A sensitive fluorometric technique for the measurement of phycobilin pigments and its application to the study of marine and freshwater picophytoplankton in oligotrophic environments

A sensitive and specific technique is described for the estimation of phycobiliprotein in freshwater and marine picophytoplankton. The method uses fluorescent properties to detect phycoerythrin concentrations as low as 40 ng L-1 from a 1 L water sample and is capable of distinguishing between R-phycoerythrin, C-phycocyanin and C-phycoerythrin. The application of the method to the study of natural picophytoplankton populations in marine and freshwater environments is described. Nitrate concentrations appear to influence picophytoplankton cellular C-phycoerythrin concentrations in surface waters and increasing cellular C-phycoerythrin fluorescence with water depth suggests that this pigment plays a role as a photosynthetic accessory pigment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation, properties and spatial site analysis of γ subunits of B-phycoerythrin and R-phycoerythrin

Polysiphonia urceolata R-phycoerythrin andPorphyridium cruentum B-phycoerythrin were degraded with proteinaseK, and then the nearly native γ subunits were isolated from the reaction mixture. The process of degradation of phycocrythrin with proteinaseK showed that the γ subunit is located in the central cavity of (αβ)₆ hexamer of phycoerythrin. Comparative analysis of the spectra of the native phycoerythrin, the phycoerythrin at pH 12 and the isolated γ subunit showed that the absorption peaks of phycoerythrobilins on α or β subunit are at 535 nm (or 545 nm) and 565 nm, the fluorescence emission maximum at 580 nm; the absorption peak of phycoerythrobilins on the isolated γ subunit is at 589 nm, the fluorescence emission peak at 620 nm which overlaps the absorption maximum of C-phycocyanin and perhaps contributes to the energy transfer with high efficiency between phycoerythrin and phycocyanin in phycobilisome; the absorption maximum of phycourobilin on the isolated γ subunit is at 498 nm, which is the same as that in native phycoerythrin, and the fluorescence emission maximum at 575 nm.     

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.     

Ultrastructural and microspectrophotometric characterization of multiple species of cyanobacterial photosymbionts coexisting in the colonial ascidian Trididemnum clinides (Tunicata,Ascidiacea, Didemnidae)

Trididemnum clinides is a multi-photosymbiotic ascidian that inhabits shallow coral reef lagoons. Three types of cyanobacteria are harboured in the tunic of the ascidian colony; of these, two are unicellular coccoid cyanobacteria and the other is a multicellular filamentous type. They also differ in ultrastructure and distribution patterns within the host tunic. Microspectrophotometric analysis revealed the composition of photosynthetic pigments in each photosymbiont. One of the coccoid types is yellowish-green and is distributed under the colony surface. This photosymbiont cell preferentially absorbs red and blue light, and therefore the dominant colour in the inner tunic is green. The other two types of coexisting photosymbionts contain the green-light-absorbing R-phycoerythrin as the major photosynthetic pigment; they exploit the wavelengths of light not used by the first type of photosymbiont. In T. clinides, the outer and inner photosymbionts in the tunic have different photosynthetic pigments, which adapt to each microhabitat, thereby sharing the incident light resources effectively.     

Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica

R-phycoerythrin is one of the three phycobiliproteins which are extensively employed as fluorescent probes, and it is prepared from red macroalgae. Phycobiliproteins in the marine red macroalga Heterosiphonia japonica were extracted in 50 mM phosphate buffer (pH 7.0) and precipitated by salting-out. The R-phycoerythrin was isolated by gel filtration with Sepharose CL-4B and Sephadex G-200. Then it was purified by ion exchange chromatography on DEAE Sepharose Fast Flow which was developed by linear ionic strength gradients. The purified R-phycoerythrin gave a ratio of A₅₆₅ to A₂₈₀ of 4.89. It showed a single band and a pI of 4.8 on the examination by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. The polypeptide analysis of the purified R-phycoerythrin by SDS–PAGE demonstrated that it contains four chromophore-carrying subunits and no colorless polypeptide and has two hexameric aggregates. The preparative procedures of the R-phycoerythrin purification established based on the experiments exhibit advantages and can offer a reference for R-phycoerythrin preparation from other marine red macroalga.     

荧光染料R-藻红蛋白标记小鼠抗人CD4单克隆抗体

以R-藻红蛋白(R-PE)标记小鼠抗人CD4单克隆抗体,形成单色或和用其他荧光染料标记的CD系列单抗组成双色、多色的荧光试剂,应用于流式细胞仪检测分析。用异双功能交联试剂SPDP和SMCC分别活化R-PE和CD4单抗,用DTT使经SPDP活化后的R-PE巯基化,再与用SMCC活化的CD4单抗交联。使用NEM终止交联反应,经Sephacryl S-300柱在AKTA FPLC快速液相色谱系统(简称AKTA)监测下分离纯化。结果用R-PE标记的抗CD4单抗,检测正常人外周血淋巴细胞表面CD4抗原的表达,经流式细胞仪(简称FACS)分析表明,R-PE标记的CD4抗体特异性保持完好,荧光强度较高,还可与用FITC标记的其它CD系列单抗配伍成双标或多标试剂。使用SPDP,SMCC异双功能交联试剂和DTT还原剂,成功地偶联了R-藻红蛋白和CD4单克隆抗体,可应用于流式细胞仪检测分析。     

用膨化柱填料的方法从红藻中规模分离纯化R-藻红蛋白

以Phenyl-sepharose作为柱填料,用streamline柱层析技术从红藻(Palmaria palmata (Lannaeus) Kuntze)中规模分离捕光色素蛋白--R-藻红蛋白.由于不是传统的从层析柱上方进样,而是用泵将样品从streamline层析柱的下方加样(从下至上),因而解决了用一般层析柱分离R-藻红蛋白时海藻抽提液中大量的粘性多糖堵塞层析柱的难题.用P.palmata粗提液上样后,分别用0.2 mol/L、 0.1 mol/L和0.05 mol/L的(NH4)2SO4溶液从相反的方向(即从上到下)洗脱层析柱,发现这些洗脱液中的藻红蛋白纯度已经较高.然后将洗脱液透析去盐,用阴离子交换柱层析(Q-sepharose)进一步纯化.经过这两次柱层析后,R-藻红蛋白的纯度(OD565/OD280)超过3.5,高于一般认可的R-藻红蛋白的纯度标准3.2;产率为每克冷冻P.palmata可纯化0.122 mg高纯度的R-藻红蛋白,比使用一般分离方法的产率要高10倍.这些结果表明,使用本文报道的方法纯化藻红蛋白,将会使作为生化检测试剂的藻红蛋白市场价格大幅度下降.     

Some observations on phycobilisomes of Pterocladiella capillacea (Gelidiaceae,Rhodophyta)

ABSTRACT

Phycobilisomes (PBSs) of the red alga Pterocladiella capillacea collected in the field, were characterized both in situ and in vitro by means of a transmission electron microscope (TEM) and an image analyzer. Ultrathin sections of thalli and negatively stained PBSs after isolation revealed hemi-ellipsoidal shapes. In situ PBS dimensions were 38.5 ± 0.2 nm (height) × 38.8 ± 0.2 nm (width) × 22.6 ± 0.2 nm (thickness) in good agreement with the in vitro measurements (mean diameters of 37.6 ± 0.2 nm). These dimensions, especially the width, are smaller than those so far reported for red algae. This could depend either on the ecophysiological conditions of the thalli when harvested and/or on a staggered, symmetrically rotated and compressed disposition of biliprotein rods with respect to the allophycocyanin (APC) core. Hydroxylapatite chromatography of biliproteic extracts and SDS-PAGE electrophoresis revealed that phycoerythrin type R-(λ_max 565 nm>540 nm>498 nm) is formed by α (18.6 kDa), β (19.9 kDa), γ (30.2kDa) and γ′ (33.8 kDa) sub-units. The presence of two γ sub-units suggests that this phycoerythrin is a set of (αβ)₆γ + (αβ)₆γ′ aggregates (R-PE). A spectroscopically distinct form of phycoerythrin with different peak ratios, also found in pure fractions, is thought to be a polydisperse form (the so called r-PE). Similarity of shape and size observed in PBSs both in situ and in vitro, and fluorescence spectral characteristics of PBSs in vitro would indicate a substantial integrity of isolated PBSs. These measurements, if compared with total biliprotein content, would seem to indicate a PE fraction not assembled into PBS. A possible role of phycoerythrin in relation to the ability for a rapid adaptation of this surface species to environmental changes is suggested.     

珊瑚藻R-藻红蛋白γ亚基基因的克隆

根据珊瑚藻(Corallina afficinalis L.)R-藻红蛋白γ亚基N末端部分氨基酸序列(P83592)设计简并引物,结合RACE方法,扩增获得g亚基的全长cDNA序列.结果表明,序列全长为2 308 bp(AY209894),5'非编码区长1 203bp,3'非编码区长145 bp,编码区长960 bp,编码320个氨基酸组成的前体,包含71个氨基酸构成的信号肽和249个氨基酸组成的成熟蛋白.成熟蛋白序列内部存在重复序列与前人的报道一致.珊瑚藻亚基cDNA序列不同克隆子的测序结果表明,g亚基cDNA序列存在不同的3'末端,说明该基因可能存在多个拷贝或存在转录后加工.此外,扩增获得g亚基DNA序列(AY308999),比较表明编码区内部没有内含子存在.本文是对珊瑚藻R-藻红蛋白g亚基基因序列的首次报道.     

Engineering a single-chain Fv antibody to alpha v beta 6 integrin using the specificity-determining loop of a foot-and-mouth disease virus

The αvβ6 integrin is a promising target for cancer therapy. Its expression is up-regulated de novo on many types of carcinoma where it may activate transforming growth factor-β1 and transforming growth factor-β3, interact with the specific extracellular matrix proteins and promote migration and invasion of tumor cells. The viral protein 1 (VP1) coat protein of the O₁ British field strain serotype of foot-and-mouth disease virus is a high-affinity ligand for αvβ6, and we recently reported that a peptide derived from VP1 exhibited αvβ6-specific binding in vitro and in vivo. We hypothesized that this peptide could confer binding specificity of an antibody to αvβ6. A 17-mer peptide of VP1 was inserted into the complementarity-determining region H3 loop of MFE-23, a murine single-chain Fv (scFv) antibody reactive with carcinoembryonic antigen (CEA). The resultant scFv (B6-1) bound to αvβ6 but retained residual reactivity with CEA. This was eliminated by point mutation (Y100bP) in the variable heavy-chain domain to create an scFv (B6-2) that was as structurally stable as MFE-23 and reacted specifically with αvβ6 but not with α5β1, αvβ3, αvβ5, αvβ8 or CEA. B6-2 was internalized into αvβ6-expressing cells and inhibited αvβ6-dependent migration of carcinoma cells. B6-2 was subsequently humanized. The humanized form (B6-3) was obtained as a non-covalent dimer from secretion in Pichia pastoris (115 mg/l) and was a potent inhibitor of αvβ6-mediated cell adhesion. Thus, we have used a rational stepwise approach to create a humanized scFv with therapeutic potential to block αvβ6-mediated cancer cell invasion or to deliver and internalize toxins specifically to αvβ6-expressing tumors.