SIRT1 facilitates amyloid beta peptide degradation by upregulating lysosome number in primary astrocytes.

Previous studies have shown that sirtuin 1 (SIRT1) reduces the production of neuronal amyloid beta (Aß) and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aß neurotoxicity in animal models of Alzheimer's disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aß in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aß, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aß degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aß was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aß degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aß degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aß degradation in primary astrocytes.

[A preliminary proteomic analysis of tauopathies].

OBJECTIVE: To investigate the molecular mechanisms of tauopathies. Comparative proteomic analysis of brain proteins was employed to study 4 patients with tauopathies as compared with 4 controls. METHODS: The brains of subjects who died without clinical or pathological involvement of nervous system and brains of patients with tauopathies were obtained at autopsy. The brain proteins were run by immobilized pH gradient (IPG) isoelectric focusing electrophoresis as the first dimension, and then run by vertical SDS-PAGE as the second dimension. The maps were visualized by silver staining or colloidal coomassie blue and analyzed with Image Master 2D Elite software. The proteins of interest were in-gel digested and identified using MALDI-TOF mass spectrometry or MALDI-TOF/TOF tandem mass spectrometry. RESULTS: 18 protein spots were differentially expressed as compared with age-matched nondemented control brains which were identified as glyceraldehyde 3-phosphate dehydrogenase, uracil DNA glycosylase, human superoxide dismutase, isocitrate dehydrogenase subunit, synaptotagmin I, thioredoxin peroxidase 1, glial fibrillary acidic protein, p25 alpha, enoyl coenzyme A hydratase short chain 1, pyridoxine-5'-phosphate oxidase, Mn-superoxide dismutase and alpha enolase, antioxidant protein 2, ferritin heavy chain, glutamate dehydrogenase precursor, peptidyl-prolyl cis-trans isomerase A, serum albumin precursor and dihydropyrimidinase-related protein 2. CONCLUSIONS: We got a number of related-proteins of tauopathies. Some proteins are quite useful for discovering the molecular mechanisms of tauopathies and may be helpful for diagnosis and of treatment tauopathies.

[Proteomic comparison between human cerebellums and frontal lobes].

OBJECTIVE: To compare the results of proteomics between the cerebellum and frontal lobe of the aged. METHOD: Proteins were isolated from human cerebellums and frontal lobes and separated by two-dimensional (2D) gel electrophoresis. The proteins were then stained with silver or colloidal coomassie blue to produce a high-resolution map of the proteome. Selected proteins from this map were in-gel digested with trypsin and the resulting tryptic peptides were analyzed by MALDI-TOF mass spectrometry and MALDI TOF/TOF tandem mass spectrometry. The mass spectrometric data were used to identify the proteins through searches of the SWISSPROT protein sequence database. RESULTS: Three up-regulated protein spots in 2D gels of cerebellums were found as compared with those of frontal lobes. These protein spots were identified as antioxidant protein 2, creatine kinase precursor, fructose-bisphosphate aldolase C. Two down-regulated proteins in cerebellums were identified as pyruvate kinase M1 isozyme and glial fibrillary acidic protein. 30 common proteins, the expressions of which were not altered between cerebellums and frontal lobes, were also identified. CONCLUSIONS: These data will be used for future studies of differential protein expression in neurological disorders. Some proteins are useful for discovering the molecular mechanisms of degenerative dementia and brain aging.

Effects of deletion and shift of the A20-B19 disulfide bond on the structure, activity, and refolding of proinsulin.

To investigate the role of the A20-B19 disulfide bond in the structure, activity and folding of proinsulin, a human proinsulin (HPI) mutant [A20, B19Ala]-HPI was prepared. This mutant, together with another proinsulin mutant previously constructed with an A19Tyr deletion, which can also be taken as shifted mutant of the A20-B19 disulfide bond, were studied for their in vitro refolding, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion in comparison with native proinsulin. The results indicate that deletion of the A20-B19 disulfide bond results in a large decrease in the alpha-helix content of the molecule and higher sensitivity to tryptic digestion. Both the deletion and shift mutations, especially the latter, cause a great decrease in the biological activity of proinsulin analogues. The folding yields of HPI analogues were much lower than that of HPI. And the shift mutant, [Delta A19Tyr]-HPI, was scarcely refolded correctly in vitro and its refolding yield was extremely low. These results suggest that the A20-B19 disulfide bond plays an important role in the structural stabilization and folding of the insulin precursor. By summarizing the refolding studies on proinsulin, a possible folding pathway is proposed.

Differential protein expression induced by transient transfection of metallothionein-3 gene in SH-SY5Y neuroblastoma cell line.

Metallothionein-3(MT-3), also known as growth inhibitory factor (GIF), is predominantly expressed in central nervous system (CNS). It belongs to the family of metallothionein(MT) but has several unique properties that are not shared by other family members such as MT-1/MT-2. In the past few years, MT-3 had been postulated to be a multipurpose protein which could play important neuromodulatory and neuroprotective roles in CNS besides the common roles of MTs. However, the primary function of MT-3 and the mechanism underlying its multiple functions were not elucidated so far. In present study, human neuroblastoma cell line SH-SY5Y was employed to study the overall cellular protein changes induced by transient transfection of MT-3 gene, based on comparative proteome analysis. Averagely about 750 spots were visualized by Coomassie staining in one 2D gel, in which 17 proteins were shown to display significant and reproducible changes by semiquantitative analysis with ImageMaster 2D Elite software. Among them, 12 proteins were up-regulated while other 5 proteins were down-regulated. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 10 proteins were further identified to be zinc finger protein, glutamate transporter, and enhancer protein, etc., which were involved in several important pathways regulating the functions of central nervous system. The results showed that MT-3 might exert its unique functions by regulating the expression of these proteins.

[Isolation and identification of proteins with anti-tumor and fibrinolysogen kinase activities from Eisenia foetida].

Proteins from Eisenia foetida possess many biological activities. A group of proteins precipitated by ethanol were isolated and purified by Sephadex G-75 and HiPrep 16/60 DEAE columns, then identified by one- or two- dimensional electrophoresis and mass spectrometry. 2D gel experiments displayed that the pI of proteins from Eisenia foetida were mainly from 3.0 to 4.0. Anti-tumor and kinase activities were determined by in vitro experiments. The enthanol fraction D2(8) showed both of the activities. These ethanol-precipitated proteins were identified further by native polyacrylamide electrophoresis, the protein spots were cut off from gels and digested by trypsin, the peptide mass fingerprints (PMFs) were determined by mass spectrometry. PMF, molecular weight, amino acid composition and N-terminus of 6 proteins were characterized, and band 9 was identified as D2(8). The results suggested that there exist proteins in Eisenia foetida possessed both anti-tumor and fibrinolysogen kinase activities. These methods can be used for identification of the natural bioactive proteins.

Expression, purification and activity measurement of soluble BACE protein in E.coli.

To express BACE (beta-site APP cleaving enzyme), an aspartic protease related to Alzheimer's disease in E.coli to obtain the active protein after refolding and purification, the sequence for soluble form of BACE was inserted into prokaryotic expression vector pET11a. After expression in E.coli BL21(DE3), the protein was obtained as inclusion bodies, after refolding and purification. The proteolytic activity of the protein was measured by HPLC and MS. After refolding and purification, the protein exhibited beta-secretase activity by cleaving the synthetic peptide, which was designed according to the beta-secretase site at Swedish mutant of APP, and according Hanes method the kinetics constants for the synthetic peptide of BACE protein were measured. The study will facilitate BACE protein structure determination and inhibitor development efforts, which may lead to the evolution of promising and effective treatments for Alzheimer's disease.

[Treatment of avascular necrosis of femoral head by impacting granular bone grafting via window in femoral neck].

OBJECTIVE: To evaluate the clinical results of continuing skeletal traction and impaction granular bone grafting via window in femoral neck for the treatment of avascular necrosis of femoral head. METHODS: From August 2000 to October 2004, 23 patients (35 hips) with femoral head necrosis were treated by continuing skeletal traction and impacting granular bone grafting via bone window on femoral neck. There were 18 males, 5 females, with an average age of 32 years ranging from 19 to 52 years, which included 7 hips of stage II, 28 hips of stage III. All patients had various degrees of hip joint pain and suffered from limited hip motion. The necrotic bone, granulation tissue and hardening zone were completely cleaned via bone window on the femoral neck. The autogenous granular iliac bone was grafted, and impacted persistently. Skeletal traction through femur condyles was applied continually after the operations. The effects before and after operation were compared by the hip pain, function, joint activity and X-ray. RESULTS: Regular follow-up was carried out after the patients were dismissed from the hospital. The follow-up period was 6 months, 1 year, 2 years, 3 years, 4 years, 5 years respectively. According to Wang's standard, the average score was increased from (52.66 +/- 12.53) preoperatively to (88.94 +/- 5.84) preoperatively at half a year, (89.78 +/- 6.18) at 1 year, (86.37 +/- 7.46) at 2 years, (84.08 +/- 7.57) at 3 years, (83.76 +/- 8.08) at 4 years, and (76.83 +/- 8.98) at 5 years. Scores of operation were greatly increased and the difference had statistical significance. CONCLUSION: Continuing skeletal traction after the operation, completely cleaning the necrotic bone and impacting granular bone grafting via window on femoral neck can greatly raise the satisfactory rate of clinical effect and delay the progression of disease for avascular necrosis of femoral head.

A comprehensive study of optimal conditions for naked plasmid DNA transfer into skeletal muscle by electroporation.

Efficient gene transfer is a key factor in gene therapy. Reducing the damage caused by gene transfer to muscle by electroporation is very important for its clinical application. Extensive investigation of optimal conditions for gene transfer by electroporation is required. The parameters used for electroporation, including plasmid concentration; injection volume; the plasmid dose of the injection; the concentration of saline media; the size of plasmid DNA; the age of the mice; the lag time between plasmid injection and electroporation; and the effect of repeated gene transfer by electroporation, were systematically investigated in the present study. The efficiencies of gene transfer by electroporation in normal and rodent models of diabetes were also evaluated. We found that electroporation used for non-viral gene transfer could be repeated in the same place in the muscle, but the expression efficiency was closely related to the muscle damage. Increasing pulse times could enhance the efficiency of gene transfer with a lower strength of electric field. It was better to use a higher plasmid concentration than to use a larger dose of plasmid and repeated injection to achieve a high level of transgene expression. Optimal conditions varied in different animal models, being milder for diabetic mice than for normal mice, and it was also shown that the conditions that worked well on these small rodents were not necessarily suitable for larger animals. Our results provide a comprehensive view of the factors that affect the efficiency of gene transfer into skeletal muscle by electroporation.

Differential display proteome analysis of PC-12 cells transiently transfected with metallothionein-3 gene.

Metallothionein-3 (MT-3), also known as growth inhibitory factor, possesses several unique properties other than the common features of metallothionein family. To investigate the mechanisms underlying its multifaceted roles in the central nervous system, we employed differential display proteomics techniques to study holistic protein changes of PC-12 cells induced by transient transfection of MT-3. Ten significantly and reproducibly changed proteins were identified and their functional implications are discussed in some detail.