Proteomic analysis of vascular smooth muscle cells in physiological condition and in pulmonary arterial hypertension: Toward contractile versus synthetic phenotypes

Vascular smooth muscle cells (VSMCs) are highly specialized cells that regulate vascular tone and participate in vessel remodeling in physiological and pathological conditions. It is unclear why certain vascular pathologies involve one type of vessel and spare others. Our objective was to compare the proteomes of normal human VSMC from aorta (human aortic smooth muscle cells, HAoSMC), umbilical artery (human umbilical artery smooth muscle cells, HUASMC), pulmonary artery (HPASMC), or pulmonary artery VSMC from patients with pulmonary arterial hypertension (PAH‐SMC). Proteomes of VSMC were compared by 2D DIGE and MS. Only 19 proteins were differentially expressed between HAoSMC and HPASMC while 132 and 124 were differentially expressed between HUASMC and HAoSMC or HPASMC, respectively (fold change 1.5≤ or ?1.5≥, p < 0.05). As much as 336 proteins were differentially expressed between HPASMC and PAH‐SMC (fold change 1.5≤ or ?1.5≥, p < 0.05). HUASMC expressed increased amount of α‐smooth muscle actin compared to either HPASMC or HAoSMC (although not statistically significant). In addition, PAH‐SMC expressed decreased amount of smooth muscle myosin heavy chain and proliferation rate was increased compared to HPASMC thus supporting that PAH‐SMC have a more synthetic phenotype. Analysis with Ingenuity identified paxillin and (embryonic lethal, abnormal vision, drosophila) like 1 (ELAVL1) as molecules linked with a lot of proteins differentially expressed between HPASMC and PAH‐SMC. There was a trend toward reduced proliferation of PAH‐SMC with paxillin‐si‐RNA and increased proliferation with ELAVL1‐siRNA. Thus, VSMCs have very diverse protein content depending on their origin and this is in link with phenotypic differentiation. Paxillin targeting may be a promising treatment of PAH. ELAVL1 also participate in the regulation of PAH‐SMC proliferation.     

Plant‐extract‐induced changes in the proteome of the soil‐borne pathogenic fungus Thielaviopsis basicola

Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non‐host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts, while hierarchical clustering analysis of 2‐DE protein profiles of T. basicola showed plant species had a larger effect on the proteome than host/non‐host status. Analysis by LC‐MS/MS of unique and differentially expressed spots and identification using cross‐species similarity searching and de novo sequencing allowed successful identification of 41 spots. These proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several “morpho” proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B₆ is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.     

Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos

Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2‐dimensional electrophoresis coupled with matrix‐assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide‐induced responses in the mosquito.     

Response to nickel in the proteome of the metal accumulator plant Brassica juncea

To gain a comprehensive understanding of the molecular mechanism of heavy metal accumulation in Brassica juncea, comparative proteomic approaches were used to analysis protein profiles in leaf tissues of 6-week-old B. juncea after exposure to 100 µM Ni. Proteomic analysis revealed that 61 protein spots showed 1.5-fold change in protein abundance after Ni exposure as compared to that of corresponding spots in control. Out of the 61 differentially expressed protein spots, 37 protein spots were ambiguously identified by matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). The majority of these identified proteins were found to be involved in sulphur metabolism, protection against oxidative stress, clearly indicated that heavy metal sequestration and antioxidant system were activated by Ni treatment. The induced expression of photosynthesis and ATP generation-related proteins were also observed in plants exposed to metals, suggesting the tolerance and accumulation is an energy-demanding process. The identification of these proteins in response to Ni can lead a deep understanding of heavy metal accumulation and tolerance in B. juncea.     

Understanding tenderness variability and ageing changes in buffalo meat: biochemical,ultrastructural and proteome characterization

Understanding of biological impact of proteome profile on meat quality is vital for developing different approaches to improve meat quality. Present study was conducted to unravel the differences in biochemical, ultrastructural and proteome profile of longissimus dorsi muscle between buffaloes (Bubalus bubalis) of different age groups (young v. old). Higher (P<0.05) myofibrillar and total protein extractability, muscle fibre diameter, and Warner-Bratzler shear force (WBSF) values was observed in old buffalo meat relative to meat from young buffaloes. Scanning electron microscopy photographs revealed reduced fibre size with increased inter-myofibrillar space in young compared with old buffalo meat. Transmission electron microscopy results revealed longer sarcomeres in young buffalo meat relative to meat from old buffaloes. Proteomic characterization using two-dimensional gel electrophoresis (2DE) found 93 differentially expressed proteins between old and young buffalo meat. Proteome analysis using 2DE revealed 191 and 95 differentially expressed protein spots after 6 days of ageing in young and old buffalo meat, respectively. The matrix assisted laser desorption ionization time-of flight/time-of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of selected gel spots helped in identifying molecular markers of tenderness mainly consisting of structural proteins. Protein biomarkers identified in the present study have the potential to differentiate meat from young and old buffaloes and pave the way for optimizing strategies for improved buffalo meat quality.     

Proteomic Analysis of Somatic Embryogenesis in <Emphasis Type="Italic">Cyclamen persicum</Emphasis> Mill

Cyclamen persicum Mill. is a widely grown ornamental species that is clonally propagated by somatic embryogenesis. To better understand the biology of somatic embryo development in C. persicum, detailed proteomic (two-dimensional gel electrophoresis) and mass spectrometric analyses of somatic embryos at globular, torpedo, and germinating stages of development, along with nonembryogenic callus and zygotic embryos, were conducted. Of ~460 proteins resolved in two-dimensional gels, 35 proteins were differentially expressed and could be reproducibly displayed across an isoelectric focusing range of 5 to 8. Among those proteins, five were constitutively expressed, 13 were upregulated, nine were downregulated, and eight were deemed as novel proteins during the torpedo stage. A total of 35 protein spots were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and only four proteins were identified and these were available in public protein databases. The remaining protein spots were subsequently analyzed by MALDI-TOF-TOF-MS, and six proteins were then identified. These findings suggested that specific proteins are involved in the regulation of somatic embryogenesis.     

Plant defense in response to chewing insects: proteome analysis of Arabidopsis thaliana damaged by Plutella xylostella

The interactions between Arabidopsis thaliana and Plutella xylostella have been considered as a model system to unravel the responses of plants to herbivorous insects. Here, we use a 2-DE proteome approach to detect protein expression changes in the leaves of Arabidopsis plants exposed to P. xylostella larval infestation at 27°C within 8?h. Approximately 450 protein spots were reproducibly detected on gels. Of these, comparing healthy and infested leaves, we identified 18 differentially expressed protein spots. Thirteen proteins were successfully identified by MALDI-TOF/MS and LC-ESI-MS/MS. Functional classification analysis indicated that the differentially identified proteins were associated with amino acid, carbohydrate, energy, lipid metabolism, and photosynthesis. In addition, their relative abundances were assessed according to larval pest feeding on Arabidopsis leaves. These data provide valuable new insights for further works in plant-biotic and environmental stress interaction.     

Proteomic analysis of schistosoma japonicum schistosomulum proteins that are differentially expressed among hosts differing in their susceptibility to the infection

Schistosomiasis is a tropical, parasitic disease affecting humans and several animal species. The aim of this study was to identify proteins involved in the growth and survival of the parasitic forms inside a host. Schistosomula of Schistosoma japonicum were isolated from three different hosts: the susceptible BALB/c mice; the Wistar rats, which have a considerably lower susceptibility; and the resistant reed vole, Microtus fortis. Soluble proteins of the schistosomula collected from the above three hosts 10 days postinfection were subjected to two-dimensional difference gel electrophoresis. Comparative proteomic analyses revealed that 39, 21, and 25 protein spots were significantly differentially expressed between schistosomula from mice and rats, mice and reed voles, or rats and reed voles, respectively (ANCOVA, p < 0.05). Further, the protein spots were identified by liquid chromatography-tandem MS. Bioinformatics analysis showed that the differentially expressed proteins were essentially those involved in the metabolism of proteins, ribonucleotides, or carbohydrates, or in stress response or cellular movement. This study represents the first attempt at profiling S. japonicum living in different states and provides a basis for a better understanding of the molecular mechanisms in the development and survival of S. japonicum in different host environments.     

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE

Identification of differentially expressed proteins during Neospora caninum tachyzoite–bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5‐fold in relative abundance, p<0.05, t‐test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde‐3‐phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.     

Effect of Seasonal Change on Testicular Protein Expression in White Roman Geese

The purpose of this study was to investigate the change in protein expression in the testes of ganders at various breeding stages. A total of nine 3-year-old male White Roman ganders were used. The blood and testis samples were collected at the nonbreeding, sexual reactivation, and breeding stages for sex hormone analysis and proteomic analysis, respectively. The testicular weight and serum testosterone observed for ganders at the breeding stage were higher than those for ganders at nonbreeding and sexual reactivation stages (P?<?0.05). There were 124 protein spots differentially expressed in the testes of ganders at various reproductive stages. A total of 107 protein spots of 74 proteins was identified through mass spectrometry. Most of the differentially expressed proteins were responsible for the molecular functions of protein binding (24%) and catalytic activity (16%). A functional pathway analysis suggested that proteins involved in steroidogenesis, metabolism, and spermatogenesis pathways changed in the White Roman geese at various reproductive stages. In conclusion, ganders at various reproductive stages exhibited different levels of testosterone and protein expression in the testes. The varied levels of the proteins might be essential and unique key factors in seasonal reproduction in ganders.     

Leaf proteomic analysis in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>, Crantz) during plant development,from planting of stem cutting to storage root formation

Tuberization in cassava (Manihot esculenta Crantz) occurs simultaneously with plant development, suggesting competition of photoassimilate partitioning between the shoot and the root organs. In potato, which is the most widely studied tuber crop, there is ample evidence suggesting that metabolism and regulatory processes in leaf may have an impact on tuber formation. To search for leaf proteins putatively involved in regulating tuber generation and/or development in cassava, comparative proteomic approaches have been applied to monitor differentially expressed leaf proteins during root transition from fibrous to tuberous. Stringent cross comparison and statistical analysis between two groups with different plant ages using Student’s t test with 95% significance level revealed a number of protein spots whose abundance were significantly altered (P < 0.05) during week 4 to week 8 of growth. Of these, 39 spots were successfully identified by ion trap LC–MS/MS. The proteins span various functional categories from antioxidant and defense, carbohydrate metabolism, cyanogenesis, energy metabolism, miscellaneous and unknown proteins. Results suggested possible metabolic switches in the leaf that may trigger/regulate storage root initiation and growth. This study provides a basis for further functional characterization of differentially expressed leaf proteins, which can help understand how biochemical processes in cassava leaves may be involved in storage root development.     

Exploring membrane and cytoplasm proteomic responses of Alkalimonas amylolytica N10 to different external pHs with combination strategy of de novo peptide sequencing

Identification of differentially proteomic responses to external pHs would pave an access for understanding of survival mechanisms of bacteria living at extreme pH environment. We cultured Alkalimonas amylolytica N10 (N10), a novel alkaliphilic bacterium found in Lake Chahannor, in media with three different pHs and extracted the correspondent membrane and cytoplasm proteins for proteomic analysis through 2‐DE. The differential 2‐DE spots corresponding to the altered pHs were delivered to MALDI TOF/TOF MS for protein identification. Since the genomic data of strain N10 was unavailable, we encountered a problem at low rate of protein identification with 18.1%. We employed, therefore, a combined strategy of de novo sequencing to analyze MS/MS signals generated from MALDI TOF/TOF MS. A significantly improved rate of protein identification was thus achieved at over than 70.0%. Furthermore, we extensively investigated the expression of these pH‐dependent N10 genes using Western blot and real‐time PCR. The conclusions drawn from immunoblot and mRNA measurements were mostly in agreement with the proteomic observations. We conducted the bioinformatic analysis to all the pH‐dependent N10 proteins and found that some membrane proteins participated in iron transport were differentially expressed as external pH elevated and most of differential proteins with increased or bell‐shape mode of pH‐dependence were involved in bioenergetic process and metabolism of carbohydrates, fatty acid, amino acids, and nucleotides. Our data thus provide a functional profile of the pH‐responsive proteins in alkaliphiles, leading to elucidation of alkaliphilic‐adaptive mechanism.     

食源性肥胖大鼠下丘脑差异表达蛋白的蛋白质组学分析

建立食源性肥胖大鼠模型,对正常大鼠和肥胖大鼠下丘脑全蛋白进行双向凝胶电泳,产生下丘脑蛋白双向凝胶电泳图谱.对图谱进行比对分析后,从凝胶上切取差异表达的蛋白点,经胶内酶解,通过基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS) 对酶解后的肽段进行分析,再经数据库（NCBInr）检索,对蛋白质进行鉴定.研究发现,正常组表达图谱可检测到1　160±15（n=5）个蛋白点,肥胖组表达图谱可检测到1　070±10 （n=5）个蛋白点,与对照组相比,匹配率大于80％.并且成功鉴定了17种差异表达蛋白质,其中有7 种在肥胖组表达上调,10种表达下调.它们分别属于代谢酶、细胞周期调控因子、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、细胞骨架蛋白以及未知蛋白等. 与正常对照组相比,肥胖组的蛋白质表达存在着较大差异,通过对差异表达蛋白的分析,提示了在肥胖发生的过程中,下丘脑神经中枢经历了一个非常复杂的信号活动和特定改变,为深入认识肥胖的发病机制奠定了基础.     

Identification of differentially expressed proteins in spontaneous thymic lymphomas from knockout mice with deletion of p53

Background

Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two-dimensional gel electrophoresis (2D-PAGE).

Results

Protein spots excised from 2D-PAGE gels, were subjected to in-gel tryptic digestion and identified by liquid chromatography – tandem mass spectrometry. A total of 47 protein spots were identified. Immunological verification was performed for several of the differentially regulated proteins where suitable antibodies could be obtained. Functional annotation clustering revealed similarities as well as differences between the tumours. Twelve proteins that changed similarly in both tumours included up-regulation of rho GDP-dissociation inhibitor 2, proteasome subunit α type 3, transforming acidic coiled-coil containing protein 3, mitochondrial ornithine aminotransferase and epidermal fatty acid binding protein and down-regulation of adenylosuccinate synthetase, tubulin β-3 chain, a 25 kDa actin fragment, proteasome subunit β type 9, cofilin-1 and glia maturation factor γ.

Conclusion

Some of the commonly differentially expressed proteins are also differentially expressed in other tumours and may be putative diagnostic and/or prognostic markers for lymphomas.     

Differential proteomic response to heat stress in thermal Agrostis scabra and heat‐sensitive Agrostis stolonifera

Knowledge of heat‐responsive proteins is critical for further understanding of the molecular mechanisms of heat tolerance. The objective of this study was to compare proteins differentially expressed in two C₃ grass species contrasting in heat tolerance, heat‐tolerant thermal Agrostis scabra and heat‐sensitive Agrostis stolonifera L., and to identify heat‐responsive proteins for short‐ and long‐term responses. Plants were exposed to 20/15°C (day/night, control) or 40/35°C (day/night, heat stress) in growth chambers. Leaves were harvested at 2 and 10 days after temperature treatment. Proteins were extracted and separated by fluorescence difference gel electrophoresis (DIGE). Thermal A. scabra had superior heat tolerance than A. stolonifera, as indicated by the maintenance of higher chlorophyll content and photochemical efficiency under heat stress. The two‐dimensional difference electrophoresis detected 68 heat‐responsive proteins in the two species. Thermal A. scabra had more protein spots either down‐ or up‐regulated at 2 days of heat stress, but fewer protein spots were altered at 10 days of heat stress compared with A. stolonifera. Many protein spots exhibited transient down‐regulation in thermal A. scabra (only at 2 days of heat treatment), whereas down‐regulation of many proteins was also found at 10 days of heat treatment in A. stolonifera, which suggested that protein metabolism in thermal A. scabra might acclimate to heat stress more rapidly than those in A. stolonifera. The sequences of 56 differentially expressed protein spots were identified using mass spectrometry. The results suggest that the maintenance or less severe down‐regulation of proteins during long‐term (10 days) heat stress may contribute to the superior heat tolerance in thermal A. scabra, including those involved in photosynthesis [RuBisCo, RuBisCo activase, chloroplastic glyceraldehydes‐3‐phosphate dehydrogenase (GAPDH), chloroplastic aldolase, oxygen‐evolving complex, photosystem I subunits], dark respiration (cytosolic GAPDH, cytoplasmic aldolase, malate dehydrogenase, hydroxypyruvate reductase, sedoheptulose‐1,7‐bisphosphatase), photorespiration [(hydroxypyruvate reductase, alanine aminotransferase (AlaAT), hydroxymethyltransferase (SHMT), glycine decarboxylase (GDC)], as well as heat and oxidative stress protection [heat shock cognate (HSC) 70 and FtsH‐like protein].     

鸭疫里默氏杆菌强、弱菌株外膜蛋白的比较蛋白质组学研究

应用双向电泳及质谱技术对血清2型鸭疫里默氏杆菌强毒株及其体外传代200代(RA200)的弱毒菌株的外膜蛋白进行比较蛋白质组学研究,借此分析鸭疫里默氏杆菌的外膜蛋白表达特点,研究差异表达蛋白与细菌毒力的关系.在实验中检测到血清2型鸭疫里默氏杆菌原代及其体外传代获得的弱毒菌株的外膜蛋白约表达60个蛋白质点(n=3),其中相差5倍以上3个.胶内酶解和肽质量指纹图谱分析后鉴定,W1为热休克蛋白Hsp20家族成员,W2、W3为转座酶,推测它们可能与里默氏杆菌的毒力密切相关.     

Protein dynamics during seed germination under copper stress in Arabidopsis over-expressing <Emphasis Type="Italic">Potentilla superoxide dismutase</Emphasis>

Copper (Cu), though an essential micronutrient for plants, poses toxicity at higher concentrations possibly by inducing oxidative stress. With the background that enzyme superoxide dismutase (SOD) ameliorates oxidative stress, the present work focused on understanding physiological and proteomic response of Arabidopsis seeds constitutively over-expressing copper–zinc SOD of Potentilla atrosanguinea (PaSOD) during germination in response to varied concentrations of copper sulphate (Cu stress). Transgenics showed higher germination percentage and required less “mean time to germination” under Cu-stress. In response to Cu stress, 39 differentially expressed protein spots were detected by 2-D electrophoresis in proteins of germinating wild type (WT) and transgenic seeds, of which 14 spots appeared exclusively in transgenics. Among the rest 25 protein spots, 14 showed down-regulation, one showed up-regulation, and 10 spots disappeared. MALDI-TOF and subsequent peptide mass fingerprinting analysis revealed that the down-regulated proteins in transgenics were related to oxidative stress, detoxification, germination, intermediary metabolism and regulatory proteins. Up-regulated proteins in WT and down-regulated proteins in transgenic during Cu stress were the same. Changes in key proteins, vis-à-vis alleviation of oxidative stress in transgenic Arabidopsis over-expressing PaSOD possibly alleviated toxicity of Cu-induced stress during seed germination, resulting in higher germination rate and germination percentage.     

Proteomic analysis of tilapia Oreochromis niloticus Streptococcus agalactiae strains with different genotypes and serotypes

Nine tilapia Oreochromis niloticus group B streptococcus (GBS) strains differing in serotype and genotype were selected and paired. Two‐dimensional difference gel electrophoresis (2D DIGE) and matrix‐assisted laser‐desorption ionization time‐of‐flight‐mass spectrometry (MALDI‐TOF‐MS) were used to analyse the protein profiles of the strain pairs. Forty‐three proteins corresponding to 66 spots were identified, of which 35 proteins were found in the seven selected strain pairs that represented pairs differing in genotype and serotype. Among the 35 proteins, numbers of differentially expressed proteins in strains of different serotypes were greater than found in strains of different genotypes, suggesting that serotype plays a more essential role than genotype in the differential protein expression among GBS strains. No distinct pattern was found with respect to genotype and the protein expression profile of GBS strains. Several proteins were identified as surface‐associated cytoplasmic proteins that possessed the typical immunity‐eliciting characteristics of surface proteins. The identified proteins were found to be involved in 16 biological processes and seven Kyoto encyclopaedia of genes and genomes (KEGG) pathways. The data, for the first time, identified differentially expressed proteins in O. niloticus GBS strains of different serotypes, which play a major role in immunogenicity of O. niloticus GBS than does genotype, offering further information for design of a vaccine against O. niloticus GBS.