Study of urinary 8-hydroxydeoxyguanosine as a biomarker of oxidative DNA damage in diabetic nephropathy patients

Increased oxidative stress induced by hyperglycemia may contribute to the pathogenesis of diabetic complications. Urinary 8-hydroxydeoxyguanosine (8-OHdG) has been reported to serve as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. This article studied oxidative DNA damage in patients with diabetic nephropathy and in healthy control subjects by urinary 8-OHdG evaluations. Contents of 8-OHdG in urine were analyzed by capillary electrophoresis with end-column amperometric detection (CE-AD) after a single-step solid-phase extraction (SPE). Levels of urinary 8-OHdG in diabetic nephropathy patients with macroalbuminuria was significant higher than in control subjects (5.72 +/- 6.89 mumol/mol creatinine versus 2.33 +/- 2.83 mumol/mol creatinine, P = 0.018). A significant difference of 24 h urinary 8-OHdG excretions exists between the patients with macroalbuminuria and the patients with nonnoalbuminuria (19.2 +/- 16.8 mug/24 h versus 8.1 +/- 1.7 mug/24 h, P = 0.015). There was a positive correlation between urinary excretion of 8-OHdG and glycosylated hemoglobin (HbA(1)c) (r = 0.287, P = 0.022). A weak correlation exists between the levels of 8-OHdG and triglyceride (r = 0.230, P = 0.074). However, the urinary 8-OHdG contents are not correlated with blood pressure and total cholesterol. The increased excretion of urinary 8-OHdG is seen as indicating an increased systemic level of oxidative DNA damage in diabetic nephropathy patients. (C) 2004 Elsevier B.V. All rights reserved.

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregrarn is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.

Sensing H2O2 with layer-by-layer assembled Fe3O4-PDDA nanocomposite film

It was found that Fe3O4 nanoparticles (Fe3O4 NPs) possess intrinsic enzyme mimetic activity similar to that found in natural peroxidase. Here, we applied Fe3O4 NPs to the construction of efficient electrochemical sensor to detect the concentration of hydrogen peroxide. The sensor was fabricated with layer-by-layer assembly of Fe3O4 NPs and poly(diallyldimethylammonium chloride) (PDDA) through the electrostatic interaction, and the multilayer film was characterized with UV-vis absorption spectra, atomic force microscopy, and cyclic voltammetry.

Sensitive, Label-Free Protein Assay Using 1-ethyl-3-methylimidazolium Tetrafluoroborate Supported Microchip Electrophoresis with Laser-Induced Fluorescence Detection

Based on the dimer-monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF(4)) instead of PBS was applied as running buffers in microchip electrophoresis.

Electrochemical and electrochemiluminescence study of Ru(bpy)(3)(2+)-doped silica nanoparticles with covalently grafted biomacromolecules

Spherical Ru(bpy)(3)(2+)-doped silica (RuSi) nanoparticles were prepared via a water-in-oil microemulsion approach. The electrochemical and electrochemiluminescent properties of the RuSi nanoparticles immobilized on an indium tin oxide (ITO) electrode were investigated. Further, electrochemiluminescence (ECL) of the RuSi nanoparticles with covalently coated biomacromolecules was studied. By covalent cross-linking with glutaraldehyde, gamma-(aminopropyl) triethoxysilane (APTES)-pretreated RuSi nanoparticles were coupled with different concentrations of bovine serum albumin (BSA), hemoglobin, and myoglobin, respectively.

One-step immobilization of tris(2,2 '-bipyridyl)ruthenium(II) via vapor-surface sol-gel deposition towards solid-state electrochemiluminescence detection

A novel method for immobilization of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)Cl-2) on electrode surfaces based on the vapor-surface sol-gel deposition strategy is first demonstrated in this paper. Ru(bpy)(3)Cl-2 immobilized sol-gel (Ru(bpy)(3)Cl-2/sol-gel) films were characterized by UV-vis spectroscopy and field-emitted scanning electron microscopy (FE-SEM). These results showed that Ru(bpy)(3)Cl-2 was successfully incorporated into the silica sol-gel film. it was found that many irregular Ru(bpy)(3)Cl-2/sol-gel clusters were formed on surfaces through one deposition and thick sol-gel films were observed after further deposition.

Direct electrochemistry of glucose oxidase and biosensing for glucose based on carbon nanotubes@SnO2-Au composite

Multiwalled carbon nanotubes@SnO2-Au (MWCNTs@SnO2-Au) composite was synthesized by a chemical route. The structure and composition of the MWCNTs@SnO2-Au composite were confirmed by means of transmission electron microscopy, X-ray photoelectron and Raman spectroscopy. Due to the good electrocatalytic property of MWCNTs@SnO2-Au composite, a glucose biosensor was constructed by absorbing glucose oxidase (GOD) on the hybrid material. A direct electron transfer process is observed at the MWCNTs@SnO2-Au/GOD-modified glassy carbon electrode. The glucose biosensor has a linear range from 4.0 to 24.0 mM, which is suitable for glucose determination by real samples. It should be worthwhile noting that, from 4.0 to 12.0 mM, the cathodic peak currents of the biosensor decrease linearly with increasing the glucose concentrations in human blood. Meanwhile, the resulting biosensor can also prevent the effects of interfering species.

Electrochemistry of glucose oxidase immobilized on the carbon nanotube wrapped by polyelectrolyte

A more stably dispersing of multi-wall carbon nanotube composite (noted as PDDA-MWNT), which was obtained by wrapping the MWNT with poly (diallydimethylammonium) chloride (PDDA), was used for the immobilization of glucose oxidase (GOD) and its bioelectrochemical studies. The morphologies and structures of the PDDA-MWNT composite were characterized by environment-canning electron microscopy (ESEM) and X-ray photoelectron spectroscopy (XPS). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were used to feature the GOD adsorbed onto the electrode modified by PDDA-MWNT composite. The immobilized GOD at the PDDA-MWNT films exhibited a pair of well-defined nearly reversible redox peaks and a fast heterogeneous electron transfer rate with the rate constant (k(s)) of 2.76 s(-1). In addition, GOD immobilized in this way retained its bioelectrocatalytic activity for the oxidation of glucose. The method of immobilizing GOD without any additional cross-linking agents presented here is easy and facile, which provides a model for other redox enzymes and proteins.

Direct electrochemistry and bioelectrocatalysis of horseradish peroxidase immobilized on active carbon

For the first time horseradish peroxidase (HRP) immobilized on the surface of active carbon powder modified at the surface of a glassy carbon electrode has been shown to undergo a direct quasi-reversible electrochemical reaction. Its formal potential, E-o/, is -0.363 V in phosphate buffer solution (pH 6.8) at a scan rate of 100 mV/s and is almost independent of the scan rate in the range of 50-700 mV/s. The dependence of E-o/ on the pH of the buffer solution indicated that the conversion of HRP-Fe(III)/HRP-Fe(II) is a one-electron-transfer reaction process coupled with one-proton-transfer. The experimental results also demonstrated that the immobilized HRP retained its bioelectrocatalytic activity to the reduction of H2O2. Furthermore, the HRP adsorbed oil the surface of the active carbon powder can be stored at 4 degreesC for several months without any loss of the enzyme activity. The method presented for immobilizing HRP can be easily extended to immobilize and obtain the direct electrochemistry of other enzymes.

Protein structural characterization by scanning tunneling microscopy with electrochemistry

The structure characterization of proteins or enzymes by STM on electrochemically prepared HOPG surface studied in this laboratory is reviewed. The serial structures of Hb were observed. The differences between the denaturation and inactivation of HRP were investigated by in situ and ex situ STM. The structural variation of Hb in an organic solvent was imaged while protein denaturation was easily observed in a polar solvent.

Detection of specific noncovalent protein-fullerenols complexes by matrix-assisted laser desorption ionization mass spectrometry

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry is difficult for the characterization of noncovalent complexes hitherto because of the limitations in acidic matrix, sample preparation, laser-induced polymerization and adduct formation with matrix. Under our experimental conditions, sinapinic acid is used as a matrix, the specific noncovalent interactions of protein with fullerenols were observed by MALDI mass spectrometry. Some mass spectrometric features, such as mass shifts, broad adduct peaks and stoichiometries, showed that the specific non-covalent complexes between protein and fullerenols have been formed at a ratio of 1 : 4 for hemoglobin-fullerenols or 1 : 1 for myoglobin-fullerenols. The results implied that fullereneols could be used to protect partly hemoglobin from decomposition in acidic media, and therefore, it is possible to realize the molecular weight determination of a quaternary protein by MALDI mass spectrometry via the addition of specific organic compound in the matrix.

A COMPARATIVE-STUDY ON STM IMAGING AND ELECTROCATALYTIC ACTIVITY OF DIFFERENT SURFACES MODIFIED WITH FLAVIN ADENINE-DINUCLEOTIDE

Flavin adenine dinucleotide (FAD) was modified onto the highly oriented pyrolytic graphite (hopg) and glassy carbon electrode (gee) surfaces with three methods, respectively. Corresponding image analysis for FAD-modified hopg surfaces has been performed by scanning tunnelling microscope (STM) for the first time. The molecular resolution STM image of FAD adsorbed on the freshly-cleaved hopg was obtained, the quantitative size determination suggests that the FAD molecules adsorb side lying on the substrate surface. The anodization treatment of hopg surface yields many pits, which were clearly observed under STM. These pits provide active sites on the hopg surface for modification and the treated hopg can strongly adsorb FAD molecules, the latter exhibiting an irregular cluster structure on such a surface. When FAD was electrochemically deposited on the substrate surface, a chain structure was successfully observed. The adsorbed FAD on anodized glassy carbon electrode (gee) surface can effectively catalyze the reduction of glucose oxidase, hemoglobin and myoglobin, with a large decrease in the overvoltage, whereas the deposited FAD film exhibits excellent electrocatalysis towards dioxygen reduction.

DIRECT ELECTRON-TRANSFER REACTION OF HEMEPROTEINS PROMOTED BY METHYLENE-BLUE MODIFIED ELECTRODE

The heterogeneous electron transfer reaction of hemeproteins including hemoglobin, myoglobin and cytochrome C at Pt mesh electrode adsorbed methylene blue has been investigated. Thin-layer spectroelectrochemical technique was used for observing the electron transfer processes of three kinds of proteins, and the corresponding electrode rate constants were measured.

评价水产动物生长性能的生理生化指标和分子生物学指标

以奥利亚罗非鱼(Oreochromis aureus)为实验对象，设计了3种不同的摄食类型，分别是鲜活饵料组、饥饿3周后饱食投喂组和人工饲料组。鲜活饵料组投喂冰冻赤子爱胜蚓，利用蚯蚓体内丰富的营养成分和活性物质，以期获得奥利亚罗非鱼良好的生长状况；饥饿后饱食组是指饥饿3周后，以人工饲料饱食投喂2周，用于研究饥饿与补偿生长获得快速生长时血液理化指标的变化情况；人工饲料组作为对照组。纯淡水条件下养殖，水温25±2℃。测定了奥利亚罗非鱼在3种摄食类型饲喂下某些血液生理生化指标变化的情况，并将指标变化情况与增重率做相关性分析，试图找出能够反映奥利亚罗非鱼生长性能的血液生理生化指标。 研究结果表明，奥利亚罗非鱼在饥饿3周后获得了补偿生长，补偿生长时的增重率和特定生长率显著高于人工饲料组(P<0.05)，高于鲜活饵料组，但差别不显著；相关性分析研究表明血清总蛋白、胆固醇、四碘甲状腺原氨酸（T4）与增重率极显著相关(P<0.01)，血红蛋白显著相关(P<0.05)，红细胞、白细胞、碱性磷酸酶高度相关(相关系数为0.580、0.551和0.557)，因此，建议血清总蛋白、胆固醇和血红蛋白可作为能够反映罗非鱼生长性能的新指标。 根据序列设计引物，PCR反应条件：变性温度：95 ℃，3 min；退火温度：57℃，20 sec；延伸温度：72℃，5 min，共36个循环，从牙鲆、黑鲪和鲈鱼中克隆出胰岛素样生长因子（IGF-Ⅰ）部分序列，首次证实了IGF-Ⅰ在3种海水鱼中的存在。 利用蛋氨酸与ZnSO4•7H2O，在pH 5.5、80℃下，反应1小时，采用蛋氨酸与硫酸锌2：1的配料比，合成出了产物蛋氨酸螯合锌，蛋氨酸螯合锌外观白色，粉状，室温下微溶于水，不溶于乙醇，并用原子吸收光谱法测定其含锌量为15%，螯合率为88.2%。

Blood-gas properties of plateau zokor (Myospalax baileyi)

Plateau zokor (Myospalax baileyi) is one of the blind subterranean mole rats that spend their life solely underground in scaled burrows. It is one of the special species of the Qinghai-Tibet plateau. In their burrows, oxygen is low and carbon dioxide is high and their contents fluctuate with the change of seasons, soil types, rain and depth of burrows. However, plateau zokors show successful adaptation to that extreme environment. In this study, their adapting mechanisms to the hypoxic hypercapnic environment were analyzed through the comparison of their blood-gas properties with that of pikas (Ochotona curzniae) and Sprague-Dawley rats. The results indicated that plateau zokors had higher red blood corpuscle counts (8.11 +/- 0.59 (10(12)/L)) and hemoglobin concentrations (147 +/- 9.85 g/L), but hematocrit (45.9 +/- 3.29%) and mean corpuscular volume (56.67 +/- 2.57 fL) were lower than the other rodents. Their arterial blood and venous blood pH were 7.46 +/- 0.07 and 7.27 +/- 0.07. Oxygen pressure in arterial blood of plateau zokors was about 1.5 times higher than that of pikas and rats, and it was 0.36 and 0.26 times in their venous blood. Partial pressure for carbon dioxide in arterial and venous blood of plateau zokors was 1.5-fold and 2.0-fold higher, respectively, than in rats and pikas. Oxygen saturation of plateau zokors was 5.7 and 9.3 times lower in venous blood than that of pikas and rats, respectively. As result, the difference of oxygen saturation in arterial blood to venous blood was 2- and 4.5-fold higher in plateau zokors as that of pikas and rats, respectively. In conclusion, plateau zokors had a high tolerance to pH changes in tissues, together with strong capabilities to obtain oxygen from their hypoxic-hypercapnic environment. (c) 2006 Published by Elsevier Inc.