCN3A, not found in mouse SCN3A, and the transcriptional factors Sp、Sp3 and GBF predicted in the region of -400 to +200 of mouse SCN3A, not found in human SCN3A. The comparison between the promoter region of human and mouse SCN3A may provide an important clue to explore the mechanisms of the regulations of human and mouse SCN3A expression.

Abstract:To investigate the effect of OGT expression inhibited by RNAi on the alteration of tau phosphorylation and glycosylation level in HEK293T cells. The siRNAs targeting OGTgene (OGT-siRNA_1～3) were designed and chemically synthesized. The OGT-siRNA_1～3 were transfected into HEK293T cells via lipofectamine2000. The efficacy of RNA interference was detected by RT-PCR. The fluorescence of GFP/OGT was counted by fluorescence microscopy after pEGFP/OGT and OGT-siRNA_1～3 cotransfected to HEK293T cells for 24 h. The level of tau phosphorylation and glycosylation were detected by Western blot after Plasmid pCI/tau₄₄₁ and the siRNA cotransfected HEK293T cells for 48 h. OGT-siRNA₃(100 nmol/L) could effectively downregulate the expression of OGT gene compared with Mock group (80.0% at mRNA level and 51.3% at protein level). The level of phosphorylation at various sites of the tau was significantly upregulated and the glycosylation was downregulated following the inhibition of OGT gene expression. There is an apparent negative correlation between the modification of phosphorylation and glycosylation of tau protein, the deficient in glucose uptaken/metabolism may be an important pathogenesis of AD.

Abstract:To explore the expressional information of PIWIL4 in human ovarian cancer, semi-quantitative reverse transcription polymerase chain reaction (semi-qRT-PCR) was used to detect the mRNA expression level of PIWIL1, PIWIL2, PIWIL3 and PIWIL4 in human ovarian clear cell carcinoma ES-2 cells. Meanwhile, in human ovarian cancer and adjacent normal tissues, the mRNA expression of PIWIL4 was determined. Then, PIWIL4-siRNA, which was designed to target PIWIL4 and chemical synthesized, was transfected into ES-2 cells with lipofectamine. MTT assay and clone formation assay were used to investigate the effects of PIWIL4-siRNA on the cell growth activity and proliferation capacity of ES-2 cells. Experimental results showed that the mRNA expression level of PIWIL4 was the highest compared to PIWIL1, PIWIL2 and PIWIL3 in ES-2 cells (P < 0.05), and the expression of PIWIL4 mRNA in ovarian cancer tissues was higher than that in adjacent normal tissues (P < 0.01). MTT and clone formation analyses demonstrated that the cell growth activity and clone formation ratio of PIWIL4-siRNA transfected ES-2 cells were markedly decreased (P < 0.05). In conclusion, the expression of PIWIL4 mRNA was highest in ovarian cancer cell line ES-2 cells and was significantly increased in ovarian cancer tissues. Moreover, PIWIL4-siRNA can efficiently inhibit the cell activity and proliferation ability of ES-2 cells. In a word, PIWIL4 may be related to the pathogenesis of human ovarian cancer.

Abstract:A cold-adapted (ca), temperature sensitive (ts), live attenuated influenza B virus strain B/Ann Arbor/1/66 was chosen for influenza virus rescue research, in which six internal gene segments, PB1, PB2, PA, NP, M, NS, were fully synthesized and nine amino acid substitutions were artificially alter by human intervention. The resultant B/Ann Arbor/1/66 plasmids were named as pAB121-PB1, pAB122-PB2, pAB123-PA, pAB124-HA, pAB125-NP, pAB126-NA, pAB127-M and pAB128-NS, respectively. A recombinant influenza A virus was previously generated entirely from cloned cDNA. An infectious recombinant influenza B virus was generated here, and designated as rMDV-B, by plasmid-based reverse genetics. The rMDV-B virus contained HA and NA genes from an epidemic influenza B virus strain B/Malaysia/2506/2004 in the background of internal genes derived from influenza B virus strain B/Ann Arbor/1/66. HA titer of rMDV-B in MDCK cells and embryonated chicken eggs ranged from 1∶64 to 1∶512. The results may allow an effective live influenza B vaccine to be produced from a single master strain, providing a model for the design of future live human influenza vaccines.

Abstract:To study the changes of the silkworm dynein light chain 8 (Dlc8) gene expression pattern in different gravitational environments. The open reading frame sequence of Dlc8 gene was amplified and analyzed. The distribution of Dlc8 gene was investigated in embryo, head, silk gland, midgut, cuticle, blood, fat, tuba malpighii of silkworm respectively. The effects of simulated gravitational environments (0 g, 1 g, 2 g) produced by large gradient high magnetic field (LGHMF) on expression of the silkworm Dlc8 gene were tested by RT-PCR and real time RT-PCR in BmN cells. Expression of the silkworm Dlc8 gene in simulated weightless environment was tested by real time RT-PCR in inverse period and in total embryo period of silkworm. The Dlc8 gene with 270 bp coding 323 aa was successfully amplified. The homology rates of amino acid sequences of Dlc8 gene between silkworm and Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, Xenopus tropicalis, Mus musculus, Homo sapiens were 67%, 96%, 91%, 95%, 92%, 92%, respectively. Signal peptide analysis showed that Dlc8 was not a secretion protein. There was not glycosyl-phosphatidyl inositol anchor site in Dlc8 amino acid sequence. Molecular mass and isoelectric point of Dlc8 were 10.34 ku and 6.81, respectively. Dlc8 gene is conservative in embryo, head, silk gland, midgut, cuticle, blood, fat, tuba malpighii of silkworm. Dlc8 gene is more sensitive to gravity than magnetic field in BmN cells. There are different responses to gravity on Dlc8 gene expression in different period of silkworm embryo. There was no significant change of Dlc8 gene expression between simulated weightless and control groups. The results showed that Dlc8 gene could be the molecular target to study gravity bioeffect. This research may contribute to reveal the mechanism of gravity bioeffect of the silkworm Dlc8 gene.

Abstract:Nogo-A is a myelin derived neurite outgrowth inhibitor which is not only highly expressed in mature oligodendrocytes, but also in various kinds of neurons in the central nervous system. However, the function of neuronal Nogo-A remains unclear. To investigate the function of Nogo-A in neurons, human embryonic kidney cell line 293FT was used as a model, along with the pathway profiling reporter system to check the involvement of Nogo-A in regulating the intracellular signaling pathway. It was found that over-expression of Nogo-A could activate NF-κB signaling specifically, then followed by using different Nogo-A alternative splicing isoforms and a serial of truncations, it was confirmed that the activation of NF-κB by Nogo-A relied on its N-terminal proline rich domain. Furthermore, by co-expressing the dominant negative mutants of some NF-κB pathway associated proteins, it revealed that the IκBα,TRAF6, Rac/Cdc42 were involved in Nogo-A inducing activation of NF-κB. Above all, these results indicate that Nogo-A could significantly activate NF-κB, and this activation depended primarily on its N-terminal proline rich domain.

Abstract:Mammalian gonadotropin-releasing hormone (termed GnRHⅠ) is a decapeptide that plays key role in the process of reproduction. In addition to its well-known endocrine function, it has become evident that GnRHⅠ and the second GnRH subtype (termed GnRHⅡ) are potentially important autocrine and/or paracrine regulators in placental trophoblast cells. The effects of GnRHⅠ and GnRHⅡ on trophoblastic cell invasion were investigated in human choriocarcinoma cell line (JEG-3) by the methods of invasion assay, RNA interference, Western blot and Real-time PCR. The data revealed that exogenous native peptide of these two forms of GnRH significantly induced the expression of typeⅠ GnRH receptor (GnRHRⅠ) in JEG-3 cells. The specific siRNA for GnRHRⅠ was capable of blocking the invasion-promoting effect of GnRHⅠ, but did not influence the effect of GnRHⅡ. The data suggested that the two hormones may be elicited by distinct receptors. Interference of the activities of ERK and JNK was capable of attenuating GnRHⅠ and Ⅱ-induced MMP-2 expression and trophoblast invasion, indicating that activation of ERK and JNK are required in both GnRHⅠ and GnRHⅡ-mediated MMP-2 production and invasion-promoting effect in human trophoblastic cells.