Abstract:Brain-derived neurotrophic factor (BDNF) is the most widely distributed neurotrophin in the central nervous system (CNS), serving many biological functions such as neural survival, differentiation, and plasticity. Treating the antidepressant drug could promote nerve cell survival and increase the synaptic plasticity and neurogenesis by improving BDNF expression.

Abstract:Ubiquitin-proteasome system, the essential mechanism for eukaryotic cellular protein degradation, plays an important role in the regulation of cellular physiological functions. In 1980s, researchers found that proteasome also reside in actinomycetes, but the function and mechanism of the prokaryotic proteasome were unknown. In 2008, the prokaryotic ubiquitin-like protein (Pup) was identified in Mycobacterium tuberculosis. With the help of accessory factors, Dop, PafA and Mpa, Pup covalently linked to the Lys ε-NH₂ in the target proteins and mediated the target protein degradation through the proteasome. The discovery of Pup-proteasome system revealed a novel mechanism of prokaryotic protein degradation, which is involved essential physiological function including the intermediary metabolism, information pathway, detoxification/virulence, cell wall and cell membrane formation and so on. Disruption of Pup-proteasome system can suppress the pathogenicity of Mycobacterium tuberculosis. Therefore it is regarded as the new therapeutic target for tuberculosis. In the present paper, the progress in the study on mechanism and function of Pup-proteasome system is reviewed.

Abstract:The innate immune system may be the first defence line to detect and counter viral and microbial invasion. There are many mechanisms working in innate system, which would be helpful to understand the pathogenesis of infectious diseases, virus-related cancers and autoimmune diseases. Despite of the understanding about the recognition signaling in response to cellular RNA, the recognition mechanisms and related innate immunological response of pathogen DNA still remain largely unclear. Recently, a few molecules in the cell were discovered to function as DNA sensors which play key roles during DNA mediated IFN production. Besides, some molecules that could regulate IFN signaling were isolated and shown to be critical for DNA mediated innate immunity. Recent progress about the detection and recognition of pathogenic DNA, as well as its regulation of innate immunity were summarized.

Abstract:This paper focuses on the role of microRNAs (miRNAs) in response to cadmium (Cd) stress in rice. A number of Cd-responsive miRNAs in rice were identified by using microarray assay and quantitative Real-time PCR. The downstream targets were mainly regulatory genes which encoded transcription factors and protein kinases. These target genes were reported to be associated with metabolic processes and signal pathways. Promoter analysis showed that metal stress-responsive cis-elements tended to occur more frequently in the promoter regions of Cd-responsive miRNAs. These findings suggested that miRNAs played an important role in Cd tolerance in rice, and highlighted a heavy metal tolerance network involving miRNAs in plants.

Abstract:Hyperphosphorylation of Tau protein and related neuron death is one of the most important characteristics of Alzheimer's disease. Our laboratory has shown that Tau protein is able to bind and protect DNA. It is still unknown whether phosphorylation affects the binding of Tau to DNA. Therefore, it is of importance to investigate the effect of phosphorylation on the interaction of Tau with DNA in cells. In this work, we treated N2a cells with formaldehyde and found that Tau protein was hyperphosphorylated in the cells under the experimental conditions. Phosphorylation was remarkably observed at both T181 and S396 of Tau protein in the cells in the presence of formaldehyde compared with those in the absence of formaldehyde. Cytoimmunofluorescence hardly showed that most of the nuclear phosphorylated Tau protein was co-localized with DNA, while Tau protein was partially co-localized with DNA in the absence of formaldehyde as control. Electrophoretic mobility shift assay (EMSA) showed that phosphorylated Tau catalyzed by GSK-3β reduced the interaction between Tau protein and DNA in vitro. These findings reveal that hyperphosphorylation declines Tau protein to protect DNA, and may thereafter lead to damage of DNA and even cell death, giving a novel viewpoint to the pathology of Alzheimer's disease.

Abstract:Caveolin-1 is a transmembrane protein and essential structural constituent of the caveolae membrane. Caveolin-1 has been involved in multiple cellular functions and oncogenesis. To investigate the roles of caveolin-1, stable transfectants were established in PANC1 pancreatic adenocarcinoma cells which had up-regulated caveolin-1 expression. The plasmid pCI-neo-cav-1 and its corresponding empty vector (pCI-neo) were transfected into PANC1 cell lines. The expression of caveolin-1 in these three cell lines was determined by RT-PCR and Western blot. Cell cycle phase distribution was determined by flow cytometry. The colony formation ability of tumor cells was detected by anchorage-independent growth assay. Cell migration and invasion were assayed in MilliCell chambers. Xenograft tumor models in nude mice were developed. Immunohistochemistry was used to characterize Ki-67 levels in residual tumors, and apoptosis was evaluated by TUNEL technique. Caveolin-1 overexpression inhibited PANC1 cell proliferation by arresting the cell cycle in the G0/G1 phases and also markedly reduced the capacity of the cells to form colonies in soft agar. Additionally, caveolin-1 overexpression dramatically inhibited cells to invade and migrate. Importantly, in vivo experiments demonstrated that overexpression of caveolin-1 resulted in significant growth inhibition of the xenograft pancreatic tumors. Immunohistochemistry analysis demonstrated both a marked decrease in the number of proliferating tumor cells and an increase in the number of apoptotic tumor cells in PANC1/cav-1 xenograft tumors. The results provide an initial demonstration that caveolin-1 can function as a tumor suppressor rather than as a tumor promoter in PANC1 cells.

Abstract:Our previous studies have showed that adipophilin promoted intracellular lipids accumulation through ERK1/2-PPARγ signaling pathway. In the study we explored that whether highexpression and knockdown adipophilin affects the activity of ERK1/2 and the expression of PPARγ and lipid accumulation in RAW264.7 cells, and further certified that adipophilin promoted intracellular lipids accumulation through this pathway. The recombinant retroviral vetors pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA were verified by the methods of enzyme-digesting. The recombinant retroviral vetors were transfected into PA317 cell by mediating Sofast^TM, which can induce retroviruses release. Then we used the collected retroviruses to infect RAW264.7 cells and achieved adipophilin gene highexpression and knockdown RAW264.7 cell lines applying puromycin screening. After the infected RAW264.7 cells incubated with 50 mg/L Ox-LDL for 24 h, the lipids accumulation were measured by Oil red O staining and HPLC, the expression of mRNA and proteins of adipophilin and PPARγ were detected by semi-quantitative RT-PCR and Western blot respectively, and phosphorylation of ERK1/2 was analyzed by Western blot too. The results of enzyme-digesting confirmed the recombinant retroviral vectors pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA as expected. In the situation of Dutch fat, transfected cells with pQCXIP-HA-Adipophilin significantly increased the accumulation of lipids, but reduced the expression of PPARγ and the phosphorylation of ERK1/2, which were reversed in cells with pSuper-retro-adipophilin siRNA transfection. Our results showed that ERK1/2 and PPARγ may be related with lipids accumulation caused by adipophilin expression in macrophages incubated with modified LDL. Therefore, adipophilin might contribute, in vivo, to lipid accumulation in the intima of the arterial wall through the ERK1/2-PPARγ signaling pathway.

Abstract:At the early stage of sepsis, it is clear that tumor necrosis factor alpha (TNF-α) will be excessively produced after mitogen-activated protein kinase is activated by lipopolysaccharide (LPS). TNF-α, which is an important early inflammatory cytokine, activates indirectly signal transducer and activator of transcription (STAT3) pathways. Activated STATs enter into the nucleus to take part in the gene expression induced by LPS. The present study was performed to explore the potential sites of STAT3 binding to TNF-α promoter. The purpose is to provide the theory basis of understanding molecular mechanism and intervention pathway of JAK/STAT signal pathway in the development of sepsis. In the present study, dose-dependent response of STAT3 on TNF-α expression was observed after Flag-STAT3 and full length TNF-α promoter reporter gene were co-transfected into COS-7 cells. The results indicated that TNF-α gene expression was enhanced along with increased doses of STAT3 with or without LPS. Flag-STAT3 (200 μg/L) and TNF-α promoter reporter gene of different length were co-transfected into COS-7 cells respectively, and LPS was added into cells 4 hours later. Compared to the controls, fold activity value of pTNF-α(95 bp) was found to be the raised 6.9-fold. Then, pTNF-α(70 bp), pTNF-α(75 bp), pTNF-α(80 bp) and pTNF-α(85 bp) deletion mutants were constructed and co-transfected with Flag-STAT3 (200 μg/L) to COS-7 cells induced by LPS. Data showed that fold activity value of pTNF-α(85 bp) was similar to that of pTNF-α(95 bp), and fold activity value of pTNF-α(80 bp) decreased to the control levels. 81 base and 82 base in the TNF-α promoter were mutated and then the activity results proved that the two sites were important to the TNF-α gene expression induced by STAT3. The potential binding site of STAT3 on TNF-α promoter was identified to be between 80 bp and 85 bp. Furthermore, the results of EMSA showed that it was wild probe tagged with γ-³²P of the TNF-α promoter 62～85 bp could bind to nucleic proein STAT3, but not mutant 62～85 bp probe. These data suggested that the effect of STAT3 on TNF-α gene expression did not depend on LPS, and the potential binding site of STAT3 on TNF-α promoter might be between 80 and 85 base radical fragment.

Abstract:Previously, we found that G protein-coupled receptor APJ endogenous ligand apelin-13 stimulates vascular smooth muscle cells (VSMC) proliferation mediated in part by PKC-PI3K-ERK1/2-cyclinD1 signaling cascades. In this study, Raf-1-14-3-3 signaling in rat VSMCs proliferation stimulated by apelin-13 was further investigated. Cell proliferation was measured with MTT assay. Expression of PI3K, phospho-PI3K, Raf-1, phospho-Raf-1, ERK1/2, phospho-ERK1/2, cyclinD1 and cyclinE were detected by Western blotting. 14-3-3 protein combining with Raf-1 was detected by immunoprecipitation. Here, we demonstrated that apelin-13 increased the expression of 14-3-3, Raf-1 phosphorylation and ERK1/2 phosphorylation in a concentration- dependent and time-dependent manner at 0～4 μmol/L and 0～48 h. 14-3-3 inhibitor Difopein decreased the apelin-13-induced Raf-1 phosphorylation, ERK1/2 phosphorylation, expression of cyclinD1 and cyclinE. Furthermore, apelin-13 promoted the combination of 14-3-3 protein and Raf-1, Difopein significantly inhibited the combination of 14-3-3 and Raf-1 stimulated by apelin-13. Similarly, Difopein significantly inhibited the VSMCs proliferation stimulated by apelin-13. Our results revealed that Raf-1+14-3-3-ERK1/2 signaling cascades mediated the effect of apelin-13 on rat VSMCs proliferation .

Abstract:Previously we reported that G protein-coupled receptor APJ endogenous ligand apelin-13 induced adhesion of monocytes to human umbilical vein endothelial cells(HUVECs).Now we investigated whether phosphatidylinositol 3-kinase (PI3K) signaling pathway mediated monocytes (MCs) adhesion to HUVECs induced by apelin-13. Human umbilical vein endothelial cell line ECV304 was cultured in DMEM medium and the monocyte cell line THP-1 was cultured in 1640 medium. Myeloperoxidase(MPO) assay was used to identify effects of monocytes adhesion to HUVECs. Expression of vascular cell adhesion molecule (VCAM) -1, phospho-PI3K and PI3K were detected by Western blotting. Apelin-13 promoted PI3K phosphorylation in concentration-dependent and time-dependent manner, which reached the peak at 1 μmol/L and 30 min respectively. The PI3K inhibitor LY294002 inhibited PI3K phosphorylation and the expression of VCAM-1 in HUVECs induced by apelin-13. And the PI3K inhibitor LY294002 inhibited the MCs adhesion to HUVECs induced by apelin-13. It can be concluded that apelin-13 promoted monocytes adhesion to HUVECs through VCAM-1 mediated by PI3K signaling pathway.

Abstract:Chronic impairments of brain caused by accumulation of endogenous formaldehyde as ageing is thought as one of the risk factors for sporadic age-related dementia. Thus, it is necessary to determine the endogenous concentration of formaldehyde in brain. Using 2, 4-dinitrophenylhydrazine (2, 4-DNPH) in the assay, the concentrations of formaldehyde in parietal lobe, frontal lobe, temporal lobe, occipital lobe, hippocampus, cerebellum and brainstem of porcine brain (n=5) were measured through UV-HPLC. Homogenizing brain tissues with 10% trichloroacetic acid (pH=1.0) not only avoided the disturbance from contaminated protein and other compounds, but also eliminated the step extraction of FA-DNPH derivative (2, 4-dinitrophenylhydrazone) and improve the methodological sensitivity (P < 0.05). The porcine brain formaldehyde was 75.5～83.4 μmol/kg fresh brain tissues measured by this improved method. Recoveries of spiked formaldehyde at low and high level were 95.96%～102.04% with relative standard deviations less than 10%. This suggests that the modified 2, 4-DNPH method is effective enough to measure the endogenous formaldehyde in brain tissues.