Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

Ribosomal DNA variation and its phylogenetic implication in the genusBrachypodium (Poaceae)

The structure of ribosomal DNA ofBrachypodium and several other grass species was investigated using a heterologous rDNA probe from wheat. Several different rDNA families were present among perennial and annual species within the genus. In contrast to the annual species the perennial species exhibited a very low degree of repeat length variation. An extra Eco RI site and a Hin dIII site were observed in the IGS, which distinguishedBrachypodium from other grass genera. The restriction fragment length polymorphism and length variation of the repeat units have taxonomic value withinBrachypodium and are correlated with the classification ofBrachypodium derived from other data.     

Towards complete analysis of the platelet proteome

Platelets exert a crucial function in haemostasis, wound repair, and the formation of vascular plugs, underlying thrombotic diseases such as stroke and myocardial infarction. Analysis of platelet biochemistry is largely dependent on protein analysis as platelets are anucleated cells providing little analytical target for DNA or RNA based strategies. Here we present data from our analysis of the human platelet proteome, the entire set of proteins building a platelet at a given point in time. Proteins were separated by two-dimensional electrophoresis (2-DE) using broad and narrow range pH gradients in the isoelectric focusing step. Consequently, a high-resolution 2-DE proteome map has been generated that comprises approximately 2300 different protein features. From the 536 protein features detected in the 4-5 pI range 284 features were identified by electrospray ionisation time of flight tandem mass spectrometry. These 284 proteins originate from 123 different open reading frames. This includes the five human proteins KIAA0193, KIAA0573, KIAA0830, WUGSC:H_DJ0777O23 protein, and cytokine receptor related protein 4, all isolated for the first time. The data are discussed with regard to proteome characteristics, protein function, and the high prevalence of signalling molecules. This study contributes to a more thorough and holistic understanding of platelet biology, helping to build the basis for future identification of new drug targets and therapeutic strategies.     

Lipidomics of the sea sponge Amphimedon queenslandica and implication for biomarker geochemistry

Demosponges are a rich natural source of unusual lipids, some of which are of interest as geochemical biomarkers. Although demosponges are animals, they often host dense communities of microbial symbionts, and it is therefore unclear which lipids can be synthesized by the animal de novo, and which require input from the microbial community. To address this uncertainty, we analyzed the lipids of Amphimdeon queenslandica, the only demosponge with a published genome. We correlated the genetic and lipid repertoires of A. queenslandica to identify which biomarkers could potentially be synthesized and/or modified by the sponge. The fatty acid profile of A. queenslandica is dominated by an unusual Δ^5,9 fatty acid (cis‐5,9‐hexacosadienoic acid)—similar to what has been found in other members of the Amphimdeon genus—while the sterol profile is dominated by C₂₇‐C₂₉ derivatives of cholesterol. Based on our analysis of the A. queenslandica genome, we predict that this sponge can synthesize sterols de novo, but it lacks critical genes necessary to synthesize basic saturated and unsaturated fatty acids. However, it does appear to have the genes necessary to modify simpler products into a more complex “algal‐like” assemblage of unsaturated fatty acids. Ultimately, our results provide additional support for the poriferan affinity of 24‐isopropylcholestanes in Neoproterozoic‐age rocks (the “sponge biomarker” hypothesis) and suggest that some algal proxies in the geochemical record could also have animal contributions.     

Biological effectiveness of very high gamma dose rate and its implication for radiological protection

Radiation and Environmental Biophysics - Many experimental studies are carried out to compare biological effectiveness of high dose rate (HDR) with that of low dose rate (LDR). The rational for...     

A tool for biomarker discovery in the urinary proteome: a manually curated human and animal urine protein biomarker database

Urine is an important source of biomarkers. A single proteomics assay can identify hundreds of differentially expressed proteins between disease and control samples; however, the ability to select biomarker candidates with the most promise for further validation study remains difficult. A bioinformatics tool that allows accurate and convenient comparison of all of the existing related studies can markedly aid the development of this area. In this study, we constructed the Urinary Protein Biomarker (UPB) database to collect existing studies of urinary protein biomarkers from published literature. To ensure the quality of data collection, all literature was manually curated. The website (http://122.70.220.102/biomarker) allows users to browse the database by disease categories and search by protein IDs in bulk. Researchers can easily determine whether a biomarker candidate has already been identified by another group for the same disease or for other diseases, which allows for the confidence and disease specificity of their biomarker candidate to be evaluated. Additionally, the pathophysiological processes of the diseases can be studied using our database with the hypothesis that diseases that share biomarkers may have the same pathophysiological processes. Because of the natural relationship between urinary proteins and the urinary system, this database may be especially suitable for studying the pathogenesis of urological diseases. Currently, the database contains 553 and 275 records compiled from 174 and 31 publications of human and animal studies, respectively. We found that biomarkers identified by different proteomic methods had a poor overlap with each other. The differences between sample preparation and separation methods, mass spectrometers, and data analysis algorithms may be influencing factors. Biomarkers identified from animal models also overlapped poorly with those from human samples, but the overlap rate was not lower than that of human proteomics studies. Therefore, it is not clear how well the animal models mimic human diseases.     

Restriction-site variation of PCR-amplified chloroplast DNA regions and its implication for the evolution and taxonomy of Actinidia

 Twenty six restriction sites from five PCR-amplified chloroplast DNA sequences (rbcL, psbA, rpoB, and two spacers flanking the trnL gene) were mapped and analysed in 20 Actinidia taxa, encompassing all four sections into which the genus is divided. At least three species out of the 20 examined have been found to have originated through natural interspecific hybridisation on the basis of the discrepancy between morphological and biochemical traits and the cpDNA profiles of pairs of species. A widely reticulate evolution has therefore been postulated in Actinidia. Wagner and weighted parsimony analysis produced consensus trees that did not match the traditional taxonomy based on morphological characters. The molecular data clearly showed that some taxa, such as A. rufa and A. kolomikta, occupy a wrong position and most, if not all, of the traditional groups represented by sections and series are weakly supported, since they appear as polyphyletic. A. chinensis and A. deliciosa were confirmed to be very closely related. Since chloroplast DNA is paternally inherited in Actinidia, A. chinensis is a paternal progenitor, if not the only one, of A. deliciosa, the domesticated kiwifruit. Received: 18 August 1997 / Accepted: 6 October 1997     

Analysis of serum proteome profiles of non-Hodgkin lymphoma for biomarker identification

Serum samples from non-Hodgkin lymphoma (NHL) patients who had not undergone chemotherapy, lymphnoditis patients, and healthy adults were analyzed using surface-enhanced laser desorption–ionization time-of-flight mass spectrometry (SELDI-TOF MS) to detect the differentially expressed serum proteins. Models were developed to distinguish between the healthy adult group and the NHL group, with a sensitivity of 69% and specificity of 90%, and between the lymphnoditis group and the NHL group with a sensitivity of 74% and specificity of 84%. A protein with the m/z of M10 197.91 u was expressed at a significantly higher level in the NHL group, compared to the other groups. Furthermore, differences were also significant among different stages of NHL and among samples with different International Prognosis Index (IPI) scores or lactase dehydrogenase (LDH) levels. The three identified proteins may offer a new serological approach for early diagnosis, differential diagnosis, and pathogenic investigation of NHL. And the protein with the m/z of M10 197.91 u may be a new serological biomarker for monitoring treatment response and evaluating the prognosis of patients with NHL.     

硒的生物学作用及其研究进展

硒在机体的生命活动过程中具有重要的生物学作用,不断有研究发现硒缺乏与许多疾病的发生密切相关。本文主要对硒在生物体内的存在形式,硒与机体抗氧化能力、免疫功能、心血管功能、遗传损伤保护以及抗衰老之间的关系方面的研究进展作了综述,同时也对生物源有机硒的转化与富硒产品的研发现状与前景进行了概要介绍。     

Serial changes in urinary proteome profile of membranous nephropathy: implications for pathophysiology and biomarker discovery

Membranous nephropathy is one of the most common causes of primary glomerular diseases worldwide. The present study adopted a gel-based proteomics approach to better understand the pathophysiology and define biomarker candidates of human membranous nephropathy using an animal model of passive Heymann nephritis (PHN). Clinical characteristics of Sprague-Dawley rats injected with rabbit anti-Fx1A antiserum mimicked those of human membranous nephropathy. Serial urine samples were collected at Days 0, 10, 20, 30, 40, and 50 after the injection with anti-Fx1A (number of rats = 6; total number of gels = 36). Urinary proteome profiles were examined using 2D-PAGE and SYPRO Ruby staining. Quantitative intensity analysis and ANOVA with Tukey post-hoc multiple comparisons revealed 37 differentially expressed proteins among 6 different time-points. These altered proteins were successfully identified by MALDI-TOF MS and classified into 6 categories: (i) proteins with decreased urinary excretion during PHN; (ii) proteins with increased urinary excretion during PHN; (iii) proteins with increased urinary excretion during PHN, but which finally returned to basal levels; (iv) proteins with increased urinary excretion during PHN, but which finally declined below basal levels; (v) proteins with undetectable levels in the urine during PHN; and (vi) proteins that were detectable in the urine only during PHN. Most of these altered proteins have functional significance in signaling pathways, glomerular trafficking, and controlling the glomerular permeability. The ones in categories (v) and (vi) may serve as biomarkers for detecting or monitoring membranous nephropathy. After normalization of the data with 24-h urine creatinine excretion, changes in 34 of initially 37 differentially expressed proteins remained statistically significant. These data underscore the significant impact of urinary proteomics in unraveling disease pathophysiology and biomarker discovery.     

Biological species and variation in arthroderma

Summary The distinctive peridial appendages ofArthroderma were of little value for the delimitation of species. The peridial appendages as well as the conidia of certain species were so similar as to be value-less for the delimitation of species. Further, morphological and physiological variation was found to be as great within, as between, species. The usual morphological methods for classifying species were, therefore, inadequate and the concept of biological species was considered.The following points were found to favor the applicability of biological species concepts. Nine of ten species were heterothallic which assured crossing. A gene pool, the result of sexual reproduction and an essential requirement of a biological species, was maintained. This was evidenced by complete fertility between isolates of the same species from diverse geographic areas. Finally, no hybridization between species was evident in over one hundred attempted crosses.Strains which were readily compatible and produced viable ascospores were considered to be members of the same species. Conversely, incompatible strains were differentiated into separate species. In this way over fifty strains from more than one hundred isolations were found to be members of ten species. Six of the species correlated with species previously described on morphological bases, and confirmed their validity. Four species were considered to be new. All four were previously confused withA. quadrifidum orA. cuniculi because they possessed similar types of conidia. Once separated on an incompatibility basis, they were found to possess characteristics of distinct species. The only reliable method for their differentiation, however, was found to be interspecific incompatibility.Several strains, some pathogens, were crossed with soil borne species ofArthroderma. Some mated but others did not. Several incompatible strains possessedArthroderma-like appendages and partial compatibility was indicated. Partial compatibility also occurred in certain interspecific crosses.     

细胞核重编程对克隆的影响

细胞核重编程是哺乳动物正常受精胚胎和克隆胚胎发育过程中的一个重要特性,主要是对表观遗传学特征进行重新编写,包括染色质重塑、组蛋白修饰、DNA甲基化、印记基因表达、X染色体失活等表观遗传修饰的改变。通过细胞核重编程,首先,受精卵和克隆胚胎的供体核停止其特有的基因表达程序,恢复为全能状态的基因表达程序;然后,受精胚胎和克隆胚胎的细胞再从全能状态重新进入分化状态,最终形成各种组织和器官。近年来,不少研究表明,克隆胚胎的细胞核重编程存在不同程度的表观遗传修饰异常,可能对克隆及其农业和医学应用有着重要影响。本文就正常和克隆胚胎细胞核重编程的研究进展以及克隆胚胎的细胞核重编程异常对克隆的影响作一综述,并对目前有关治疗性克隆前景的不同看法进行了讨论。     

蛋白质组研究技术及其进展

蛋白质组学是在后基因时代出现的一个新的研究领域．它是对机体或组织或细胞的全部蛋白质的表达和功能模式进行研究。介绍并总结了蛋白质组研究的主要技术,包括双向凝胶电泳、质谱技术、蛋白质芯片和生物信息学等。     

乳腺癌蛋白质组研究进展

乳腺癌是发生在乳房腺上皮组织的恶性肿瘤,其发病率占女性恶性肿瘤首位。近年来,蛋白质组研究广泛深入乳腺癌发病机理、临床早期诊断和治疗等各个方面。本文比较系统地综述了乳腺蛋白质组研究的样品处理、研究技术和研究成果。     

Geographical variation in heading characters among wheat landraces,Triticum aestivum L., and its implication for their adaptability

Summary Heading time and its constituent traits, photoperiodic response, narrow-sense earliness and vernalization requirement, were surveyed for 158 wheat landraces. Wide varietal variation was observed in each character. Nearly half of the variation for each character was explained by a geographical difference in origin. Based on these data and the growing environments in each locality, we analyzed adaptation strategy, seen as the adjustment of heading time in terms of differences in the constituent traits, both individually and combined. The difference among localities indicated that wheat landraces had been selected for early heading as an adaptation strategy to water stress and/or high temperature in early summer. This change was caused by a reduction in photoperiodic response and narrow-sense earliness. The vernalization requirement was also reduced for adaptation to relatively mild winters. Adaptation strategy deduced from the variation within each locality was also different amongst localities. In the central region of wheat evolution, where wide variations existed in both photoperiodic response and narrow-sense earliness, the late-heading trait was achieved by either one of these traits individually or both of them combined. On the contrary, in the eastern and the western regions, wide variation in heading time was achieved by the unique combination of photoperiodic response and narrowsense earliness. A sampling strategy for wheat germ plasm is also discussed.     

Comparative proteome analysis of serum from acute pulmonary embolism rat model for biomarker discovery

Pulmonary embolism (PE) is a common, potentially fatal disease and its diagnosis is challenging because clinical signs and symptoms are nonspecific. In this study, to investigate protein alterations of a rat PE model, total serum proteins collected at different time points were separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bioinformatics analysis of 24 differentially expressed proteins showed that 20 had corresponding protein candidates in the database. According to their properties and obvious alterations after PE, changes of serum concentrations of Hp, Fn, DBP, RBP, and TTR were selected to be reidentified by western blot analysis. Semiquantitative RT-PCR showed DBP, RBP, and TTR to be down-regulated at mRNA levels in livers but not in lung tissues. The low serum concentrations of DBP, RBP, and TTR resulted in the up-regulation of 25(OH)D3, vitamin A, and FT4 (ligands of DBP, RBP, and TTR) after acute PE in rat models. The serum levels of Hp and Fn were detected in patients with DVT/PE and controls to explore their diagnostic prospects in acute PE because the mRNA levels of Hp and Fn were found to be up-regulated both in lung tissues and in livers after acute PE. Our data suggested that the concentration of serum Fn in controls was 79.42 +/- 31.57 microg/L, whereas that of PE/DVT patients was 554.43 +/- 136.18 microg/L (P < 0.001), and that the concentration of serum Hp in controls was 824.37 +/- 235.24 mg/L, whereas that of PE/DVT patients was 2063.48 +/- 425.38 mg/L (P < 0.001). The experimental PE rat model selected in this study was more similar to the clinical process than the other existing PE animal models, and the findings indicated instant changes of serum proteins within 48 h after acute PE. The exploration of these differentially expressed proteins or their combination with existent markers such as D-dimer may greatly improve the accuracy of the diagnosis of acute PE, but diagnostic tests are still needed to evaluate the sensitivity and specificity of these markers and also the number of false positives and false negatives.     

Advances in plant biotechnology and their implication for forestry research

Summary Over the past few years, techniques of cell biology, genetic screening, and gene manipulation have been developed to the extent that their impact on commercial development of improved plant varieties is predicted to have a measurable impact on agriculture by the year 2000 and beyond. A review will be given of progress that has been made in each of these areas toward the manipulation of crop plants for improved field performance and product quality. There are now several opportunities in which these techniques can be employed for the improvement of forestry species. In the light of the long-time scales involved in the generation of forestry products, it is important to focus on targets that are worthwhile pursuing commercially using appropriate technical routes. Selected examples will be given of the application of plant biotechnology techniques that promise potentially significant improvement for forestry species. Presented in the Keynote address Toward the Forest of Tomorrow at the 5th Meeting of the Conifer Biotechnology Working Group, Siltingbourne, England, July 8–13, 1990.     

Intragenomic variation in nuclear ribosomal markers and its implication in species delimitation,identification and barcoding in fungi

Intragenomic variation is the molecular variation within the genome among repetitive DNA. As a multigene family, nuclear ribosomal DNA (rDNA) has been widely used in fungal taxonomy for their ease in amplification and suitable variability to attain various levels of taxonomic resolution. At the intraspecific level, rDNA is believed to be under concerted evolution and the internal transcribed spacers (ITS) region is actually accepted as a universal barcoding marker for fungi. However, documentation of intragenomic variation of rDNA indicated that it can be problematic in species delimitation and identification. Fungal taxonomic studies have not generally taken into account the intragenomic variation of rDNA in a systematic manner. In this review, our objective is to address the definition, the origin and the mechanisms for maintenance of intragenomic variation, as well as its implication in the domain of fungal molecular taxonomy, particularly for species delimitation, identification and DNA barcoding. With advanced sequencing technologies (second and third generations), we also addressed how these technologies can be used to study the intragenomic variation of rDNA and also how the intragenomic variation will impact on DNA barcoding via high-throughput sequencing.