Arabinogalactan as a carrier for contrast agent in magnetic resonance imaging

Arabinogalactan-Gd-DTPA was synthesized by the reaction of diethylenetriaminepenta-acetic acid (DTPA) bisanhydride with polysaccharide in dry DMSO and characterized by FTIR, elemental analysis and ICP-AES. Its stability was investigated by competition with Ca2+, EDTA, DTPA. The t(1)-relaxivity is 8.06 mmol(-1) . L . s(-1) in D2O, 8.48 mmol(-1) . L . s(-1) in 0.725 mmol . L-1 BSA, respectively. t(1)-weighted MR imaging of rat kidney and liver showed a remarkable enhancement post injection of Arabinogalactan-Gd-DTPA. The results indicate that the arabinogalactan-Gd-DTPA is a potential contrast agent for MRI.

Matrixassisted laser desorption/ionization mass spectral study of saccharides

Matrixassisted laser desorption/ionization (MALDI) mass spectra of saccharides with three common matrixes were studied here. It is shown that Na+ and K+ play important roles in the determination of oligasaccharide molecule weight and the molecular weights (MW) of glucans(dextran) with MW more than 10 000 is determined successfully with the help of column chromatography, By comparing the effects of three matrixes in saccharide analysis and comparing positive-ion and negative-ion matrixassisted laser desorption/ionization mass spectra of oligo- and polysaccharides, the most suitable matrix for saccharide analysis, 2,5-dihydroxylbezonic acid (DHB), is selected and the ion formation processes of saccharides under matrix-assisted laser adsorption/ionization condition are clarified.

Positive- and negative-ion matrix-assisted laser desorption/ionization mass spectrometry of saccharides

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of oligosaccharides and polysaccharides has been investigated in detail, It is demonstrated that cationized species of oligosaccharides, [M+Na](+) and [M+K](+), are dominant products under the MALDI condition, and negative ions of oligosaccharides are not formed to any significant extent in this process, The molecular masses of polysaccharides are similarly determined by positive- and negative-ion MALDI-MS with the help of column chromatography. The distinction between positive- and negative-ion MALDI mass spectra of oligo-and polysaccharides indicates that the MALDI processes for saccharides vary with molecular mass. The matrix plays a more important role in the ionization process for oligosaccharides, while in the desorption process for polysaccharides. (C) 1998 John Wiley & Sons, Ltd.

In vivo growth-inhibition of S 180 tumor by mixture of 5-Fu and low molecular lambda-carrageenan from Chondrus ocellatus

POLYSACCHARIDES; ANTICOAGULANT; SURVIVAL

Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis

A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.

Improved RNA isolation from Laminaria japonica Aresch (Laminariaceae, Phaeophyta)

RNA isolation is difficult in some plants and algae because phenolics, polysaccharides, or other compounds can bind or co-precipitate with RNA, and because the success of RNA isolation can be strain-specific and species-specific. To create an improved RNA isolation protocol for Laminaria japonica Aresch (Laminariaceae, Phaeophyta), four methods for extracting RNA were tested. A cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocol was developed that clearly showed 28S and 18S ribosomal RNA bands and produced RNA with high yield (68 mu g g(-1) fresh weight) and high quality (A (260/280) ratio 1.96 +/- 0.05). The isolated RNA was intact, and RT-PCR analysis confirmed that further molecular application is feasible.

A simple method for DNA extraction from sporophyte in the brown alga Laminaria japonica

A simple method was developed for extracting DNA from brown algae Laminaria japonica, which possess large amounts of acidic polysaccharides. Firstly, the sporophyte were washed by eliminating polysaccaride buffer to remove the polysaccharides and then ground in liquid nitrogen. Secondly, the powders were treated with lysing buffer. Thirdly, KAc was used to eliminate the remaining acidic polysaccharides. The extracted DNA was purified using a chloroform-isoamyl alcohol ( 24: 1 v/v), and precipitated in cold isopropanol. The yield was from 18.7 to 37.5 mu g g(-1) (wet weight) and the purity of total DNA was determined spectrophotometrically as the ratio of A(260)/A(280), which was about 1.7 - 1.9. The extracted DNA was of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion. This method is a reproducible, simple, and rapid technique for routine DNA extraction from sporophyte in Laminaria japonica. Furthermore, the low cost of this method makes it attractive for large-scale studies.

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of total genomic DNA from 15 red algal species. It resulted in 0.1 mug high quality DNA from 1 mg fresh algal material, with an A(260)/A(280) ratio of 1.68 - 1.90. Using this rapidly isolated DNA, the 18S ribosomal RNA genes ( rDNA) and the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. The tested DNA was suitable for restriction endonuclease digestion, genetic marker analysis and polymerase chain reaction (PCR) amplification, and may be valid for other genetic manipulation.

Isolation and purification of phycoerythrin from red alga Garcilaria verrucosa by expanded-bed-adsorption and ion-exchange chromatogaphy

R-phycoerythrin was isolated and purified from Gracilaria verrucosa on an expanded-bed adsorption column combined with ion-exchange chromatography, which can effectively solve the problem of blockage of chromatographic columns due to polysaccharides during isolation and purification of phycobiliproteins. 0.1 M (NH4)(2)SO4 proved best to elute R-phycoerythrin from the expanded-bed column, and desalted 0.1 M (NH4)(2)SO4 eluate was used on an ion-exchange column to purify the R-phycoerythrin. Using this two-stage chromatography, the purity (OD565/OD280) of the R-phycoerythrin from G. verrucosa is increased to 4.4, and the yield of purified R-phycoerythrin can reach 0.141 mg . g(-1) of the frozen alga.

Large-scale isolation and purification of R-phycoerythrin from red alga Palmaria palmata using the expanded bed adsorption method

R-phycoerythrin, a light-harvesting protein in some marine algae, and can be widely used in medicine, was isolated and purified from a red alga, Palmaria palmata (Lannaeus) Kuntze, using the streamline column (expanded bed adsorption) combined with ion-exchange chromatography. Because the crude extract was applied to the column upwardly, the column would not be blocked by polysaccharides usually very abundant in the extract of marine alga, this kind of blockage could hardly lie overcome in ordinary chromatographic column. After applying the crude extract containing 0.5 mol/L (NH4)(2)SO4, (NH4)(2)SO4 solution of different concentrations (0.2 mol/L, 0.1 mol/L and 0.05 mol/L) was used to elute the column downwardly and the eluates were collected and desalted. The desalted eluates were then applied onto all ion-exchange chromatographic column loaded with Q-sepharose for further purification of the R-phycoerythrin. Through these two steps, the purity (OD565/OD280) of the R-phycoerythrin from P. palmata was up to 3.5, more than 3.2, the commonly accepted criterion for purity, and the yield of the purified R-phycoerythrin could reach 0.122 mg/g of frozen P. palmata, much higher than that of phycobiliproteins purified with the previous methods. The result indicated that the cost of R-phycoerythrin will drop down with the method reported in this article.

Algal biotechnology industries and research activities in China

In old China there were very few people engaged in the study of the algae, but in new China, freshwater and marine algae are studied by over one hundred old and new phycologists. There is now an algal biotechnology industry consisting of an aquaculture industry, producing large amounts of the seaweeds Laminaria, Porphyra, Undaria, Gracilaria, eucheumoids, and the microalgae Dunaliella and Spirulina. There is also a phycocolloid industry, producing algin, agar and carrageenan; an industry producing chemicals and drugs, such as iodine, mannitol, phycocyanin, beta -carotene, PSS (propylene glycol alginate sulfate) and FPS (fucose-containing sulfated polysaccharides) and an industry producing food, feed and fertilizer. The Laminaria cultivation industry produces about 900,000 t dry Laminaria, probably the largest producer in the world and 13,000 t algin, undoubtedly one of the largest algin producer in the world.

Chemical species of iodine in some seaweeds II. Iodine-bound biological macromolecules

The distribution of iodine in various biological macromolecules in Sargassum kjellmanianum was studied using neutron activation analysis combined with chemical and biochemical separation techniques. The results indicate that iodine is mainly bound with protein, part of iodine with pigment and polyphenol, and little with polysaccharides, such as algin, fucoidan and cellulose. This result is significant for the mechanism of enriching iodine of algae and utilization of alga iodine.

Purification of High-quality Plastid DNA from the Brown Alga Laminaria japonica Sporophytes

Extracting DNA from a variety of algae is rather difficult because of high levels of polysaccharides, tannins, and phenolics as these interfere with DNA isolation and downstream applications. High-quality plastid DNA (ptDNA) purification is particularly difficult because of its small proportion in total genomic DNA. This report describes an improved protocol for ptDNA purification that efficiently produces high-quality ptDNA from sporophytes of Laminaria japonica and several other algae. This improved protocol simplifies procedures for ptDNA purification and improves yield to 150-200 mu g of ptDNA per 100 g of frozen algal tissue. Polymerase chain reaction (PCR) amplification of conserved sequences has been used to verify purity of the ptDNA product.

羟基自由基降解琼胶及其产物的活性研究

琼胶是一种从石花菜等红藻中提取的，目前生产工艺和结构等方面研究比较成熟的海藻多糖，广泛应用于医药、仪器等行业。但是，海藻多糖因为具有分子量大，粘度大，溶解度较小的等特点，而使其应用范围受到限制。利用降解的手段对其进行修饰，降低分子量和粘度，改善溶解性，可以拓展其应用范围。并且根据文献报道，琼 胶寡糖具有一些特殊的生物活性，如抗氧化性，抗炎症等。因此，对琼胶降解的研究具有生要意义。本研究中，为了选择一种合适的降解方法，进行了几种水解方法的尝试，其中包括在不同湿度和酸度下盐酸水解，过氧化氢和醋酸催化水解，Fenton体系羟基自由基降解。对于酸水解和Fenton体系氧化还原降解方法，通过粘度法对反应的速度进行了比较，表明氧化还原降解反应中琼胶的粘度降低比较快，并且具有代表性和新意，确定为本实验的降解琼胶的方法并对氧化还原降解所得的产物进行了活性实验。通过模仿自然界普遍存在的氧化还原降解反应，利用Vc诱导的Fenton体系产生的羟基自由基氧化还原降解琼胶得到低分子量的琼胶。降解产物经过高速离心、60％乙醇沉淀，除去分子量比较大的降解产物和磷酸盐，得到可溶于60％乙醇的分子量估计小于3000的降争产物，其产率为85％。利用经Sephadex-G25凝胶色谱分离所香的不同分子量的级分进行分子量和α－葡萄糖苷酶抑制活性关系的实验。降解产物对α－葡萄糖苷酶的抑制率和各级分的浓度呈线性正相关，并且各级分的IC_(50)则随着分子量的降低而降低。另外，对所得的降解产物混合物进行了红外吸收光谱、质子去偶核磁共震碳谱和负离子基质辅助激光诱导－飞行时间质谱结构分析。结果表明，氧化还原降解反应的专一性差，在得到寡糖的同时，在光谱图中出现一些比较复杂的副产物的结构信息。最后，根据MTT法的原理，以有体皮肤成纤维细胞为材料，通过紫外线辐射产生自由基造成氧化损伤，研究降解产物对成纤维细胞的保护作用。当无紫外线辐射时，降解产物对成纤维细胞具有显著的促进生长增殖作用：当经UVa、UBb辐射时则可以显著地表现出对损伤的保护作用，并且这种促进生长和保护作用呈显著的量效关系，表明降解产物具有清除基自由基的作用。但是，因为氧化还原降解以应的机理尚不十分明的以及琼羟胶的特殊结构，使得反应的副产物很难预测，也就使得分离工作难以进行，所以，根据目前所得的信息，尚不能确定是降解产物的什么级分产生的以上两种生物活性。

不同分子量坛紫菜多糖的制备及其抗衰老活性研究

坛紫菜（Porphyra haitanensis），属红藻门（Rhodophyta），红毛菜科（Bangiacease）紫菜属（Porphyra），是我国大规模养殖的重要经济海藻之一，主要产于我国南方沿海如福建、广东、浙江等地。紫菜作为中药，具有“补肾养心，利水消肿”的功效，实验室前期所作的工作表明，坛紫菜多糖具有良好的抗衰老作用。分子量对多糖生物活性有重大影响，本文以福建沿海的坛紫菜为原料，对坛紫菜多糖（P）的降解方法和该紫菜多糖的抗衰老活性与分子量的关系展开了研究。 本论文首次将植物生长发育过程中发生的抗坏血酸和过氧化氢反应体系应用于坛紫菜多糖的降解。抗坏血酸和过氧化氢的浓度及两者的比例是该降解方法的主要影响因素。采用该法制备了分子量分别为50、30、13、8.2和2.7 kD 的P1、P2、 P3、P4和P5五种产品。其中P1、P2和P3产品的红外图谱与原料多糖的红外图谱一致，表明这两种多糖的化学结构在降解过程中并未发生改变，而P4和P5样品的红外图谱显示该样品中出现了少量的羧基。 本论文还首次对各产物进行了抗氧化、免疫调节、抗疲劳、延长寿命、抗老年痴呆等活性的筛选与研究，具体结果如下： 采用化学法对五种降解多糖和原料多糖的抗氧化活性进行研究。结果表明分子量对坛紫菜多糖清除各种自由基能力的影响是不同的。降解之后，坛紫菜多糖清除超氧阴离子的能力有所下降，与金属离子的螯合能力几乎全部丧失，但是多糖清除羟基自由基的能力变化甚微。降解之后，坛紫菜多糖清除有机自由基的能力和还原能力显著提高，随分子量的降低这两种能力明显加强，其中P4样品清除有机自由基能力最强，而分子量最小的P5样品还原能力最突出。 通过测定T、B淋巴细胞增殖率考察了五种降解多糖和原料多糖的免疫调节作用。研究表明：不同分子量坛紫菜多糖均具有免疫调节作用，可显著促进小鼠脾脏T淋巴细胞增殖，显著抑制小鼠脾脏B淋巴细胞增殖。P2和P4样品效果强于其它降解产品和原料。 通过测定小鼠游泳和爬杆时间考察了三种降解多糖P1、P2、P4和原料多糖的耐疲劳作用。研究表明：坛紫菜多糖降解产品耐疲劳作用要优于原料多糖P。P1、P2和P4通过提高小鼠肝糖原储备从而达到显著延长小鼠爬杆时间和负重游泳时间的功效。 通过测定D. melanogaster果蝇寿命、热耐受时间和性活力考察了三种降解多糖P1、P2、P4和原料多糖对寿命及生命活力的影响。研究表明：分子量对坛紫菜多糖对果蝇的寿命和生命活力有很大影响。降解使坛紫菜原料多糖P对果蝇的延长寿命和提高生命活力作用得到加强。在果蝇正常代谢状态下，降解产品中分子量较大的P1和P2作用更显著，而在热应激状态下，降解产品中分子量较小的P4作用更突出。 采用小鼠侧脑室注射Aβ1-40致痴呆模型考察了样品P1改善认知功能的作用。研究表明：P1通过降低皮层和海马组织乙酰胆碱酯酶活性，显著提高乙酰胆碱转移酶的活性，有效改善痴呆小鼠的认知能力，使之水迷宫游泳时间缩短，使避暗潜伏期延长。 为探讨P1样品改善小鼠认知功能的作用机制，本文采用Aβ25-35损伤神经母细胞瘤株SH-SY5Y模型对P1样品的神经细胞的保护作用进行了研究。结果显示低浓度下P1对该损伤模型具有显著保护作用，高浓度下表现出显著的抑制作用。 本文的研究表明坛紫菜多糖在降解之后生物活性得到显著改善。分子量对坛紫菜多糖活性影响很大。总的看来，在体外实验中，分子量越低其体外抗氧化活性和免疫调节作用越显著，但是，在体内实验中，降解产品中分子量最大的P1样品能更有效地改善小鼠疲劳程度、延长果蝇的寿命及热耐受时间，P1样品还能显著改善痴呆小鼠记忆功能。不同分子量坛紫菜多糖体内和体外生物活性的这种差异还需要进行深入研究。