Tandem mass spectrometry identifies proteins phosphorylated by cyclic AMP-dependent protein kinase when sea urchin sperm undergo the acrosome reaction

The exocytotic acrosome reaction (AR), which is required for fertilization, occurs when sea urchin sperm contact the egg jelly (EJ) layer. Among other physiological changes, increases in adenylyl cyclase activity, cAMP and cAMP-dependent protein kinase (PKA) activity occur coincident with the AR. By using inhibitors of PKA, a permeable analog of cAMP and the phosphodiesterase inhibitor IBMX, we show that PKA activity is required for AR induction by EJ. A minimum of six sperm proteins are phosphorylated by PKA upon exposure to EJ, as detected by a PKA substrate-specific antibody. The phosphorylation of these proteins and the percentage of acrosome reacted sperm can be regulated by PKA modulators. The fucose sulfate polymer (FSP), a major component of EJ, is the molecule that triggers sperm PKA activation. Extracellular Ca(2+) is required for PKA activation. Six sperm proteins phosphorylated by PKA were identified by tandem mass spectrometry (MS/MS) utilizing the emerging sea urchin genome. Based on their identities and localizations in sperm head and flagellum, the putative functions of these proteins in sperm physiology and AR induction are discussed.     

High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry.

We describe an integrated workstation for the automated, high-throughput, and conclusive identification of proteins by reverse-phase chromatography electrospray ionization tandem mass spectrometry. The instrumentation consists of a refrigerated autosampler, a submicrobore reverse-phase liquid chromatograph, and an electrospray triple quadrupole mass spectrometer. For protein identification, enzymatic digests of either homogeneous polypeptides or simple protein mixtures were generated and loaded into the autosampler. Samples were sequentially injected every 32 min. Ions of eluting peptides were automatically selected by the mass spectrometer and subjected to collision-induced dissociation. Following each run, the resulting tandem mass spectra were automatically analyzed by SEQUEST, a program that correlates uninterpreted peptide fragmentation patterns with amino acid sequences contained in databases. Protein identification was established by SEQUEST_SUMMARY a program that combines the SEQUEST scores of peptides originating from the same protein and ranks the cumulative results in a short summary. The workstation's performance was demonstrated by the unattended identification of 90 proteins from the yeast Saccharomyces cerevisiae, which were separated by high-resolution two-dimensional PAGE. The system was found to be very robust and identification was reliably and conclusively established for proteins if quantities exceeding 1-5 pmol were applied to the gel. The level of automation, the throughput, and the reliability of the results suggest that this system will be useful for the many projects that require the characterization of large numbers of proteins.     

A global analysis of the determinants of alien geographical range size in birds

Aim Determining the causes of range size variation in the distributions of alien species is important for understanding the spread of invasive species. Factors influencing alien range size have been explored for some species at a regional level, but to date there has been no global analysis of an entire class. Here, we present such an analysis for birds, testing for the effects of introduction event, location and species‐level variables on alien range sizes. Location Global. Methods We used a novel dataset on the global distributions of alien bird species to test for relationships between alien range size and colonization pressure, residence time, extent of the global climatic niche, native range size, body mass and specialization, using a statistical approach based on phylogenetic generalized least squares models. We performed this analysis globally, and for separate biogeographical realms. Results Approximately half of the variation in alien bird range size is explained by colonization pressure in univariate analysis. We identified consistent effects of higher colonization pressure at global and realm levels, as well as support for effects of native range size and residence time. We found less support for effects of body mass, specialization or extent of the global climatic niche on alien range size. Main conclusions Alien bird range sizes are generally small relative to their native range sizes, and many are continuing to expand. Nevertheless, current variation is predictable, most strongly by the event‐level factor of colonization pressure. Whether a species is widespread is a better predictor of alien range size than whether a species could be widespread (estimated by global climatic niche extent), while we also find effects of residence time on alien range size. These relationships may help to identify those alien species that are more likely to spread and hence have greater environmental and economic impacts where they have been introduced.     

Is CD36 gene polymorphism in region encoding lipid-binding domain associated with early onset CAD?

CD36 is a fatty acid translocase in striated muscle cells and cardiomyocytes. Some study suggested that alterations in CD36 gene may be associated with coronary artery disease (CAD) risk. The aim of the current study was to compare the frequency of CD36 variants in region encoding lipid-binding domain in Caucasian patients with early-onset CAD, no-CAD adult controls and neonates. The study group comprised 100 patients with early onset CAD. The genetic control groups were 306 infants and 40 no-CAD adults aged over 70 years. Exons 4, 5 and 6 including fragments of flanking introns were studied using the denaturing high-performance liquid chromatography technique and direct sequencing. Changes detected in analyzed fragment of CD36: IVS3-6 T/C (rs3173798), IVS4-10 G/A (rs3211892), C311T (Thr104Ile, not described so far) in exon 5, G550A (Asp184Asn, rs138897347), C572T (Pro191Leu, rs143150225), G573A (Pro191Pro, rs5956) and A591T (Thr197Thr, rs141680676) in exon 6. No significant differences in the CD36 genotype, allele and haplotype frequencies were found between the three groups. Only borderline differences (p = 0.066) were found between early onset CAD patients and newborns in the frequencies of 591T allele (2.00% vs 0.50%) and CGCGCGT haplotype (2.00% vs 0.50%) with both IVS3-6C and 591T variant alleles. In conclusion, CD36 variants: rs3173798, rs3211892, rs138897347, rs5956, rs143150225 rs141680676 and C311T do not seem to be involved in the risk of early-onset CAD in Caucasian population.     

Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.     

Aerated subsurface irrigation water gives growth and yield benefits to zucchini, vegetable soybean and cotton in heavy clay soils

Inadequate oxygen concentration in the root zone is a constraint to plant performance particularly in heavy, compacted and/or saline soils. Sub-surface drip irrigation (SDI) offers a means of increasing oxygen to plant roots in such soils, provided irrigation water can be hyper-aerated or oxygenated. Hydrogen peroxide (HP) at the rate of 5 litre ha⁻¹ at the end of each irrigation cycle was injected through SDI tape to a field-grown zucchini (courgette) crop (Cucurbita pepo) on a saturated heavy clay soil in Queensland, Australia. Fruit yield, number and shoot weight increased by 25%, 29% and 24% respectively due to HP treatment compared to the control. Two pot experiments with vegetable soybean (Glycine max) and cotton (Gossypium hirsutum) compared the effectiveness of HP and air injection using a Mazzei air injector (a venturi), throughout the irrigation cycle in raising crop yield in a heavy clay soil kept at saturation or just under field capacity. Fresh pod yield of vegetable soybean increased by 82–96% in aeration treatments compared with the control. The yield increase was associated with more pods per plant and greater mean pod weight. Significantly higher above ground biomass and light interception were evident with aeration, irrespective of soil water treatment. Similarly cotton lint yield increased by 14–28% in aeration treatments compared with the control. The higher lint yield was associated with more squares and bolls per plant which accompanied greater above ground biomass and an increase in root mass, root length and soil respiration. Air injection and HP effected greater water use, but also brought about an enhancement of water use efficiency (WUE) for pod and lint yield, and increased leaf photosynthetic rate in both species but had no effect on transpiration rate and stomatal conductance per unit leaf area. Aeration-induced enhanced root function was arguably responsible for greater fruit set and yield in all three crops, while in vegetable soybean greater canopy cover, radiation interception and total vegetative biomass were responsible for additional yield benefit. Increased aeration of the root zone in heavy clay soils employing either air injection or HP proved beneficial to SDI irrigated crops, irrespective of the soil water conditions, and can add value to grower investments in SDI.     

苹果蠹蛾性信息素的研究和应用进展

苹果蠹蛾Cydia pomonella (L.)是我国重要的检疫害虫, 在我国仅分布于新疆和甘肃以西局部地区, 但一直保持向我国东部扩张的趋势。在国际上, 利用性信息素监测和迷向防治苹果蠹蛾已经成为一种切实可行并广泛应用的害虫管理技术。本文综述了苹果蠹蛾性信息素的成分鉴定、人工合成和应用情况的研究进展, 指出了目前存在的问题和应用前景, 以期为我国苹果蠹蛾的防控策略的研究和应用提供参考。     

基于电子捕获裂解/电子转运裂解串联质谱技术的蛋白质组学研究

蛋白质组学的兴起带动了质谱技术的快速发展,而质谱技术的进步则拓宽了蛋白质组学研究问题的广度.最近10年内,肽段或完整蛋白质在质谱仪中的裂解技术——电子捕获裂解(electron capture dissociation,ECD)与电子转运裂解(electron transfer dissociation,ETD)逐渐发展起来.ECD和ETD在蛋白质组学中的应用,特别是在蛋白质的翻译后修饰鉴定和自顶而下(Top-down)的完整蛋白质裂解研究中已经展示出了诱人的前景.对ECD和ETD的基本原理、质谱特点、仪器实现、数据解析算法与软件开发,以及在蛋白质组学中的应用进展等方面进行了比较系统全面的阐述,并对当前的研究问题、面临的技术挑战与未来的发展趋势等方面作了深入剖析.     

中国野生笃斯越橘花青素的初步分离和分析

采用反相高效液相色谱法和电喷雾质谱法对我国野生笃斯越橘中花青素组分进行了分离和分析．制备野生越橘花青素的粗提物,经大孔树脂柱层析得到花青素样品,其得率为0.72%．用香草醛法测得其总黄烷醇含量为95%．采用Alltima C18反相色谱柱(250 mm×4.6 mm,5 μm),以2 %甲酸水溶液(A)和2 %甲酸/甲醇溶液(B)作为流动相,分2个梯度对花青素组分进行洗脱,将其主要洗脱峰在电喷雾正离子模式下进行分析,根据质谱的准分子离子［M+H］(m/z)检测其聚合度．结果表明,我国野生越橘花青素的提取物被展开成8个主要的洗脱峰,以峰面积计算,主要洗脱峰总含量达85%．经质谱分析,判断为2个单体,3个二聚体,1个三聚体,2个四聚体．结果表明,我国野生笃斯越橘中含有较丰富的花青素,低聚体是其中的主要成分．本文首次报道了我国大兴安岭野生笃斯越橘花青素的组成情况,对于开展其后续的结构、功能、质量检测以及生产开发研究都具有一定的参考价值．