两种藻胆蛋白对人喉癌Hep-2细胞的光动力杀伤效果

从条斑紫菜中提取高纯度R-藻红蛋白(R-PE)和R-藻蓝蛋白(c-pc),采用MTT法测定主要研究了不同浓度(10、25、50和100μg/ml)R-PE和C-PC分别介导的光动力效应对人喉癌Hep-2细胞的生存率的影响.实验结果显示,两种藻胆蛋白的光动力作用对Hep-2细胞具有杀伤作用.在浓度为100μg/ml,照射剂量为5OJ/cm2的条件下,藻蓝蛋白对应的细胞存活率为64%.藻红蛋白对应的仅为57%;单用碘钨灯处理,Hep-2细胞的存活率达到86.9%;而单独使用这两种藻胆蛋白处理Hep-2细胞,培养24h后,低浓度藻胆蛋白(10、25μg/ml)对细胞的抑制效果不明显,高浓度对细胞生长具有一定的抑制效果,抑制率为68%.实验证明条斑紫菜R-藻红蛋白和C-藻蓝蛋白具有可开发人喉癌治疗光敏剂应用前景.     

藻胆蛋白复合物的合成及其分子内能量传递

通过偶联剂3-(2-吡啶联巯基)丙酸N-羟基琥珀亚胺酯(SPDP)及改变配料比, 合成了两个R-藻红蛋白(R-PE)与C-藻蓝蛋白(C-PC)的复合物A和B.利用吸收光谱确定了分子内R-PE与C-PC的摩尔比为6∶1和2∶1. 通过荧光光谱, 观察到能量传递现象, 并计算出能量传递效率为63%和88%.证明分子内能量传递效率很高. 二硫苏糖醇(DTT)还原连接R-PE与C-PC的二硫桥键后, 能量传递被阻断. 这一现象进一步证明复合物中存在分子内能量传递.     

不同pH条件下R-藻红蛋白和C-藻蓝蛋白荧光寿命的研究

采用自己研制的时间分辨毫微秒荧光谱仪对R-藻红蛋白(R-PE )和C-藻蓝蛋白(C-PC)进行了荧光寿命的测定和研究,并对能量传递过程进行了分析和讨论.测得R-PE的荧光寿命为3.1±0.1ns,C-PC的荧光寿命为1.3±0.1ns,且在pH5—pH9的范围内不变;当pH<5时,两者的荧光寿命都有变短的趋势.我们还测定了R-PE和C-PC混合溶液中R-PE的荧光寿命不变,而C-PC的荧光寿命变长,从而表明存在着R-PE向C-PC的辐射能量传递.     

多管藻R—藻红蛋白的激光光敏作用对体外培养的肿瘤细胞生存…

用１００μｇ／ｍｌ藻红蛋白联合激光处理８１１３人口腔上皮癌细胞,ＭＴＴ比色法测定癌细胞存活率。实验表明,先用藻红蛋白处理再经波长为４８８ｎｍ,２５．６Ｊ／ｃｍ＾２氩离子激光辐照,细胞存活率为２５％;而单用藻红蛋白,单用激光组分别为４３％和１０７％。结果证明,藻红蛋白的光敏作用对体外培养的肿瘤细胞具有较强的杀伤作用。     

多管藻R-藻红蛋白的激光光敏作用对体外培养的肿瘤细胞生存率的影响

用１００μｇ／ｍｌ藻红蛋白联合激光处理８１１３人口腔上皮癌细胞,ＭＴＴ比色法测定癌细胞存活率。实验表明,先用藻红蛋白处理再经波长为４８８ｎｍ,２５．６Ｊ／ｃｍ２氩离子激光辐照,细胞存活率为２５％;而单用藻红蛋白,单用激光组分别为４３％和１０７％。结果证明,藻红蛋白的光敏作用对体外培养的肿瘤细胞具有较强的杀伤作用。     

条斑紫菜R-藻红蛋白提纯工艺研究

对条斑紫菜叶状体R-藻红蛋白提纯方法进行了研究和分析。首先分别采用冻融法、化学试剂法、溶胀法等单一及组合细胞破碎方法对条斑紫菜细胞进行破碎,结果表明溶胀 组织捣碎法效果最佳。其次用25%～60%饱和度范围内的硫酸铵沉淀法粗提藻红蛋白,发现25%～45%硫酸铵分步沉淀法效果最好,再经结晶法盐析纯度(D565nm/D280nm)达到2.088。盐析液用SephadexG-25层析柱脱盐,再经羟基磷灰石(HA)柱层析,纯度可达到4.98。R-藻红蛋白的最大吸收峰在565nm,其室温荧光发射峰为578nm。SDS-PAGE结果显示,R-藻红蛋白可分为α、β两个亚基,Mr分别为17.0×103和19.0×103。     

条斑紫菜R-藻红蛋白荧光探针制备条件优化

通过化学方法使条斑紫菜R-藻红蛋白与抗体进行交联以制备荧光探针, 并对制备条件进行优化。首先采用异双功能试剂SPDP (N-琥珀酰亚氨基-3-2-吡啶基二硫丙酸醇)和DTT（二硫苏糖醇）分别使 R-PE(R-藻红蛋白)衍生化、IgG（单克隆抗体）巯基化, 其次测定了SPDP与R-PE不同摩尔比对R-PE衍生化的影响、DTT与IgG不同摩尔比对IgG巯基化的影响, 结果表明：SPDP与R-PE的最佳摩尔比为40：1, DTT与IgG的最佳摩尔比为500:1。在此基础上, 建立了R-PE与IgG交联的制备技术, 并应用全波长扫描吸收光谱、电泳分析和荧光显微镜观察等监测和分析技术, 证实了藻红蛋白与抗体已成功交联形成了复合物。     

硫酸铵三步盐析对藻胆蛋白纯化的影响

主要研究了多次硫酸铵盐析对条斑紫菜藻胆蛋白提取纯化效果。对分离提取的对条斑紫菜藻胆蛋白溶液进行了3次硫酸氨溶液盐析,实验结果表明:55%饱和度可以将绝大部分藻胆蛋白盐析;采用不同组合(15%、20%、25%、30%、35%、40%、45%7个饱和度分别与50%、55%、60%3个饱和度两两组合)二步硫酸铵盐析沉淀藻胆蛋白,使R-藻红蛋白和C-藻蓝蛋白的盐析后纯度(A564/A280)分别达到了1.0和0.45以上,得率分别为1.4%和0.95%;第3次硫酸铵盐析使R-藻红蛋白、C-藻蓝蛋白的纯度分别达到了1.4和0.4以上,最终产率分别为1.3%和0.8%,而变藻蓝蛋白产率有所下降(从0.65%到0.49%),但纯度变化不大。实验证明了采用多次盐析方法可以很大程度提高藻胆蛋白纯度。     

三种藻胆蛋白在复合累积LB膜中的能量传递

制备了R-藻红蛋白(R-PE)、C-藻蓝蛋白(C-PC)及变藻蓝蛋白(APC)的单组分累积LB膜及三组分复合累积LB膜. 吸收及荧光光谱测定表明三种蛋白的LB膜的光谱重叠性质与他们在水溶液中的性质相同. 结构测定表明, 三种蛋白在其LB膜中排列有序, 且由它们组装的复合累积LB膜结构类似于藻类植物中的藻胆体结构. 通过稳态荧光光谱及其光谱解叠, 观察到了三种蛋白在复合累积LB膜中的激发能量传递现象. 根据给体荧光峰的猝灭, 计算出在复合累积LB膜中从给体R-PE经C-PC到受体APC的能量传递效率为51%.     

条斑紫菜藻红、藻蓝蛋白α和β亚基基因序列测定及分析

为了获得江苏吕泗的条斑紫菜藻红蛋白α(Pea)和β(Peb)亚基、藻 蓝蛋白α(Pca)和β(Pcb)亚基基因的DNA序列,本文对其基因进行了克隆、 序列测定及分析.根据已发表的基因序列(DQ666487.1)设计引物,对提取自 条斑紫菜叶状体的DNA进行PCR和电泳鉴定,产物经测序证实获得藻红蛋白 序列1 357 bp (HM008263.1)和藻蓝蛋白序列1 335 bp (HM008262.1).上述 两段序列与已报道的条斑紫菜相关序列(DQ666487.1)同源性均为99％,与 其它几种紫菜相关序列同源性为88％～98％.两段序列均采用多顺反子转录 策略,排布顺序为5′Untranslated Regions(UTR)- Peb -间隔区- Pea -3 ′UTR 和 5′UTR- Pcb -间隔区- Pca -3′UTR.文中对基因翻译所得氨基 酸序列做了理化参数、功能位点及空间构型的预测.基于对序列开放阅读框 、启动子、Shine-Dalgarno (SD)序列的分析,本文对条斑紫菜分类地位进 行了讨论.     

Probing the pH sensitivity of R-phycoerythrin: Investigations of active conformational and functional variation

Crystal structures of phycobiliproteins have provided valuable information regarding the conformations and amino acid organizations of peptides and chromophores, and enable us to investigate their structural and functional relationships with respect to environmental variations. In this work, we explored the pH-induced conformational and functional dynamics of R-phycoerythrin (R-PE) by means of absorption, fluorescence and circular dichroism spectra, together with analysis of its crystal structure. R-PE presents stronger functional stability in the pH range of 3.5-10 compared to the structural stability. Beyond this range, pronounced functional and structural changes occur. Crystal structure analysis shows that the tertiary structure of R-PE is fixed by several key anchoring points of the protein. With this specific association, the fundamental structure of R-PE is stabilized to present physiological spectroscopic properties, while local variations in protein peptides are also allowed in response to environmental disturbances. The functional stability and relative structural sensitivity of R-PE allow environmental adaptation.     

海洋红藻多管藻R—藻红蛋白的体外聚集特性

将海洋红藻R 藻红蛋白 (R PE)吸附到刚揭开的高定向石墨 (HOPG)表面上 ,然后用扫描隧道显微镜 (STM)在纳米尺度上进行直接观察 ,发现纯化的R 藻红蛋白在体外自然聚集时 ,能够“面对面”的聚集在一起 ,形成非常规则的类似藻胆体的杆状结构。将R PE溶于 2 %酒精 /水的混合液中 ,然后滴加于空气 /水界面上 ,具有很好的成膜性能。STM观察结果表明 ,R PE的分子在Langmuir Blodgett膜中的排列方式与其在自然状态下的聚集方式类似 ,圆盘状的R PE分子面对面的聚集在一起形成类似藻胆体的杆状结构 ,这些“杆”状结构进一步聚集在一起形成膜。以上结果表明 ,藻胆蛋白分子在体内以藻胆体的形式存在 ,除了连接肽的作用之外 ,藻胆蛋白自身的结构特性而导致的分子与分子之间的相互作用 ,在藻胆体的组装过程中也起着重要的作用     

Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry

BACKGROUND: Phycobiliproteins play an important role in fluorescent labeling, particularly for flow cytometry. The spectral properties of R-phycoerythrin (R-PE) and allophycocyanin (APC) have made them the dominant reagents in this class of fluorochromes. In this study, we evaluate a lesser-known but potentially important series of low-molecular weight cryptomonad-derived phycobiliproteins (commercially termed the CryptoFluortrade mark dyes) for their applicability to flow cytometry, both in extracellular and intracellular labeling applications. METHODS: Several cell lines were labeled with biotin-conjugated antibodies against expressed extracellular surface proteins, followed by streptavidin conjugates of three cryptomonad phycobiliproteins (CryptoFluor-2, CryptoFluor-4, and CryptoFluor-5). Cells were then analyzed by flow cytometry using a variety of laser lines and emission filters to establish the optimal excitation/emission characteristics for each fluorochrome. Some cells were permeabilized and labeled for intracellular antigens, also using the cryptomonad fluorochromes. Where appropriate, parallel samples were labeled with other fluorochromes (including R-PE, APC, the cyanin dyes Cy3 and Cy5, and others) to gauge the performance of the cryptomonad fluorochromes against fluorescent labels previously evaluated for flow cytometry. RESULTS: CryptoFluor-2 possessed excitation/emission maxima similar to those of APC and Cy5, with good excitation in the red (HeNe laser 632 nm) and strong emission in the far red (660 nm). CryptoFluor-4 possessed excitation/emission maxima similar to those of Cy3, with optimal excitation in the green (Kr 530 nm) and strong emission in the yellow/orange (585 nm). CryptoFluor-5 possessed excitation/emission maxima similar to those of lissamine rhodamine, with optimal excitation in the yellow (Kr 568 nm) and emission in the orange (610 nm). All cryptomonad fluorochromes gave satisfactory results for both intracellular and extracellular labeling, with detection sensitivities that were comparable or better than traditional phycobiliproteins and low- molecular weight synthetic fluorochromes such as the cyanin dyes. CONCLUSIONS: The CryptoFluor fluorochromes were applicable to flow cytometric immunodetection, with excitation and emission conditions commonly found on multilaser instruments. Performance of several of these dyes was at least comparable to existing fluorescent labels. The low molecular weights (30-60 kd) of phycobiliproteins may make them particularly useful in intracellular antigen detection. Cytometry 44:16-23, 2001. Published 2001 Wiley-Liss, Inc.     

Extracting and purifying R-phycoerythrin from Mediterranean red algae Corallina elongata Ellis & Solander

R-Phycoerythrin (R-PE) is a protein acting as a photosynthetic accessory pigment in red algae (Rodophyta). This protein has gained importance in many biotechnological applications in food science, immunodiagnostic, therapy, cosmetics, protein and cell labelling, and analytical processes. In this paper we report on a new, one step procedure for the extraction and purification of R-PE from a new source: the Mediterranean red algae Corallina elongata Ellis & Solander. This red algae contains mainly R-PE and is suitable for the production in culture. No other contaminating phycobiliproteins could be detected in the extracts. The method we propose for the purification is based on the use of hydroxyapatite, a chromatographic resin that can be produced in the laboratory at very low cost and can be used batch-wise with large amounts of extracts, alternative to chromatography, and therefore can be scaled up. Both the yield and the purity of R-PE are very good.     

Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions

Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (lambda>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (1O2) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of 1O2. In addition, the generation of 1O2 was verified further in the presence of D2O and specific singlet oxygen quencher 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN3). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of 1O2 by PBPs are as follows: APC > C-PC > R-PE.     

R-藻红蛋白介导的光敏反应对DNA分子的生物学效应

藻红蛋白(phycoerythrin, PE)是海藻中的重要捕光色素蛋白,具有强荧光性,易溶于水.在藻体内能将捕获的光能传递给光合反应中心; 在体外则能将光能传递给周围环境中的氧分子,产生如单线态氧等活性氧组分,可用来介导光动力效应治疗癌症.将纯化的藻红蛋白加入到瘤细胞培养基中,数小时后,采用488 nm波长的氩离子激光辐照,MTT法检测细胞存活数,计算细胞存活率. ³H-TdR掺入实验观察细胞DNA的合成.结果表明,藻红蛋白介导的光动力反应能够有效地抑制肿瘤细胞DNA合成并杀伤癌细胞.随着藻红蛋白浓度增加,DNA合成下降,瘤细胞存活率降低.将藻红蛋白加入到pUC18质粒溶液中,随之进行激光辐照,琼脂糖电泳结果可见pUC18构象由超螺旋（supercoiled）向带切口的环形构象(relax)转换.结果提示：通过改变或影响DNA构象,抑制细胞DNA合成可能是藻红蛋白介导肿瘤光动力治疗的途径之一.     

二氢杨梅素对人胃癌MKN45细胞迁移和侵袭的影响及其机制*

目的:探讨二氢杨梅素(DHM )对人胃癌MKN45细胞迁移和侵袭的作用及其分子机制。方法:培养人低分化胃癌MKN45细胞,用不同浓度的DHM(0,10,20,30,40,50 μmol/L)分别处理细胞24及48 h,每组实验重复3次,采用CCK8实验检测癌细胞增殖活力;划痕实验检测细胞迁移能力;Transwell小室检测细胞侵袭能力;免疫印迹分析细胞迁移和侵袭相关蛋白表达情况。结果:不同浓度DHM干预可降低MKN45细胞活力。20,30及40 μmol/L的DHM处理48 h可明显抑制细胞的迁移能力(P＜0.01)和侵袭能力(P＜0.05及0.01)。20及30 μmol/L的DHM处理48 h可增加E-cadherin蛋白表达(P＜0.01)、降低Vimentin表达(P＜0.01),从而逆转EMT过程;10,20及30 μmol/L的DHM处理48 h可明显降低pJNK的活性表达水平(P＜0.05及0.01),及MMP-2蛋白表达(P＜ 0.01);JNK通路特异性抑制剂SP600125预处理可明显促进DHM对癌细胞侵袭能力的抑制作用(P＜0.01)及降低MMP-2表达(P＜0.01)。结论:DHM具有抑制人胃癌MKN45细胞的迁移及侵袭的作用,其机制可能与通过JNK通路下调MMP-2蛋白表达水平、逆转上皮间质转化有关。     

Optimization of IgG conjugation with R-phycoerythrin from <Emphasis Type="Italic">Porphyra yezoensi</Emphasis>s

To optimize the chemical conjugation between R-phycoerythrin (R-PE) and antibody, different molar ratios of the heterobifunctional reagent N-succinimidyl-3-2-pyridyldithio propionate (SPDP) to R-PE were tested for R-PE derivation into R-PE-PDP, and different molar ratios of dithiothreitol (DTT) to IgG were tested for IgG thiolation. The results showed that in terms of best product yields determined by ultraviolet (UV) spectrophotometry, the optimal molar ratio of SPDP to R-PE was 40:1 for PE derivation, and that of DTT to IgG was 500:1 for thiolation. R-PE-labeled secondary antibody was produced by cross-linking PE-PDP and thiolated IgG. After further purification, UV spectra and native polyacrylamide gel electrophoresis determined its high purity and molecular weight. Finally, in conjunction with antigen-specific first antibodies, the R-PE-labeled IgG was applied in fluorescence immunoassays as secondary antibody and successfully detected antigens spotted on nitrocellulose membrane as well as intracellular antigen in SMCC-7721 cells. This study provides a feasible method of fluorescence antibody preparation from R-PE of Porphyra yezoensis and demonstrates high fluorescent labeling efficiency and good immunologic reactivity of the product.     

Oncolytic viral therapy for human colorectal cancer and liver metastases using a multi-mutated herpes simplex virus type-1 (G207).

G207 is a multi-mutated, replication-competent type-1 herpes simplex virus designed to target, infect, and lyse neurological tumors. This study examines the feasibility of using G207 in the treatment of human colorectal cancer and defines the biological determinants of its antitumor efficacy. This virus was tested on five human colorectal cancer cell lines in vitro to determine efficacy of infection and tumor cell kill. These results were correlated to measures of tumor cell proliferation. In vivo testing was performed through direct injections of G207 into xenografts of human colorectal cancer tumors grown in flanks of athymic rats. To evaluate an alternate method of administration, hepatic portal vein infusion of G207 was performed in a syngeneic model of liver metastases in Buffalo rats. Among the five cell lines tested, infection rates ranged between 10% and 90%, which correlated directly with S-phase fraction (8.6%-36.6%) and was proportional to response to G207 therapy in vitro (1%-93%). Direct injection of G207 into nude rat flank tumors suppressed tumor growth significantly vs. control (0.58 +/- 0.60 cm(3) vs. 9.16 +/- 3.70 cm(3), P<0. 0001). In vivo tumor suppression correlated with in vitro effect. In the syngeneic liver tumor model, portal infusion resulted in significant reduction in number of liver nodules (13 +/- 10 nodules in G207-treated livers vs. 80 +/- 30 nodules in control livers, P<0.05). G207 infects and kills human colorectal cancer cells efficiently. In vitro cytotoxicity assay and tumor S-phase fraction can be used to predict response to treatment in vivo. This antineoplastic agent can be delivered effectively by both direct tumor injection and regional vascular infusion. G207 should be investigated further as therapy for colorectal cancer and liver metastases.