hHO-1结构预测及突变体的构建、表达、纯化和活性检测

人血红素加氧酶-1(human heme oxygenase-1,hHO-1)是血红素代谢的限速酶,直接调节体内胆红素水平。利用软件Spdbv程序对hHO-1进行结构模拟预测,以Ala取代第25位His残基,hHO-1活性部位的结构有较明显的改变,但据模拟推测突变后hHO-1与血红素仍然具有结合性。依据模拟结果,构建野生型和突变体表达载体pBHO-1和pBHO-1(M),并分别转化大肠杆菌DH5α,IPTG诱导表达目的蛋白。表达产物经30％-60％(NH4)2SO4盐析后纯度提高3.6倍,再经两次Q-Sepharose Fast Flow阴离子交换树脂分离则表达产物的纯度提高30倍。酶活性测定显示,突变体hHO-1(△hHO-1)较野生型hHO-1(whHO-1)活性下降了91．21％。本研究显示hHO-1的第25位His在酶与底物血红素氧化反应中起着重要作用,为有效调控酶活性发挥其生物学作用提供依据。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

用siRNA沉默人血红素加氧酶-1基因降低胆红素水平

拟通过RNA干扰技术特异下调人血红素加氧酶-1(human heme oxygenase-1,hHO-1)基因的表达,减少hHO-1的产量从而降低胆红素的产生,探讨在胆红素产生前就阻断其产生,为临床早期防治新生儿高胆红素血症及胆红素中毒性脑病探索一种新的有效手段。针对hHO-1基因设计并化学合成三对小分子干扰RNA(small interfering RNA,siRNA)。采用脂质体转染法将siRNA转染入人肝脏细胞株HL-7702;荧光显微镜检测siRNA转染细胞的效率;转染siRNA1~2天后经RT-PCR和Western印迹方法检测hHO-1表达水平和蛋白质量;并采用HO-1诱导剂血红素诱导或hHO-1表达质粒转染细胞以上调hHO-1表达,检测siRNA干扰后hHO-1产量和酶活性。结果显示:设计的三对siRNA能不同程度的特异下调hHO-1表达,筛选获得抑制效果最佳的siRNA-3。siRNA-3抑制hHO-1呈现浓度与时间依赖性。与非特异对照siRNA及未处理组比较,血红素诱导和hHO-1表达质粒转染均能上调HL-7702细胞内hHO-1表达,提高hHO-1产量,但转染siRNA-3后hHO-1表达明显抑制,同时hHO-1活性随着基因表达下调而下降。实验表明设计合成的siRNA-3抑制效果明显。siRNA-3通过降解hHO-1,减少hHO-1产量,降低酶活性,最终减少胆红素产生,从而使RNA干扰技术成为降低新生儿高胆红素血症和胆红素中毒性脑病发生的一种候选方法。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

枯草蛋白酶E的突变体

Ser236位于横贯枯草蛋白酶E的α螺旋末端,远离催化活性中心,Ser236的突变不会对酶的活性产生大的影响。用定点突变的方法对枯草蛋白酶E的基因进行改造引入Ser236Cys,可能会形成分子间二硫键,有利于提高酶的稳定性。Ser236Cys变体酶(BP1)活性是野生型蛋白酶E的15倍,热稳定性提高3倍;进一步在其他位点引入突变的变体酶BU1(A1a15Asp/Gly20His/Ser236Cys)和BW1(Ser24His/Lys27Asp/Ser236Cys)活性都比野生型蛋白酶E低,但BW1的稳定性稍高于野生型蛋白酶E。     

转染人血红素加氧酶-1基因对大鼠血管平滑肌细胞抗氧化损伤的作用

本实验构建含人血红素加氧酶-1（hHO-1)基因的逆转录病毒载体XM-6/hHO-1,将其导入离体培养的大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC),观察外源性hHO-1基因在VSMC内的表达及其抗活性氧损伤作用,结果表明：（1）hHO-1基因可在靶细胞中明显表达,转染VSMC的HO-1蛋白表达和HO酶活性分别比非转染细胞高1.8倍和2.0倍;（2）转染hHO-1的VSMC可对抗大剂量H2O2对细胞的损伤作用,表现为细胞存活率增加和乳酸脱氢酶（LDH）漏出减少,上述保护作用可被HO特异性抑制剂锌原卟啉IX（Zinc-proto-porphyrinIX,ZnPP-IX)所阻断,研究结果提示,外源性HO-1的过量表达可增加VSMC对抗氧化损伤的能力。     

基于易错PCR的假密环菌Armillariella tabescens MAN47β-甘露聚糖酶耐高温定向进化

本研究利用易错PCR技术突变假密环菌Armillariella tabescens MAN47β-甘露聚糖酶野生型基因,PCR产物与大肠杆菌-酿酒酵母穿梭表达载体pYCα上连接,在大肠杆菌DH5α中扩增后电转入酿酒酵母Saccharomyces cerevisiae,构建了库容为10_4的初级突变体库,筛选得到耐高温最佳突变株M262.DNS法测得80℃处理30 min后最大酶活力为25 U/mL,较之野生型最适条件酶活力提高了4.3倍.序列分析表明,突变体有3个碱基发生了突变:T343A/C827T/T1139C,相应的氨基酸改变为Ser115Thr/Thr276Met/Val380Ala,利用SWISS-MODEL数据库同源建模显示,这3个突变氨基酸分别位于第4个13折叠的第6个氨基酸、第6个α螺旋的第1个氨基酸、第10个α螺旋和第11个β折叠之间的转角.     

高活性β-内酰胺酶的分离、表达和性质

应用DNA家族改组方法制备TEM型β-内酰胺酶基因突变文库,利用平皿初筛和琼脂稀释法复筛获得一株酶活力增高的突变体blaTEMm.带有该突变基因的大肠杆菌,其最低抑菌浓度(MIC)比带有野生型blaTEM的大肠杆菌高2～4倍(氨苄青霉素)、4～8倍(头孢唑啉)、4～8倍(头孢呋辛)、16～32倍(头孢他啶)、8倍以上(头孢曲松)和4～8倍(头孢噻肟).在E.coli BL21(DE3)高效表达blaTEM和blaTEMm,利用离子交换柱层析和凝胶过滤层析对包涵体进行柱上复性和纯化,获得纯度达90%的重组蛋白质.酶动力学分析显示,与野生型蛋白相比,突变蛋白酶促反应的kcat/Km值分别增加1.5倍(青霉素G)、4.4倍(氨苄青霉素)、3.1倍(头孢唑啉)、2.8倍(头孢他啶),但是对头孢噻肟的kcat/Km下降了2.5倍.DNA序列分析显示易blaTEMm发生7处碱基置换,造成4个氨基酸改变,即S59G、R164S、A237T和E240K.应用Swiss-Pdb Viewer3.7软件预测蛋白质的三维结构,显示这些突变不影响酶的活性中心,但是变异造成的蛋白质空间结构的细微变异增加了酶和底物的亲和性.     

人血红素加氧酶-1的原核表达、纯化及其中和抗体的制备

目的确定人血红素加氧酶-1（human heme oxygenase-1,hHO-1）在大肠埃希菌中的表达条件与纯化方法,利用纯化的蛋白制备具有中和活性的hHO—1多克隆抗体。方法将hHO-1原核表达质粒pMW172／hHO-1转人大肠埃希菌菌株BL21,通过改变摇床转速、诱导剂IPTG浓度和培养时间确定hHO-1蛋白的最佳可溶性表达条件;利用超声破碎、高速离心、分级盐析、分子筛层析等方法纯化hHO-1蛋白,建立体外HO-1活性测定方法检测hHO-1蛋白的活性;利用纯化的hHO-1活性蛋白作为抗原免疫新西兰兔,制备多克隆抗体;利用ELISA方法和Western印迹技术分别测定抗体的效价和特异性,通过HO-1活性测定检测抗体的中和活性。结果确定hHO-1最佳可溶性表达条件为：37℃、200r／min培养3h后,0．1 mmoL／LIPTG诱导培养4h。超声破碎菌体,上清经30％～40％盐析纯化及分子筛层析纯化,获得活性hHO-1蛋白,收得率为30．3％,纯化倍数为2．83倍,纯度为90％。制备的抗hHO-1的兔血清效价达到10^6,并能中和掉46％hHO-1的催化活性。结论为hHO-1蛋白的表达和纯化以及多克隆抗体制备确立了可行的技术方案;获得了高纯度活性hHO-1蛋白及hHO-1多克隆抗体,为HO-1功能、结构研究,以及相关疾病研究奠定了基础。     

嗜热酯酶APE1547催化活性的定向进化研究

对来源于嗜热古菌Aeropyrum pernix的酯酶(APE1547)催化活性进行定向进化研究。利用APE1547特殊的稳定性,建立了准确的高通量高温酯酶筛选方法。对第一代随机突变库筛选获得了催化活性较野生型提高1.5倍的突变体M010,序列分析表明其氨基酸突变为R526S。从第二代突变库中筛选出的总活力提高5.8倍突变体M020,突变位点为R526S/E88G/A200T/I519L,其比活力与M010一致,但表达量比野生型提高约4倍。对M020酶学性质表征发现,其最适pH为8.5,比野生型向碱性偏移0.5;活性中心残基酸性基团的解离常数(pK1)由野生型的7.0提高至7.5。晶体结构分析表明,突变位点R526距离活性中心较近,将其突变为Ser降低了活性中心的极性,抑制了催化残基His的解离,使酸性基团的解离常数升高。     

CHANCE,PURPOSE, AND PROGRESS IN EVOLUTION AND CHRISTIANITY

Evolutionary biology has a complex relationship with ideas of chance, purpose, and progress. Probability plays a subtle role; strikingly, founding figures in statistics were motivated by evolutionary questions. The findings of evolutionary biology have been used both in support of narratives of progress, and in their deconstruction. Likewise, professional biologists bring to their scientific work a set of preconceptions about chance and progress, grounded in their philosophical, religious, and/or political views. From the religious side, questions of purpose are ever‐present. We explore this interplay in five broad categories: chance, progress, intelligence, eugenics, and the evolution of religious practices, each the subject of a semester long symposium. The intellectual influence of evolutionary biology has had a broad societal impact in these areas. Based on our experience, we draw attention to a number of relevant facts that, while accepted by experts in their respective fields, may be unfamiliar outside them. We list common areas of miscommunication, including specific examples and discussing causes: sometimes semantics and sometimes more substantive knowledge barriers. We also make recommendations for those attempting similar dialogue.     

ECOLOGY AND PHYSIOLOGY OF HIBERNATION AND OVERWINTERING AMONG FRESHWATER FISHES, TURTLES, AND SNAKES

1. Freshwater fishes are the most northerly of freshwater ectotherms, followed by frogs. North American freshwater snakes, turtles, and salamanders do not range farther north than southernmost Canada. 2. Freezing and desiccation are the main challenges during terrestrial hibernation of ectotherms. Oxygen depletion, water balance, and ionic balance are the major problems for air breathing ectotherms that hibernate underwater. 3. The importance of accumulation of energy stores for overwintering among fishes depends upon the length and severity of the winters, whether or not there is springtime reproduction, body size, latitude, and the availability and use of food during overwintering. 4. Fishes can decrease energy demands during the winter by reductions in activity, metabolic depression, and entrance in semi-torpidity. 5. Adaptations for coping with hypoxia and anoxia among overwintering freshwater fishes may include metabolic depression, a decrease in blood O₂ affinity, microhabitat selection, air breathing, short-distance migration, biochemical modifications aimed at adjusting glycolytic rates, and alcoholic fermentation. 6. Freshwater turtles have a worldwide northern limit of approximately 50° N, which means that some species spend about half of their lives hibernating. 7. Aquatic turtles normally hibernate underwater, although occasionally they hibernate on land. In water they usually hibernate in a hypoxic or anoxic (mud) environment and in relatively shallow water. Wintertime movements of unknown frequency occur in some species. 8. The hatchlings of many turtle species can overwinter in the nest. Among northern species this behaviour is most common among painted turtles, whose hatchlings can withstand freezing. 9. Mortality among adult turtles is probably highest during the hibernation cycle. 10. Temperature appears to the most important cue for entry and exit from hibernation among freshwater turtles. 11. Little is known of the energetics of overwintering turtles. Energy stores for overwintering may be more important at lower latitudes than at higher ones, due to the higher metabolic rates of overwintering, but non-feeding, southern turtles. 12. The ability of turtles to tolerate submergence is a function of temperature, degree of water oxygenation, latitude of origin, efficacy of extrapulmonary respiratory pathways, and metabolic rate. 13. For turtles that hibernate in an anoxic hibernaculum, and for those without sufficient extrapulmonary uptake of O₂ to allow metabolism to be completely aerobic, the most important physiological perturbation is an acidosis developed from a continuing production of lactate. If sufficient O₂ can be obtained, the most likely factors limiting hibernation time are water balance and ion balance. 14. Mechanisms of turtles for coping with acidosis include metabolic depression, integumental CO₂ loss, bicarbonate buffering, and changes in ion concentrations that minimize the decrease in SID (strong ion difference). The most important among the latter are a decrease in plasma [Cl^-] and large increases in plasma calcium and magnesium. 15. Turtles are unique among reptiles in their ability to maintain both cardiovascular and nervous system function during prolonged anoxia. 16. Turtles gain weight from water uptake during submerged hibernation, but apparently maintain some kidney function; however, osmoregulation is one of the least known areas of the physiology of hibernation. 17. Recovery of turtles upon emergence commences with a rapid hyperventilatory compensation of pH, followed by a slower adjustment of ion levels. Basking speeds recovery greatly. 18. While hibernation of turtles in the northern parts of their ranges is most likely very stressful physiologically, northern range limits are more likely to be determined by reproductive restraints than by the rigors of extended hibernation. 19. The superior ability of turtles to tolerate anoxia may be more the result of an annual hibernation than of their diving habits during active periods of the year. 20. Freshwater snakes usually hibernate on land. However, they appear to be capable of aquatic hibernation and may not do so because of the risk of death from anoxia. 21. Some species of terrestrial snakes are known to hibernate underwater, and are able to do so in the laboratory for months. In the field, this behaviour is considered opportunistic, as there is no evidence to suggest that any snakes can tolerate extended anoxia.     

THE MORPHOLOGY AND RELATIONSHIPS OF RHODEA,TELANGIUM, TELANGIOPSIS,AND HETERANGIUM

This study deals with four form or organ genera from the Upper Mississippian (Chester Series) of the Illinois Basin, and provides evidence that they were produced by a single natural genus with gymnospermous affinity. The plant remains—compressions, impressions, petrifactions, and specimens that combine compression or impression with petrifaction—allow examination of both external morphology and internal anatomy. The specimens include foliage corresponding to Rhodea, stems and petioles corresponding to Heterangium, and synangiate fructifications corresponding to either Telangium or Telangiopsis. The stems and foliage are considered parts of the same plant because of the identity of the anatomical and cuticular features of petioles attached to stem and those petioles with attached foliage. The fertile material is regarded as part of the same plant because: (1) The anatomy of axes of the fertile specimens is like that of the sterile specimens. (2) A single specimen may contain both sterile Rhodea-type axes and fertile regions. (3) Axes bearing synangia have the same size and patterns of divisions as the sterile foliage. Features that indicate lyginopterid affinities include: (1) Equal forking of the petiole. (2) Presence of fiber bands in the outer cortex and sclerotic nests in the inner part of the cortex. (3) Crowded circular bordered pits on the lateral walls of the metaxylem tracheids. (4) The presence of a small amount of secondary xylem. A variety of structural details of the stem and petiole suggest the genus Heterangium. The phyletic position of the plant that produced Rhodea, Telangium, Telangiopsis, and Heterangium is reviewed in light of such discoveries as the presence of a planated frond that lacks a lamina and the presence of both monolete and trilete microspores in a single synangium.     

MASS FRAGMENTOGRAPHY OF PHENYLETHYLAMINE, m- AND p-TYRAMINE AND RELATED AMINES IN PLASMA, CEREBROSPINAL FLUID, URINE, AND BRAIN

—A mass fragmentographic method for the assay of phenylethylamine (PEA) and a number of related amines in several biological materials is described. The gas chromatographic column employed for this analysis is a 12ft 1/8 in. o.d. steel column packed with 0.5% OV₂₂+ 2% SE54 + 1% OV₂₁₀ coated on 80/100 mesh chromosorb W (HP). The mass spectral characteristics of these amines are illustrated, compared, and discussed. Of the various monoamines which could be measured, only PEA, m- and p-tyramine were detected in measurable quantities. Phenylethanolamine and p-octopamine were found in trace amounts in urine, plasma, cerebrosponal fluid, and rat brain. No diurnal variation in the urinary excretion of PEA, m- and p-tyramine was observed. Plasma concentration of PEA or p-tyramine did not significantly change 1 h after eating a breakfast. Furthermore, consuming 200 g of Cadbury milk chocolate containing about 1 mg of PEA, 0.1 mg of phenylethanolamine and 10 mg of p-tyramine did not significantly alter urine excretions of these three amines. In the brain, as has been reported by others, we found that PEA and p-tyramine are not evenly distributed and that the highest concentrations are found in the hypothalamus and caudate. From the results obtained we concluded that PEA, m- and p-tyramine are probably produced from endogenous sources and that the direct contribution of diet to their urine excretion is small.