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Although it has been well-recognized that microcystin-leucine-arginine (MCLR), the most common form of microcystins, induces neurotoxicity, little is currently known about the underlying mechanism for this neurotoxicity. Here, we found that MCLR (10 ng/μL/mouse, i.c.v.) induces significant neuronal loss in the hippocampus of mice. MCLR-induced neurotoxicity was accompanied by oxidative stress, as shown by a significant increase in the level of 4-hydroxynonenal, protein carbonyl, and reactive oxygen species (ROS). Superoxide dismutase-1 (SOD-1) activity was significantly increased, but glutathione peroxidase (GPx) level was significantly decreased following MCLR insult. In addition, MCLR significantly inhibited GSH/GSSG ratio, and significantly induced NFκB DNA binding activity. Because reduced activity of GPx appeared to be critical for the imbalance between activities of SODs and GPx, we utilized GPx-1 overexpressing transgenic mice to ascertain the role of GPx-1 in this neurotoxicity. Genetic overexpression of GPx-1 or NFκB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly attenuated MCLR-induced hippocampal neuronal loss in mice. However, PDTC did not exert any additive effect on neuroprotection mediated by GPx-1 overexpression, indicating that NFκB is a neurotoxic target of MCLR. Combined, these results suggest that MCLR-induced neurotoxicity requires oxidative stress associated with failure in compensatory induction of GPx, possibly through activation of the transcription factor NFκB.

Cyanobacterial toxins, such as microcystins, which exist in Korean lakes, are strongly toxic in fish, cattle, and humans. This study performs a quantitative analysis of cyanobacterial toxins in water by comparing the strip method and the HPLC method. Because the detection ranges of the strip method and the HPLC method are different, the water samples were diluted. The comparison of the strip method and the HPLC method was made using seven samples that contained different concentrations of microcystin. The quantitative results produced by the strip analysis were significantly aligned with the results of the HPLC analysis. The results of correlation analysis were r

For the quantitative detection of algal toxin, microcystin, a chemiluminescence immunochromatographic assay method was developed. The developed system consists of four parts, chemiluminescence assay strip (nitrocellulose membrane), horse radish peroxidase labeled microcystin monoclonal antibodies, chemiluminescence substrate (luminol and hydrogen peroxide), and luminometer. The performance of the chemiluminescence immunochromatographic assay system was compared with high performance liquid chromatography (HPLC) detection. The detection limit of chemiluminescence immunochromatographic assay system is several orders of magnitude lower than with HPLC. The chemiluminescence immunochromatography and HPLC results correlated very well with the correlation coefficient (r 2 ) of 0.979.

Cyanobacterial toxins, microcystins which exist in Korean lakes show strong toxicity to fish, cattles and human. In this study, we tried to analyze cyanobacterial toxin, microcystin in the Microcystis cultivation solution using test strip, although the most common analytical methods for the detection of microcystin are HPLC and ELISA. This new anlytical method used the advantages of high specifisity and rapidness of test strip, high sensitivity of fluorescence reader. Therefore, we could analyze the trace amount of microcystin existed in various water samples without using the microcystin standards.

A new chemiluminescence immunochromatographic analysis system with high sensitivity and high reproducibility was developed for the determination of microcystins (MCs) in water. Horse radish peroxidase (HRP) labeled microcystin monoclonal antibody was used for the sensitive chemiluminescence detection. The chemiluminescence immunochromatographic analysis system was composed of microcystin LR (MCLR)-monoclonal antibody (mAb)-Horse Radish Peroxidase (HRP) conjugate, MCLR-BSA conjugate, luminol, hydrogen peroxide mixture solution, an immunochromatographic assay strip and luminometer. To detect the concentration of microcystins in water, we utilized one spot analysis of the strip instead of flow type analysis. We could detect the microcystins in water at a concentration as low as 9.45 pg/mL with the chemiluminescence (CL) detection.

Microcystins are a family of cyclic polypeptides produced by different species of cyanobacteria (blue green algae), which can form blooms in lakes and water reservoirs. However, it is difficult to detect microcystins directly in the water since the concentration of the toxins in water is usually too low. It is necessary to develop a simple and quick method to detect microcystins. In this paper, different detection characteristics of fluorescence immunochromatography and gold colloidal immunochromatography for analysis of cyanobacterial toxins were studied. These two immunochromatography assays are easy to perform, rapid, sensitive, and their quantitative range is within detectable microcystin concentrations in water samples. The fluorescence immunochromatographic system has the unique advantages of low detection limit, and satisfactory accurate results are obtained. The gold colloidal immunochromatographic system has the strong advantage of direct detection of microcystins at the test site withou...

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is low and the detection method is usually complicated. We developed a rapid analytical detection method of microcystins gold colloidal immunochromatogeraphic strip. The sensitivity of the strip is about 1 ng/mL for microcystin LR; it is able to distinguish visually among different concentrations of microcystin solutions. The developed gold colloidal strip can detect microcystins within 15 min and does not require either a complicated extraction system, or trained or qualified experts.

Blooms of toxic microorganisms are commonly found in freshwater in several areas of the world. Freshwater poisonings are mainly caused by species of cyanobacteria, also known as blue-green algae. They develop in eutrophic water wherever proper conditions for their growth are found, including a mild wind, a water temperature between 15 and 30 C, a neutral or alkaline pH and a rather high level of mineral nutrients. As a consequence of the extended use of nitrates and phosphates, blooms occur more and more frequently. There is a risk that these blooms contaminate water supplies that are used as recreational areas or as drinking water reservoirs. This is a serious water quality problem because many of the cyanobaterial species are able to produce potent toxins. The most frequently reported toxins are hepatotoxins. They are classified as microcystins and nodularins, the former representing the largest group. All microcystins have a common cyclic heptapeptide structure consisting of (-DAla-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), where MeAsp stands for erythro-β-methylaspartic acid, Mdha for Nmethyldehydroalanine, Adda for 3-amino-9-methoxy-2,6,8trimethyl-10-phenyldeca-4,6-dienoic acid, and X and Z for the variable amino acids that give its name to the molecule. For example, microcystin LR has X = Leusine and Z = Arginine, microcystin RR has X = Arginine and Z = Arginine. Microcystins can be synthesized by various genera of cyanobacteria such as Microcystis, Oscillatoria, Anabaena or Nostoc. They have been responsible for the poisoning of fish, birds, wild and domestic animals in many countries. Adverse effects on human health have also been recognized. Recent researches have actually shown that microcystins act as type 1, 2A and 3 protein phosphatase inhibitors, as well as tumor promoters when present in nanomolar concentrations. Nowadays, WHO guidelines have been established for the monitoring of microcystins in water. Maximal values of 0.1 μg/L in drinking water for a long term exposure or 1 μg/L for a short term exposure have been proposed on the basis of laboratory experiments of toxicity on mice and pigs. Blooms of blue-green algae, which have been increasing in recent years, have the potential to cause problems for the water supply industry. Recent occurrences of toxic algal blooms have highlighted the potential risk of toxins entering drinking water supplies, and posing a threat to public health. This necessitates a need for research to determine the effectiveness of different water treatment processes for removing algal toxins. At present, slow sand filtration has shown some potential to remove toxins by biodegradation. However, not all types of toxin were successfully removed. The limited amount of work on ozonation has shown that it is an effective process for removing toxins, even with relatively low ozone doses. Ozonation does produce by-products, but these have been shown to be non-toxic. Activated carbon adsorption has been shown to be capable of effectively removing different toxins. With powdered activated carbon (PAC), high PAC doses were generally required. However, most of the tests were conducted with higher toxin concentrations than would probably be found in raw waters. Granular activated carbon (GAC) can effectively remove toxins, but the studies have not fully assessed the most suitable GAC. In addition, the PAC doses required may be impractical to dose at a treatment works. For GAC adsorption to be effective, contact times of more than 15 minutes should be considered, assuming that microcystin does not biodegrade on the GAC. Activated Carbon Fiber (ACF) is a recently developed adsorbent which has more superior adsorbing capacity to active carbons. Recently, Chungbuk National University (Chungju, South Korea) has developed a manufacturing process of ACF by using various precursors of fabrics, cottons, viscous rayons and mixed fabrics etc. ACF made by fabrics and cottons has never been used to remove microcystins in water sample. There is no published data available that has investigated water treatment processes using ACF made by fabrics and cottons, In this study we aimed to obtain basic data on the adsorption of the toxic peptide, microcystin LR to the ACF that was prepared in Chungbuk National University. Our future goal would be to identify and develop ACF treatment processes suitable for removal of algal toxins and to provide a basis for estimating potential water treatment usage.

For the detection of cyanobacterial toxin, an Enzyme-linked immunosorbent assay (ELISA) was integrated into a PDMS microchip. The conjugates of microcystin-LR (MCLR) and keyhole limpet hemocyanin (KLH) were adsorbed on the surface of polystyrene beads and these MCLR-KLH polystyrene beads were introduced into a microchamber. MCLR on the surface of polystyrene beads reacted with horseradish peroxides (HRP) conjugated anti-MCLR monoclonal antibody (mAb) which had a competitive reaction with MCLR in water sample. After the enzyme substrate 3,3,5,5-tetramethyl benzidine (TMB) was injected into the chamber and catalyzed by HRP, the color change was detected with a liquid-cord waveguide. This integration shortened the conventional ELISA analysis time from several hours to about 30 min with only 4.2 L MCLR sample consuming which was useful for the environmental analysis. More over, troublesome operations required for ELISA could be replaced by simple operations. The microchip based detection system showed a good sensitivity of 0.05 g/L and maintained good reliability through its quantitative range with low coefficients of variation (2.5-10.5%).

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin (KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. In the ELISA test, a microtiter plate coated with MCLR-bovine serum albumin conjugate was incubated with standard microcystin samples. The amount of antibody bound was determined by the reaction of peroxidase-labeled anti-mouse IgG with its substrate, 3,3′,5,5′-tetramethyl benzidine (TMB). Since the ELISA test was highly sensitive, the newly developed ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 30 and 1600 pg/mL.

Different detection characteristics of fluorescence immunochromatography method and high performance liquid chromatography (HPLC) method for the analysis of cyanobacterial toxins were studied. In particular, low and high limits of detection, detection time and reproducibility and detectable microcystin species were compared when fluorescence immunochromatography method and high performance liquid chromatography method were applied for the detection of microcystin (MC), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa. A Fluorescence immunochromatography assay system has the unique advantages of short detection time and low detection limit, and high performance liquid chromatography detection method has the strong advantage of individual quantifications of several species of microcystins.

An efficient method for the simultaneous separation and purification of microcystins (LR), (RR), and (YR) from the cyanobacterium is described. The method involves supercritical fluid extraction for the fast extraction and column chromatography for the purification of the microcystins LR, RR, and YR. A new method for the simultaneous extraction of microcystins LR, RR, and YR was developed by using supercritical CO2. Three kinds of microcystins were successfully extracted with a ternary mixture (90% CO2, 9.4% acetic acid, 0.6% water). The separation and purification procedure results in a purity of up to 95% for microcystins RR and YR without the need for a preparative high performance liquid chromatography (HPLC). Only for the purification of microcystin LR, we used a further preparative HPLC purification step. The advantage of the method developed here is that the sample handling steps were minimized, thus reducing possible losses of microcystins and saving extraction and purification time.

New monoclonal antibodies (mAbs) against microcystin (MC), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. Using these mAbs, an immunochromatographic assay system was designed for the detection of cyanobacterial hepatotoxins, MCs in water. The immunochromatographic assay system is composed of anti‐MCs‐mAb, fluorescence conjugates in detection solution, an immunochromatographic assay strip, and a laser fluorescence scanner. The performance of the immunochromatographic assay system was compared with high performance liquid chromatography (HPLC) detection. As the detection limit of HPLC is several orders of magnitude higher than with immunochromatographic assay systems, attention was also given to preconcentration of samples with solid phase extraction cartridges.

During its relativelyshort history, supercritical fluid chromatography (SFC) hasbecome an attractive alternative to GC and LC in certainindustrially important applications. SFC gives the advantageof high efficiency and allows the analysis of non-volatile orthermally labile mixtures.The density of the mobile phase in SFC is about 200-500times that in gas chromatography. The effect of shorterintermolecular distances and the resulting increase inmolecular interactions is an enhanced solubilizing capabilityof the solvent towards various solutes. Compounds withmuch higher molecular weights than gas chromatographynormally allows can therefore be chromatographed. How-ever, the most commonly used mobile phases in SFC are allrelatively non-polar fluids. Carbon dioxide, the most widelyused fluid, is no more polar than hexane

Reversed-phase liquid chromatography (RPLC) separation factors for racemic 2,4-dinitrophenyl (DNP) amino acids (Phe and Trp) having various substituents at different positions were measured on native β-CD and heptakis(3-O-methyl)-β-CD as the chiral stationary phase in order to compare enantioselectivity of these phases. Both native β-CD and heptakis(3-O-methyl)-β-CD showed good enantioselectivity for the DNP–amino acids investigated. Enantioselectivities of the two CD phases for the DNP–amino acids vary with the type and position of the substituent on the DNP moiety of the amino acids. Heptakis(3-O-methyl)-β-CD, the cavity of which is more electron-rich than that of native β-CD, showed in general much better enantioselectivity for the amino acid derivatives studied.

The syntheses, structures, and spectroscopic properties of 6(A),6(B)-bis-O-[p-(allyloxy)phenyl]-substituted beta-cyclodextrins have been investigated. Selective activation of the 6(A),6(B)-hydroxy groups was carried out by treating heptakis(2,3-di-O-methyl)-beta-cyclodextrin (1) with 2,4-dimethoxybenzene-1,5-disulfonyl chloride to give 6(A),6(B)-bissulfonate ester 2 in a yield of only 3%. This material was treated with sodium p-(allyloxy)phenoxide in DMF to form 6(A),6(B)-bis-O-[p-(allyloxy)phenyl]-heptakis(2,3-di-O-methyl)-beta-cyclodextrin (3), which had two isomers. One (3A) has the two p-(allyloxy)phenyl arms directed away from the cyclodextrin cavity, and the other (3B) has one of the p-(allyloxy)phenyl groups through the cavity to form a self-inclusion complex. When either 3A or 3B was treated with methyl iodide and sodium hydride, the resulting permethylated 6(A),6(B)-bis-O-[p-(allyloxy)phenyl]heptakis(2,3-di-O-methyl)-6(C),6(D),6(E),6(F),6(G)-penta-O-methyl-beta-cyclodextrin (4) was composed of two isomers, in which 4B is a self-inclusion complex. 3A and 3B also can be converted into a mixture of 3A and 3B in strong base but not when melted in the absence of base. 4A and 4B do not isomerize. Detailed 1D and 2D NMR spectroscopic studies were carried out to characterize the structures of these new compounds, and molecular mechanics techniques were used to explain the experimental facts.

Department of Chemistry, Kwangwoon University, Seoul 139-701, KoreaReceived September 30, 2002Key Words : Microcystin, Monoclonal antibody, CyanobacteriaMicrocystins are a family of cyclic polypeptides producedby different species of cyanobacteria (blue-green algae),which can form blooms in lakes and water reservoirs.

The polarity range of solutes that can be separated by supercritical fluid chromatography (SFC) can be greatly extended by the addition of polar modifiers to supercritical CO2. In this paper, a new mixing device was introduced, in which highly porous stainless-steel filters were used to generate water-modified or methanol-modified carbon dioxide mobile phases. When supercritical CO2 goes through the mixing device, modifiers held within the small pores of filters can be dissolved in the pressurized supercritical fluids. Among the many stainless-steel filters with different porosities whichwere tested, 0.5 and 2μm stainless-steel filters were more effective than the others. To measure the amount of modifiers dissolved in supercritical CO2 after the mixing device, a modifier sensor which consists of a polymer film, i.e. a film of a perfluorosulfonate ionomer (PFSI), was used.

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a microchip based enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin(KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. Since the ELISA test was highly sensitive, the newly developed microchip based ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 0.025 and 0.3 ng/mL.

Cyanobacterial toxins, microcystins, are very potent hepatotoxins and their occurrence has been reported all over the world. They could threaten human health when toxic Microcystis occurs in water supply reservoirs. In this study, the effects of several environmental factors on production and degradation of toxins produced by cyanobacteria in Lake Soyang have been studied. A new rapid quantification method of microcystins, using fluorescence for a detection signal and a lateral-flow-type immunochromatography as a separation system, was used. Chlorine, potassium permanganate, and hydrogen peroxide were used as chemical oxidants for the degradation of microcystin LR. When chlorine was used, the efficiency of degradation was the highest. The degradation reaction took 40 minutes.

6 A ,6 B -Bis-O-[p-(allyloxy)phenyl]per-O-methyl-β-cyclodextrin, prepared from β-cyclodextrin (CD) by a five-step process, has two isomeric forms. Form A has the two p-(allyloxy)phenyl substituents directed away from the CD cavity, while form B is a rotoxane with one p-(allyloxy)-phenyl group in the CD cavity. CD-hexasiloxane copolymers were prepared by treating each form with 1,1,3,3,5,5,7,7,9,9,11,11-dodecamethylhexasiloxane (with Si-H functions on each end) using a platinum catalyst. These copolymeric stationary phases were coated on fused silica capillary columns and found to separate enantiomeric solutes in gas chromatography (GC). The copolymer prepared from form A separated both enantiomeric hydrocarbons and polar solutes. Rotoxane copolymer prepared from form B separated only polar solutes. Since form B copolymer is a rotoxane with the benzene part of the copolymer in the cavity, these results show that an open CD cavity is necessary for interaction and enantiomeric resolution of nonpolar hydrocarbons ; however, polar solutes interact effectively with the polar CD rim or exterior surface.

A new method for the fast extraction of microcystins RR and LR in cyanobacterium was developed using supercritical CO2. The microcystins were successfully extracted with aqueous methanol modified supercritical fluid (90% CO2, 9.0% methanol, 1.0% water). The method developed here has several advantages over solid-phase extraction sample preparation for the analysis of microcystins. Sample handling steps are minimized, thus reducing possible losses of analytes and saving analysis time. No organic solvent extractions are involved in this method, and no cleanup steps are employed.