喜旱莲子草在青藏高原对模拟增温的可塑性: 引入地和原产地种群的比较

气候变暖背景下植物可通过关键性状的表型可塑性来适应环境温度的增加。表型可塑性增强进化假说预测定植到新环境中的入侵植物种群具有演化出更强表型可塑性的潜力。此前对可塑性进化的研究涵盖了外来植物性状对水分条件、光照变化、土壤养分、邻体根系以及天敌防御等的响应, 而较少有研究关注增温条件下植物重要性状的可塑性进化。已有的部分研究多集中在温带和热带地区, 而较少关注入侵植物在高寒地区对增温的响应; 且研究多集中在植物生长相关性状, 较少关注功能性状和防御性状。本研究采用同质园实验比较了喜旱莲子草6个引入地(中国)种群和6个原产地(阿根廷)种群, 在西藏拉萨模拟全天增温2℃处理下的适合度性状、功能性状和防御性状的响应差异。结果表明: (1)高寒地区模拟全天增温显著提高了喜旱莲子草总生物量(+36.4%)、地上生物量(+34.5%)、贮藏根生物量(+51.4%)和毛根生物量(+33.6%), 降低了分枝强度(-19.8%)和比茎长(-30.2%); (2)模拟全天增温使引入地种群的比叶面积和黄酮含量增加, 而原产地种群则相反。这些结果表明高寒地区全天增温2℃对喜旱莲子草可能是一种有利条件。引入地种群的适合度性状对模拟全天增温2℃的响应比原产地种群更强, 而其光能利用相关性状和防御性状的响应可能提升了其在高寒地区的适合度。因此, 在未来全球气候变暖的背景下, 高寒地区温度升高可能更有利于喜旱莲子草引入地种群的定植和扩散。     

不同干预时机下复方嗜酸乳杆菌片联合四联疗法在根除幽门螺杆菌失败补救治疗中的应用

目的探讨不同干预时机下复方嗜酸乳杆菌片联合四联疗法在首次根除幽门螺杆菌(H. pylori)失败患者补救治疗中的作用。方法选择西安市第一医院消化内科90例经标准四联疗法根除H. pylori失败的患者为研究对象,随机分为研究组A(A组)、研究组B(B组)与对照组(C组),各30例。A组患者使用复方嗜酸乳杆菌片联合补救四联疗法治疗。B组患者在补救四联疗法结束后序贯使用复方嗜酸乳杆菌片2周。C组患者单独采用补救四联疗法治疗。比较3组患者临床症状、不良反应发生率及H. pylori根除率。结果A组、B组、C组患者总有效率分别为93.33%(28/30),90.00%(27/30),66.67%(20/30),差异有统计学意义(χ²=9.120,P=0.010)。A组、B组、C组患者总不良反应发生率分别为13.33%(4/30)、16.67%(5/30)、66.67%(20/30),差异有统计学意义(χ²=24.522,P<0.001)。A组、B组、C组患者H. pylori根除率分别为60.00%(18/30)、63.33%(19/30)、33.33%(10/30),差异有统计学意义(χ² =6.502,P=0.039)。A组与B组患者H. pylori根除率相比差异无统计学意义(χ²=0.071,P=0.791)。结论复方嗜酸乳杆菌片联合四联疗法可提高首次四联疗法根除H. pylori失败患者H. pylori根除率,改善患者临床症状,降低总体不良反应发生率,但不同干预时机下复方嗜酸乳杆菌片对H. pylori根除率无明显影响。     

5种相思树和尾巨桉人工林土壤养分和酶活性特征

为揭示固氮树种土壤养分转化的酶学机制,对固氮树种[厚荚相思(Acacia crassicarpa)、黑木相思(A. melanoxylon)、卷荚相思(A.cincinnata)、大叶相思(A.auriculiformis)和马占相思(A.mangium)]及非固氮树种尾巨桉(Eucalyptusurophylla×E.grandis)人工林的土壤养分含量、酶活性及其相关性进行研究。结果表明,相思林40～60cm土层的pH均高于尾巨桉林;5种相思林土壤各土层的TP、TK含量均低于尾巨桉林,而20～40 cm土层的TC、TN含量均高于尾巨桉林,黑木相思林和马占相思林各土层的有效养分均显著高于尾巨桉林(P0.05)。0～10 cm土层中,相思林的土壤酸性磷酸酶和纤维素酶活性均高于尾巨桉林,大叶相思林的土壤脲酶、蔗糖酶、纤维素酶和芳基硫酸酯酶活性显著高于尾巨桉林(P0.05),卷荚相思林的土壤脲酶、纤维素酶、几丁质酶和淀粉酶活性显著高于尾巨桉林(P0.05)。相关分析结果表明,土壤脲酶、蔗糖酶和几丁质酶活性与AP显著负相关(P0.05),蔗糖酶和纤维素酶活性与NH4+-N显著负相关(P 0.05),脲酶、纤维素酶、芳基硫酸酯酶与土壤TK显著负相关(P0.05),几丁质酶活性与TN含量呈显著正相关(P0.05),土壤淀粉酶活性与NH4+-N呈显著正相关(P 0.05),过氧化氢酶活性与土壤TK含量呈显著正相关。可见,与尾巨桉人工林相比,在我国南方退化山地引种相思树可提高土壤关键酶的活性,对土壤有效养分具有明显改良作用,有利于退化地土壤的生态修复及人工林长期生产力的维持。     

Arabidopsis CSN5B Interacts with VTC1 and Modulates Ascorbic Acid Synthesis

Light regulates ascorbic acid (AsA) synthesis, which increases in the light, presumably reflecting a need for antioxidants to detoxify reactive molecules produced during photosynthesis. Here, we examine this regulation in Arabidopsis thaliana and find that alterations in the protein levels of the AsA biosynthetic enzyme GDP-Man pyrophosphorylase (VTC1) are associated with changes in AsA contents in light and darkness. To find regulatory factors involved in AsA synthesis, we identified VTC1-interacting proteins by yeast two-hybrid screening of a cDNA library from etiolated seedlings. This screen identified the photomorphogenic factor COP9 signalosome subunit 5B (CSN5B), which interacted with the N terminus of VTC1 in yeast and plants. Gel filtration profiling showed that VTC1-CSN5B also associated with the COP9 signalosome complex, and this interaction promotes ubiquitination-dependent VTC1 degradation through the 26S proteasome pathway. Consistent with this, csn5b mutants showed very high AsA levels in both light and darkness. Also, a double mutant of csn5b with the partial loss-of-function mutant vtc1-1 contained AsA levels between those of vtc1-1 and csn5b, showing that CSN5B modulates AsA synthesis by affecting VTC1. In addition, the csn5b mutant showed higher tolerance to salt, indicating that CSN5B regulation of AsA synthesis affects the response to salt stress. Together, our data reveal a regulatory role of CSN5B in light-dark regulation of AsA synthesis.     

Association of the SNP rs1800925(C/T) in the Interleukin-13 Gene Promoter with Pulmonary Function in Chinese Han Patients with Idiopathic Pulmonary Fibrosis

The present report studied potential association of the rs1800925(C/T) single nucleotide polymorphism (SNP) of the Interleukin (IL)-13 gene promoter with idiopathic pulmonary fibrosis (IPF) in patients of Chinese Han ethnicity. Seventy patients with IPF were enrolled and divided into three subgroups: group A (61–79 % pred. D_LCO; n = 22), group B (51–60 % pred. D_LCO; n = 20), and group C (≤50 % pred. D_LCO; n = 28). Control group consisted of 80 healthy individuals of Chinese Han ethnicity. The SNP rs1800925(C/T) was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis. The IL-13 CC genotype was present in 28/70 (40.0 %), homozygous TT in 6/70 (8.6 %) and heterozygous CT in 36/70 (51.4 %) patients with IPF. In control group, these genotypes were present in 30/80 (37.5 %), 11/80 (13.75 %), 39/80 (48.75 %), respectively, indicating that the distribution of the above three genotypes was not significantly different between patients with IPF and healthy controls. When the patients were stratified according to their D_LCO and D_LCO/VA, the frequencies of genotypes CT and TT in the groups A, B, and C were, respectively, 40.9 % (9/22), 50 % (10/20), and 82.1 % (23/28). Thus, significant differences in the distribution of alleles at ?1112 region of IL-13 gene were observed among the study groups A, B, and C, with the highest frequency in group C (p < 0.05). In conclusion, the rs1800925 T allele of the IL-13 gene is associated with worse pulmonary function in patients with IPF of Chinese Han ethnicity.     

Molecular Cloning, Co-expression, and Characterization of Glycerol Dehydratase and 1,3-Propanediol Dehydrogenase from Citrobacter freundii

1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.     

Multi-dimensional Co-separation Analysis Reveals Protein–Protein Interactions Defining Plasma Lipoprotein Subspecies

The distribution of circulating lipoprotein particles affects the risk for cardiovascular disease (CVD) in humans. Lipoproteins are historically defined by their density, with low-density lipoproteins positively and high-density lipoproteins (HDLs) negatively associated with CVD risk in large populations. However, these broad definitions tend to obscure the remarkable heterogeneity within each class. Evidence indicates that each class is composed of physically (size, density, charge) and compositionally (protein and lipid) distinct subclasses exhibiting unique functionalities and differing effects on disease. HDLs in particular contain upward of 85 proteins of widely varying function that are differentially distributed across a broad range of particle diameters. We hypothesized that the plasma lipoproteins, particularly HDL, represent a continuum of phospholipid platforms that facilitate specific protein–protein interactions. To test this idea, we separated normal human plasma using three techniques that exploit different lipoprotein physicochemical properties (gel filtration chromatography, ionic exchange chromatography, and preparative isoelectric focusing). We then tracked the co-separation of 76 lipid-associated proteins via mass spectrometry and applied a summed correlation analysis to identify protein pairs that may co-reside on individual lipoproteins. The analysis produced 2701 pairing scores, with the top hits representing previously known protein–protein interactions as well as numerous unknown pairings. A network analysis revealed clusters of proteins with related functions, particularly lipid transport and complement regulation. The specific co-separation of protein pairs or clusters suggests the existence of stable lipoprotein subspecies that may carry out distinct functions. Further characterization of the composition and function of these subspecies may point to better targeted therapeutics aimed at CVD or other diseases.Lipoproteins are circulating emulsions of protein and lipid that play important roles, both positive and negative, in cardiovascular disease (CVD).¹ Historically defined by their density as separated by ultracentrifugation, the major lipoprotein classes include the neutral lipid ester-rich very low-density and low-density lipoproteins (VLDLs and LDLs, respectively), which function to transport triglyceride and cholesterol from the liver to the peripheral tissues. Significant epidemiological evidence, in vitro studies, animal experiments, and human clinical trials have shown that high-LDL cholesterol is a bona fide causative factor in CVD (1). In contrast, protein- and phospholipid-rich high-density lipoproteins (HDLs) are thought to mediate the reverse transport of cholesterol from the periphery to the liver for catabolism and to perform anti-oxidative and anti-inflammatory functions (reviewed in Refs. 2 and 3). A host of human epidemiology and animal studies indicate that HDLs are atheroprotective (4). However, recent clinical trials of therapeutics that generically raise HDL, at least as measured by its cholesterol levels, have failed to confer the expected CVD protections (5–7).Although these traditional density-centric definitions have been used for nearly 40 years, accumulating evidence indicates that they are not particularly reflective of lipoprotein compositional and functional complexity. With respect to most physical traits (size, charge, lipid content, protein content, etc.), one can demonstrate significant heterogeneity within each density class. This suggests that particle subspecies exist with unique functions and effects on disease. For example, LDL can be resolved into large, buoyant and small, dense forms (8), with subjects carrying more cholesterol in the small, dense LDL exhibiting a greater CVD risk (9). HDL is particularly noted for heterogeneity, as it can be separated into numerous subfractions by density (10), diameter (11), charge (12), and major apolipoprotein content (13). Most strikingly, recent applications of soft-ionization mass spectrometry (MS) have identified upward of 85 HDL proteins with functions that go well beyond the structural apolipoproteins, lipid transport proteins, and lipid-modifying enzymes known from previous biochemical studies (14, 15). Many of these proteins imply functions as diverse as complement regulation, acute phase response, protease inhibition, and innate immunity (16). Individual HDL subspecies can apparently draw from this palette of proteins to produce distinct particles of distinct function. One well-defined HDL subfraction, termed trypanosome lytic factor, contains apolipoprotein apoA-I, haptoglobin-related protein, and apoL-I. Working together, these proteins enter the trypanosome brucei brucei and kill it via lysosomal disruption (17). There are numerous other instances of on-particle protein cooperation in HDL related to CVD (reviewed in Ref. 15). Furthermore, two-dimensional electrophoresis studies by Asztalos and colleagues (18), as well as our own work (11, 19), strongly support the concept that certain apolipoproteins segregate among different HDL particles. These observations present the intriguing possibility that the phospholipids of HDLs act as an organizing platform that facilitates the assembly of specific protein complexes (20). Such subspecies could have important functional implications in the context of CVD protection, inflammation, or even innate immune function. Furthermore, this subspeciation may explain why therapeutics that raise HDL cholesterol levels across the board have not yet shown promise with regard to CVD.To address this hypothesis, we began to think of lipoproteins as a continuum of phospholipid platforms that support the assembly of specific protein complexes analogous to those in cells that perform coordinated biological functions (i.e. ribosomes, centrosomes, etc.). Two common methods for characterizing protein complexes are tandem affinity purification (21) and immunoprecipitation. Both rely on the specific pull-down of a target protein (by either an introduced affinity tag or an antibody) followed by the identification of co-precipitated proteins via MS. Unfortunately, tandem affinity purification strategies are impractical in humans, and we have found that immunoprecipitation experiments with human plasma lipoproteins result in a high false-positive rate due to the low abundance of most of these proteins, particularly those in HDLs. Therefore, we took an alternative approach called co-separation analysis, a method based on the principle that stable protein complexes can be identified by tracking their co-migration as they undergo biochemical separation by multiple orthogonal approaches (22). Native proteins are analyzed in an unbiased manner without affinity tags or antibodies, and purification to homogeneity is not necessary for the identification of putative protein complexes.Most current studies of the lipoprotein proteome utilize samples isolated via density ultracentrifugation because contaminating lipid-unassociated lipoproteins, which can be highly abundant and obscure the identification of targeted lipid-associated proteins, are thus removed prior to the analysis. In previous work, we characterized the use of a calcium silica hydrate (CSH) resin that allowed the specific isolation of phospholipid-associated proteins and their subsequent MS identification without ultracentrifugation (11). This advance enabled the use of a variety of non-density-based separation methods for the study of plasma lipoproteins. Here, we take advantage of this to analyze the proteome of human plasma lipoproteins separated via three separation techniques that exploit different physicochemical properties: (i) gel filtration chromatography (size), (ii) anion exchange chromatography (charge interaction), and (iii) isoelectric focusing. By tracking the co-migration of specific proteins across these separations (Fig. 1), we identified a host of putative protein pairings, including the previously known trypanosome lytic factor HDL fraction, for further biochemical verification and characterization.Open in a separate windowFig. 1.Overview of the multi-dimensional separation co-migration analysis used in this study (see “Experimental Procedures” for details).