A proteomic investigation into adriamycin chemo-resistance of human leukemia K562 cells

This study aimed to explore the mechanism of adriamycin resistance in human chronic myelogenous leukemia cells. Proteomic approach was utilized to compare and identify differentially expressed proteins between human chronic myelogenous leukemia K562 cells and their adriamycin-resistant counterparts. The differentially expressed proteins were analyzed by 2-DE (two-dimensional gel electrophoresis), and protein identification were performed on ESI-Q-TOF MS/MS instrument. Out of the 35 differentially expressed proteins between the two cell lines, 29 were identified and grouped into 10 functional classes. Most of identified proteins were related to the categories of metabolism (24%), proteolysis (13%), signal transduction (21%) and calcium ion binding (6%), suggesting that alterations of those biological processes might be involved in adriamycin resistance of K562 cells. We believe this study may provide some clues to a better understanding of the molecular mechanisms underlying adriamycin resistance.     

Role of lutoid membrane transport and protein synthesis in the regeneration mechanism of latex in different <Emphasis Type="Italic">Hevea</Emphasis> clones

Natural rubber (cis-1,4 polyisoprene) is synthesised in the milky cytoplasm, the latex, of specialized cells called laticifers in the bark tissues of the rubber tree (Hevea brasiliensis). Regeneration mechanism of latex after each tapping (controlled wounding of the bark) was studied in relation to lutoid membrane enzymes and protein synthesis in twelve rubber clones with varying yield potentials during the peak rubber yielding season. High activity of membrane enzymes and better availability of biochemical energy [ATP] were observed in clones viz; RRII 105, RRIM 600, PB 260, RRII 422 and RRII 430. The highest protein biosynthetic capacity was noticed in clone PB 260 and RRIM 600. However, high ATP content, increased invertase activity and protein biosynthesis were observed in the medium yielding clone GT1 compared to clones with low rubber yield potential. Very low sugar content and increased invertase activity in the latex of clone PB 260 indicated intense latex metabolism with high protein turnover that implies fast recouping of the cellular metabolites lost during latex harvesting. Clone PB 217 was characterized by very high sucrose and low ATP concentration and ATPase activity in latex indicating slow metabolism and hence be suitable for inducing latex metabolism using ethylene stimulant. Low rubber yielding clones such as RRII 33 and RRII 38 were consistently recorded a high sucrose content but very low activity of membrane enzymes, reduced ATP concentration and low protein biosynthesis in latex. Among the recently released modern clones (RRII 400 series), latex regeneration capacity was higher in RRII 422 and RRII 430. The significance of lutoid membrane transport and protein synthesis is discussed in relation to general latex metabolism of these rubber clones. The outcome of this study would be helpful to design suitable latex harvesting systems and yield stimulation methods for optimizing latex production in each clone based on metabolic profiling.     

Isolation of cDNA clones differentially accumulated in the placenta of pungent pepper by suppression subtractive hybridization

Capsaicinoids responsible for pungency of chili pepper are synthesized exclusively in the placenta tissue of the fruit. As an elementary step in the molecular genetics study of capsaicinoid biosynthesis, a cDNA library was constructed from the placenta of a highly pungent pepper, Capsicum chinense cv. Habanero using the suppression subtractive hybridization (SSH). Thirty-nine cDNA clones from about 400 subtracted clones were selected through dot blot analysis and according to their nucleotides sequence. Sequence information of the chosen clones was evaluated by comparing it with DNA and protein databases. Results showed that the cDNA clones could be divided into 4 groups; cDNAs with similarities in genes encoding metabolic enzymes including acyl transferase and fatty acid alcohol oxidase (Group I), putative cell wall proteins (Group II), biotic and abiotic stress-inducible proteins (Group III), and lastly, cDNAs with no similarity (Group IV). Northern blot analysis was performed to confirm that these clones are differentially expressed in pungent pepper. The results revealed that all cDNA clones were differentially expressed in pungent pepper. In addition, the cDNA clones of Groups I and IV were differentially or preferentially expressed in the placenta of pungent pepper.     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

Proteomic Analysis of High Temperature Stress-Responsive Proteins in Chrysanthemum Leaves

Chrysanthemum is one of the most important ornamental flowers in the world, and temperature has a significant influence on its field production. In the present study, differentially expressed proteins were investigated in the leaves of Dendranthema grandiflorum ‘Jinba’ under high temperature stress using label-free quantitative proteomics techniques. The expressed proteins were comparatively identified and analyzed. A total of 1,463 heat-related, differentially expressed proteins were successfully identified by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS), and 1,463 heat-related, differentially expressed proteins were successfully identified by mass spectrometry after a high temperature treatment. Among these, 701 proteins were upregulated and 762 proteins were downregulated. The in-depth bioinformatics analysis of these differentially expressed proteins revealed that these were involved in energy metabolism pathways, protein metabolism, and heat shock. In the present study, the investigators determined the changes in the levels of some proteins, and their expression at the protein and molecular levels in chrysanthemum to help reveal the mechanism of heat resistance in chrysanthemum. Furthermore, the present study elucidated some of the proteins correlated to heat resistance in chrysanthemum, and their expression changes at the protein and molecular levels to help reveal the mechanism of heat resistance in this flower species. These results provide a theoretical basis for the selection of new heat resistant varieties of chrysanthemum in the field.     

Quantitative Global Proteome and Lysine Succinylome Analyses Reveal the Effects of Energy Metabolism in Renal Cell Carcinoma

In light of the increasing incidence of renal cell carcinoma (RCC), its molecular mechanisms have been comprehensively explored in numerous recent studies. However, few studies focus on the influence of multi‐factor interactions during the occurrence and development of RCC. This study aims to investigate the quantitative global proteome and the changes in lysine succinylation in related proteins, seeking to facilitate a better understanding of the molecular mechanisms underlying RCC. LC‐MS/MS combined with bioinformatics analysis are used to quantitatively detect the perspectives at the global protein level. IP and WB analysis were conducted to further verify the alternations of related proteins and lysine succinylation. A total of 3,217 proteins and 1,238 lysine succinylation sites are quantified in RCC tissues, and 668 differentially expressed proteins and 161 differentially expressed lysine succinylation sites are identified. Besides, expressions of PGK1 and PKM2 at protein and lysine, succinylation levels are significantly altered in RCC tissues. Bioinformatics analysis indicates that the glycolysis pathway is a potential mechanism of RCC progression and lysine succinylation may plays a potential role in energy metabolism. These results can provide a new direction for exploring the molecular mechanism of RCC tumorigenesis.     

A high‐confidence reference dataset of differentially expressed proteins in elongating cotton fiber cells

In our previous study, we used a comparative proteomic approach based on 2DE to profile dynamic proteomes of cotton fibers and found 235 protein spots differentially expressed during the elongation process ranging from 5 to 25 days post‐anthesis. Of them, only 106 differentially expressed proteins (DEPs) were identified by MS due to database limitations at the time. In the present work, we successfully identified the remaining 129 DEPs from the same experimental system using high‐resolution MS with an updated database. Bioinformatic analysis revealed that proteins involved in carbohydrate and protein metabolism, transport, and redox homeostasis are the most abundant, and glycolysis was found to be the most significantly regulated process during fiber elongation. Our high‐confidence reference dataset, composed of 235 DEPs, provides a valuable resource for future studies on the molecular mechanism of cotton fiber elongation.     

Candidate genes involved in tanshinone biosynthesis in hairy roots of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> revealed by cDNA microarray

Salvia miltiorrhiza is a valuable Chinese herb (Danshen) that is widely used in traditional Chinese medicine. Diterpene quinones, known as tanshinones, are the main bioactive components of S. miltiorrhiza; however, there is only limited information regarding the molecular mechanisms underlying secondary metabolism in this plant. We used cDNA microarray analysis to identify changes in the gene expression profile at different stages of hairy root development in S. miltiorrhiza. A total of 203 genes were singled out from 4,354 cDNA clones on the microarray, and 114 unique differentially expressed cDNA clones were identified: six genes differentially expressed in 45-day hairy root compared with 30-day hairy root; 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root; and 12 genes unstably expressed at different stages. Among the 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root, a total of 57 genes were up-regulated, and 26 genes represent 29 metabolism-related enzymes. Copalyl diphosphate synthase, which catalyzes the conversion of the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate to copalyl diphosphate, was up-regulated 6.63 fold, and another six genes involved in tanshinone biosynthesis and eight candidate P450 genes were also differentially expressed. These data provide new insights for further identification of the enzymes involved in tanshinone biosynthesis.     

Quantitative proteomics of pomegranate varieties with contrasting seed hardness during seed development stages

In pomegranate (Punica granatum), seed hardness is an important trait directly affecting fruit marketability. However, seed formation in pomegranate has not been well studied. We investigated the genetic mechanism underlying pomegranate seed hardness by comparing protein expression profiles between soft- and hard-seeded varieties 60 and 120 days after flowering. We identified 1940 proteins, of which 399 were differentially expressed. Most of the differentially expressed proteins were involved in posttranslational modification and carbohydrate metabolism. Cell wall biosynthesis, which showed positive correlations with seed hardness, was selected as the candidate pathway. The mRNA levels of 14 proteins involved in cell wall biosynthesis were further analyzed by qPCR. Lignin biosynthesis-related differentially expressed proteins showed lower expression at protein and gene levels in a soft-seeded variety at the early stages. Moreover, cellulose biosynthesis-related differentially expressed proteins showed higher expression levels in the soft-seeded variety at 60 days after flowering. Thus, the soft-seeded variety showed lower lignin but higher cellulose biosynthesis at the early fruit developmental stage, suggesting that lignin and cellulose play opposing roles in cell wall formation in pomegranate seeds. Moreover, differentially expressed proteins involved in cell wall degradation showed higher expression levels in the soft-seeded variety at both developmental stages. These results suggested that differences in seed hardness between soft- and hard-seeded pomegranates might result from cell wall biosynthesis and also be affected by cell wall degradation. The present proteome-wide profiling of pomegranate genotypes with contrasting seed hardness adds to the current knowledge base of the molecular basis of seed hardness development.     

Identification and characterization of latex-specific proteins in opium poppy

Latex from the opium poppy, Papaver somniferum L., was analyzed by polyacrylamide gel electrophoresis (PAGE). Two latex-specific bands were identified in protein samples of poppy latex using one-dimensional native PAGE. Second dimension analysis with SDS-PAGE indicates that these proteins have a relative molecular weight of approximately 20 kilodaltons. We have termed these polypeptides the major latex proteins (MLPs). Polyclonal antibodies prepared against the MLPs were used to probe protein gel blots of latex and poppy tissues known to lack laticifers. Laticifer-free tissues showed no reaction with anti-MLP immunoglobulin G indicating that MLPs are found only in poppy latex. MLP distribution was also examined in mature opium poppy tissues by immunocytochemistry. Laticifers were differentially labeled by fluorescein isothiocyanate secondary labeling of anti-MLP immunoglobulin G and could easily be identified in both transverse and longitudinal section. Fractionation studies of isolated latex showed that MLPs are concentrated in the latex cytosol and not in alkaloidal vesicles. Analysis of latex proteins by conventional two-dimensional electrophoresis indicates that the two MLP bands are composed of several distinct polypeptides with similar relative molecular weights. The pIs of these molecules range from 6.0 to 3.5. The role(s) of MLPs in laticifer metabolism has not been determined.     

Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions

Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.     

Comparison of the longissimus muscle proteome between obese and lean pigs at 180 days

Production of high-quality meat is important to satisfy the consumer and make the pig industry competitive. Obese and lean breeds of pig show clear differences in adipogenic capacity and meat quality, but the underlying molecular mechanism remains unclear. We have compared protein expression of the longissimus muscle between Lantang (LT, obese) and Landrace (LR, lean) pigs at the age of 180 days using two-dimensional fluorescence difference gel electrophoresis. Of the 1,400 protein spots detected per gel, 18 were differentially expressed between the two breeds. Using peptide mass fingerprint and tandem mass spectrometry, 17 protein spots were identified, corresponding to ten different proteins that could be divided into four groups: metabolism-related, structure-related, stress-related, and other (unclassified). Among the metabolism-related proteins, COX5A and ATP5B, which participate in oxidative phosphorylation, were highly expressed in LT, whereas ENO3, which is involved in glycolysis, was highly expressed in LR. These results may contribute valuable information to our understanding of the molecular mechanism responsible for differences between obese and lean pigs, such as growth rate and meat quality.     

Characterization,using comparative proteomics,of differentially expressed proteins in the hippocampus of the mesial temporal lobe of epileptic rats following treatment with valproate

The objective of the study was to explore the pathogenesis of mesial temporal lobe epilepsy (MTLE) and the mechanism of valproate administration in the early stage of MTLE development. We performed a global comparative analysis and function classification of differentially expressed proteins using proteomics. MTLE models of developmental rats were induced by lithium-pilocarpine. Proteins in the hippocampus were separated by 2-DE technology. PDQuest software was used to analyze 2-DE images, and MALDI-TOF-MS was used to identify the differentially expressed proteins. Western blot was used to determine the differential expression levels of synapse-related proteins synapsin-1, dynamin-1 and neurogranin in both MTLE rat and human hippocampus. A total of 48 differentially expressed proteins were identified between spontaneous and non-spontaneous MTLE rats, while 41 proteins between MTLE rats and post valproate-treatment rats were identified. All of the proteins can be categorized into several groups by biological functions: synaptic and neurotransmitter release, cytoskeletal structure and dynamics, cell junctions, energy metabolism and mitochondrial function, molecular chaperones, signal regulation and others. Western blot results were similar to the changes noted in 2-DE. The differentially expressed proteins, especially the proteins related to synaptic and neurotransmitter release function, such as synapsin-1, dynamin-1 and neurogranin, are probably involved in the mechanism of MTLE and the pharmacological effect of valproate. These findings may provide important clues to elucidate the mechanism of chronic MTLE and to identify an optimum medication intervention time and new biomarkers for the development of pharmacological therapies targeted at epilepsy.     

日本血吸虫期别差异表达基因文库的构建及分析

为从期别差异表达基因分析入手研究血吸虫的生长发育机制,应用抑制性消减杂交 (suppressed subtractive hybridization , SSH) 技术首次构建了日本血吸虫尾蚴、虫卵和成虫的期别差异表达基因文库 . 经消减效率分析和三种文库克隆的 EST 的期别差异性鉴定,表明所建文库质量较高,为在整个基因组水平分离血吸虫的差异表达基因提供了重要材料 . 由三个文库选择 257 个插入片段大于 500 bp 的克隆测定了 EST 序列 . 同源性分析结果表明 257 个 EST 代表 182 种血吸虫基因,其中有 22 种为血吸虫已知基因,有 128 种为血吸虫已知 EST ,有 32 种为新发现的血吸虫基因 . 对 EST 编码蛋白的功能预测结果显示：尾蚴消减文库的基因多与运动、能量代谢、转录调节及致病性相关;虫卵消减文库的基因可能参与信号转导、细胞粘附、蛋白质和碳水化合物的代谢以及抗氧化反应;成虫消减文库的基因多参与蛋白质的合成、转运及分解代谢,参与虫体的运动等 . 大规模分离、分析血吸虫期别差异表达基因将对从分子水平去解读血吸虫的生长发育机制,筛选高效疫苗候选抗原、药物靶标及诊断制剂有重要意义 .     

Proteomic analysis of stargazer mutant mouse neuronal proteins involved in absence seizure

The stargazer ( stg ) mutant mouse, having mutation in stargazin, the calcium channel γ2 subunit, exhibited several neurological disorders including spontaneous absence seizure, cerebellar ataxia, and head tossing. To understand the molecular pathogenic mechanism of the absence seizure resulted from the loss of stargazin function, the thalamic proteomes between control mouse and stg mouse were compared. We identified 12 proteins expressed differentially (> 1.6-fold) by fluorescence two-dimensional difference gel electrophoresis and tandem mass spectrometry. Six of them are involved in basic metabolism including energy metabolism, three in stress response, two in axonal growth regulation, and one in the endoplasmic reticulum processing. All except mortalin showed decreased level of expression in stg mouse. Two stress-related proteins, mouse stress induced phosphoprotein 1 and peroxiredoxin 6 exhibited reduced levels of expression in stg mouse, while the level of another stress protein, mortalin was increased. Analysis of oxidative protein carbonylation in thalamic proteome of stg mouse showed higher level of carbonylated proteins in stg mouse than in control mouse. Interestingly, down-regulation of stress protein mouse stress induced phosphoprotein 1, metabolic enzyme isovaleryl-CoA dehydrogenase, and the two in neuronal axon growth, collapsin response mediator protein 2 and fascin homolog 1 coincides with the results of our previous study on γ-butyrolactone-induced transient absence seizure. Our results suggest that the pathogenesis mechanism underlying absence seizure may involve the molecular events contributed by these proteins.