Pressure-assisted field-amplified sample injection with reverse migrating micelles for analyzing trace steroids in MEKC

This paper describes a novel method that applies pressure-assisted field-amplified sample injection with reverse migrating micelles (PA-FASI-RMM) for the online concentration of neutral analytes in MEKC with a low-pH BGE. After injection of a plug of water into the separation capillary, negative voltage and positive pressure were simultaneously applied to initialize PA-FASI-RMM injection. The hydrodynamic flow generated by the positive pressure compensated the reverse EOF in the water plug and allowed the water plug to remain in the capillary during FASI with reverse migrating micelles (FASI-RMM) to obtain a much longer injection time than usual, which improved stacking efficiency greatly. Equations describing this injection mode were introduced and were supported by experimental results. For a 450-s online PA-FASI-RMM injection, three orders of magnitude sample enhancement in terms of peak area could be observed for the steroids and an achievement of detection limits was between 1 and 10 ng/mL.

Genetic diversity of plankton community as depicted by PCR-DGGE fingerprinting and its relation to morphological composition and environmental factors in lake donghu

To collect information about the genetic diversity of the plankton community and to study how plankton respond to environmental conditions, plankton samples were collected from five stations representing different trophic levels in a shallow, eutrophic lake (Lake Donghu), and investigated by PCR-DGGE fingerprinting. A total of 100 bands (61 of 16S rDNA bands and 39 of 18S rDNA bands) were detected. The DGGE bands unique to any single station accounted for 38% of the total bands, whereas common bands detected at all five stations accounted for only 11%. Using UPGMA clustering and MDS ordination of DGGE fingerprints, stations I and II were found to initially group together into one cluster, which was later joined by station V. Stations III and IV were isolated into two separate groups of one station each. Some differences in grouping relationships were found when analysis was completed on the basis of chemical characteristics and morphological composition, with zooplankton composition showing the greatest variability. However, the most similar stations (I and II) were always initially grouped into one cluster. Moreover, stations that exhibited the same or similar trophic level (stations III and IV), but different concentrations of heavy metals, were further differentiated by the DGGE method. Results of the present study indicated that PCR-DGGE fingerprinting was more sensitive than the traditional methods, as other studies suggested. Additionally, PCR-DGGE appears to be more appropriate for diversity characterization of the plankton community, as it is more canonical, systematic, and effective. Most importantly, fingerprinting results are more convenient for the comparative analyses between different studies. Therefore, the use of the described fingerprinting analysis may provide an operable and sensitive biomonitoring approach to identify critical, and potentially negative, stress within an aquatic ecosystem.

Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.)

The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 mu g L-1) for 2, 4, 8, 16 and 24 h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC50 of microcystins in carp hepatocytes was 169.2 mu g L-1. The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 mu g L-1) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 mu g L-1) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 mu g L-1), they died in the form of necrosis. (C) 2006 Elsevier Inc. All rights reserved.

Induction of oxidative stress and apoptosis by PFOS and PFOA in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus)

Perfluorinated organic compounds (PFOCs) are emerging persistent organic pollutants (POPs) widely present in the environment, wildlife and human. We studied the cellular toxicology of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on oxidative stress and induction of apoptosis in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to PFOS or PFOA (0, 1, 5, 15 and 30 mg L-1) for 24 h, and a dose-dependent decrease in cell viability was determined using trypan blue exclusion method. Significant induction of reactive oxygen species (ROS) accompanied by increases in activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were found, while activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were decreased. Glutathione (GSH) content was reduced following treatment of PFOA and PFOS. A dose-dependent increase in the lipid peroxidation (LPO) level (measured as maleic dialdehyde, MDA) was observed only in the PFOA exposure groups, whereas LPO remained unchanged in the PFOS exposure groups. Furthermore, a significant activation of caspase-3, -8, -9 activities was evident in both PFOS and PFOA exposure groups. Typical DNA fragmentation (DNA laddering) was further characterized by agarose gel electrophoresis. The overall results demonstrated that PFOS and PFOA are able to produce oxidative stress and induce apoptosis with involvement of caspases in primary cultured tilapia hepatocytes. (c) 2007 Elsevier B.V. All rights reserved.

Isolation and characterization of Scophthalmus maximus rhabdovirus

A rhabdovirus associated with a lethal hemorrhagic disease in cultured turbot Scophthalm us maximus Linnaeus was isolated. The virus induced typical cytopathogenic effects (CPE) in 9 of 15 fish cell lines examined and was then propagated and isolated from infected carp leucocyte cells (CLC). Electron microscopy observations revealed that the negatively stained virions had a typical bullet-shaped morphology with one rounded end and one flat base end. The bullet-shaped morphology was more obvious and clear in ultrathin sections of infected cells. Experimental infections also indicated that the S. maximus rhabdovirus (SMRV) was not only a viral pathogen for cultured turbot, but also had the ability to infect other fish species, such as freshwater grass carp. A partial nucleotide sequence of the SMRV polymerase gene was determined by RT-PCR using 2 pairs of degenerate primers designed according to the conserved sequences of rhabdovirus polymerase genes. Homology analysis, amino acid sequence alignment, and phylogenetic relationship analysis of the partial SMRV polymerase sequence indicated that SMRV was genetically distinct from other rhabdoviruses. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified SMRV revealed 5 major structural proteins, and their molecular masses were estimated to be about 250, 58, 47, 42, and 28 kDa. Significant serological reactivity differences were also observed between SMRV and its nearest neighbor, spring viremia of carp virus (SVCV). The data suggest that SMRV is likely a novel fish rhabdovirus, although it is closely related to rhabdoviruses in the genus Vesiculovirus.

Purification and characterization of hydrosoluble components from the sap of Chinese lacquer tree Rhus vernicifera

Continuous gradient elution chromatography (CGEC) was employed to purify and separate enzymes and polysaccharides from the sap of Rhus vernicifera Chinese lacquer tree. There are three different molecules with laccase enzyme activity. Two are enzymes of each other (L1, and L2), whereas the third (RL) is an entirely separate entity. Two polysaccharides (GP1 and GP2) were also found. The Rhus laccase (RL), and isoenzymes L1 and L2, have peak molecular masses of 109,100, 120,000, 103,000 respectively; each has four copper atoms per molecule, and the pI values were 8.2, 8.6, and 9.1, respectively. The structure of the laccases was studied by Fourier-transform infrared (FT-IR) and Matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry. The typical amide I (1646 cm(-1)) and amide II (1545 cm(-1)) bands were observed. The results from MALDI-TOF were similar to those from CGEC, but the molecular mass from the MALDI-TOF was significantly different from that obtained from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (c) 2006 Elsevier B.V. All rights reserved.

Vitellogenin in rare minnow (Gobiocypris rarus): identification and induction by waterborne diethylstilbestrol

Rare minnow (Gobiocypris rarus) is a tiny Chinese carp that has a short life cycle and is easily cultured in the laboratory. In this study, juvenile rare minnows were exposed to waterborne diethylstilbestrol (DES) at 0.05, 0.5 and 5 mug/l in laboratory aquaria. After exposure for 4, 8, 13 and 21 days, juvenile fish were collected and vitellogenin (Vtg) was measured in whole body homogenates. Native and SDS electrophoresis followed by Western blotting were performed for Vtg identification, and a non-competitive ELISA was developed. In the DES exposure groups (0.5 and 5 mug/l DES), Vtg appeared after 4 days, increased significantly after 8 days and reached a maximum on day 13. Further, a significant increase in the hepatosomatic index (HSI) was found in the 5 mug/l DES exposure group after 21 days. These results indicate that rare minnow provides a good model for assessing endocrine disruption by environmental estrogens. (C) 2004 Elsevier Inc. All rights reserved.

Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus

A rhabdovirus was observed from the diseased turbot (Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells. (C) 2004 Published by Elsevier B.V.

Artificial interspecific hybridization between Macrobrachium species

Viable F-1 hybrids were obtained from crosses of female Macrobrachium nipponense and male Macrobrachium hainanense involving spermatophore transfer and artificial insemination. This represents the first successful known case of hybridization of two Macrobrachium species by means of artificial insemination. The hatching rate was over 90%. About 20-60% of newly hatched larvae metamorphosed to postlarvae. The morphological characteristics of the hybrids resembled a combination of features of both parents. Malate dehydrogenase (MDH) and esterase (EST) isozyme electrophoresis indicated parents and F-1 hybrids showed co-dominant expression of the paternal and maternal alleles controlling the isozymes and confirmed the hybridization. (C) 2004 Elsevier B.V. All rights reserved.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.

Characterization of an iridovirus from the cultured pig frog Rana grylio with lethal syndrome

Three virus isolates, RGV-9506, RGV-9807 and RGV-9808, were obtained from cultured pig frogs Rana grylio undergoing lethal infections. Previously, the first isolate, RGV-9506, was shown to be an iridovirus based on ultrastructural and morphological studies. In the present study, the original isolate, along with 2 recent ones, were more extensively characterized by experimental infection studies, histopathology, electron microscopy, serological reactivity, gel electrophoresis of viral polypeptides and DNA restriction fragments, PCR amplification, and nucleic acid sequence analysis of the major capsid protein (MCP) gene. The 3 isolates were shown to be identical to each other, and very similar to FV3, the type species of the genus Ranavirus (family Iridoviridae). These results suggest that RGV should be considered a strain of FV3, and indicate that FV3-like iridoviruses are capable of causing widespread, severe disease among cultured frogs.

A detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription polymerase chain reaction

A rapid, sensitive and highly specific detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription-polymerase chain reaction (RT-PCR) has been developed. Two pairs of PCR primers were synthesized according to the cloned cDNA sequences of the GCHV-861 strain. For each primer combination, only one specific major product was obtained when amplification was performed by using the genomic dsRNA of GCHV-861 strain. The lengths of their expected products were 320 and 223 bp, respectively. No products were obtained when nucleic acids other than GCHV-861 genomic RNA were used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified GCHV-861 dsRNA total genome (0.01 pg up to 1000 pg) were amplified and quantities of as little as 0.1 pg of purified dsRNA were detectable when the amplification product was analyzed by 1.5% agarose gel electrophoresis. This technique could detect GCHV-861 not only in infected cell culture fluids, but also in infected grass carp Ctenopharyngodon idellus and rare minnow Gobiocypris rarus with or without hemorrhagic symptoms. The results show that the RT-PCR amplification method is useful for the direct detection of GCHV.

Continuous-flow polymerase chain reaction microfluidics by using spiral capillary channel embedded on copper

This paper presents a novel method for performing polymerase chain reaction (PCR) amplification by using spiral channel fabricated on copper where a transparent polytetrafluoroethylene ( PTFE) capillary tube was embedded. The channel with 25 PCR cycles was gradually developed in a spiral manner from inner to outer. The durations of PCR mixture at the denaturation, annealing and extension zones were gradually lengthened at a given flow rate, which may benefit continuous-flow PCR amplification as the synthesis ability of the Taq polymerase enzyme usually weakens with PCR time. Successful continuous-flow amplification of DNA fragments has been demonstrated. The PCR products of 249, 500 and 982 bp fragments could be obviously observed when the flow rates of PCR mixture were 7.5, 7.5 and 3.0 mm s(-1), respectively, and the required amplification times were about 25, 25, and 62 min, respectively. Besides, the successful segmented-flow PCR of three samples ( 249, 500 and 982 bp) has also been reported, which demonstrates the present continuous-flow PCR microfluidics can be developed for high-throughput genetic analysis.

毛细管电泳电化学检测联用的应用研究

本论文探讨了毛细管电泳电化学检测器的应用。主要包括以下六个方面的内容：1．毛细管电泳与电化学检测联用测定别嗓呤醇及其代谢产物氧化镖吟醇。该方法简单、快速、灵敏。并且应用于加标尿样的分析时也取得了令人满意的结果。2．采用毛细管电泳一安培检测来定量测定中草药中的活性组分姜黄素，用自制的磷酸三丁酷树脂萃取柱来预处理样品，同时实现对姜黄素的浓缩。经毛细管电泳分离后，姜黄素可以用碳纤维电极检测。3．毛细管电泳电化学发光对尿液中的丙环定的检测。用离子交换柱尽可能除去尿中的离子的干扰，电化学发光试剂印比淀钉采用柱后加入的方式，具有三级胺结构的丙环定可被定量检测到。4．溶胶一凝胶一碳复合材料电极作为毛细管电泳安培检测器的表征。我们报道了这种电极的制作，探讨了不同尺寸的电极在毛细管电泳中的应用。当电极直径100μm，一些分析指标如：检测限、线性范围以及重现性都较好，可以做为安培检测器与毛细管电泳联用。5，毛细管电泳脉冲安培检测生物胺。分离并定量检测了四种生物胺，腐胺、尸胺、亚精胺及精胺。并用该方法测定了牛奶中的生物胺。该方法比已报道的毛细管电泳间接紫外检测的检测限低，比气相色谱及液相色谱需样品体积小，不需要烦琐的衍生步骤。6．溶胶一凝胶一碳复合材料电极作为电泳芯片的安培检测器。我们报道了一种简单，重现性好的芯片上的盘电极的制作方法。由于该安培检测器最大的特点就是稳定性好，重现性高所以我们在其上又沉积了一层铜，以扩大该芯片的应用范围。用集成了该安培检测器的芯片，我们分别测定了肾上腺素和葡萄糖。

ITO导电玻璃和PDMS微芯片毛细管电泳电化学发光检测的研究

微全分析系统是目前很前沿的研究领域，尽管现在还没有真正意义的微全分析系统出现，但它代表了分析科学的发展趋势。本文主要研究了ITO导电玻璃和PDMS微芯片毛细管电泳和电化学发光检测方法。微芯片毛细管电泳对与其联用的检测器有相当高的要求，一些传统的检测方法很难适应于微芯片毛细管电泳。电化学发光检测是一种新兴的检测技术，在化学、生物、医学诊断以及免疫分析中展现出良好的应用前景。如何实现和完善微芯片毛细管电泳与电化学发光检测联用技术是本论文的重点。我们采用聚二甲基硅氧烷（poly（dimethylsiloxone），简称PDMS）和玻璃作为芯片材料，以锢锡氧化物（indium桩n oXide，简称工T0）导电玻璃为工作电极设计了一种集成化的微芯片毛细管电泳电化学发光检测器。其中，芯片的底片由工TO导电玻璃经光刻、化学腐蚀等方法处理后得到。ITO是一种透明的导电材料，作为工作电极集成到芯片的底片上，PDMS层与芯片底片采用可逆键合的方式键合，大大简化了操作并提高了电化学发光信号的采集效率。我们采用脯氨酸作为被测物对检测器进行了表征。在实验过程中，微芯片毛细管电泳及工T0工作电极都表现出良好的稳定性。我们还提出了电化学和电化学发光同时检测技术，应用于微芯片毛细管电泳和常规毛细管电泳。在这种电化学和电化学发光双检测模式中，三联吡陡钉（Ru（bpy）32+既作为电化学发光检测所需的发光试剂与被分析物反应生成激发态的Ru（bpy）32+＊产生电化学发光信号，又在电极表面平行催化电化学反应得到增强的电流响应，提高电化学检测的灵敏度。电化学信号与电化学发光信号同时产生并分别记录，从而实现了电化学和电化学发光同时检测。我们将这种检测技术与芯片或常规毛细管电泳结合，以多巴胺及三种药物分子山蓖若碱、氧氟沙星和利多卡因作为被测物对其进行了表征。这种同时检测方法与其它多检测模式相比更为简单、方便，比单一的电化学或电化学发光检测可以获得更多的被分析物信息，扩大单一检测方式的应用范围。