Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm ± 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of “normal” early pregnancy.     

五氯酚在污染沉积物泥浆固液两相中厌氧生物降解

研究了污染沉积物泥浆液、固两相五氯酚(PCP)厌氧生物降解.结果表明,投加10g·kg^-1厌氧颗粒污泥,经31d处理泥浆液、固两相PCP降解率达98.9%,平均降解速率达到80mg·kg^-1·d^-1,对照处理平均降解速率仅为4.4mg·kg^-1·d^-1,颗粒污泥生物强化作用明显.作为泥浆修复过程的调控因子,有机溶剂、共基质和表面活性剂对PCP降解效应不同,投加乙醇,可提高PCP解吸和降解速率,4d内两相PCP降解速率达到54.3mg·kg^-1·d^-1;而投加共基质和非离子表面活性剂乙二醇丁醚后,液、固两相PCP降解均出现迟滞,两者均不同程度地抑制PCP降解.     

Proteomic analysis in peritoneal dialysis patients with different peritoneal transport characteristics

Peritoneal membranes can be categorized as high, high average, low average, and low transporters, based on the removal or transport rate of solutes. In this study, we used proteomic analysis to determine the differences in proteins removed by different types of peritoneal membranes. Peritoneal transport characteristics in patients who received peritoneal dialysis therapy were assessed by a peritoneal equilibration test. Two-dimensional differential gel electrophoresis technology followed by quantitative analysis was performed to study the variation in protein expression from peritoneal dialysis effluents (PDE) among different groups. Proteins were identified by MALDI-TOF-MS/MS analyses. Further validation in PDE or serum was performed utilizing ELISA analysis. Proteomics analysis revealed ten protein spots with significant differences in intensity levels among different groups, including vitamin D-binding protein, complement C3, apolipoprotein-A1, complement factor C4A, haptoglobin, alpha-1 antitrypsin, immunoglobulin kappa light chain, alpha-2-microglobulin, retinol-binding protein 4 and transthyretin. The levels of vitamin D-binding protein, complement C3, and apolipoprotein-A1 in PDE derived from different groups were greatly varied (P < 0.05). However, no significant difference was found in the serum levels of these proteins among different groups (P > 0.05 for all groups). This study provides a novel overview of the differences in PDE proteomes of four types of peritoneal membranes. Vitamin D-binding protein, complement C3, and apolipoprotein-A1 showed enhanced expression in PDE of patients with high transporter.     

Sample preparation and digestion for proteomic analyses using spin filters

We describe the use of commercially available microcentrifugation devices (spin filters) for cleanup and digestion of protein samples for mass spectrometry analyses. The protein sample is added to the upper chamber of a spin filter with a > or = 3000 molecular weight cutoff membrane and then washed prior to resuspension in ammonium bicarbonate. The protein is then reduced, alkylated, and digested with trypsin in the upper chamber and the peptides are recovered by centrifugation through the membrane. The method provides digestion efficiencies comparable to standard in-solution digests, avoids lengthy dialysis steps, and allows rapid cleanup of samples containing salts, some detergents, and acidic or basic buffers.     

A simple method to evaluate the concentration of pentachlorophenol degraders in contaminated soils

A new most probable number (MPN) method for the determination of pentachlorophenol (PCP) degraders in soil using the change in pH due to PCP degradation is compared with a well documented MPN method using radiolabeled PCP. The results of all MPN counts were similar within a 95% confidence limit. The results obtained in MPN per gram of dry soil using pH measurements were 1.8 (+3.1, -1.03) x10 (4) compared to 0.64 (+1.34, -0.42) x 10(4) when using production of [(14)C]CO(2).     

Involvement of aldolase A in X-ray resistance of human HeLa and UV(r)-1 cells

To find novel proteins involved in radio-resistance of human cells, we searched for nuclear proteins, whose expression levels alter after X-ray irradiation in HeLa cells, using agarose fluorescent two-dimensional differential gel electrophoresis following mass spectrometry. We identified 6 proteins, whose levels were increased in nuclei 24 h after irradiation at 5 Gy, including aldolase A. Nuclear aldolase A levels increased twofold after the irradiation, however, total aldolase A levels did not change. When the expression of aldolase A was suppressed by its specific siRNA, sensitization of the suppressed cells to X-ray-induced cell death was observed. In addition, UV^r-1 cells with higher aldolase A expression exhibited lower sensitivity to X-ray-induced cell death than the parental RSa cells with lower aldolase A expression. These results suggest that aldolase A may play a role in the radio-response of human cells, probably in nuclei, in addition to its glycolytic role in the cytosol.     

Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques―photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis—are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.     

Proteome response of an extraintestinal pathogenic Escherichia coli strain with zoonotic potential to human and chicken sera

A subset of extraintestinal pathogenic Escherichia coli is zoonotic and has developed strategies to adapt to different host-specific environments. However, the underlying mechanisms of these adaptive strategies have yet to be discerned. Here, the proteomic response of an avian pathogenic E. coli strain, which appears indistinguishable from neonatal meningitis E. coli, was compared following growth in human and avian sera to determine whether it uses the same mechanisms to overcome the antibacterial effects of sera from different host species. Proteins involved in biosynthesis of iron receptors were up-regulated under both sera, suggesting that serum, regardless of the host of origin, is an iron-limited environment. However, several proteins involved in synthesis of nucleic acids, sulfur-containing amino acids and fatty acids, were differentially expressed in response to the sera from different hosts. Mutational analysis showed that this APEC strain required nucleotide biosynthesis during incubation in human, but not avian serum, and deletion of genes involved in the biosynthesis of sulfur-containing amino acids increased its resistance to human serum. Continued investigation of the proteome of 'zoonotic' ExPEC strains, grown under other 'dual' host conditions, will contribute to our understanding of ExPEC pathogenesis and host specificity and development of effective therapies and control strategies.     

Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization

Shed membrane microparticles (MPs) are microvesicles generated from the plasma membrane when cells are submitted to stress conditions. Although MPs reflect the cell state (at least in vitro), little is known on their protein composition. We describe the first set of experiments aiming to characterize the MP proteome. Two ways of triggering MP formation from a T-lymphocytic cell line were analyzed using a 1-D gel approach coupled with LC-MS/MS and the results were compared with those obtained from a classic membrane preparation. In total, 390 proteins were identified in MPs, among which 34% were localized to the plasma membrane. The MPs revealed a broad representation of plasma membrane proteins including 17 hematopoietic clusters of differentiation. This approach was successfully applied to one human chronic B-cell lymphoid malignancy. In all, 413 proteins were identified, including 117 membrane proteins, many of them being pathology associated. The sequence coverage in identified proteins was improved combining both nano-LC-MS/MS and MALDI-MS data. The suppression effect, observed on very complex peptide mixtures, was remediated by chromatographic fractionation. MPs may represent a new tool for studying plasma membrane proteins, displaying the advantages of reproducibility, minimal organelle contamination, and being potentially applicable to most cell types.     

石油降解菌HX-2耐盐机制及甜菜碱转运蛋白基因的研究

【背景】修复石油烃污染的高盐水体及土壤是具有挑战性的,因此探究石油烃降解菌株的耐盐机制尤为重要。【目的】对石油降解菌HX-2的耐盐机制及与耐盐性相关的基因进行研究。【方法】通过GC分析菌株HX-2在不同石油加入量及高盐条件下的烃降解情况;利用电导率仪及原子吸收光谱对细胞内离子含量进行分析;比较外源添加甜菜碱前后对胞外多糖(extracellular polysaccharide,EPS)及高盐土壤中石油降解情况的影响;最后对耐盐相关基因进行了qPCR分析研究。【结果】石油降解菌Rhodococcus sp. HX-2可以对10 000-100 000 mg/L的石油进行降解,3 d降解率均达到70%以上,并可在1%-10%NaCl存在下降解石油,在6%Na Cl浓度下仍有43.8%的降解率。对HX-2菌株耐盐机制的研究表明,细胞内阳离子浓度随着盐浓度的变化没有显著差异,而积累相容性物质甜菜碱并促进EPS的合成才是石油降解菌HX-2的耐盐机制。同时,扫描电镜结果表明,外源甜菜碱的添加通过刺激EPS的合成提高菌株的耐盐性。由HX-2菌株得到4种甜菜碱转运蛋白基因H0、H1、H3、H5和1种...     

Effects of pentachlorophenol on the bacterial denitrification process

The use of pentachlorophenol (PCP) was banned or restricted in many countries worldwide because of its adverse influences on the ecological environment and humans. However, the potential disrupting effects of PCP on denitrifying microorganisms have warranted more analysis. In this study, the impacts of PCP on denitrification were investigated by using Paracoccus denitrificans as a model denitrifying bacterium. Compared with the control, the presences of 10 and 50 μM of PCP were found to significantly decrease the denitrification efficiencies from 98.5 to 87.2% and 68.7%, respectively. The mechanism studies showed that PCP induced the generation of reactive oxygen species, which decreased the vital enzymes activities related to glycolysis process, causing the disturbance of the metabolism of P. denitrificans utilizing carbon source (glucose) and the growth of the cell, and subsequently the generation of electron donor (NADH) for denitrification via NAD⁺ reduction was severely depressed. Further studies indicated that PCP also decreased the genes expression of several key enzymes responsible for denitrification, such as napA of nitrate reductase (NAR), nirS of nitrite reductase, norB of nitric oxide reductase, and nosZ of nitrous oxide reductase; however, there was only the enzyme activity of NAR was remarkably inhibited.