Glutathione peroxidase-1 overexpressing transgenic mice are protected from neurotoxicity induced by microcystin-leucine-arginine.

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.

Understanding the structural differences between spherical and rod-shaped human insulin nanoparticles produced by supercritical fluids precipitation.

In this study, the thermal denaturation mechanism and secondary structures of two types of human insulin nanoparticles produced by a process of solution-enhanced dispersion by supercritical fluids using dimethyl sulfoxide (DMSO) and ethanol (EtOH) solutions of insulin are investigated using spectroscopic approaches and molecular dynamics calculations. First, the temperature-dependent IR spectra of spherical and rod-shaped insulin nanoparticles prepared from DMSO and EtOH solution, respectively, are analyzed using principal component analysis (PCA) and 2D correlation spectroscopy to obtain a deeper understanding of the molecular structures and thermal behavior of the two insulin particle shapes. All-atom molecular dynamics (AAMD) calculations are performed to investigate the influence of the solvent molecules on the production of the insulin nanoparticles and to elucidate the geometric differences between the two types of nanoparticles. The results of the PCA, the 2D correlation spectroscopic analysis, and the AAMD calculations clearly reveal that the thermal denaturation mechanisms and the degrees of hydrogen bonding in the spherical and rod-shaped insulin nanoparticles are different. The polarity of the solvent might not alter the structure or function of the insulin produced, but the solvent polarity does influence the synthesis of different shapes of insulin nanoparticles.

New trends in fluorescence immunochromatography.

During the last decade, immunochromatographic techniques, in particular fluorescence labeled immunochromatography, have become more popular for the determination of low concentrations of analytes. It offers several potential advantages including highly sensitive detection and wide applications in clinical chemistry, bioanalysis, and environmental analysis. Currently, fluorescence labeled immunochromatography is not the exclusive preserve for a few specialists who are well experienced with antigen-antibody analysis, since the recent developments in fluorescence labeled immunochromatography have become widespread, from simple screening application to quantitative analysis of analytes. In this review, we assembled recent advances in the development and applications of fluorescence labeled immunochromatography techniques, considering their potential benefits in the future.

Quantitative analysis of a prostate-specific antigen in serum using fluorescence immunochromatography.

A quantitative analysis of prostate-specific antigen (PSA) in samples of human blood serum by fluorescence immunochromatography using monoclonal antibodies to PSA was developed. The fluorescence immunochromatographic analysis system is composed of anti-PSA-monoclonal antibody (mAb), fluorescence conjugates in detection solution, a immunochromatographic assay strip, and a laser fluorescence scanner. A fluorescence immunochromatographic analysis system was employed to detect PSA on the basis of the area ratio between the control line and the test line of the strip. Under optimal conditions, the area ratio was proportional to PSA concentration ranging from 0.72 to 46.0 ng/mL with a detection limit of 0.72 ng/mL.

Determination of trace amount of cyanobacterial toxin in water by microchip based enzyme-linked immunosorbent assay.

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.

Degradation of cyanobacterial toxin, microcystin LR, using chemical oxidants.

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.

Genetic overexpression of glutathione peroxidase-1 attenuates microcystin-leucine-arginine-induced memory impairment in mice.

Microcystin-leucine-arginine (MCLR) is the most common form of microcystins, which are environmental toxins produced by cyanobacteria, and its hepatotoxicity has been well-documented. However, the neurotoxic potential of MCLR remains to be further elucidated. In the present study, we investigated whether intracerebroventricular (i.c.v.) infusion of MCLR induces mortality and neuronal loss in the hippocampus of mice. Because we found that MCLR impairs memory function in the hippocampus at a low dose (4 ng/µl/mouse, i.c.v.) without a significant neuronal loss, we focused on this dose for further analyses. Results showed that MCLR (4 ng/µl/mouse, i.c.v.) significantly increased oxidative stress (i.e., malondialdehyde, protein carbonyl, and synaptosomal ROS) in the hippocampus. In addition, MCLR significantly increased superoxide dismutase (SOD) activity without corresponding induction of glutathione peroxidase (GPx) activity, and thus led to significant decrease in the ratio of GPx/SODs activity. The GSH/GSSG ratio was also significantly reduced after MCLR treatment. GPx-1 overexpressing transgenic mice (GPx-1 Tg) were significantly protected from MCLR-induced memory impairment and oxidative stress. The DNA binding activity of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) in these mice was significantly enhanced, and the ratios of GPx/SODs activity and GSH/GSSG returned to near control levels in the hippocampus. Importantly, memory function exhibited a significant positive correlation with the ratios of GPx/SODs activity and GSH/GSSG in the hippocampus of MCLR-treated non-transgenic (non-Tg)- and GPx-1 Tg-mice. Combined, our results suggest that MCLR induces oxidative stress and memory impairment without significant neuronal loss, and that GPx-1 gene constitutes an important protectant against MCLR-induced memory impairment and oxidative stress via maintaining antioxidant defense system homeostasis, possibly through the induction of Nrf2 transcription factor.

Self-Inclusion Complexes Derived from Cyclodextrins: Synthesis and Characterization of 6(A),6(B)-Bis-O-[p-(allyloxy)phenyl]-Substituted beta-Cyclodextrins.

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.

Extraction and analysis of microcystins RR and LR in cyanobacteria using a cyano cartridge.

A new method for the extraction of microcystins RR and LR in cyanobacteria was developed using a cyano cartridge. Lyophilized cells (100 mg) were extracted with 5% (v/v) acetic acid. The extract was centrifuged and then the supernatant was applied to a CN cartridge. The cartridge that contained microcystins was rinsed with 5 ml of water and 5 ml of 0.5 M acetic acid, followed by 5 ml of 5% acetonitrile in water. Microcystins were finally eluted from the CN cartridge with 70% acetonitrile in water and were determined by HPLC. Better recoveries and chromatograms were observed than with ODS cartridges.

Size prediction of recombinant human growth hormone nanoparticles produced by supercritical fluid precipitation.

Solution-enhanced dispersion by supercritical fluids (SEDS) was applied to produce nano-sized recombinant human growth hormone (hGH) particles. Ethanol was used to help the supercritical carbon dioxide to extract water from the aqueous protein solution. Various sizes of hGH nanoparticles were successfully prepared with a narrow particle size distribution from aqueous ethanol solution without using any additive. The theoretical particle sizes were deduced from the calculated droplet sizes based on a modified Jasuja's equation. The calculated mean particle sizes and the experimentally obtained ones were compared and the results showed an excellent correlation coefficient (R (2)) of 0.995.

Rapid analytical detection of microcystins using gold colloidal immunochromatographic strip.

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.

Enzyme-linked immunosorbent assay detection of microcystins using new monoclonal antibodies.

New monoclonal antibodies (mAbs) against the microcystin-leucine-arginine variant (microcystin-LR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. Using these mAbs, an enzyme-linked immunosorbent assay (ELISA) experiment was made for the detection of cyanobacterial hepatotoxins, microcystins in water sampled in Soyang Lake, Korea. The performance of the ELISA test with mAbs established in this study was evaluated. The ELISA detection was compared with HPLC detection. Since the detection limit of HPLC is several orders of magnitude higher than with ELISA, attention was also paid to concentration of samples with solid phase extraction cartridges.