An ellipsometry-based Alzheimer plaque mimic: Effect of beta-amyloid, lipoprotein identity and apolipoprotein E isoform

Ca2+-induced deposition of low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL) at proteoheparan sulfate-modified surfaces was investigated as a function of beta-amyloid (Abeta) presence and apolipoprotein E isoform. Presence of beta-amyloid resulted in an increased deposition, as did the E4/E4 isoform compared to the corresponding E3/E3 isoform. The results are compatible with findings reported in literature on plaque formation in Alzheimer's disease, and suggest that, although simplistic, the present model system may have some potential in biosensor studies of Alzheimer plaque formation.     

Oxidation-dependent effects of oxidized LDL: proliferation or cell death

Oxidized low-density lipoprotein (oxLDL) induces a wide range of cellular responses to produce atherosclerotic lesion, but key factors determining the response are not understood. In this study, purified LDL was oxidized with copper sulfate, and its physical properties and the related biological responses were investigated. The average hydrodynamic diameter of the lightly oxidized LDL was approximately 25 nm and its Rf value relative to nLDL on agarose gel was between 1.0 and 1.25. The diameter of the extensively oxidized LDL was over 30 nm, the Rf value was over 2.0. A 24 h-exposure of resting RAW264.7 macrophage cells to 100 microg/ml of the lightly oxidized LDL induced proliferation and macrophage activation whereas the extensively oxidized LDL induced cell death at the same concentration. In contrast, 200 microg/ml of oxLDL caused cell death regardless of oxidation degree. Short incubation (4-6 h) of the highly oxidized LDL (100 microg/ml) also resulted in cell proliferation. OxLDL-induced cell death showed mixed characteristics of apoptosis and/or necrosis depending on the strength and duration of the insult. These results suggest that cellular responses induced by oxLDL be dependent on the oxidation degree, the duration of exposure, and the concentration of oxLDL.     

改性固体表面的吸附作用——甲基化硅胶的热稳定性和吸附性能

本文利用三甲基氯硅烷与硅胶表面羟基反应的方法制备了甲基化硅胶。测定了亲水硅胶和甲基化硅胶的热处理对其自四氯化碳中吸附乙酸的等温线的影响,并配合有热重分析(TG)和红外光谱(IR)的测定。结果表明:(1)甲基化后的硅胶对乙酸的吸附能力大大下降;(2)甲基化硅胶的热处理温度达500℃时吸附能力完全恢复到甲基化前的硅胶的水平,甲基化层明显开始破坏的温度为450℃;(3)甲基化硅胶高温处理后吸附能力得以恢复的主要原因是重新形成表面自由羟基。     

The Thermal Stability and Adsorbability of the Mechylated Silica Gel

The methylated silica gel has been produced by the reaction between the surface silanol groups and Clsi(CH3)3 vapor.The adsorption isotherms of acetic acid from carbon tetrachloride onto silica gel and emthylated silica gel heated at various temperatures have been determined at 25℃,and the above mentioned silica gels have been studied by TC and IR.The results indicate:(1) The adsorption of acetic acid from carbon tetrachloried onto methylated silica gel decreased greatly,probably because the concentration of the free hydroxyl groups for methylated silica gel decreased greatly as the IR shows.(2) As the methylated surface was destroyed at 450℃,the adsorption ability was restored when the methylated silica gel treated at>500℃.(3) So long as the methylated silica gel was treated at high temperature,the adsorption ability could be restored owing to that surface free hydroxyl groups were reproduced.     

聚乙二醇、聚丙二醇和环氧乙烷-环氧丙烷嵌段共聚物在固-液界面上的吸附 III:硅烷化活性炭/水和甲基化硅胶/水界面

测定了25℃时硅烷化不同时间(1至30天)的活性炭及甲基化硅胶自水溶液中吸附四种聚乙二醇(PEG)、三种聚丙二醇(PPG)和环氧乙烷(EO)-环氧丙烷(PO)嵌段共聚物pluronic-L64的等温线。结果表明, 在各活性炭样品上的等温线均为Langmuir型的; 同一炭样对不同PEG的极限吸附量(g·g^-^1)与分子量无关; 极限吸附时每个PEG分子所占面积(A)与分子中所含EO数(nEO)间有直线关系, 直线的斜率与硅烷化时间有关, 这一结果可用硅烷化时间延长时吸附分子的EO基可能以其氧原子向水, 碳氢链节靠近固体表面取向的模型解释。根据PPG的极限吸附量与分子量有关和极限吸附时的分子面积推断PPG分子不是以平躺方式吸附。甲基化硅胶对PEG的吸附量极小, 对PPG的吸附量随分子量减小急剧降低, 而对L64的吸附量明显大于在亲水硅胶上的。文中对所得结果给出了初步的解释。     

聚二甲基硅氧烷(PDMS)/玻璃微流控芯片电泳快速分离血清高密度脂蛋白亚类及临床应用研究

经梯度密度超速离心,高密度脂蛋白(HDL)分为HDL2和HDL3两亚型。HDL2抑制低密度脂蛋白(LDL)氧化功能受损是冠心病(CHD)发生发展的关键因素。因此,通过对HDL亚类进行分离,从而达到预测和诊断CHD的目的。本研究建立了用PDMS/玻璃微流控芯片快速电泳分离HDL亚类的方法。选择N-十二烷基-β-D-麦芽糖苷(DDM)、十二烷基硫酸钠(SDS)和羟丙基纤维素(HPC)共同修饰脂蛋白和泳道表面。在以含0.3 mmol/L SDS的50 mmol/L 3-(N-吗啉代)丙磺酸(MOPS)(pH 8.0)为样品缓冲液,含0.6%HPC的50 mmol/L MOPS(pH 8.0)为分离缓冲液,分离电压为260 V/cm的优化条件下,HDL2和HDL3在4 min内得到基线分离,二者的出峰时间和峰面积的相对标准差(RSD)分别是2.0%和2.7%,2.0%和2.9%,具有较好的重复性。临床标本研究发现,正常人血清标本可分离出HDL2和HDL3双峰,而CHD患者的HDL2峰面积显著减小,甚至消失。PDMS/玻璃微流控芯片分离HDL亚类是一种简单、快速、高效的用于分析CHD危险因子的方法。     

Application of poly(dimethylsiloxane)/glass microchip for fast electrophoretic separation of serum small,dense low-density lipoprotein

Due to the mounting evidence of altered low-density lipoprotein (LDL) size in several disease states, there has been an increasing interest in developing new analytical methods for small, dense low-density lipoprotein (sdLDL) for diagnosis. The present report demonstrates that sdLDL analysis can be performed in a poly(dimethylsiloxane) (PDMS/glass) microchannel. n-Dodecyl β-d-maltoside (DDM) was utilized to alter channel surface to make it become hydrophilic and nonionic, thus reducing the interaction between the protein and the surface. Moreover, hydroxypropylcellulose (HPC) was added into the running buffer to suppress the adsorption of analytes and also to serve as a sieving matrix. Under optimal conditions, two baseline separations of lipoproteins including high-density lipoprotein (HDL), sdLDL, and lLDL were achieved with different selectivity. LDL particles shown on the electropherogram were also identified by several procedures. This method affords high separation speed and high reproducibility. The intraassay and interassay RSDs of lipoprotein migration times were in the range of 2.01–2.45%. The variation of serum sdLDL of a patient between prior treatment and post-treatment was assessed by this method. This system has the potential for rapid and sensitive detection of different LDL forms, and thus will be applicable to clinical diagnosis.     

Energetics of lysozyme adsorption on mesostructured cellular foam silica: effect of salt concentration

The heat of lysozyme adsorption on mesostructured cellular foam (MCF) silica was measured using flow microcalorimetry (FMC) to investigate the influence of a neutral salt, sodium sulfate. At concentrations up to 0.5 M sodium sulfate, a complex initial exotherm was followed by an endotherm. Protein surface coverage, the magnitudes of the exothermic heat signals and the magnitudes of the net heat of adsorption increased with sodium sulfate concentration. These observations suggest that electrostatic interactions are the principal driving force at low ionic strengths; van der Waals interactions become dominant at higher salt concentrations. Each exotherm could be deconvoluted into two exotherms, indicating multiple modes of lysozyme attachment to the silica surface. The endothermic peak, associated with protein desorption, disappeared at the highest sodium sulfate concentration (1.0 M), indicating irreversible adsorption of the protein on the MCF silica surface. The data are consistent with an adsorption mechanism in which the initial attachment of lysozyme to the surface is followed by a reorientation and formation of a secondary or stronger attachment to the surface.     

Microchip-based small, dense low-density lipoproteins assay for coronary heart disease risk assessment

Small, dense low-density lipoprotein (sdLDL) has been accepted as an emerging cardiovascular risk factor, and there has been an increasing interest in analytical methods for sdLDL profiling for diagnosis. Serum sdLDL may be measured by different laboratory techniques, but all these methods are laborious, time-consuming, and costly. Recently, we have demonstrated that a low-temperature bonding of quartz microfluidic chips for serum lipoproteins analysis (Zhuang, G., Jin, Q., Liu, J., Cong, H. et al., Biomed. Microdevices 2006, 8, 255-261). In contrast to this previous study, we chose SDS as anionic surfactant to modify both lipoproteins and the channel surface to minimize lipoprotein adsorption and improve the resolution of lipoprotein separation. Two major LDL subclass patterns including large, buoyant LDL (lLDL), sdLDL, and high-density lipoprotein (HDL) were effectively separated with high reproducibility. RSD values of the migration time (min) and peak areas of standard LDL and HDL were 6.28, 4.02, 5.02, and 2.5%, respectively. Serum lipoproteins of 15 healthy subjects and 15 patients with coronary heart disease (CHD) were separated by microchip CE. No peaks of sdLDL were detected in serum samples of healthy subjects while sdLDL fractional peaks were observed in patients' entire serum samples. These results suggested that the microchip-based sdLDLs assay was a simple, rapid, and highly efficient technique and significantly improved the analysis of CHD risk factors.     

Lipoproteins modulate growth and differentiation of cultured human epidermal keratinocytes

The effects of various lipoproteins on the growth and the differentiation of cultured normal human keratinocytes were investigated. Primary cultures of human epidermal keratinocytes were obtained from neonatal foreskin, and then added with lipoproteins, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Cell growth potential was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. VLDL and LDL enhanced keratinocytes growth and LDL receptor expression at the plasma membrane level. These effects were more remarkably observed in cells cultured with VLDL than in cells cultured with LDL. Apolipoprotein E (ApoE) was highly increased in VLDL treated cells. These results suggest that VLDL binds with high affinity to cell surface receptors and induces cell proliferation.     

Ellipsometry studies of fibronectin adsorption

The adsorption of fibronectin on a series of different surfaces was investigated with in situ ellipsometry. For silica and methylated silica, the adsorbed amount (Γ), the adsorbed layer thickness (δ_el) and the mean adsorbed layer refractive index (n_f) were obtained by a procedure involving studies of the bare substrate at two different ambient refractive indices, as well as four-zone averaging. It was found that the adsorbed amount of fibronectin was the same (1.9 ± 0.1 mg m⁻²) on both silica and methylated silica surfaces. However, the adsorbed layers formed on methylated silica were more extended and had a lower average protein concentration than those formed on silica. Furthermore, on both silica and methylated silica, an increasing adsorbed amount is achieved both by a denser packing of the fibronectin molecules and by a growth of the adsorbed layer normal to the surface. Furthermore, the adsorption of fibronectin on lipid surfaces was investigated. It was found that the adsorption of fibronectin on phosphatidic acid was quite significant (2.2 ± 0.2 mg m⁻²), while that on phosphatidylcholine, phosphatidylinositol and phosphatidylserine was much smaller (all 0.1 ± 0.05 mg m⁻²). These results are correlated to findings on the adsorption of fibrinogen on these surfaces, as well as on the opsonization of lipid-stabilized colloidal particles.     

Characterization of plasma apolipoproteins by capillary electrophoresis.

The main apolipoproteins of plasma high-density lipoproteins (HDL) and low-density lipoproteins (LDL) were analyzed by capillary electrophoresis. Where possible the results were compared with slab sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Addition of the detergent SDS to the running buffer was essential for separation. Separations were carried out in bare silica and polyacrylamide-coated capillaries. The main apolipoproteins of HDL could be separated in an uncoated capillary filled with borax buffer containing 0.1% SDS. Using the coated capillary, a mixture of HDL and LDL apolipoproteins was resolved in less than 12 min. These preliminary studies indicate that capillary electrophoresis is a promising technique for screening plasma apolipoproteins.     

苯基化硅胶的物理结构和表面性质的研究

本文利用苯基三氯硅烷和硅胶表面羟基反应制备了苯基化硅胶。测定了苯基化硅胶的物理结构、水蒸气吸附等温线、润湿热、差热分析(DTA)和红外光谱(IR)。结果表明,所有苯基化硅胶的真密度(dT)、比表面(S)和比孔体积(V)均减少, 而表观密度(dA)增加, 但苯基化对不同硅胶的平均孔半径(r)有不同的影响; 苯基化硅胶对水蒸气吸附, 对水、苯和环己烷的润湿热均显著减少; 苯基化硅胶的热稳定性大于甲基化硅胶, 甲基化硅胶的表面是高度憎水的, 但苯基化硅胶的憎水性则很弱。     

Adsorption of Poly(ethylene oxide)-Poly(lactide) Copolymers. Effects of Composition and Degradation

The effect of chemical degradation of two diblock copolymers of poly(ethylene oxide) (E) and poly(lactide) (L), E(39)L(5) and E(39)L(20), on their adsorption at silica and methylated silica was investigated with in situ ellipsometry. Steric stablization of polystyrene dispersions was investigated in relation to degradation. Hydrolysis of the poly(lactide) block of the copolymers was followed at different temperatures and pH by using HPLC to measure the occurrence of lactic acid in solution. The block copolymers were quite stable in pH-unadjusted solution at low temperature, whereas degradation was facilitated by increasing temperature or lowering of the pH. Lower degradation rates of E(39)L(20) where observed at low temperature in comparison with those of E(39)L(5), whereas the degradation rates of the copolymers were quantitatively similar at high temperature. The adsorption of the copolymers at methylated silica substrates decreased with increasing degree of degradation due to the reduction in the ability of hydrophobic block to anchor the copolymer layer at the surface. At silica the adsorption initially increased with increasing degradation, particularly for E(39)L(20) due to deposition of aggregates onto the surface. After extensive degradation the adsorption of the copolymers at both silica and methylated silica resembled that of the corresponding poly(ethylene oxide) homopolymer. Overall, it was found that the eventual reduction in adsorption occurred at a lower degree of degradation for E(39)L(5) than for E(39)L(20). Mean-field calculations showed a reduced anchoring for the block copolymers with decreasing poly(lactide) block length at hydrophobic surfaces. In accordance with this finding, it was observed that polystyrene dispersions were stabilized by E(39)L(20) or E(39)L(5) in a way that depended on both the lactide block length and the degree of degradation. Upon degradation of the hydrophobic block, stabilization of the polystyrene dispersions was maintained initially, but eventually degradation resulted in destabilization. The average residual copolymer concentration required for stabilization of the polystyrene dispersions was much higher than the corresponding concentration of intact copolymer required for stabilization. Copyright 2001 Academic Press.     

Enantioselective binding analysis of verapamil to plasma lipoproteins by capillary electrophoresis-frontal analysis

Capillary electrophoresis coupled with frontal analysis was applied to the study of enantioselective binding of verapamil (VER) to plasma lipoproteins. The drug-lipoprotein mixed solution, which had been in the binding equilibrium, was hydrodynamically introduced into a non-coated fused-silica capillary. Since VER is positively charged in the neutral run buffer (pH 7.4), the unbound VER enantiomers migrated toward the cathodic end much faster than negatively charged lipoproteins and their bound forms. Once unbound VER migrated apart from lipoprotein, the bound VER was quickly released from the protein to maintain the binding equilibrium. Thus, VER migrated as a zone through the capillary and gave a trapezoidal peak with a plateau region on the electropherogram. The VER concentration in this plateau region was equal to the unbound VER concentration in the initial sample solution. It was found that the bindings of VER to high-density lipoprotein (HDL), low-density lipoprotein (LDL) and oxidized LDL were not site-specific and not enantioselective. Partition-like binding to lipid part of these lipoproteins seemed to be dominant. The total binding affinities of LDL to VER were about seven-times stronger than those of HDL, and the oxidation of LDL by copper ion enhanced the binding affinities significantly.     

热处理对憎水硅胶吸附水蒸气的影响

用甲基氯硅烷蒸气或溶液处理硅胶,均可制成憎水硅胶。关于憎水硅胶的吸附性能和热稳定性的研究,文献中时有报道。本文主要探讨以下3个问题:(1)在水蒸气吸附中,硅胶表面自由羟基和缔合羟基究竟哪种起主要作用;(2)从吸附水蒸气等数据讨论硅胶表面有机基团—OSi(CH_3)_3的热稳定性;(3)用二甲基二氯硅烷(DMCS)和三甲基氯硅烷(TMCS)处理的憎水硅胶,哪种硅胶的热稳定性较高。这些基本问题,不仅具有学术意义,对研究氧化物表面改性也有参考价值。     

Comparative analysis of foetal calf and human low density lipoprotein: relevance for pharmacodynamics of photosensitizers

The effects of differences in lipoprotein content on the distribution of the novel hydrophobic photosensitizer n-butyl-3-[18-(2-butylcarbamoyl-ethyl)-3,7,12,17-tetramethyl-18,13-divinyl-22,24-dihydro-porphin-2-yl]propionamide (PP-N-3) and haematoporphyrin ester (HpE), a relatively hydrophilic photosensitizer, in human (HS) and foetal calf sera (FCS), were investigated. The binding characteristics of human and foetal calf low-density lipoprotein (LDL) were characterised using a human fibroblast line (Vag 12). The uptake into cells of HpE and PP-N-3 was also examined. A comparison of the lipoprotein content, composition and receptor-binding characteristics of foetal calf and human serum was also carried out. LDL content was measured directly using sequential ultracentrifugation to isolate LDL. In our study, we found haematoporphyrin ester to bind to human very low-density lipoprotein (VLDL), LDL and high-density lipoprotein (HDL) in the ratio 2:31:65. In the case of PP-N-3 this ratio was 56:10:33. As VLDL was not detected in foetal calf serum, only binding to LDL and HDL was observed. Using the sequential ultracentrifugation technique, foetal calf serum was found to contain LDL which in turn did bind to human LDL receptors. The uptake of PP-N-3 and HpE in the presence of low density lipoprotein from foetal calf serum (FC-LDL) was not significantly different to values observed in the presence of human serum low density lipoprotein (HS-LDL).     

Low-density lipoprotein analysis in microchip capillary electrophoresis systems

Due to the mounting evidence for altered lipoprotein and cholesterol-lipoprotein content in several disease states, there has been an increasing interest in analytical methods for lipoprotein profiling for diagnosis. The separation of low- and high-density lipoproteins (LDL and HDL, respectively) has been recently demonstrated using a microchip capillary electrophoresis (CE) system [1]. In contrast to this previous study, the present report demonstrates that LDL analysis can be performed in an uncoated glass microchannel. Moreover, by adding sodium dodecyl sulfate (SDS) to the sample at a concentration well below the critical micellar concentration prior to injection, the LDL peak undergoes a focusing effect and exhibits an apparent efficiency of 2.2 x 10(7) plates/m. Laser light scattering experiments demonstrate that the low concentration of SDS used does not significantly alter lipoprotein particle size distribution within the time course that the analysis is performed. It is thus hypothesized that SDS nondisruptively coats LDL particles. The peak sharpening effect, observed only when SDS is added solely to the sample, is probably due to a mobility gradient created between the sample and the running buffer. The chip-based method demonstrated here has the potential for rapid analysis and sensitive detection of different LDL forms of clinical relevance.     

Surface properties of hen egg yolk low-density lipoproteins spread at the air–water interface

Hen egg yolk is largely used as food ingredient notably because of its exceptional emulsifying properties. Low-density lipoproteins (LDL) are the main egg yolk constituent. LDL and particularly apoLDL are thought to control largely emulsifying properties of egg yolk-based products. Nevertheless, few studies have concerned the interfacial behaviour of these lipoproteins at the oil–water interface and nothing has been published about the air–water interface. Controversies still remain about LDL adsorption mechanism at the oil–water interface even if a widely spread theory suggests their breaking at the interface, allowing then their constituents to spread. The Langmuir film balance and atomic force microscopy (AFM) were used in this study in the aim to characterise LDL surface behaviour in dynamic conditions at the air–water interface. The understanding of LDL adsorption mechanism and surface organisation at the air–water interface should provide useful information about LDL behaviour at the oil–water interface. LDL and lipids extracted from LDL—neutral lipids, phospholipids and total lipids (mixture of the two previous species)—were spread at the air–water interface to clarify the role of each constituent in the lipoprotein film. Results clearly show that LDL are disrupted at the interface to release notably neutral lipids from the lipoprotein core, enabling then their spreading. Each lipid class has been identified on the LDL film isotherm and seems to behave independently and individually at the interface within the lipoprotein film.     

The Biological and Chemical Ameliorative Effects of Bread Substituted with Dried Moringa Leaves

This study conducted to evaluate the effect of additional dried moringa leaves on bread and their effects on the chemical and biological of rats fed it. Wheat flour with dried moringa leaves powder (DMLB) is used at different levels (6 and 9%) to produce moringa bread. We performed a sensory evaluation of wheat bread substituted with dried moringa leaves, chemical properties (approximate composition and antioxidants), microbiology (total bacteria, spore-forming bacteria, fungi, and yeast) and biological analysis (Triglycerides, total cholesterol, HDL and LDL, ALT, AST, urea, creatinine). Therefore, histopathology examination for kidney and livers of male albino rats fed fortified bread with dried moringa leaves compared with the control sample. The results of the approximate analysis showed significant differences by adding moringa leaves and the percentage of ash, protein, fat, and fiber increased with the moisture content. Moreover, carbohydrates decreased from fortified bread with dried moringa leaves compared to the control sample. The best treatment was fortified bread with 6% dried moringa leaves for all sensory evaluations compared to the other samples and moringa bread (DMLB 9%) helped reduce the microbial load during storage. The effect of moringa bread on liver cirrhosis was evaluated in rats induced by carbon tetrachloride CCl₄. We found out that the effect of moringa leaves on liver and kidney functions in a state of improvement, as for high-density lipoprotein (HDL) and low-density lipoprotein (LDL), the proportion of LDL was less than HDL compared to the control sample. At the same time, moringa leaves have a significant effect on reducing cholesterol, triglycerides, urea, and creatinine in the serum of rats, which may be attributed to the presence of biologically active plant components. It can be concluded that moringa leaves can improve biological and histological of rats feed it.