坛紫菜R-藻蓝蛋白和变藻蓝蛋白的亚基组成和发色团含量

坛紫菜中的Ｒ－藻蓝蛋白在Ｂｉｏ－Ｒｅｘ７０柱上用脲溶液（ｐＨ３．０）解离和层析，得到了α和β两个亚基。用ＳＤＳ－ＰＡＧＥ测定α和β亚基的分子量分别为１８４００和２０５００，变藻蓝蛋白的α和β两亚基的分子量经测定，分别为１８８００和１９７００。Ｒ－藻蓝蛋白和变藻蓝蛋白的分子量分别确定为１１７０００和１２２０００，两藻胆蛋白中α和β两个亚基的摩尔比都为１：１，确定两藻胆蛋白的亚基组成都为（αβ）３。各     

坛紫菜R-藻蓝蛋白和变藻蓝蛋白的亚基组成和发色团含量

坛紫菜(Porphyra haitanensis T.J.Chang et B.F.Zheng)中的R-藻蓝蛋白在Bio-Rex 70柱上用脲溶液(pH3.0)解离和层析,得到了α和β两个亚基,用SDS-PAGE测定α和β亚基的分子量分别为18 400和20 500。变藻蓝蛋白的α和β两亚基均分子量经测定,分别为18 800和19 700。R-藻蓝蛋白和变藻蓝蛋白的分子量分别确定为117 000和112 000,两藻胆蛋白中α和β两个亚基的摩尔比都为1:1,确定两藻胆蛋白的亚基组成都为(αβ)_3。各亚基的发色团含量为:R-藻蓝蛋白中,α亚基含1个藻蓝胆素,β亚基含1个藻蓝胆素和1个藻红胆素。变藻蓝蛋白中,α和β亚基各含1个藻蓝胆素。     

藻蓝蛋白及其酶解产物的抗肿瘤效应研究进展

从海洋生物中寻找高效、低毒副作用的抗肿瘤药物是近年天然药物研究的新领域。随着海洋生物药学研究的不断深入,发现许多海洋生物的活性物质源于其食物。因此对于海洋生物食物链基层藻类的生物活性物质研究,更具有实际的意义。藻蓝蛋白(C-phycocyanin,C-PC)是存在于一些藻体中的光合色素蛋白,具有抗肿瘤、抗病毒、抗氧化等多种生物学功能,有重要的开发利用价值。国内外关于藻蓝蛋白的抗肿瘤作用及其机制的研究已有报道,而酶解后的藻蓝蛋白多肽抗肿瘤效应研究国内外报道很少。随着酶工程技术的发展和应用,利用蛋白酶水解藻蓝蛋白获取生物活性肽更具研究开发潜力,围绕藻蓝蛋白及其酶解产物的抗肿瘤活性及机制进行了综述。     

钝顶螺旋藻C-藻蓝蛋白和多管藻R-藻红蛋白的分离纯化及摩尔消光系数的测定

１９９３年１１月￣１９９４年１月用简单的羟基磷灰石柱层析法从多管藻中分离纯化了Ｒ－藻红蛋白和从钝顶螺旋藻中纯化了Ｃ－藻蓝蛋白，它们的纯度（指可见光部分的最大吸收与２８０ｎｍ处吸收值之比）可分别达到６（Ｒ－藻红蛋白）和５．５（Ｃ－藻蓝蛋白）。由于不同藻种的同种藻胆蛋白的摩尔消光系数不同，所以应用凯氏定氮法并结合可见光的吸收值测定了上述两种藻胆蛋白的摩尔消光系数，钝顶螺旋藻Ｃ－藻蓝蛋白为１．８５３×１     

螺旋藻藻蓝蛋白对酒精性肝损伤大鼠肠道菌群的影响

酒精性肝病(alcoholic liver disease, ALD)是中国常见的肝脏疾病之一, 严重危害人民健康。目前, 缺乏治疗酒精性肝病的有效方法。螺旋藻藻蓝蛋白(phycocyanin, PC)是一种天然蛋白质, 具有抗炎、抗氧化等作用。本文研究了PC对大鼠酒精性肝损伤的保护作用及其对肠道菌群的影响。利用酒精诱导酒精性肝损伤模型, 将大鼠随机分为4组, 分别为对照组, 模型组, 阳性对照组, 藻蓝蛋白组, 灌胃PC干预之后, 通过肝脏切片的Masson和HE染色的病理分析判断肝损伤的程度, 之后通过16S rRNA高通量测序技术检测大鼠肠道菌群的组成和结构。模型组大鼠肠道菌群发生了紊乱, 显著降低了双歧杆菌(Bifidobacteriaceae)的丰度, 该菌种可以减少肝脏炎症和肝酶, 口服PC后Bifidobacteriaceae的丰度显著增加。此外, 酒精诱导后, 乳酸杆菌(Lactobacillaceae)的丰度略微增加, 促进酒精代谢。藻蓝蛋白能够通过改变酒精性肝损伤大鼠的肠道菌群构成, 从而起到缓解酒精诱导的肝损伤的效果。     

钝顶螺旋藻C-藻蓝蛋白和多管藻R-藻红蛋白的分离纯化及摩尔消光系数的测定

１９９３年１１月～１９９４年１月用简单的羟基磷灰石柱层析法从多管藻中分 离纯化了Ｒ－藻红蛋白和从钝顶螺旋藻中纯化了Ｃ－藻蓝蛋白，它们的纯度（指可见 光部分的最大吸收与 ２８０ｎｍ处吸收值之比）可分别达到 ６（Ｒ－藻红蛋白）和 ５． ５（Ｃ－ 藻蓝蛋白）。由于不同藻种的同种藻胆蛋白的摩尔消光系数不同，所以应用凯氏定 氮法并结合可见光的吸收值测定了上述两种藻胆蛋白的摩尔消光系数，钝顶螺旋 藻Ｃ－藻蓝蛋白为１．８５３ × １０６ｍｏｌ－１ｃｍ－１（６２０ｎｍ），多管藻Ｒ－藻红蛋白为１．７９６ × １０６ｍｏｌ－１ｃｍ－１（４９８ｎｍ），为藻胆蛋白的浓度测定提供了方便。     

光学活性藻蓝蛋白β亚基的体内重组

在Synechococcus sp.PCC 7002中,首先发现了特异性催化脱辅基藻蓝蛋白β亚基(153位)与藻蓝胆素(phycocyanobiling, PCB)连接的裂合酶基因cpcT.本实验在Synechocystis sp.PCC 6803中找到与cpcT具有高度同源性的基因slr1649.构建2组相容的重组质粒pCDF-cpcB(C82I)或(C153I)-ho1-pcyA和pRSF-slr1649,cpcB(C82I)和cpcB(C153I)分别编码82和153位被突变的脱辅基藻蓝蛋白β亚基,slr1649编码可能的特异性裂合酶,在大肠杆菌体内生成PCB必需的2个基因(ho1编码血红素氧化酶,pcyA编码藻蓝胆素合成酶),将这些基因共同转化到大肠杆菌诱导表达,利用亲和层析柱对可溶性蛋白进行纯化,产物进行SDS-PAGE、色素蛋白锌电泳和光谱检测.结果表明基因slr1649能够特异性催化CpcB(153位)与PCB的连接,产生了具有光学活性的CpcB(C82I)-PCB,为进一步研究螺旋藻藻蓝蛋白的生物活性奠定了基础.     

藻蓝蛋白对博来霉素肺纤维化小鼠肠道菌群的影响

博来霉素(bleomycin,BLM)是目前用于治疗头颈癌等多种癌症的药物之一,但会产生一系列副作用,例如肺纤维化(pulmonary fibrosis,PF),这大大降低了患者的生活质量。藻蓝蛋白(phycocyanin,PC)作为一种色素蛋白对包括PF在内的多种疾病具有调节作用。研究发现,慢性肠道疾病通常伴有肺部疾病,患者的肠道菌群会发生紊乱。针对上述线索,研究了PC对BLM诱导的PF小鼠肠道菌群的影响。通过尾静脉将BLM注入小鼠体内,以构建肺纤维化模型。然后通过16S r RNA高通量基因测序技术,分析了PC对BLM引起的PF小鼠肠道菌群的影响。结果表明,PC降低了BLM诱导的PF程度并调节了小鼠的肠道菌群。通过尾静脉注射BLM后,小鼠肺部受到损伤,肠道菌群发生紊乱。口服藻蓝蛋白干预后,小鼠的肺组织切片显示其胶原蛋白沉积减少,肺泡间隔和炎性细胞浸润减少,小鼠PF程度减轻。此时,小鼠肠道菌群中Alistipes、Lachnospiraceae等有益菌的丰度增加,Rikenella等有害菌的丰度降低,肠道菌群趋于正常。推测PC可以通过增加肠道中有益细菌的数量并减少肠道中有害细菌的数量...     

钝顶螺旋藻藻蓝蛋白提取纯化新工艺

对钝顶螺旋藻（Spirulina platensis）中藻蓝蛋白的提取和纯化方法进行了改进。用磷酸盐缓冲液循环冻融联合超声波破碎法,50％硫酸铵沉淀获得藻蓝蛋白（phycocyanin,PC）,提取率达到13．1%。粗蛋白提取液再经过两次羟基磷灰石柱（HA）层析和sephacryl-HR-200凝胶层析对其进行纯化,纯度达到4．71％。纯化后的PC在12％十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中得到两条带,分别是α和β两个亚基,分子质量分别为21．4ku和17．0ku。结果表明,通过上述分离纯化过程得到了较高提取率和电泳纯度的藻蓝蛋白。     

乳糖诱导重组别藻蓝蛋白基因在大肠杆菌中的表达

以重组别藻蓝蛋白表达工程菌JM109（DE3）／pET28-APC作为研究对象，对于JM109（DE3）菌株在乳糖诱导下表达重组蛋白的规律进行了研究。比较分析了不同生长阶段进行诱导，最佳乳糖浓度、诱导持续时间和诱导温度等参数对重组蛋白表达的影响。实验结果表明，采用JM109（DE3）为宿主菌，经过条件优化，乳糖诱导重组蛋白的表达量可以达到IPTG诱导的水平；较低的诱导培养温度能有效提高可溶性重组蛋白的表达。最后在摇瓶发酵结果的基础上，实现了乳糖诱导工程菌的5L全自动发酵罐培养。研究结果为在大肠杆菌大规模发酵中，运用廉价、无毒的乳糖作为诱导剂提供了一定的借鉴。     

藻蓝蛋白对四氯化碳诱导的大鼠肝纤维化及肠道微生物的影响

为开发藻类活性物质的药用价值,探究了藻蓝蛋白(phycocyanin,PC)对四氯化碳(CCl4)诱导的大鼠肝纤维化的影响.试验中将大鼠随机分为3组,分别为对照组、肝纤维化组、CCl4+PC干预组.利用CCl4腹腔注射诱导肝损伤模型,在干预后,通过RT-PCR技术检测了大鼠肝脏中纤维化标志物I型胶原蛋白(collage...     

Protective effects of Ecklonia cava extract on the toxicity and oxidative stress induced by hair dye in in-vitro and in-vivo models

Oxidative hair dyes containingρ-phenylenediamine(PPD)are reported to induce an allergic reaction by promoting oxidative stress when absorbed through the skin.Despite the associated risk,these hair dyes remain popular owing to their convenience and sharpness of color.This makes it important to minimize the cytotoxicity and oxidative stress induced by PPD-containing hair dyes.Ecklonia cava extract has been evaluated in different studies for its protective effects against external stress in fibroblasts and keratinocytes.Our study was aimed at using in-vitro and in-vivo models to investigate the extract’s effects on cytotoxicity of and oxidative stress induced by PPD-containing hair dyes.Analysis of CIEL*a*b*Color space was first used to determine the range of E.cava extract that would not interfere with the coloring ability of the dye upon addition.Subsequently,the set ranges of E.cava extract(5% and 7%)were added to the hair dye and their toxicity assessed by evaluating the viability of fibroblasts and keratinocytes.The effects on developmental phenotypes and induction of oxidative stress by hair dye were evaluated and compared with those of hair dyes containing different contents of E.cava extract using an in-vivo zebrafish model.Our results showed that E.cava extract in hair dye could significantly decrease the cytotoxicity and levels of oxidative stress caused by hair dyes containing PPD in both in-vitro and in-vivo models.These results suggest that the addition of 7% E.cava extract to 250μg/mL hair dye does not interfere with the coloring ability of the dye while showing significant protective eff ects against the hair dye.The study proposes that the use of E.cava extract as an adduct to hair dyes containing PPD reduces the cytotoxicity and oxidative stress induced by these hair dyes.     

蓝隐藻藻蓝蛋白结构与功能稳定性研究

以蓝隐藻(Chroomonas placiodea)藻蓝蛋白 PC-645为材料,通过改变环境 pH 和尿素质量浓度,监测其变性和复性过程中特征荧光谱和吸收谱的动力学变化,以期了解隐藻藻蓝蛋白的色基和蛋白结构稳定性及其与功能的关系。结果表明, PC-645在很宽的 pH 范围和一定质量浓度的尿素中维持结构和功能的稳定,空间结构具有很强的柔性。pH诱导的 PC-645蛋白构象与功能变化可分为3个不同的区段。(1)稳定区(pH 3.5~7)：吸收和荧光光谱都比较稳定,显示蛋白质构象和功能在此区域都保持正常。(2)次稳定区(pH 7~10)：光吸收依然保持平稳,亚基内部的色基的状态和疏水微环境都没有改变,但荧光传递效率降低,可能是由亚基表面局部构象变化、解离(四级结构变化)或者色素基团间的空间距离变化引起。(3)不稳定区(pH<3.5和 pH>10),吸光度和荧光强度都呈快速下降,色基在近紫外和可见光区的吸收峰位变动,蛋白构象处于快速崩溃期。PC-645在酸性条件下的稳定性高于在碱性环境下,是与隐藻藻蓝蛋白所处的特殊环境及生理功能相适应的。     

隐藻藻蓝蛋白与类囊体膜的动态结合模型

隐藻藻胆蛋白在类囊体腔中的存在状态始终存在争议,为了深入了解这一问题,作者以含有藻蓝蛋白(PC645)的蓝隐藻(Chroomonas placoidea)活体细胞及充分洗涤过的类囊体膜为材料,测定其室温吸收光谱、荧光谱以及77K低温荧光光谱,并根据光谱数据分析了PC在类囊体腔中的存在状态。结果显示,蓝隐藻的类囊体膜上始终紧密结合着一定量的PC,并且很可能是以PC的β亚基与膜相接触,证实藻胆蛋白在类囊体腔中不是完全游离的,与膜的接触也并非完全无倾向性排布;PC既可将激发能传递给光系统(PS)II,但也可传递给PSI,相较于叶绿素(Chl)a和Chl c,在PSI长波荧光中PC的贡献更明显,而Chl c吸收的光能,更倾向于传递给PSII或者短波长Chl a;蓝隐藻细胞中表现出超比例的PC荧光发射,说明除了结合在膜上的PC外,有一部分PC是游离的,其存在对于隐藻捕光功能来说可能是有冗余的;游离的PC可与类囊体膜重新接触而恢复能量传递功能。根据实验结果以及前人的报道,提出了隐藻藻胆蛋白与类囊体膜的结合是动态的观点,并给出了相应的模型。     

Ship-of-opportunity based phycocyanin fluorescence monitoring of the filamentous cyanobacteria bloom dynamics in the Baltic Sea

Distribution of cyanobacteria cannot be evaluated using chlorophyll a (Chla) in vivo fluorescence, as most of their Chla is located in non-fluorescing photosystem I. Phycobilin fluorescence, in turn, is noted as a useful tool in the detection of cyanobacterial blooms. We applied phycocyanin (PC) fluorometer in the monitoring of the filamentous cyanobacterial bloom in the Baltic Sea. For the bloom forming filamentous cyanobacteria Aphanizomenon flos-aquae and Nodularia spumigena, PC fluorescence maximum was identified using the excitation–emission fluorescence matrix. Consequently, the optical setup of our instrument was noted to be appropriate for the detection of PC, and with minor or no interference from Chla and phycoerythrin fluorescence, respectively.During summer 2005, the instrument was installed on a ferryboat commuting between Helsinki (Finland) and Travemünde (Germany), and data were collected during 32 transects providing altogether 200 000 fluorescence records. PC in vivo fluorescence was compared with Chla in vivo fluorescence and turbidity measured simultaneously, and with Chla concentration and biomass of the bloom forming filamentous cyanobacteria determined from discrete water samples.PC fluorescence showed a linear relation to the biomass of the bloom forming filamentous cyanobacteria, and the other sources of PC fluorescence are considered minor in the open Baltic Sea. Estimated by PC fluorescence, cyanobacterial bloom initiated late June at the Northern Baltic Proper, rapidly extended to the central Baltic Proper and the Gulf of Finland, and peaked in the mid-July with values up to 10 mg l⁻¹ (fresh weight). In late July, bloom vanished in most areas.During single transects, or for the whole summer, the variability in Chla concentrations was explained more by PC fluorescence than by Chla fluorescence. Thus, filamentous cyanobacteria dominated the overall variability in phytoplankton biomass. Consequently, we show that during the cyanobacterial blooms, the estimation of Chla concentration using only Chla in vivo fluorescence is not applicable, but PC in vivo fluorescence is required as a predictor as well.     

重组藻蓝蛋白与水稻类囊体膜之间光能传递的初步研究

为了研究藻蓝蛋白和高等植物类囊体膜之间的光能传递,本实验将诱导表达的重组藻蓝蛋白和水稻类囊体混合孵育后,检测其77K荧光发射光谱以及类囊体膜的电子传递速率和光化学量子产量,结果表明重组藻蓝蛋白和水稻类囊体膜光系统Ⅰ之间存在能量传递使得光化学量子产量提高.本研究为利用藻胆蛋白构建新型光合系统提供了理论基础和实验依据.     

Seasonal changes of antioxidant and oxidative parameters in the coral Pocillopora capitata on the Pacific coast of Mexico

The physiological responses of the coral Pocillopora capitata to environmental conditions common in winter and summer were studied in 2007 during February–March (winter) and June–July (summer) at La Boquita reef (Manzanillo, Colima, Mexico). Shallow and deep sampling stations were established at different distances from a small jetty built next to the Juluapan Lagoon. We analyzed superoxide radicals () and lipid peroxidants (TBARS); the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione‐S‐transferase (GST); chlorophyll a (Chl a), zooxanthellae density (ZD); and mycosporine‐like amino acids (MAAs). Our results showed that the , TBARS, CAT, GST, MAAs, and Chl a, levels were significantly higher in summer (P < 0.05); no seasonal difference was found for GPx, GR or ZD. We found significant differences (P < 0.05) in winter only for Chl a and ZD at shallow sites and, in contrast, for at deeper sites. The results of this study indicate that increasing temperature and radiation associated with seasonal changes (from winter to summer), the efficiency of the enzymes GST, CR and GPX, and the production of MAAs together form a powerful mechanism for P. capitata to offset the detrimental effects of environmental change.