Abstract:MAPK (mitogen-activated protein kinase) family, which is conserved throughout high eukaryotes, is implicated in multiple cellular processes including cell growth, migration, proliferation, differentiation, survival and development. Pathogen hijacks hosts' MAPK pathways to facilitate its pathogenesis using diverse strategies. To further explore the mechanism underlying interactions between pathogens and hosts' MAPK pathway, is of benefit to our understanding of nature as well as to our fight against pathogen infection.

Abstract:Geminin is a multifunctional protein which localizes in nucleus, it has complicated structural and functional domains. Geminin plays very important roles in cell proliferation, embryonic development, tumorigenesis and so on. Geminin affects cell proliferation through influence on the important events in cell cycle phases. There are many mechanisms by which Geminin can control DNA replication, inhibit centrosome over-duplication, promote G2/M phase progression, maintain proper cytokinesis. At different development stages, Geminin acts as an inhibitor or inducer in regulating embryonic development, especially in nervous system formation. Geminin also plays a regulative role in eye development and embryonic development through the interactions with homeobox genes or proteins Six3 and Hox, Geminin functions as a coordinator of cell proliferative and differentiative control. Recently, Geminin was found to have relationship with cancer, the study on the function of Geminin in cancer has been a meaningful aspect. Geminin can act as a marker to evaluate progression and prognosis in cancer. It may be a novel molecular target in therapy of cancer. The activity of Geminin is regulated by transcription level and post-transcription level, the post-transcriptional regulation of Geminin protein may take the main position.

Abstract:Cell adhesion molecules (CAMs) play important roles in specifying cell-cell interactions during development, regeneration, and modification of synaptic activity. The close homolog of L1 (CHL1), a recently identified member of the immunoglobulin superfamily of cell adhesion molecules, is localizably expressed in the nervous system. CHL1 interacts with like molecules (homophilic interaction) and non-like molecules (heterophilic interaction) on neighboring cells or the extracellular matrix to regulate axon outgrowth and fasciculation, neuronal migration and survival, synaptic plasticity and regeneration after trauma.

Abstract:The DNA damage response is a cornerstone of genomic stability. The cell utilizes mutiple mechanisms including damage detection, cell cycle regulation, damage repair and apoptosis to keep cell homeostasis. The DNA damage response include several biochemical pathways: first, the recognition and repair of damaged DNA; second, the activation of DNA damage checkpoint, which arrests cell cycle progression so as to provides time for DNA repair and prevention of the transmission of genomic abnormalities to the daughter cells; third, apoptosis, which eliminates serious damaged cells. The double strand break (DSB) is believed to be one of the most severe types of DNA damage, and errors in DSB repair could result in genomic instability that might lead to malignancy. It has been reported recently that constitutive activation of the ATM-Chk2-p53 pathway and phosphorylation of histone H2AX acts as an inducible anti-cancer barrier in the early stages of human tumorigenesis. This ATM-regulated DNA damage response network maintains genomic integrity and delays or prevents cancer by eliciting growth arrest or cell death. In context with a recent report, the ATM-dependent DNA-damage cellular signaling has also been shown to be involved in the integration of human immunodeficiency virus type-1 (HIV-1) into host genomes, and KU55933, a specific ATM inhibitor, attenuated the infection of HIV-1 into host cells. The regulation and mechanisms of the signaling pathways of DSB response, and its role in HIV-1 infection and malignancy genesis were reviewed.

Abstract:Tetrodotoxin-resistant Nav1.5 Na⁺ channel has been considered as the cardiac sodium channel. Na⁺ currents with tetrodotoxin resistance (TTX-R) and Nav1.5/SCN5A mRNA have been observed in neurons, but the cDNA encoding the TTX-R Nav1.5 Na⁺ channels in human central nervous system (CNS) has not been identified. Nav1.5/SCN5A cDNA was first cloned from human brain cortex by using RT-PCR method. Two variants of Nav1.5/SCN5A were found and tentatively named hB1 and hB2.Full sequence of cDNA encoding the α-subunit of TTX-R Nav1.5 Na⁺ channel in human brain cortex was 6 201 nucleotides long and was designated hB1. The longest open reading frame of hB1 (accession number EF629346 ) encodes 2 016 amino acid residues. Sequence analysis has indicated that hB1 is highly homologus with human cardiac Nav1.5/SCN5A with >98% amino acid identity. There are 28 different amino acids between them, with 7 of which locating in the region encoded by exon6A or exon6. Alternative splicing of exon18 was not found in the gene cloning of human brain Nav1.5/SCN5A, which was different from human heart Nav1.5/SCN5A, but a novel alternative splicing lacking exon24 was first found. The two variants were detected in similar ratio in brain, but they were proved to relate to age development in heart tissue. The exon24 of human Nav1.5/SCN5A has 54 nucleotides, encoding 30 amino acid residues, and are located in human chromosome 3P21. This alternative splicing was also found in other tissues other than heart and brain. The expression pattern of the two variants in different tissues was different when detected by competitive PCR method and it was also changing with age development. Furthermore,Nav1.5/SCN5A mRNA was detected in 16 different tissue types of Wistar rats (P80) by reverse polymerase chain reaction (RT-PCR). These results suggest that Nav1.5 Na⁺ channels in human brain are encoded by new variants of Nav1.5/SCN5A and its mRAN is more widely expressed than previously thought. The study is useful for making further investigation in the functional analysis of Nav1.5 Na⁺ channels in different tissues.

Abstract:Tau is a microtubule associated protein in neuron. The biological functions of tau are to stimulate microtubule assembly and to stabilize microtubule structure. These functions are regulated by its phosphorylation status. Abnormally phosphorylated tau is a major component of neurofibrillary tangles. As one of the major tau kinases, glycogen synthase kinase-3β (GSK-3β) can phosphorylate tau at several sites. Site-specific phosphorylation of tau by GSK-3β and the kinetics of GSK-3β on tau phosphorylation was investigated. Tau₄₄₁ was phosphorylated by recombinant GSK-3β in vitro, and the site-specific phosphorylation was detected by Western blots using site-specific and phosphorylation-dependent tau antibodies. The kinetics of phosphorylation by GSK-3β to total tau and to individual site was studied by incubating GSK-3β with various concentration of tau at 30℃ for 10 min. These velocities of phosphorylation reaction were determined at various concentrations of tau by measuring ₃₂P incorporation to tau and phosphorylation level at individual site detected with immuno-dot blots using site-specific and phosphorylation dependent anti-tau antibodies, respectively. The K_m values of GSK-3β toward total tau and toward individual site were calculated by using Lineweaver-Burk double-reciprocal method. The site-specific phosphorylation of tau by GSK-3β was further confirmed in cultured CHO cells by co-transfection of tau₄₄₁ with GSK-3β. It was found that GSK-3β phosphorylated tau at several sites, including Thr181, Ser199, Ser202, Thr205, Thr212, Thr 217, Thr231, Ser396, and Ser404. The Km value of GSK-3β toward total tau was 42 μmol/L, but the K_m value of GSK-3β to every site was different. Among all the phosphorylation sites detected here, Ser396 phosphorylation catalyzed by GSK-3β showed the lowest K_m, only 16 μmol/L. In cultured CHO cells, GSK-3β over-expression also induced tau phosphorylation at Ser396 the most dramatically. These results suggested that GSK-3β catalyzed tau phosphorylation at several sites with different efficiency, and Ser396 was the most efficient site for phosphorylation by GSK-3β .

Abstract:The transcriptional repressor RE1 silencer transcription factor (NRSF/REST) is an important factor that restricts some neuronal traits in neurons. Since these traits are also present in pancreatic islet cells, NRSF-regulated genes involved in islet function are searched. A NRSE-like motif was analysed in human insulin promoter. The role of NRSE was evaluated by generating a model of insulin-secreting cells that firmly express NRSF. The presence of NRSF led to a decrease in activity of human insulin promoter by stable or transient transfection with human insulin-promoter luciferase. The predicted NRSE-like motif also confers NRSF-dependent transcriptional repression in the context of a surrogate gene promoter. Specific binding activity of NRSF/REST to the NRSE-like motif was confirmed by EMSA. Moreover, the binding activity is competed by consensus NRSE sequence. These data showed that human insulin promoter is regulated by the transcriptional repressor NRSF/REST via the NRSE-like motif.

Abstract:Dehydrins accumulate during the late stages of embryo genesis or in response to ABA application, low temperature, drought, or any environmentally imposed dehydrative force. Despite the abundance and widespread occurrence in cell of dehydrins, the biochemical role of dehydrins remains elusive. In order to study the expression and functional characteristics of dehydrin gene during different growth, and make the polycolonal antibody, using wheat plumelet under the water deficit condition as experiment material, and its total RNA was extracted. The target dehydrin gene through RT-PCR was got, connect to the cloning vector PUC_M-T, recombination expression plasmid PET-32a(+)-wzy1-1 was constructed according to recombination of cloned vector PUC_M-T-WZY1-1 of wheat dehydrin gene in this experiment. Then the recombination was transformed into the host strain E. coli BL21(DE3), was induced by IPTG and target protein was got. The expression production was detected by SDS-PAGE, and Western blot assays revealed that the fusion protein was expressed in soluble form with a relative molecular mass 37 ku, and the fusion protein was expressed at high level. After purification by Ni-NTA resin affinity chromatography and electroelution in bagfilter, the fusion protein was used to induce the production of polyclonal antibody in rabbits and ELISA detection showed the antigenicity of the fusion protein was satisfactory, Western blot showed the antiserum raised against the recombination dehydrin protein in rabbits could react to the protein expressed specifically, and the protein of zheng yin 1# wheat under drought stress was extracted, through the SDS-PAGE, Western blot assays revealed that there is a protein band with a relative molecular mass 28 ku, this revealed that antiserum could react to the dehydrin protein expressed in wheat leaf specifically and demonstrated its good antigenicity.

Abstract:Integration is a critical step in the retroviral life cycle. HIV-1 integrase is involved in the integration of HIV DNA into host chromosomal DNA and appears to have no functionally equivalent in human cells. It has become an attractive and rational target for selective anti-AIDS therapy. A random linear heptapeptides phage display library was panned on the recombinant HIV-1 integrase protein. After five rounds of panning, 13 positive phage clones were selected and sequenced. Two consensus peptides (TPSHSSR and HPERATL) were chemically synthesized. The non-radioactive ELISA-based HIV-1 integrase assay showed that the synthetic peptides TPSHSSR and HPERATL were able to inhibit the 3'cleavage or strand transfer activity of HIV-1 integrase to some extent (IC₅₀=(54.56±5.18) μmol/L, IC₅₀=(28.29±1.32) μmol/L, respectively). These heptapeptides could be used for developing new anti-HIV drug candidates, as well as for structural studies of the three-dimensional structure of the entire integrase molecule.

Abstract:S1 gene targeted libraries containing the major immunodominant region (1～2 367 bp) of PEDV spike glycoprotein were constructed by phage-display vectors based on filamentous phage strain fd-tet, in which the exogenous polypeptides were expressed in the N-terminal of gene Ⅲ coat protein. The S1 libraries were panned three times using the purified rabbit sera against PEDV. Three peptides, displayed on recombinant phages, showed strong binding affinity with the PEDV antisera, and were designated as S1P1 (248～280aa), S1P2 (442～499aa) and S1P3 (697～742aa) respectively. In ELISA and Western blot, the three peptides were all recognized by the PEDV antisera, but S1P3 showed strong binding activity. To further determine antigenicity of the three peptides, the antisera of S1P1-GST, S1P2-GST, S1P3-GST and their ligations GST fusion protein were prepared. In ELISA, S1P1-GST, S1P2-GST, S1P3-GST and S1P123-GST fusion proteins were able to induce the S1-specific antisera. The result of indirect immunofluorescence assay (IFA) demonstrated that the antisera induced by S1P2-GST, S1P3-GST and S1P123-GST fusion proteins possessed binding ability to the native S protein of PEDV cultured in Vero cells.

Abstract:Ca²⁺ is a ubiquitous second messenger which plays a key role in early development of embryos. Ca²⁺ probes (Fluo-4 or Indo-1) were injected into zebrafish eggs to detect the distribution of free Ca²⁺ during their first cleavage using confocal microscopic or dual-wavelength ratiometric imaging. A high Ca²⁺ zone was first observed in the animal pole right before the first cleavage, then it extended along the cleavage furrow and the Ca²⁺ signal remained high in this region throughout the first cleavage. Intracellular Ca²⁺ concentration ([Ca]_i) was measured via Indo-1 dual-wavelength system, and it was shown to be homogeneous within the whole embryo before the first cleavage. During the first cleavage, [Ca]_i increased significantly near the cleavage furrow, while it remained unchanged in other areas. As the dual-wavelength ratiometric imaging eliminates the artifacts due to indicator inhomogeneity, the results provided an unequivocal quantification for the Ca²⁺ dynamics associated with the first cleavage of embryonic development.

Abstract:In order to construct the yeast display system of the human proteasome subunit alpha 6 (α6) and obtain its specific monoclonal antibodies for epitope analysis and mechanism investigation of ubiquitin-proteasome pathway, and set up a new rapid efficient way for the preparation of specific monoclonal antibodies (MAbs) without proteantigens which applicated recombinant antigen into detection directly, the gene PSA6_HUMAN coding human proteasome subunit alpha 6 was cloned into a yeast-displaying expression vector, pICAS-H, which had been inserted a His.tag marker for expression level detection. As probed with a His.tag monoclonal antibody and a specific monoclonal antibody generated by hybridoma, a recombinant yeast strain, α6-MT8, was selected by flow cytometry and fluorescence microscopy analysis. Combining with enzyme-linked immunosorbent assay (ELISA), ‘yeast-ELISA’ detection was established by basing on Saccharomyces cerevisiae cell surface engineering and applied to examined monoclonal antibody and its valance. The yeast-displaying recombinant antigen α6 with highly specific affinity was expressed efficiently after 48 h cultivation. The ‘yeast-ELISA’ was demonstrated primarily to detect and screen monoclonal antibody successfully.

Abstract:A novel method for assaying the enzymatic activity of ribosome-inactivating proteins (RIPs) has been developed. The principle of the method is based on that RIP can remove some adenine bases from double-stranded supercoiled DNA molecules, subsequently, the deadenylated DNA was cleaved into nicked and linear form. After treatment with acidic aniline, the deadenylated DNA was degraded into many small fragments, and run out of the gel. The enzymatic activities of two RIPs (trichosanthin and cinnamomin) were tested using this method, the limit of sensitivity is about 50 ng (trichosanthin) and 5 ng (reduced cinnamomin). It should be emphasized that the merit of this method is to avoid the preparation of ribosome.