A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae

Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 run. An optimized procedure yielded a high correlation coefficient (R-2 = 0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 mu g/mL and 20 mu g/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 mu g/mL and 2.0 mu g/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production. (C) 2009 Published by Elsevier B.V.

LIPID MICRORESISTOR AS MIMICKING OLFACTORY SENSOR - THE RULE OF THE RESPONSE OF LIPIDS TO C-1-C-4 ALCOHOLS

Phosphatidylcholine (PC) and six other PC-similar lipids are coated on interdigital electrodes, IEs, as sensitive membranes. Eight alcohols (C-1-C-4) are tested in a flow system at room temperature. It is found that all responses are log(response)-log(concentration) linear relations. These results agree with Steven's law in psychophysics. Moreover, the thresholds of the sensors are coincident with human olfactory thresholds. The authors have analysed the data of the lipid hypothesis suggested by Kurihara et al. We have found that this hypothesis is also in agreement with Steven's law. Lipid microresistors are real mimicking olfactory sensors. A definition of an olfactory sensor is suggested.

Interaction between alpha-actinin and negatively charged lipids membrane investigated by surface plasmon resonance and electrochemical methods

alpha-Actinin has been shown to be capable of interacting with some special membrane phospholipids directly, which is important for its function. In this study, hybrid bilayer membranes composed of negatively charged lipids are constructed on the surface plasmon resonance gold substrate and on the gold electrode, respectively, and the interaction between alpha-actinin and negatively charged lipids membrane is investigated by surface plasmon resonance, cyclic voltammetry and electrochemical impedance spectroscopy methods. alpha-Actinin is proved to be able to interact with the negatively charged lipids membrane directly. It can also insert at least partly into the membrane or lead to some defect or lesion in the membrane, which increase the permeability of the membrane. This study would bring some insight on the interaction between the alpha-actinin and the cell membranes in vivo.

Carbon isotopic studies of individual lipids in organisms from the Nansha sea area, China

Carbon isotopes of individual lipids in typical organisms from the Nansha sea area were measured by the GC-IRMS analytical technique. delta(13)C values of saturated fatty acids in different organisms examined are from -25.6parts per thousand to -29.7parts per thousand with the average values ranging from -26.4parts per thousand to -28.2parts per thousand and the variance range of 11.8parts per thousand, between different organisms is also observed. Unsaturated fatty acids have heavy carbon isotopic compositions and the mean differences of 2.9%.9-6.8parts per thousand compared to the same carbon number saturated fatty acids. delta(13)C values of n-alkanes range from -27.5%o to -29.7parts per thousand and their mean values, ranging from -28.6parts per thousand, to -28.9parts per thousand, are very close in different organisms. The mean difference in delta(13)C between the saturated fatty acids and n-alkanes is only 1.5parts per thousand, indicating that they have similar biosynthetic pathways. The carbon isotopic variations between the different carbon-number lipids are mostly within +/-2.0parts per thousand, reflecting that they experienced a biosynthetic process of the carbon chain elongation. At the same time, the carbon isotopic genetic relationships between the biological and sedimentary lipids are established by comparative studies of carbon isotopic compositions of individual lipids in organisms and sediments from the Nansha sea area, which provides scientific basis for carbon isotopic applied research of individual lipids.

Bacterial roles in the formation of high-molecular-weight dissolved organic matter in estuarine and coastal waters: Evidence from lipids and the compound-specific isotopic ratios

High-molecular-weight dissolved organic matter (HMW-DOM, > 1,000 Daltons) is actively involved in the global biogeochemical cycling of many elements, but its carbon sources and detailed formation pathways are still not well understood. In this study, we measured bulk stable carbon and nitrogen isotopic ratios, lipid composition, and compound-specific carbon isotopic ratios of HMW-DOM samples collected from four U.S. estuaries (Boston Harbor/Massachusetts Bay, Delaware/Chesapeake Bay, San Diego Bay, and San Francisco Bay). Analytical results show (1) a fraction of HMW-DOM (lipid associated) in estuarine and coastal waters is derived from bacteria and phytoplankton; (2) this fraction of HMW-DOM is formed by various release processes of bacterial membrane components and bacterial reworking of phytoplankton-derived material; (3) this fraction of HMW-DOM is generally present in all samples from different coastal systems despite variable organic matter inputs and environmental conditions, suggesting an important bacterial role in HMW-DOM formation.

膜脂在类囊体不同功能膜区的分布及其与植物抗寒性的研究

一． 通过对黄瓜类囊体及其基粒片层、间质片层、PS II放氧颗粒和LHC II 复合体脂类成分的分析，得 到以下结果：各膜区均含有类囊体膜的五种脂类成分，在类囊体及其基粒片层、间质片层和PS II放氧颗粒中，MGDG含量最高，分别为42.5%、40.5%、46.3%、和35.7%，其次是DGDG，含量在31-35%之间。值得注意的是黄瓜类囊体间质片层MGDG含量高于基粒片层，而且DGDG/DGDG分子比也较高。这在其它植物材料中还未见报道。在黄瓜LHC II中，PG含量最高，为35.5%，约是类囊体膜PG含量的3倍。从基粒片层、PS II放氧颗粒到LHC II， PG含量呈逐渐增加的趋势，而在间质片层中，PG含量最低。SQDG除在LHC II中含量稍低外，在其它膜区中的分布没有明显的差异。脂类脂肪酸组成分析结果表明：MGDG主要含亚麻酸，含量在90%以上。DGDG也主要含亚麻酸，含量在90%左右，DGDG所含棕榈酸多于MGDG中的含量。SQDG中主要脂肪酸组分为棕榈酸和亚麻酸。不同膜区MGDG、DGDG和SQDG脂肪酸组成没有明显差异在PG中含量最高的脂肪酸是叶绿体特有的反式十六碳一烯酸（trans-16:1)。此外，PG还含有较多的棕榈酸、硬脂酸和油酸。在不同膜区PG的脂肪酸组成有较明显的差异。 根据以上结果，我们推测脂类除了形成膜的流动性基质外，还可能选择性地结合在膜蛋白周转形成特 异的脂质微区，通过膜脂膜蛋白的相互作用，以行使其特殊的生理功能。 二、通过比较两种不同抗寒性小麦品种在低温锻炼前后类囊体脂类及其脂肪酸成分、LHC II复合体及类囊体吸收光谱、低温荧光发射光谱，发现经低温锻炼后：（1）抗寒与不抗寒小麦品种类囊体PG的trans-16:1含量均明显降低，抗寒品种类囊体MGDG/DGDG比值也明显降低，而不抗寒品种这一比值变化不明显。（2）抗寒品种脂/色素比值明显增高，而不抗寒品种滑明显增加。（3）抗寒与不抗寒品种LHC II宏聚体含量均降低而单体含量增加。（4）抗寒与不抗寒品种类囊体吸收光谱四阶导数光谱A_(683)/A_(652)比值均升高。（5）不抗寒品种低温荧光发射光谱F_(685)/F_(738)比值上升，而抗寒品种这一比值没有变化。通过对上述结果的分析，我们认为低温锻炼过程中类囊体膜流动性增强是使抗寒品种抗寒力增强的主要原因之一，此外，MGDG含量降低对膜的稳定性可能起重要作用。trans-16:1含量的降低和LHC II寡聚体解聚可能是植物对于低温的一种适应性反应。

细胞色素b6f蛋白复合体的结构与功能色素的结构与功能及膜脂的作用

采用改进的的分离纯化方法从菠菜叶绿体中获得了纯度和活性、Chl a 和β-carotene含量较高的Cyt b6f制剂。利用吸收光谱、CD光谱和共振喇曼光谱对Cyt b6f蛋白复合体中的β-carotene分子的构型及构象进行了研究。结果表明，无论是结合在蛋白复合体上还是处于自由状态，该β-carotene都是一种特殊的9-顺式构型的分子;在蛋白复合体中，该9-顺式β-carotene存在于一种不对称的蛋白环境中，但从喇曼光谱中没有观察到构象扭曲现象。SDS处理前、后的荧光激发光谱和光破坏的研究表明Cyt b6f中的β-carotene不能有效地向Chl a传递光能，但对Chl a具有明显的光保护功能，因而说明β-carotene分子是Cyt b6f的天然结构和功能成分。根据这些实验结果和文献的有关报道，提出了β-carotene在Cyt b6f中可能的光保护机理模型，，即在Cyt b6f中，Chl a不是单线态氧的主要发生源，单线态氧主要由Rieske[Fe-s]中心产生，β-carotene不具有直接淬灭Chl a三线态的功能，其主要功能是作为抗氧化剂抵御Rieske[Fe-s]中心产生的单线态氧对Chl a的攻击，从而保护Cyt b6f中Chl a免受强光照射的破坏。 利用非变性凝胶电泳和硫酸铵沉淀对Chl a在Cyt b6f中的结合和解离现象进行了研究，观察到Chl a的解离与Rieske蛋白的解离及Cyt b6f复合体单体化的关系比较密切，膜脂能够有效地抵制这三个过程的发生，推测膜脂的作用位置位点可能位于Cyt b6f二聚体中两个单体之间的交界处。色素重组实验表明缺失Rieske[Fe-S]蛋白的Cyt b6f单体不能有效的和Chl a重组。 利用从菠菜叶绿体中分离纯化出的缺脂Cyt b6f与从菠菜类囊体分离纯化的膜脂进行体外重组，检测了不同膜脂对Cyt b6f催化电子传递活性的影响。结果表明：被检测的五种膜脂单半乳糖基甘油二酯（MGDG）、双半乳糖基甘油二酯 （DGDG），磷脂酰胆碱（PC），磷脂酰甘油（PG）和硫代异鼠李糖基甘油二酯（SQDG）对Cyt b6f催化电子传递的活性均有明显的促进作用，但促进的程度各不相同。不带电荷的MGDG和DGDG及分子整体呈电中性的PC对促进Cyt b6f催化电子传递的活性非常用效，最适条件下可分别使其活性提高89%、75%和77%;而带负电荷的PG和SQDG的活性促进作用则相对较弱，最适条件下仅可使其活性分别提高43%和26%。由此可见，膜脂对Cyt b6f活性的促进作用可能与这些膜脂分子的带电性质密切相关。

小麦光系统I的分离纯化及其光合特性的研究

从不同基因型的小麦京411和小偃54中分离纯化了PSI颗粒并研究了PSI颗粒的一些光合特性： 1. 测定了两种基因型小麦PSI颗粒的室温吸收光谱、低温荧光光谱，并进行了SDS-PAGE多肽分析。吸收光谱显示了红区680nm和蓝区439nm的两个最大吸收峰，低温荧光光谱显示了PSI特征的位于735nm左右的发射峰，同时SDS-PAGE也显示我们的制备物包括PsaA、PsaB、LHCI以及一些其它小分子量蛋白亚基。这些都表明我们从不同基因型小麦中获得了比较理想的PSI颗粒制备物。 2. 测定了京411类囊体膜和PSI颗粒的脂类组成和脂肪酸成分，发现PSI颗粒中也存在着类囊体膜中的五种膜脂，即：MGDG、DGDG、PG、SQDG、PC，但PSI颗粒的MGDG含量比类囊体膜高，而DGDG含量较类囊体低。PSI颗粒和类囊体膜的脂肪酸组成也有差异。 3. 运用光谱学手段，研究两种基因型小麦PSI颗粒光破坏过程的异同。发现在经过强光破坏后，两种小麦PSI颗粒都发生叶绿素漂白现象，在各种状态叶绿素中，683nm状态的Chl a对强光最为敏感，受到光破坏的程度最大，而649nm Chl b和667nm Chl a分子变化较小。结合吸收光谱和低温荧光光谱我们提出了PSI中可能存在的能量传递途径。比较两种小麦PSI颗粒光破坏在低温荧光光谱上的不同，我们初步认为，小偃54可能通过将能量较多地分配给予长波长Chl而一定程度的避免过多能量向P700反应中心传递，从而起到对P700的保护作用。 4. 研究了不同表面活性剂SDS和Triton X-100对PSI颗粒色素结合状态和能量传递的影响。发现表面活性剂对色素状态和PSI中的能量传递都有很大的影响。并且Triton X-100的作用较SDS强烈。紫外荧光显示PSI蛋白结构也发生了显著变化。