Proteomic analysis of rat pheochromocytoma PC12 cells

PC12 cell line is well documented and widely applied as many kinds of models in neurobiological and neurochemical studies. Yet a thorough proteomic analysis has not been performed so far. Here we report the construction of a large-scale 2-D protein database for PC12 cells. The proteins extracted from PC12 cells were separated by 2-DE and identified by MALDI-TOF/TOF MS. A total of 1080 protein spots, excised from three different 2-D gels, were identified with high confidence. These proteins represent 474 different gene products, mainly binding proteins and enzymes. Three hundred and seven identified protein spots were located in the low-molecular weight region below 20 kDa. This database today represents one of the largest 2-D databases for higher eukaryotic cell proteomes and for low-molecular weight proteins. In addition, fragment ion spectra obtained by TOF/TOF confirmed that calcylin in PC12 cells was N-acetylated. The database of PC12 proteome is expected to be a powerful tool for neuroscientists.     

东北红豆杉开发研究进展

系统论述了东北红豆杉的生物学和生态学特性,野生资源的分布现状,蕴藏量及开发前景,人工繁殖现状和技术,资源开发存在的问题及发展对策.     

Proteomic signature of human embryonic stem cells

Human embryonic stem cells (hESC) represent a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics. Proteomics provides a powerful approach for studying the characteristics of hESC and discovering molecular markers. We have analyzed proteome profiles of three hESC lines using 2-DE and MALDI TOF-TOF. Out of 844 spots analyzed with MALDI TOF-TOF, 685 proteins were identified of which 60 proteins were classified as the most abundant proteins on 2-D gels. A large number of proteins particularly high abundant ones were identified as chaperones, heat shock proteins, ubiquitin/proteasome, and oxidative stress responsive proteins underscoring the ability of these cells to resist oxidative stress and increase the life span. Several proteins involved in cell proliferation and differentiation were also among the highly expressed proteins. Although overall expression pattern of three hESC were similar, 54 spots changed quantitatively and 14 spots changed qualitatively among the hESC cell lines. Most of these proteins were identified as proteins involved in cell growth, metabolism and signal transduction, which may affect the self-renewal and pluripotency. To our knowledge, this study represents the first proteomic dataset for hESC and provides a better insight into the biology of hESC. Proteome maps of hESC are accessible at http://www.RoyanProteomics.ir.     

Proteomic analysis of bovine skeletal muscle hypertrophy

Myostatin plays a major role in muscle growth and development and animals with disruption of this gene display marked increases in muscle mass. Little is known about muscle physiological adaptations in relation to this muscle hypertrophy. To provide a more comprehensive view, we analyzed bovine muscles from control, heterozygote and homozygote young Belgian blue bulls for myostatin deletion, which results in a normal level of inactive myostatin. Heterozygote and homozygote animals were characterized by a higher proportion of fast-twitch glycolytic fibers in Semitendinosus muscle. Differential proteomic analysis of this muscle was performed using two-dimensional gel electrophoresis followed by mass spectrometry. Thirteen proteins, corresponding to 28 protein spots, were significantly altered in response to the myostatin deletion. The observed changes in protein expression are consistent with an increased fast muscle phenotype, suggesting that myostatin negatively controls mainly fast-twitch glycolytic fiber number. Finally, we demonstrated that differential mRNA splicing of fast troponin T is altered by the loss of myostatin function. The structure of mutually exclusive exon 16 appears predominantly expressed in muscles from heterozygote and homozygote animals. This suggests a role for exon 16 of fast troponin T in the physiological adaptation of the fast muscle phenotype.     

Two new taxanes from the needles and branches bark of Taxus cuspidata

Two new taxanes were isolated from the MeOH extract of the needles and branches bark of the Japanese yew, Taxus cuspidata. The structures were established as (2alpha,5alpha,7beta,9alpha,10beta,13alpha)-5,10,13,20-tetraacetoxytax-11-ene-2,7,9-triol (1) and (2alpha,5alpha,9alpha,10beta)-2,9,10-triacetoxy-5-[(beta-D-glucopyranosyl)oxy]-3,11-cyclotax-11-en-13-one (2) on the basis of in-depth 1D- and 2D-NMR analyses. Compound 2 is the first example of a transannular taxane glycoside isolated from a natural source.     

东北红豆杉细胞两液相培养中紫杉醇释放行为研究

在东北红豆杉细胞悬浮培养中,分别研究了稀土化合物(硫酸铈铵)、有机溶剂(油酸和邻苯二甲酸二丁脂)和稀土化合物与有机溶剂的协同作用对紫杉醇释放的影响。在此基础上深入研究了在东北红豆杉细胞两液相培养中,紫杉醇释放率随不同的有机溶剂(烷烃、有机酸、醇和脂)、有机溶剂的体积分数、有机溶剂的加入时间和有机溶剂相毒性的变化规律。结果表明分别加入稀土化合物和有机溶剂都明显促进紫杉醇的释放,特别是有机溶剂更显著促进紫杉醇的释放。但在东北红豆杉细胞两液相培养中,稀土化合物加入不能进一步促进紫杉醇的释放。因此两液相培养中有机溶剂本身就是很好的产物释放剂。紫杉醇的释放率由对照组的40%提高到75%以上。     

水杨酸作用下东北红豆杉细胞的二维凝胶电泳分析

东北红豆杉悬浮培养体系中加入适量浓度的水杨酸,染色结果表明,水杨酸可以增加细胞膜通透性,并可诱导部分细胞发生核凝集或核碎裂。提取细胞染色体DNA进行琼脂糖凝胶电泳,发现染色体DNA发生了部分降解。应用蛋白质双向凝胶电泳技术,研究了水杨酸处理后东北红豆杉细胞发生应激反应过程中蛋白质的表达情况,分析了处理细胞与正常细胞的蛋白质组差异,发现在水杨酸处理48h后的样品中有7个蛋白质差异点,而且有6个蛋白点仅在对照中检测到。结果表明,外加水杨酸改变了东北红豆杉细胞的基因表达,抑制部分蛋白合成的同时也合成了部分新蛋白质,这些蛋白可能与水杨酸的作用有关。     

Proteomic analysis of salt stress-responsive proteins in rice root

Salt stress is one of the major abiotic stresses in agriculture worldwide. We report here a systematic proteomic approach to investigate the salt stress-responsive proteins in rice (Oryza sativa L. cv. Nipponbare). Three-week-old seedlings were treated with 150 mM NaCl for 24, 48 and 72 h. Total proteins of roots were extracted and separated by two-dimensional gel electrophoresis. More than 1100 protein spots were reproducibly detected, including 34 that were up-regulated and 20 down-regulated. Mass spectrometry analysis and database searching helped us to identify 12 spots representing 10 different proteins. Three spots were identified as the same protein, enolase. While four of them were previously confirmed as salt stress-responsive proteins, six are novel ones, i.e. UDP-glucose pyrophosphorylase, cytochrome c oxidase subunit 6b-1, glutamine synthetase root isozyme, putative nascent polypeptide associated complex alpha chain, putative splicing factor-like protein and putative actin-binding protein. These proteins are involved in regulation of carbohydrate, nitrogen and energy metabolism, reactive oxygen species scavenging, mRNA and protein processing, and cytoskeleton stability. This study gives new insights into salt stress response in rice roots and demonstrates the power of the proteomic approach in plant biology studies.     

Proteomic analysis of Burkholderia cepacia MBA4 in the degradation of monochloroacetate

Burkholderia cepacia MBA4 is a bacterium that degrades 2-haloacids by removing the halogen and subsequent metabolism of the product for energy. In this study, 2-DE, MS/MS, and N-terminal amino acid sequencing were used to investigate the protein expression profiles of MBA4 grown in a 2-haloacid (monochloroacetate, MCA) and in the corresponding metabolic product (glycolate). Glycolate was used as a control to eliminate the proteins induced by it. Five proteins were found to be up-regulated and five proteins were down-regulated in response to MCA. The differentially expressed proteins were examined, seven of them were identified by MS/MS and two of them were sequenced by Edman degradation. Our results definitely provide an insight for understanding the physiology of B. cepacia MBA4 in response to organohalide contaminated site.     

Proteomic analysis of anti-Francisella tularensis LVS antibody response in murine model of tularemia

Francisella tularensis live vaccine strain infection of mice has been established as an experimental model of tularemia that is suitable for studies of immune mechanisms against the intracellular pathogen. In this study, the model was used to explore immunogenic repertoire of F. tularensis with the aim of identifying new molecules able to activate the host immune system, potential bacterial markers with vaccine, and diagnostic applications. Immunoproteomic approach based on the combination of two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry was applied. Globally, 36 different proteins were identified, which strongly reacted with sera from experimentally infected mice, including several putative virulence markers of intracellular pathogens as nucleoside diphosphate kinase, isocitrate dehydrogenase, RNA-binding protein Hfq, and molecular chaperone ClpB. Of them, 27 proteins are described for the first time as immunorelevant Francisella proteins. When comparing murine immunoproteome of F. tularensis with our previous data from human patients, 25 of the total of 50 identified murine sera immunoreactive spots were recognized by human sera collected from patients suffering from tularemia, as well. Immune sera from two Lps gene congenic strains of mice, C3H/HeN (Lpsn) and C3H/HeJ (Lpsd), represented murine immunoproteome in this study. The spectrum of immunoreactive spots detected by two-dimensional immunoblotting varied throughout the course of infection depending on murine strain. Nevertheless, the antibody patterns of the two strains showed significant homogeneity in being directed against almost identical subset of antigens.     

东北红豆杉可再生部位紫杉醇含量时空动态变化规律

利用ODS-C18反相高效液相色谱体系研究了东北红豆杉(Taxus cuspidata)叶、茎、果肉及种子中紫杉醇含量的时空动态变化。结果表明,东北红豆杉的4个部位均有紫杉醇存在,但存在的时间和部位具有较大差异。紫杉醇的含量最大值为0.043mg.g-1(FW)出现在10月份种子中,最小值0.001mg.g-1(FW)出现在11月份果肉中。叶与茎中紫杉醇的含量变化较为一致,叶和茎中紫杉醇含量在7月份达到最高,分别为0.032mg.g-1(FW)和0.029mg.g-1(FW),在8月份降至最低,分别为0.011mg.g-1(FW)和0.010mg.g-1(FW)。在新叶萌发期,老茎叶比新茎叶中紫杉醇的含量高约2.5倍。     

A New Taxane with an Opened Oxetane Ring from the Needles of Taxus cuspidata

A new taxoid metabolite was isolated from the MeOH extract of Taxus cuspidata needles. The structure was established as (2α,5α,7β,9α,10β,13α)‐10,13,20‐tris(acetyloxy)‐1,4,5,7,9‐pentahydroxytax‐11‐en‐2‐yl benzoate ( 1 ) on the basis of spectral analyses including ¹H‐ and ¹³C‐NMR, HMQC, HMBC, and NOESY, and confirmed by HR‐FAB‐MS.     

Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non-growth related (higher in the stationary phase) and at relatively low levels (up to 4 mug/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4mug/mL) and another during the late (0.1-mug/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 mug/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. (c) 1994 John Wiley & Sons, Inc.     

聚乙二醇对东北红豆杉培养细胞的生长及紫杉醇生产的影响

在改良的B5培养基中加入不同浓度的聚乙二醇对东北红豆杉培养细胞进行摇瓶培养,通过不同时期取样并测定细胞鲜,干重及用HPLC测定紫杉醇的含量,发现聚乙二醇对东北红豆杉培养细胞的生长及紫杉醇生产均有明显的促进作用,聚乙二醇为10g/L时,对细胞生长最为有利,细胞培养16d可达到最大生物量,其平均鲜重为28．73g/瓶,增重3．8倍,平均干重为2．14g/瓶,增重3．1倍,聚乙二醇为20g/L,对紫杉醇的生产最有利;细胞培养25d时,培养基中紫杉醇的含量达到最高水平,其含量为2350ug/L,是不加聚乙二醇的11倍。     

Proteomic analysis of a filamentous fungal endophyte using EST datasets

Proteomic analysis of many species of fungi, particularly filamentous fungi, is difficult due to the lack of publicly available genome sequence data and the problems associated with cross‐species comparisons. Furthermore, the detection of fungal proteins in biological systems where there are a greater number of proteins present from other eukaryote species provides additional challenges. We present an EST‐based approach for identifying proteins from a fungal endophyte of temperate grasses and demonstrate that this method is well suited for fungi with minimal sequence data.     

紫外辐射对东北红豆杉鲜叶中紫杉醇及三尖杉宁碱含量的影响

分别采用254、365 nm两种波长的紫外光对东北红豆杉鲜叶进行辐射,研究了波长、辐射时间以及样品处理方式对东北红豆杉鲜叶中紫杉醇及三尖杉宁碱含量变化的影响。结果表明,东北红豆杉鲜叶经匀浆处理后接受紫外辐射,两种波长的紫外光都可以使紫杉醇及三尖杉宁碱的含量增加,但不同波长对紫杉醇及三尖杉宁碱含量提高的趋势却不相同。365 nm的紫外光辐射2 h时使紫杉醇和三尖杉宁碱含量均提高到了最大值,两种物质含量分别提高了44.6%和53.0%,而254 nm的紫外光在辐射8 h时才达到最大值,两种物质含量分别提高了39.2%和24.3%。可以选取365 nm的紫外光对东北红豆杉鲜叶水匀浆体系进行辐射,快速高效地提高鲜叶内紫杉醇和三尖杉宁碱的含量。     

Administering cultured Taxus cells with early precursors reveals bifurcations in the taxoid biosynthetic pathway

Administering Taxus suspension cells with labeled 5alpha-hydroxytaxadiene and 5alpha,10beta-dihydroxytaxadiene, and the corresponding 5alpha-acetate esters, demonstrated that acetylation at C5 of the monool precursor promotes the formation of 14beta-hydroxy taxoids, such as taxuyunnanine C, at the expense of 13alpha-hydroxy taxoids, including Taxol and its congeners, but that the major bifurcation in taxoid biosynthesis, toward 13alpha- or 14beta-hydroxy taxoids, occurs after 10beta-hydroxylation of the taxane core.