利用大肠杆菌高效表达人载脂蛋白AI及其性质分析

载脂蛋白AI(apolipoproteinAI,apoAI)是高密度脂蛋白HDL的主要组成成分 ,流行病学研究表明apoAI的含量决定血浆中HDL的高低 ,而HDL具有胆固醇逆向转运的功能 ,起到降低冠心病发生概率的作用。通过大肠杆菌表达系统来生产apoAI的蛋白原proapoAI,蛋白的表达形式为包涵体 ,通过疏水柱进行柱上复性。同时在proapoAI和apoAI之间设计一个酸水解位点 ,通过将蛋白原proapoAI进行酸水解来得到成熟蛋白apoAI。最终得到的成熟蛋白apoAI在结构分析和脂结合特性上都与天然的apoAI相似 ,从而为该蛋白的工业生产上提供了一个良好的基础。     

Quorum sensing inhibitors as antipathogens: biotechnological applications

The mechanisms through which microbes communicate using signal molecules has inspired a great deal of research. Microbes use this exchange of information, known as quorum sensing (QS), to initiate and perpetuate infectious diseases in eukaryotic organisms, evading the eukaryotic defense system by multiplying and expressing their pathogenicity through QS regulation. The major issue to arise from such networks is increased bacterial resistance to antibiotics, resulting from QS-dependent mediation of the formation of biofilm, the induction of efflux pumps, and the production of antibiotics. QS inhibitors (QSIs) of diverse origins have been shown to act as potential antipathogens. In this review, we focus on the use of QSIs to counter diseases in humans as well as plants and animals of economic importance. We also discuss the challenges encountered in the potential applications of QSIs.     

Macrophage apoAI protects against dyslipidemia-induced dermatitis and atherosclerosis without affecting HDL

Tissue cholesterol accumulation, macrophage infiltration, and inflammation are features of atherosclerosis and some forms of dermatitis. HDL and its main protein, apoAI, are acceptors of excess cholesterol from macrophages; this process inhibits tissue inflammation. Recent epidemiologic and clinical trial evidence questions the role of HDL and its manipulation in cardiovascular disease. We investigated the effect of ectopic macrophage apoAI expression on atherosclerosis and dermatitis induced by the combination of hypercholesterolemia and absence of HDL in mice. Hematopoietic progenitor cells were transduced to express human apoAI and transplanted into lethally irradiated LDL receptor^−/−/apoAI^−/− mice, which were then placed on a high-fat diet for 16 weeks. Macrophage apoAI expression reduced aortic CD4⁺ T-cell levels (−39.8%), lesion size (−25%), and necrotic core area (−31.6%), without affecting serum HDL or aortic macrophage levels. Macrophage apoAI reduced skin cholesterol by 39.8%, restored skin morphology, and reduced skin CD4⁺ T-cell levels. Macrophage apoAI also reduced CD4⁺ T-cell levels (−32.9%) in skin-draining lymph nodes but had no effect on other T cells, B cells, dendritic cells, or macrophages compared with control transplanted mice. Thus, macrophage apoAI expression protects against atherosclerosis and dermatitis by reducing cholesterol accumulation and regulating CD4⁺ T-cell levels, without affecting serum HDL or tissue macrophage levels.     

Chromosome Numbers and Karyotypes of Six Oxytropis Species (Fabaceae) from the Qinghai Tibetan Plateau,China

Chromosome numbers and karyotypes of 13 populations of six Oxytropis species (Fabaceae) from the Qinghai Tibetan Plateau, China, were presented. The chromosome numbers and karyotypes in O.ochrocephala, O.tatarica, O.kansuensis and O.humifusa (2n=16) were reported for the first time. B chromosomes were found from O.stracheyana (2n=48). The basic chromosome number of x=8 is confirmed for the genus. The available chromosomal data indicate that polyploidy may have played an important role in the evolution of the genus, with the incidence of polyploidy in the genus reaching 58%. However, our results indicated that among the populations here examined only one was a hexaploid with 2n=48. Such a chromosomal pattern indicates that the karyotypic repatterning at the diploid level seems to be the predominant feature of chromosomal evolution in the Oxytropis species from the Qinghai Tibetan Plateau, and that sympatric speciation via hybridization and polyploidization has played a minor role in the species diversification of the genus from this area.     

南沙群岛西南部和北部湾口海域鱼类物种多样性

根据2012-2013年在南沙群岛西南部和北部湾口海域春秋两个航次的调查资料, 分析了该海域鱼类种类组成、相对重要性指数和物种多样性等特征。结果表明, 两个航次调查共鉴定鱼类504种, 隶属于2纲31目129科294属; 其中北部湾口海域出现鱼类301种, 南沙群岛西南部海域出现鱼类357种。优势种数量较少, 多以中小型鱼类为主, 且季节间变化较大。春季多样性指数高于秋季, 这主要是因为春季出现的大量鱼类为补充群体, 而许多种类在秋季有向较深海区移动的趋势; 南沙群岛西南部海域多样性指数高于北部湾口海域, 这主要是由于南沙群岛西南部海域受水温和洋流的影响较大造成的。更替指数和迁移指数显示, 秋季鱼类群落结构稳定性要低于春季, 而且两个季节的鱼类群落结构都偏离平衡状态, 主要是由鱼类的洄游和不同适温性鱼类的迁入迁出造成的。综合来看, 南沙群岛西南部海域鱼类物种多样性和群落结构稳定性均高于北部湾口海域, 在努力控制资源可捕量范围的同时, 可合理开发南海中南部海域的渔业资源。     

用酵母双杂交系统研究载脂蛋白AI(apoAI)和清道夫受体BI(SR-BI)间的相互作用

目的:利用酵母双杂交系统验证在胆固醇逆转运过程中起关键作用的大鼠载脂蛋白AI(apoAI)和清道夫受体BI-(SR-BI)间存在着相互作用,为初步筛选具有降脂活性组分提供1对新的靶点。方法:首先分别克隆了Wistar大鼠的apoAI和SR-BI基因的cDNA,并构建了酵母表达载体,利用共转化技术观察到apoAI和SR-BI间存在着相互作用,并在酵母交配实验中证实了这个结果。结果:经共转化后的实验组与阳性对照组可在SD/-Leu/-Trp/-His/-Ade/X-α-Gal平板上生长且菌斑呈蓝色,经测定α、β半乳糖苷酶活力可知酶活分别为8～12U和10～40U。酵母交配后的二倍体实验组、阳性对照组可在SD/-Leu/-Trp/-His/-Ade/X-α-Gal平板上生长且菌斑呈蓝色。结论:apoAI和SRBI间的确存在相互作用。     

Apolipoprotein AI tertiary structures determine stability and phospholipid‐binding activity of discoidal high‐density lipoprotein particles of different sizes

Human high‐density lipoprotein (HDL) plays a key role in the reverse cholesterol transport pathway that delivers excess cholesterol back to the liver for clearance. In vivo, HDL particles vary in size, shape and biological function. The discoidal HDL is a 140–240 kDa, disk‐shaped intermediate of mature HDL. During mature spherical HDL formation, discoidal HDLs play a key role in loading cholesterol ester onto the HDL particles by activating the enzyme, lecithin:cholesterol acyltransferase (LCAT). One of the major problems for high‐resolution structural studies of discoidal HDL is the difficulty in obtaining pure and, foremost, homogenous sample. We demonstrate here that the commonly used cholate dialysis method for discoidal HDL preparation usually contains 5–10% lipid‐poor apoAI that significantly interferes with the high‐resolution structural analysis of discoidal HDL using biophysical methods. Using an ultracentrifugation method, we quickly removed lipid‐poor apoAI. We also purified discoidal reconstituted HDL (rHDL) into two pure discoidal HDL species of different sizes that are amendable for high‐resolution structural studies. A small rHDL has a diameter of 7.6 nm, and a large rHDL has a diameter of 9.8 nm. We show that these two different sizes of discoidal HDL particles display different stability and phospholipid‐binding activity. Interestingly, these property/functional differences are independent from the apoAI α‐helical secondary structure, but are determined by the tertiary structural difference of apoAI on different discoidal rHDL particles, as evidenced by two‐dimensional NMR and negative stain electron microscopy data. Our result further provides the first high‐resolution NMR data, demonstrating a promise of structural determination of discoidal HDL at atomic resolution using a combination of NMR and other biophysical techniques.     

Synchronization and resynchronization of inseminations in lactating dairy cows with the CIDR insert and the Ovsynch protocol

Pregnancy per artificial insemination (AI) was evaluated in dairy cows (Bos taurus) subjected to synchronization and resynchronization for timed AI (TAI). Cows (n = 718) received prostaglandin F_2α (PGF) on Days –38 and –24 (Days 39 and 53 postpartum), gonadotropin-releasing hormone (GnRH) on Day –10, PGF on Day –3, and GnRH and TAI on Day 0. Between Days –10 and –3, cows received a progesterone intravaginal insert (CIDR group) or no CIDR (Control group). Between Days 14 and 23, cows received a CIDR (Resynch CIDR group) or no CIDR (Resynch control group), GnRH on Day 23, with pregnancy diagnosis on Day 30. Cows in estrus (between Days 0 and 30) were re-inseminated at detected estrus (RIDE). Nonpregnant cows received PGF on Day 30 and GnRH and TAI on Day 33. Plasma progesterone was determined to be low or high on Days –24 and –10. Pregnancy rates were evaluated 30 and 55 d after AI. The CIDR insert included in the Presynch-Ovsynch protocol did not increase overall pregnancy per AI for first service (36.1% and 33.6% for CIDR; 34.1% and 28.8% for Control) but did decrease pregnancy loss (7.0% for CIDR and 15.6% for Control). The CIDR insert increased pregnancy per AI in cows with high progesterone at the time the CIDR insert was applied. Administration of a CIDR insert between Days 14 and 23 of the estrous cycle after first service did not increase overall pregnancy per AI to second service (24.7% and 22.7% for Resynch CIDR; 28.6% and 25.3% for Resynch control). For second service, RIDE cows had lower pregnancy rates in the Resynch CIDR group than in the Resynch control group. Cows with a CL (corpus luteum) at Day 30 had higher pregnancy rates in the Resynch CIDR group than those in the Resynch control group.     

60例活动性肝硬变患者血清载脂蛋白AI与B的临床研究

目的：为了探讨活动性肝硬变患者的血清载脂蛋白AI（APOAI）、载脂蛋白B（APOB）与肝细胞损害的严重程度及其预后的关系。方法：采和速度比浊法检测60例活动性肝硬变患者的血清APOAI、APOB水平,并与其他肝脏生化试验进行比较。结果：活动肝硬变代偿性与失代偿性两组APOAI,APOB均有着显著性差异（P＜0．01,P＜0．05）。活动性肝硬变患者的APOAI与总胆红素（TB）、凝血酶原时间（PT）呈负相关性（P＜0．01,P＜0．01,与白蛋白（ALB）,胆碱酯酶（CHE）、纤维蛋白原（FIB）呈显著正相关性（P＜0．01,P＜0．01,P＜0．05）。APOX仅与FIB呈显著相关性（P＜0．05）,好转组与死亡组两组APOAI有显著性差异（P＜0．05）,APOB无显著性差异,结论：检测活动性肝硬变患者血清APOAI,APOB水平有助于判断肝硬变的严重程度,且APOAI比APOB更敏感、可靠。