Response of Wild Type of Arabidopsis thaliana to Copper Stress

Wild type of Arabidopsis thaliana plants were cultivated hydroponically in Hoagland and Arnon nutrient solution and treated with copper (5 – 100 µM) for 2, 4, 7 and 14 d. A progressive decrease of the root length and biomass was observed at increasing Cu concentration in the nutrient solution. Roots accumulated higher amounts of Cu than shoots at all Cu treatments. Changes of cell and chloroplast ultrastructure of Cu-treated plants were also observed. Cu application did not induce formation of Cu-phytochelatin complexes. Changes in glutathione and glutathione disulfide content observed in roots and shoots of Cu-treated plants suggest their participation in amelioration of metal-induced oxidative stress.     

Monoclonal antibody disulfide reduction during manufacturing

Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.     

The effects of reduced and oxidized glutathione on white spruce somatic embryogenesis

Summary The glutathione-glutathione disulfide redox pair was utilized to improve white spurce somatic embryo development. Mature cotyledonary-stage somatic embryos were divided into two groups (A and B) based on morphological normality and the ability of the mature somatic embryos to convert into plantlets. Group A embryos had four or more cotyledons and converted readily upon germination after a partial drying treatment. Group B embryos had three or fewer cotyledons with a low conversion frequency. The addition of reduced glutathione (GSH) at a concentration of 0.1 mM resulted in an increase in embryo production (total population) with a mean total number of 64 embryos per 100 mg embryogenic tissue as well as an increase in post-embryonic root growth. However, at a higher concentration (1 mM), GSH inhibited embryo formation. The manipulation of the tissue culture environment via the inclusion of glutathione disulfide (GSSG), at concentrations of 0.1 and 1.0 mM, enhanced the development of better-quality embryos. This quality was best exemplified when embryos forming four or more cotyledons increased by at least twofold to 73.9% when treated with 1.0 mM GSSG, compared to 38% in control. Furthermore, this improved quality was reflected by an increased conversion frequency. A 20% increase in the ability of the somatic embryo to produce both root and shoot structures during post-embryonic development was noted when embryos were matured on maturation medium supplemented with 1.0 mM GSSG over the control.     

Understanding nicotinamide dinucleotide cofactor and substrate specificity in class I flavoprotein disulfide oxidoreductases: crystallographic analysis of a glutathione amide reductase

Glutathione reductase (GR) plays a vital role in maintaining the antioxidant levels of the cytoplasm by catalyzing the reduction of glutathione disulfide to reduced glutathione, thereby using NADPH and flavin adenine dinucleotide as cofactors. Chromatiaceae have evolved an unusual homolog that prefers both a modified substrate (glutathione amide disulfide [GASSAG]) and a different cofactor (NADH). Herein, we present the crystal structure of the Chromatium gracile glutathione amide reductase (GAR) both alone and in complex with NAD⁺. An altered charge distribution in the GASSAG binding pocket explains the difference in substrate specificity. The NADH binding pocket of GAR differs from that of wild-type GR as well as that of a low active GR that was engineered to mimic NADH binding. Based on the GAR structure, we propose two attractive rationales for producing an efficient GR enzyme with NADH specificity.     

无胶筛分毛细管电泳中核酸的迁移行为及分离的研究

无胶筛分毛细管电泳分析小于1kb的核酸,其迁移率与碱基数的对数成线性关系,长度大于1kb核酸的迁移率不是仅由其分子大小决定。据此可推测小于1kb核酸片段的大小。采用不更换聚合物法分析核酸,迁移时间的变异系数小于1.3％,适于大量样本的快速测定。考虑温度对核酸迁移行为的影响时,观察到22℃时,柱效最高。电进样与压力进样相比,分析大于300bp核酸的柱效提高,但不适于定量分析。     

Electrochemiluminescence detection with integrated indium tin oxide electrode on electrophoretic microchip for direct bioanalysis of lincomycin in the urine

In this article, an antibiotic, lincomycin was determined in the urine sample by microchip capillary electrophoresis (CE) with integrated indium tin oxide (ITO) working electrode based on electrochemiluminescence (ECL) detection. This microchip CE-ECL system can be used for the rapid analysis of lincomycin within 40s. Under the optimized conditions, the linear range was obtained from 5 to 100 microM with correlation coefficient of 0.998. The limit of detection (LOD) of 3.1 microM was obtained for lincomycin in the standard solution. We also applied this method to analyzing lincomycin in the urine matrix. The limit of detection of 9.0 microM was obtained. This method can determine lincomycin in the urine sample without pretreatment, which demonstrated that it is a promising method of detection of lincomycin in clinical and pharmaceutical area.     

Rapid monitoring of autolysis process of proteases by capillary electrophoresis

A protease, MCP-01, produced by a deep-sea psychrotrophic strain of Pseudoaltermonas sp. SM9913 was purified and its autolysis reaction at 20 °C–50 °C was monitored by capillary electrophoresis. Capillary electrophoresis provides a rapid assay because the degree and state of autolysis of protease MCP-01 could be observed within 6 min. The autolysis rate increased as the temperature rose in the tested range. After 30 min incubation at 30 °C, 77% of MCP-01 autolyzed into peptides. However, its activity for the hydrolysis of casein was reduced by only 4%. The rate of loss of activity of MCP-01 was thus slower than that of autolysis of MCP-01 at 30 °C. Similar results were obtained when MCP-01 was incubated at 20 °C, 40 °C and 50 °C. Large peptides produced by autolysis of MCP-01 therefore still have catalytic activity. When these large peptides autolyzed further into smaller peptides, the enzyme conformation that retained its catalytic activity was destroyed and activity was lost.     

Escherichia coli ß-galactosidase is heterogeneous with respect to a requirement for magnesium

Commercially obtained E. coli ß-galactosidase was stored at 25 °C in buffer containing 1 mM MgCl₂ and in buffer containing no added MgCl₂. Samples were removed at set times and the activity of individual enzyme molecules assayed. When stored in the presence of 1 mM magnesium, the number of active molecules did not change over a 2.5-h period. When stored in the absence of added MgCl₂, over half the enzyme molecules became inactive within the first hour. However, those molecules which retained activity remained active for the duration of the experiment. This indicates that there may exist two populations of E. coli ß-galactosidase, one which requires storage in the presence of the higher concentration of Mg²⁺ in order to remain active. There was no observed correlation between this requirement for magnesium and reaction rate. Additionally, the presence of the 1 mM MgCl₂ was found to decrease the average activity of the ß-galactosidase molecules under the conditions employed.     

Vascular Oxidant Stress and Hepatic Ischemia/Reperfusion Injury

The objective of this study was to test the hypothesis that the extracellular oxidation of glutathione (GSH) may represent an important mechanism to limit hepatic ischemia/reperfusion injury in male Fischer rats in vivo. Basal plasma levels of glutatione disulfide (GSSG: 1.5 ± 0.2μM GSH-equivalents), glutathione (GSH: 6.2 ± 0.4 μM) and alanine aminotransferase activities (ALT 12 ± 2U/I) were significantly increased during the l h reperfusion period following l h of partial hepatic no-flow ischemia (GSSG: 19.7 ± 2.2μM; GSH 36.9 ± 7.4μM; ALT: 2260 ± 355 U/l). Pretreatment with 1,3-bis-(2-chloroethyl)-I-nitrosourea (40mg BCNU/kg), which inhibited glutathione reductase activity in the liver by 60%. did not affect any of these parameters. Biliary GSSG and GSH efflux rates were reduced and the GSSG-to-GSH ratio was not altered in controls and BCNU-treated rats at any time during ischemia and reperfusion. A 90% depletion of the hepatic glutathione content by phorone treatment (300 mg/kg) reduced the increase of plasma GSSG levels by 54%, totally suppressed the rise of plasma GSH concentrations and increased plasma ALT to 4290 ± 755 U/I during reperfusion. The data suggest that hepatic glutathione serves to limit ischemialreperfusion injury as a source of extracellular glutathione, not as a cofactor for the intracellular enzymatic detoxification of reactive oxygen species.     

Copper-glutathione complexes under physiological conditions: structures in solution different from the solid state coordination

The physiologically important copper complexes of oxidized glutathione have been examined by electron spin resonance (ESR) spectroscopy in aqueous solution at neutral pH. Low temperature measurements show that the Cu(II) binding site in oxidized glutathione has the same ligand arrangement as in the copper complexes of S-methylglutathione, glutamine, glutamate and glycine. The site is composed of the amino nitrogens and the carboxyl oxygens of two -glutamyl residues; there is no interaction with amide nitrogens, the sulphur bond or the glycyl carboxyl groups. At high metal to ligand ratios a binuclear species exists, in which each Cu(II) binds only to one -glutamyl residue. The previously reported forbidden transition detected at g = 4 is due to non-specific aggregation and not to spin coupling of intramolecular sites. Liquid solution ESR spectra show the Cu(II)-glutathione complex has a lower mobility than the corresponding Cu(II)-S'-methylglutathione species. From the degree of spectral anisotropy the complex with glutathione is calculated to exist as a dimer. These results demonstrate that the physiologically relevant complex between copper and oxidized glutathione in solution is completely different from the known solid state structure determined by crystallography.     

Estimation of receptor-ligand interactions by the use of a two-marker system in affinity capillary electrophoresis

The study of receptor-ligand interactions by affinity capillary electrophoresis (ACE) requires an accurate form of analysis. Here, we examine the use of two noninteracting standards (markers) in the analysis of binding constant data in ACE studies. This concept is demonstrated using two model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and the dipeptide N-acetyl-d-Ala-d-Ala. In this procedure a plug of receptor and noninteracting standards is injected, and analysis of the change in the relative migration time ratio of the receptor, relative to the noninteracting standards, as a function of the concentration of the ligand yields a value for the binding constant. The findings described here demonstrate that data from ACE studies can best be analyzed using two noninteracting standards, yielding values comparable to those estimated using other binding and ACE techniques.     

无胶筛分毛细管电泳分离DNA片段的现状与展望

毛细管电泳已DNA片段分离分析的重要手段。本简述了毛细管电泳中采用无胶筛分介质分离DNA片段的机理研究,介绍了筛分介质近年的研究发展状况,依据分离介质的化学组成,分单聚物、共聚物和混聚物等3个部分进行了评述,并对其发展前景进行了展望。     

Determination of ions, amino acids/amines in the haemolymph of fairy shrimps by capillary electrophoresis with indirect UV and laser-induced fluorescence detection

We identified and quantified the major inorganic ions present in a haemolymph of four species of fairy shrimp, to determine its ionic strength, using capillary electrophoresis, non-destructive micro-technique for the analysis of small amounts of body fluids. This is the first speciation and quantitative analysis of inorganic ions in the haemolymph of fairy shrimps.     

毛细管电泳-激光诱导荧光分析血清多胺的研究

为进一步探讨多胺的生物学作用,建立了毛细管电泳-激光诱导荧光(λ_ex=488 nm,λ_em=513 nm)分析血清多胺方法.在碱性介质中,多胺与荧光素异硫氰酸酯进行衍生化反应,硼酸盐(pH 8.6)作为运行缓冲液,运行电压20 kV,腐胺、精胺、精脒和1,6-己二胺（内标）在8 min内达到基线分离.考察了方法的线性范围、稳定性、检测限和方法的回收率等,方法具有样品处理简单,灵敏度高,速度快等特点.用于正常对照大鼠和肿瘤大鼠血清多胺的测定.结果提示：实验组肿瘤大鼠血清精胺和精脒水平显著高于正常对照大鼠和实验组未出现肿瘤大鼠血清精胺和精脒水平（P＜0.05）,正常对照组大鼠和实验组未出现肿瘤大鼠血清精胺和精脒水平间无显著性差异（P＞0.05）,各组间血清腐胺水平均无显著性差异（P＞0.05）.     

Enantioseparations with the macrocyclic antibiotic ristocetin a using a countercurrent process in CE

The chiral selectivity of ristocetin A was examined in a countercurrent process in CE using a coated column to suppress electroosmotic flow. Excellent enantioseparations of several nonsteroidal antiinflammatories, dansylamino acids, dinitrophenyl‐derivatized amino acids, and other optically active compounds were achieved. The chiral selectivity of ristocetin A also was examined as a function of antibiotic concentration and pH. Enantioresolution was found to significantly improve with a slight increase in migration time upon increasing chiral selector concentration. Enantioselectivities were found to be greatly influenced by pH of the running buffer. Chirality 11:663–668, 1999. © 1999 Wiley‐Liss, Inc.     

Analysis of the DNA Sequencing Quality and Efficiency of the Apollo100 Robotic Microcycler in a Core Facility Setting

Sanger, or dideoxynucleotide sequencing, is an important tool for biomolecular research. An important trend in DNA sequencing is to find new and innovative ways to provide high-quality, reliable sequences in a more efficient manner, using automated capillary electrophoresis. The Apollo100 combines Sanger cycle sequencing and solid-phase reversible immobilization for product purification in a single instrument with robotic liquid handling and microfluidic (Microscale On-chip Valve) chips that have onboard thermal cycling and pneumatic mixing. Experiments were performed to determine how the DNA sequencing results from the Apollo100 compared with conventional, manual methods used in a core facility setting. Through rigorous experimentation of multiple baseline runs and a dilution series of template concentration, the Apollo100 generated sequencing that exceeded 900 bases with a quality score of 20 or above. When comparing actual client samples of amplicons, plasmids, and cosmids, Apollo100 sequencing results did not differ significantly from those reactions prepared manually. In addition, bacterial genomic DNA was sequenced successfully, directly with the Apollo100, although results were of lower quality than the standard manual method. As a result of the microscale capabilities, the Apollo100 offers valuable savings with respect to the quantity of reagents consumed compared with current manual sequencing methods, thereby continuing the demand for smaller template and reagent requirements. In conclusion, the Apollo100 can generate high-quality DNA sequences for common templates equivalent to those produced using manual sequencing methods and increases efficiency through reduced labor and reagents.