GST-SOD1-R9融合蛋白的表达、纯化、稳定性与跨膜效应

穿膜肽TAT介导的双效抗氧化酶GST(谷胱甘肽巯基转移酶)-TAT-SOD1(Cu,Zn超氧化物歧化酶),可有效清除胞内自由基,其预防氧化损伤的效果强于SOD1-TAT,但前者的跨膜能力不如后者。为增强双效抗氧化酶的跨膜效率,本研究融合了SOD1和穿膜肽R9,合成SOD1-R9全基因序列,并将其插入带有GST的原核表达载体pGEX-4T-1中,成功构建了GST-SOD1-R9融合蛋白表达质粒。然后,将重组质粒pGEX-4T-1-SOD1-R9转化大肠杆菌BL21(DE3),用IPTG诱导表达融合蛋白,通过改变诱导温度和诱导时间,确定了融合蛋白在25℃下表达11 h,可得到高表达量的可溶性GST-SOD1-R9融合蛋白。利用80%硫酸铵沉淀和GST琼脂糖树脂纯化得到纯蛋白,应用SDS-PAGE和酶活性鉴定纯化的蛋白为正确表达的目标蛋白。GST-SOD1-R9融合蛋白的温度和pH稳定性实验结果证实,该蛋白在生理条件下具有良好的SOD和GST活性。细胞跨膜实验结果证明其跨膜能力与GST-TAT-SOD1融合蛋白相比显著增强(P0.05)。这些工作为深入研究GST-SOD1-R9的抗氧化损伤效应建立了基础。     

全长SOD2重组蛋白的可溶表达、纯化、稳定性与跨膜效应

超氧化物歧化酶(SOD)家族是保护细胞免受正常代谢过程中产生的活性氧(ROS)毒性所必需的,含Mn2+离子的超氧化物歧化酶(Mn-SOD,SOD2)是其中最重要的一种。本研究合成了人源SOD2全基因序列,并将其插入带有GST的原核表达载体p GEX-4T-1中,成功构建了GST-SOD2融合蛋白表达质粒。然后,将重组质粒p GEX-4T-1-SOD2转化大肠杆菌BL21(DE3),用IPTG在25℃下诱导表达融合蛋白,得到可溶性GST-SOD2融合蛋白,经GST亲和树脂纯化得到比活为1 788 U/mg的纯蛋白,分子量约为46 k Da。利用凝血酶切去GST标签后经肝素亲和柱纯化得到了电泳纯的SOD2重组蛋白,该蛋白分子量约为25 k Da,与SOD2全长序列的理论分子量相符,比活为2 000 U/mg。两种重组SOD2蛋白在生理条件下都具有良好的SOD活性,且都具有显著的跨膜能力(P0.05)。这些工作为深入研究两种全长重组SOD2蛋白的结构与生物效应建立了基础。     

通过细胞穿膜肽和皂苷增强一种核糖体失活蛋白抗肿瘤活性

将核糖体失活蛋白类鼻疽伯克霍尔德菌致死因子1(BLF1)与细胞穿膜肽(CPP)HBP融合表达并与商陆皂苷甲(EsA)联合使用,提高BLF1重组蛋白抑制肿瘤细胞生长的活性。通过原核表达及NI-NTA亲和层析纯化BLF1、BLF1-HBP融合蛋白,以HepG2、MCF-7、A549和HeLa细胞为检测模型,MTT法检测BLF1重组蛋白对肿瘤细胞的毒性,激光共聚焦显微镜观察重组蛋白进入细胞效率,流式细胞术分析抗肿瘤效应。结果显示,穿膜肽HBP可有效提高BLF1对HepG2、MCF-7、A549和HeLa四种肿瘤细胞的生长抑制作用,其中对HeLa细胞的药效增强效果最显著,可达47.5倍;皂苷EsA的使用进一步显著提高了BLF1-HBP对上述4种肿瘤细胞生长抑制活性,且对MCF-7细胞的IC50值从6 840 nmol/L降至0.57 nmol/L。激光共聚焦观察揭示EsA可有效促进BLF1重组蛋白进入肿瘤细胞效率,流式结果分析表明EsA可大大强化BLF1-HBP诱导肿瘤细胞凋亡的能力。将BLF1与穿膜肽HBP融合表达并在EsA存在下,可显著提升BLF1对肿瘤细胞的毒性,强化其诱导肿瘤细胞凋亡的能力。     

多聚精氨酸融合增强型绿色荧光蛋白制备方法及穿膜效果

为了方便细胞穿膜肽R9融合蛋白的可溶性表达及功能上的研究,构建了pSUMO (小分子泛素样修饰蛋白) -R9-EGFP (增强型绿色荧光蛋白) 原核表达载体。分别纯化EGFP及R9-EGFP蛋白后,作用于HepG2,细胞经流式细胞仪及激光共聚焦检测R9细胞穿膜肽的作用效果。实验结果显示在SUMO分子伴侣的作用下,R9-EGFP融合蛋白获得可溶性表达。经流式细胞仪检测,R9细胞穿膜肽可以快速有效的携带目的蛋白进入细胞内部且呈时间、剂量依赖性,大约1.5 h以后荧光强度进入平台期。共聚焦显微镜检测结果表明R9细胞穿膜肽可以有效携带EGFP进入HepG2细胞,并显示主要聚集在细胞浆内。同时体外经肝素抑制实验显示,肝素抑制R9-EGFP穿膜的效率达到50％。这些结果表明,可以利用pSUMO-R9/Ni-NTA表达纯化系统,快速、有效地表达出可溶性多聚精氨酸融合蛋白,同时R9细胞穿膜肽可以有效地携带目的蛋白进入细胞内,为进一步研究多聚精氨酸的穿膜机制提供了基础。     

CXCL1促进内质网应激在肝癌肿瘤微环境中传递

目的:研究趋化因子1(CXCL1)对肝癌肿瘤微环境中肿瘤细胞间及肿瘤细胞和巨噬细胞间内质网应激(ERS)的作用。方法:收集受衣霉素(TM)刺激的HepG2细胞的上清作为条件培养基,用于培养未受TM刺激的HepG2和THP-1细胞,检测培养基中未折叠蛋白反应(UPR)相关蛋白IRE1、PERK和ATF6的mRNA表达,构建ERS在细胞间传递的模型;合成si-CXCL1,瞬时转染HepG2细胞,RT-PCR检测HepG2细胞的CXCL1的转染效率,ELISA检测上清中CXCL1的表达水平;TM刺激CXCL1敲低的HepG2细胞,收集其上清用于培养未受TM刺激的HepG2和TPH-1细胞,RT-PCR检测HepG2细胞和THP-1细胞中UPR相关的IRE1、PERK及ATF6的mRNA表达。结果:用TM刺激后的条件培养基培养HepG2和TPH-1细胞时,2种细胞中的IRE1、PERK、ATF6表达均上调,构建了ERS在细胞间传递的模型;si-CXCL1转染HepG2细胞后,与对照组相比,si-CXCL1组HepG2细胞中CXCL1的表达降低,同时ELISA检测培养基中的CXCL1也降低;用TM刺激CXCL1敲低的HepG2细胞的上清培养未受TM刺激的HepG2和TPH-1细胞后,与对照组相比,CXCL1血清敲低组培养的细胞中IRE1、PERK和ATF6的mRNA水平均下降。结论:CXCL1能够促进肝癌肿瘤微环境中内质网应激在细胞间传递。     

纤黏连蛋白重组多肽CH50抑制黑色素瘤MMP-2和MMP-9表达、激活的研究

目的 研究纤黏连蛋白重组多肽CH50对黑色素瘤B16细胞侵袭能力的影响,探讨CH50多肽抑制肿瘤生长、侵袭的机制。方法 体外培养小鼠黑色素瘤B16细胞、小鼠腿部皮下注射B16细胞建立肿瘤动物模型。采用明胶电泳法检测B16细胞和黑色素瘤组织中基质金属蛋白酶MMP-2和MMP-9的表达和激活;以CH50多肽体外处理B16细胞或体内表达CH50,观察CH50下调MMPs表达以及对肿瘤细胞侵袭能力的抑制作用。结果 B16细胞在体外培养条件下主要表达MMP-2,而在肿瘤微环境中则同时表达MMP-2和MMP-9。肿瘤组织中MMPs的表达明显高于体外培养B16细胞。CH50多肽对体外培养B16细胞的MMPs表达和激活无明显抑制作用,但处理后的B16细胞进入体内后表达MMPs的能力受到明显抑制。体内转染表达的CHSO多肽亦可明显抑制肿瘤表达MMPs、并抑制肿瘤侵袭能力。结论 纤黏连蛋白重组多肽CHSO可以抑制肿瘤微环境中基质金属蛋白酶MMP-2和MMP-9的表达和激活,从而抑制黑色素瘤生长及侵袭能力。     

重组EpoAB-NGF模拟肽的神经营养功能

目的:研究重组融合蛋白红细胞生成素AB肽-神经生长因子模拟肽(EpoAB-NGF9和EpoAB-NGF/12)的神经营养作用。方法:构建pET-42a-EpoAB-NGF9/12原核表达质粒,转化大肠杆菌BL21(DE3),以IPTG诱导表达,亲和层析纯化重组蛋白,显微镜观察PC12细胞诱导分化,流式细胞仪检测凋亡细胞。结果:大肠杆菌表达的重组融合蛋白GST-EpoAB-NGF9/12分子量约为30kDa,抗GST抗体免疫印迹反应呈阳性。融合蛋白可以诱导PC12细胞分化,促进轴突生长。R2L1细胞去血清培养,凋亡细胞占(31.7±0.60)%,去血清后加入融合蛋白GST-EpoAB-NGF9/12,细胞凋亡率分别为(25.2±3.52)%、(25.7±1.46)%,呈现一定程度的抑制细胞凋亡的活性。结论:重组EpoAB-NGF模拟肽融合蛋白具有与NGF类似的神经营养作用。     

基质金属蛋白酶2抑制剂的筛选及鉴定

以原核表达的具有明胶水解活性的人基质金属蛋白酶 2的催化区 (MCD)为靶标 ,筛选噬菌体随机环七肽库和十二肽库 .找到 6种与MCD特异结合的小肽 ,将 6种小肽基因分别与GST表达质粒重组 ,进行GST融合表达 ,制备融合蛋白 .采用Glutathione Sepharose 4B亲和层析法纯化融合蛋白 ,通过酶抑制实验、体外侵袭实验检测融合蛋白的活性 .结果表明 ,GST C71能够抑制MCD水解 β酪蛋白的活性 ,并且对人纤维肉瘤细胞HT10 80的体外侵袭有明显的抑制作用     

9R穿膜肽可增强P53抗肿瘤效果

为解决P53蛋白难以进入细胞内部发挥治疗作用的瓶颈难题.将p53基因融合插入带有9个精氨酸作为穿膜肽的表达载体中表达融合蛋白CPPs-P53,并与没有穿膜肽的P53蛋白进行比较,利用Western blotting方法检测蛋白的表达情况,MTT及Annexin V/PI双染法检测细胞生长抑制率及细胞凋亡率.Western blotting检测表明已成功在原核表达系统中表达融合蛋白CPPs-P53和P53蛋白,且蛋白纯度均已达到90％以上;MTT检测表明,P53蛋白对肿瘤细胞的生长虽有一定的抑制作用,但融合蛋白CPPs-P53与之相比,对肿瘤细胞生长的抑制效果显著增强,细胞生长抑制率有明显的提升,并且细胞生长抑制率呈现剂量依赖性;Annexin V/PI双染检测细胞凋亡情况也表明P53虽可以在一定程度上诱导肿瘤细胞的凋亡,但与P53蛋白相比较,融合蛋白CPPs-P53诱导的凋亡细胞明显增加,凋亡率是P53蛋白的2～3倍.由此说明在抑制肿瘤细胞的生长和诱导细胞凋亡方面,CPPs-P53比没有穿膜肽的P53蛋白的效果更显著.     

肝脱细胞基质溶液包被对HepG2细胞增殖及基质金属蛋白酶活性的影响

目的:建立由猪肝组织制备肝脱细胞基质溶液的新方法,并研究基质溶液包被对人肝癌细胞HepG2增殖及基质金属蛋白酶活性的影响。方法:对猪肝脏进行脱细胞处理,经研磨、冻干及胃蛋白酶消化制备肝脱细胞基质溶液,利用此基质溶液包被聚苯乙烯培养表面,进行HepG2细胞体外培养,并使用CCK-8、实时定量PCR、明胶酶谱等检测细胞增殖以及基质金属蛋白酶的基因表达和活性。结果:建立了由猪肝组织制备肝脱细胞基质溶液的新方法。与未包被组相比,脱细胞基质溶液包被培养显著促进HepG2细胞增殖,其细胞周期蛋白cyclin D1的基因表达增高,为未包被组的1.95倍(P0.01)。此外,脱细胞基质溶液包被组HepG2细胞的MMP14基因表达为未包被组的2.01倍(P0.05),明胶酶谱检测基质溶液包被组细胞的MMP9活性为未包被组的6.66倍(P0.01)。结论:肝脱细胞基质溶液包被培养可显著促进HepG2细胞增殖并提高其MMP9的活性,在构建模拟肝癌微环境培养体系中具有一定的应用潜力。     

Further studies of the sieve-element plastids of theCaryophyllales includingBarbeuia,Corrigiola, Lyallia,Microtea, Sarcobatus,andTelephium

The sieve-element plastids of 69 species of theCaryophyllales were investigated by transmission electron microscopy. All contained the specific subtype-P3 plastids characterized by a peripheral ring of protein filaments. The presence or absence of an additional central protein crystal and their shape being either polygonal or globular as well as the average sizes of the sieve-element plastids are useful features in the characterization of some families.—Barbeuia contains sieve-element plastids that confirm its placement within thePhytolaccaceae. Lyallia differs fromHectorella by including small starch grains in their sieve-element plastids, which otherwise by their globular crystals negate a closer connection to theCaryophyllaceae. The lack of a central protein crystal in its form-P3fs plastids placesMicrotea best within theChenopodiaceae. Sarcobatus, a so far uncontested member of theChenopodiaceae, contains form-P3cf plastids, i.e., including a central crystal not found elsewhere in this family.Telephium andCorrigiola, shifted back and forth betweenMolluginaceae andCaryophyllaceae, have form-P3cf(s) plastids with a polygonal crystal which favor their placement within theCaryophyllaceae.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Novel phenylanthraquinones,isofuranonaphthoquinones, homoisoflavonoids,and biflavonoids from African plants in the genera Bulbine,Scilla, Ledebouria,and Rhus

The sale of non-cultivated plants poses a real threat to the survival of a large number of plant species. On the other hand, the fact that a given plant has become a traded item is an important lead in the effort to investigate plants for the purpose of discovering novel secondary metabolites that may have marketable properties. This report is largely based on the investigations of plants collected from vendors of medicinal plants. The identification is based on botanical specimens derived from the purchased plant parts either by growing them in a garden or through micropropagation using tissue culture techniques. Bulbine species yielded novel phenyl anthraquinones and isofuranonaphthoquinones, which were found to exhibit antiparasitic and antioxidant properties. Scilla nervosa subsp. rigidifolia and Ledebouria graminifolia yielded twenty homoisoflavonoids and two xanthones. Bulbs derived from tissue culture based specimens of L. graminifolia contained all the compounds isolated from the marketed samples. Some of the homoisoflavonoids were found to possess activity against colon and breast cancer cell lines. Rhus pyroides furnished six novel bichalcones some of which exhibited insect antifeedant, cytotoxic and antiproliferative activities. Structures of the compounds described in this report were elucidated by spectroscopic means including HRMS and 1D-, and 2D-NMR experiments. Interconversion of some of the phenyl anthraquinones was accomplished as part of their structure proof. The total synthesis of three of the biflavonoids was achieved using a synthetic route which involves a microwave irradiation promoted Ulmann synthesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synopsis of the tribe Bocageeae (Annonaceae), with revisions of Cardiopetalum,Froesiodendron, Trigynaea,Bocagea, and Hornschuchia

The tribal name Bocageeae Endlicher is reestablished and the tribe is circumscribed on the basis of solitary internodal ebracteate pedicels that are articulated at the base, and pollen shed in polyads of eight or more grains. Septate anther locules, large pollen size, and seed appendages are prevalent in the tribe. Intectate pollen with free-standing columellae, rare in Annonaceae, occurs not only in the genusTrigynaea but also in some species ofBocagea andHornschuchia. As defined here, the Bocageeae include seven neotropical genera:Cymbopetalum, Porcelia, Bocagea, Cardiopetalum, Froesiodendron, Hornschuchia, andTrigynaea. The latter five genera are revised and the treatments include ten new species:Cardiopetalum plicatum, Froesiodendron urceocalyx, Hornschuchia lianarum, H. santosii, H. leptandra, Trigynaea cinnamomea, T. lanceipetala, T. triplinervis, T. lagaropoda, andT. axilliflora, all from tropical South America. A new combination,Froesiodendron longicuspe, changes the rank of that taxon from subspecies to species.Cardiopetalum surinamense is removed fromFroesiodendron and reassigned toCardiopetalum on the basis of its connate petals, dehiscent monocarps, and seeds with bilobed arils. A cladogram provides an explicit hypothesis of intergeneric relationships in the tribe. The new combinationOnychopetalum periquino, based onTrigynaea periquino, is made.     

Gametogenesis,endosperm development,and embryogeny inMicrocarpaea muscosa (Scrophulariaceae,Gratioleae)

InMicrocarpaea muscosa gametogenesis, embryo sac development (Polygonum type), endosperm development (cellular), embryogeny (Onagrad type), and seed structure are described. The different modes of endosperm development withinGratioleae are compared.     

rbcL sequences support exclusion ofRetzia,Desfontainia, andNicodemia from theGentianales

The taxonomic positions ofRetzia, Desfontainia, andNicodemia have been much discussed, and all three genera have been included inLoganiaceae (Gentianales). We have made a cladistic analysis ofrbcL gene sequences to determine the relationships of these taxa toGentianales. Four newrbcL sequences are presented; i.e., ofRetzia, Desfontainia, Diervilla (Caprifoliaceae), andEuthystachys (Stilbaceae). Our results show thatRetzia, Desfontainia, andNicodemia are not closely related toLoganiaceae or theGentianales. Retzia is most closely related toEuthystachys and is better included inStilbaceae. The positions ofDesfontainia andNicodemia are not settled, butDesfontainia shows affinity for theDipsacales s.l. andNicodemia for theLamiales s.l.     

A reconsideration of the genusRabdosiella (Lamiaceae,Nepetoideae, Ocimeae)

The two generaPlectranthus andIsodon are compared and found to be very dissimilar.Isodon ist considered to be misplaced inOcimeae subtribePlectranthinae and apparently is more closely related to subtribeHyptidinae. The disjunct genusRabdosiella is compared to these two genera and regarded to be polyphyletic. The AfricanR. calycina (Benth.)Codd is returned toPlectranthus and calledP. calycinus Benth., while the AsianR. ternifolia (D. Don)Codd is placed inIsodon sect.Pyramidium and calledI. ternifolius (D. Don)Kudo.     

Pseudomonas aeruginosa, Candida albicans, and device-related nosocomial infections: implications, trends, and potential approaches for control

For many years, device-associated infections and particularly device-associated nosocomial infections have been of considerable concern. Recently, this concern was heightened as a result of increased antibiotic resistance among the common causal agents of nosocomial infections, the appearance of new strains which are intrinsically resistant to the antibiotics of choice, and the emerging understanding of the role biofilms may play in device-associated infections and the development of increased antibiotic resistance. Pseudomonas aeruginosa and Candida albicans are consistently identified as some of the more important agents of nosocomial infections. In light of the recent information regarding device-associated nosocomial infections, understanding the nature of P. aeruginosa and C. albicans infections is increasingly important. These two microorganisms demonstrate: (1) an ability to form biofilms on the majority of devices employed currently, (2) increased resistance/tolerance to antibiotics when associated with biofilms, (3) documented infections noted for virtually all indwelling devices, (4) opportunistic pathogenicity, and (5) persistence in the hospital environment. To these five demonstrated characteristics, two additional areas of interest are emerging: (a) the as yet unclear relationship of these two microorganisms to those species of highly resistant Pseudomonas spp and Candida spp that are of increasing concern with device-related infections, and (b) the recent research showing the dynamic interaction of P. aeruginosa and C. albicans in patients with cystic fibrosis. An understanding of these two opportunistic pathogens in the context of their ecosystems/biofilms also has significant potential for the development of novel and effective approaches for the control and treatment of device-associated infections.     

Puccinia carici-adenocauli, a new rust fungus onCarex, and its anamorph,Aecidium adenocauli

Field observations and inoculation experiments showed thatAecidium adenocauli onAdenocaulon himalaicum was an anamorph of aPuccinia onCarex hakonensis andC. uda. From comparative morphology with caricicolous puccinias, the rust on theseCarex species was considered as a new species and was named asPuccinia carici-adenocauli. Contribution No. 142, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Novel glycopeptides,containing desmosine and isodesmosine,isolated from porcine aorta

Intima-media of porcine thoracic aorta were digested with pronase, after extraction of the saline-soluble matters and fat. A glycopeptide fraction was precipitated with 90% (vol/vol) ethanol from the 80% ethanol-soluble fraction of the trichloroacetic acid (7%)-soluble fraction of the pronase digest. The glycopeptide fraction was fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose 4B, yielding 4 fractions (FA, FB, FC and FD). The most carbohydrate-rich fraction (FB) was further purified to a homogeneous state. The purified FB (FB-0.1) and all other fractions contained desmosine and isodesmosine. The major sugars in the fractions without or with low affinity for Con A (FA, FB, and FB-0.1) were glucosamine, galactose, mannose and sialic acid, while those in the fractions with high affinity for this lectin (FC and FD) were glucosamine, glucose and mannose. All the fractions contained glycine, aspartic acid (and/or asparagine), serine, proline, threonine, glutamic acid (and/or glutamine) and alanine as the major amino acids, amounting to approximately 80% of the total.