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Objective To summarize the characteristic of projects accepted by human genetic resources management office in 2016 and put forward further measures.Methods Statistic analyses were carried out based on the overall projects application,then Chinese application units and partners were analyzed separately.Results 1 138 international cooperation projects were approved in total,mainly on clinical research.24 countries were involved with increasing trend of globalization.Conclusions The human genetic resources management played a positive role in the development of the biomedical research and industry.
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Objective@#To construct a Tat-dependent MazF expression vector pcDNA3.1-GFP-HA-MazF-U3TAR.@*Methods@#HIV-1 U3TAR and MazF gene were amplified from pCR2.1-U3TAR and pET28a-MazF, respectively. Two gene fragments were linked together to form U3TAR-MazF by an overlapping PCR, and then cloned into pMD18T. An N-terminal HA tag was added to MazF to form U3TAR-MazF-HA. After double enzyme digestion using EcoR I and Hind Ⅲ, U3TAR-MazF-HA was reversely inserted into pcDNA 3.1 to form pcDNA3.1-HA-MazF-U3TAR. Then, a fluorescent reporter gene GFP was inserted into the downstream of U3TAR to form pcDNA3.1-GFP-HA-MazF-U3TAR.@*Results@#The co-transfection experiments with pcDNA3.1-tat-flag showed that pcDNA3.1-GFP-HA-MazF-U3TAR is Tat dependent. MazF was expressed only under the presence of Tat. In addition, compared with the cells transfected with pcDNA3.1-GFP-HA-MazF-U3TAR, less green fluorescent signal was observed in the cells co-transfected with pcDNA3.1-GFP-HA-MazF-U3TAR and pcDNA3.1-tat-flag, indicating that expressed MazF down-regulated the expression of GFP.@*Conclusions@#The expression vector pcDNA3.1-GFP-HA-MazF-U3TAR will provide an important tool for the development of MazF-based AIDS gene therapy strategies.
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Lewy body (LB), an eosinophilic inclusion localized in the neuronal perikaryon, consists of a wide range of proteins, including the consistent organization and the selective composition. Treatment of PC12 cells with synthetic proteasome inhibitor (PSI) at 10 μmol/L for 48 hours induced the formation of inclusions, which were detected by eosin staining and immunostaining for α-synuclein. To investigate the potential new components of PSI-induced inclusions in vitro, pure intact inclusions were successfully obtained by fractionation and subjected to two-dimensional electrophoresis (2-DE) then analyzed with unequivocal matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Eukaryotic translation initiation factor 3 subunit 5 (eIF-3ε), eukaryotic elongation factor 2 (eEF-2) and mitochondrial elongation factor Tu (EF-Tumt) were identified. The results suggest that 3 eukaryotic translation factors recruited in PSI-induced inclusions may influence formation of the intermediate organelles following the inhibition of proteasomes.
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Proteomic analysis is an effective way to identify protein constituent in Lewy bedy-like inclusions (or aggresome) in vitro. Exposure to synthetic proteasome inhibitor (PSI, 10 μmol/L) for 48 hours was used to induce the formation of cytoplasmic proteineous inclusions (termed as PSi-induced inclusions) in PC12 cells.The proteomic approaches of biochemical fractionation, two-dimensional electrophoresis (2-D) and identification via peptide mass fingerprints (PMF) were deployed, and 20 protein components of LBs were identified,i ncluding 2 proteins involved in the production of synaptic neurotransmitter, 6 subunits of the 26 S proteasome,2 cytoskeleton proteins, 2 subunits of mitochondrial complexes, 1 anti-oxidant protein, and 7 chaperone proteins and (or) chaperone-like proteins. The results suggested that these LB protein components might had been recruited in PSI-induced inclusions formed in PC12 cells under the condition of proteasome inhibition.
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Objective To investigate the pathogenetic mechanism of ubiquitin-proteasome dysfunction in a model of Parkinson's disease(PD),which can provide the theoretical basis for PD.Methods After establishment of PD model induced by PSI in PC12 cells,proteins of untreated(DMSO) and PSI-treated PC12 cells were extracted 36 h after incubation,and then the maps of the extracted proteins were established by DIGE system.The altered protein spots were identified with MALDI-TOF Pro MS and database searching.Results Thirty-six treatment of PC12 cells with PSI induced the appearance of cytoplasmic Lewy body-like eosinophilic inclusions and apoptosis.The percentage of apoptotic cells was 25.53%.ERp29 were identified by MALDITOF Pro MS.The expression of ERp29 decreased in treatment group,compared with normal group(P
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0.05).The symptom of peripheral neuropathy emerged 1 w after poisoning,the numbers of gait abnormal were 3,4 and 4 respectively in groups A,B and C,and there were no significant difference among those groups.It showed that the necrosis of segments of muscle fibers and macrophages infiltration scattering distribution were observated in the necrosis area and muscular interstition.At the end of 3 w,4 w and 8 w,there were significant differences in the area of muscular necrosis between group A and C(P