Hepatotoxin (microcystin) and neurotoxin (anatoxin-a) contained in natural blooms and strains of cyanobacteria from Japanese freshwaters

Amounts of hepatotoxic microcystin and neurotoxic anatoxin-a were estimated in natural blooms and strains of cyanobacteria from freshwaters in Japan. A simultaneous analysis method of anatoxin-a and microcystin was applied to natural bloom samples, which has been dominated by several species and the strains of cyanobacteria which produced simultaneously both toxins. The natural blooms examined in the present study were mainly composed of Anabaena and Oscillatoria, but most also contained Microcystis and other cyanobacteria. Only one sample was almost unialgal, Anabaena spiroides, collected from Lake Sagami. The toxins in 14 samples collected from nine different natural blooms during 1988-1992 were identified as microcystins-RR, -YR, and -LR; desmethyl-7-microcystin-LR (7-DMLR); and anatoxin-a. Microcystins were the main toxins contained in these natural blooms, with anatoxin-a not being detected or of very little quantity. 7-DMLR was detected in samples only from Lake Kasumigaura. Five strains of Anabaena isolated from waters in Japan produced a small amount of anatoxin-a, but no microcystins. One half of the strains of Microcystis produced microcystins and/or anatoxin-a. This is the first study showing Microcystis producing both anatoxin-a and microcystins.     

Role of microcystins in poisoning and food ingestion inhibition of Daphnia galeata caused by the cyanobacterium Microcystis aeruginosa

The effects of microcystins on Daphnia galeata, a typical filter-feeding grazer in eutrophic lakes, were investigated. To do this, the microcystin-producing wild-type strain Microcystis aeruginosa PCC7806 was compared with a mcy- PCC7806 mutant, which could not synthesize any variant of microcystin due to mutation of a microcystin synthetase gene. The wild-type strain was found to be poisonous to D. galeata, whereas the mcy- mutant did not have any lethal effect on the animals. Both variants of PCC7806 were able to reduce the Daphnia ingestion rate. Our results suggest that microcystins are the most likely cause of the daphnid poisoning observed when wild-type strain PCC7806 is fed to the animals, but these toxins are not responsible for inhibition of the ingestion process.     

Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806

Several bloom-forming cyanobacterial genera produce potent inhibitors of eukaryotic protein phosphatases called microcystins. Microcystins are hepatotoxic cyclic heptapeptides and are presumed to be synthesized non-ribosomally by peptide synthetases. We identified putative peptide synthetase genes in the microcystin-producing strain Microcystis aeruginosa PCC 7806. Non-hepatotoxic strains of M. aeruginosa lack these genes. Strain PCC 7806 was transformed to chloramphenicol resistance. The antibiotic resistance cassette insertionally inactivated a peptide synthetase gene of strain PCC 7806 as revealed by Southern hybridization and DNA amplification. This is the first report of genetic transformation and mutation, by homologous recombination, of a bloom-forming cyanobacterium. Chemical and enzymatic analyses, including high-performance liquid chromatography (HPLC), mass spectrometry, amino acid activation, and protein phosphatase inhibition, revealed the inability of derived mutant cells to produce any variant of microcystin while maintaining their ability to synthesize other small peptides. The disrupted gene therefore encodes a peptide synthetase (microcystin synthetase) that is specifically involved in the biosynthesis of microcystins. Our results confirm that microcystins are synthesized non-ribosomally and that a basic difference between toxic and non-toxic strains of M. aeruginosa is the presence of one or more genes coding for microcystin synthetases.     

High-performance liquid chromatographic separation of microcystins derivatized with a highly fluorescent dienophile

Microcystins are potent hepatotoxins produced by cyanobacteria, and are also tumor promoters as well as potent inhibitors of the catalytic subunits of protein phosphatases 1 and 2A. In order to establish a physicochemical method for individual detection and determination of trace amounts of microcystins, we developed a derivatization method for fluorescence (FL) and chemiluminescence (CL) detection, in which a highly fluorescent dienophile, DMEQ-TAD (4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3,5-dione), was used as the labeling reagent. DMEQ-TAD reacted smoothly with the conjugated diene of the Adda moiety to give 2 stereoisomers of the adducts. As a result of the extensive experiments, the following reaction conditions were optimized for the labeling: sample amount, 10 micrograms; reaction solvent, DMF:acetonitrile (1:1); reaction time, 15 minutes; reaction temperature, 70 degrees C; amount of DMEQ-TAD used relative to that of microcystin, 80 equivalent. The resulting 6 adducts from microcystins-LR, -YR, and -RR can be separated from one another using the following reversed phase HPLC conditions in combination with a clean-up using ODS silica gel: column, Cosmosil 5C18-AR (150 x 4.6 I.D. mm); mobile phase, methanol:0.05M phosphate buffer (pH 3) (1:1); flow rate, 1.0 ml/min; detection, FL lambda ex 370 nm, lambda em 440 nm. The detection limits of the DMEQ-TAD derivatives were estimated to be 100 and 500 pg for LR, and 65 and 2,500 pg for RR using FL and CL detections, respectively; and the detection behavior was different from that of the Dns-Cys derivatives, which were more sensitive to CL than FL.     

Determination of butorphanol in horse race urine by immunoassay and gas chromatography-mass spectrometry

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC-MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC-MS system. The ELISA test (20 microl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC-MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC-MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10-250 ng/ml), recovery (+/-100%), intra-assay (4.1-14.9%) and inter-assay (9.3-45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).     

Development of monkey C-reactive protein (CRP) assay methods

Monkey-specific C-reactive protein (CRP) assay methods (enzyme-linked immunosorbent assay (ELISA) and turbidimetric immunoassay (TIA)) were developed. The anti-monkey CRP serum was prepared by immunization of rabbits with the immune complex formed between the acute-phase serum from turpentine oil-inoculated monkeys and goat anti-human CRP serum. The specificity of the rabbit anti-monkey CRP serum was confirmed by immunoelectrophoresis and Western blotting. The purity of monkey CRP prepared by chromatography procedures was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The serum CRP levels in nine normal monkeys, as measured by sandwich ELISA were ranged from 0.26 to 1.42 microg/ml (mean 0.71+/-0.37). The CRP levels in five acute-phase sera of turpentine oil-inoculated monkeys were 248-451 microg/ml (mean 371.2+/-73.8). This monkey-specific CRP assay method was found more sensitive than the human-specific CRP assay method in detecting monkey CRP by TIA.     

Serological diagnosis of experimental Enterococcus faecalis endocarditis

A modified rat model of endocarditis with catheterization for 2 days was established in female Lewis rats using different inocula of Enterococcus faecalis (strain no. EF 19) in order to measure IgG antibodies in serum during the course of infection. Increasing the inocula intravenously resulted in an increase in the CFU/g vegetation and the CFU/g spleen, the ID50 being about 10 CFU/ml and the ID90 about 1x10(2) CFU/ml. The lowest bacterial inoculum infecting 100% of the rats was 3x10(3) CFU/ml, and for further investigations we used this inoculum size. Rats were sacrificed on day 2, 5, 7, 9, 11 and 28 after infection. The CFU/g vegetation and the CFU/g spleen increased until day 7 and then decreased. Serum samples were collected from 129 rats at different times after challenge. Three different ELISA systems were established to measure the IgG antibody responses: E. faecalis sonicate ELISA (a pool of four sonicates of strain no. EF 10, EF 11, EF 19 and EF 48), E. faecalis whole cell ELISA (strain no. EF 19) and E. faecalis purified cell wall ELISA (strain no. EF 19). An IgG antibody response was detected already on day 2, and except for a minor decrease on day 6/7 the antibody response continued to increase until day 14 (whole cell ELISA and sonicate ELISA) and day 21 (purified cell wall ELISA) when a plateau was reached. Significant increases in IgG antibody responses (p<0.05) were found between groups of rats from days 0-2, 2-8/9 and 8/9-14 in the E. faecalis whole cell and sonicate ELISAs and from days 0-2, 2-10/11 and 10/11-21 in the E. faecalis purified cell wall ELISA. In conclusion, we established a model of endocarditis in rats with catheterization for 2 days and were able to demonstrate an increase in IgG antibodies during the course of infection.     

Echo contrast-enhanced three-dimensional power Doppler of intracranial arteries

Microcystins are a family of more than 50 structurally similar hepatotoxins produced by species of freshwater cyanobacteria, primarily Microcystis aeruginosa. They are monocyclic heptapeptides, characterised by some invariant amino acids, including one of unusual structure which is essential for expression of toxicity. Microcystins are chemically stable, but suffer biodegradation in reservoir waters. The most common member of the family, microcystin-LR (L and R identifying the 2 variable amino acids, in this case leucine and arginine respectively) has an LD50 in mice and rats of 36-122 microg/kg by various routes, including aerosol inhalation. Although human illnesses attributed to microcystins include gastroenteritis and allergic/irritation reactions, the primary target of the toxin is the liver, where disruption of the cytoskeleton, consequent on inhibition of protein phosphatases 1 and 2A, causes massive hepatic haemorrhage. Microcystins are tight-binding inhibitors of these protein phosphatases, with inhibition constants in the nanomolar range or lower. Uptake of microcystins into the liver occurs via a carrier-mediated transport system, and several inhibitors of uptake can antagonise the toxic effects of microcystins. The most effective of these is the antibiotic rifampin (a drug approved for clinical use), which protects mice and rats against microcystin-induced lethality when given prophylactically and, in some cases, therapeutically.     

Education and health in the Czech Republic

An enzyme linked immunosorbent assay, specific for prothymosin alpha (ProT alpha) was developed using an antibody against the synthetic C-terminal peptide ProT alpha[101-109] and isolated bovine ProT alpha for the preparation of standard solutions and immunoplates. Due to the antibody used, the ELISA developed was capable of fully discriminating between ProT alpha, the naturally occuring and partially homologous peptide parathymosin alpha (ParaT alpha) and the peptide thymosin alpha1 (T alpha1), whose sequence is identical to the [1-28] sequence of ProT alpha, and its in vivo occurrence is under question. Moreover, due to its improved sensitivity, the ELISA was capable of directly determining ProT alpha concentration in human serum and tissue extracts, without any pretreatment of the samples. ProT alpha levels were directly measured in sera obtained from 48 apparently healthy individuals and 27 patients with diagnosed breast cancer and found to range from 0.67 to 2.34 microg/ml (mean value 1.27 +/- 0.49 microg/ml) and from 0.47 to 1.74 microg/ml (mean value 1.02 +/- 0.29 microg/ml), respectively. ProT alpha levels were also measured in four breast tumor and adjacent normal breast tissue extracts and found to be elevated in the tumor extracts.     

Prevalence of obesity with increased blood pressure in elementary school-aged children

A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.     

The effects of L-deprenyl treatment, alone and combined with GM1 ganglioside, on striatal dopamine content and substantia nigra pars compacta neurons

A novel amperometric HPLC detection method for the cyanobacterial (blue-green algal) peptide toxins microcystin-LR, -YR and -RR was developed. Purified microcystins and cyanobacterial extracts were chromatographed using an internal surface reversed-phase column with acetate- and phosphate-based mobile phase systems. Electrochemical oxidation reactions at 1.20 V vs. Ag/AgCl (glassy carbon working electrode) were show to originate in arginine and tyrosine residues of microcystins.     

Regulation of IRF and STAT gene expression by retinoic acid

Ex vivo production of cytokines as determined by whole blood stimulation and supernatant ELISA is partly determined by heritability. To assess the ability of this system to distinguish between high and low producers the laboratory error and individual variation were investigated. Whole blood samples from healthy volunteers were collected using endotoxin-free tubes and were incubated with 0 to 1000 ng/ml lipopolysaccharide concentrations for 4 and 24 h, and subsequently centrifuged. In the supernatants, TNF-alpha and IL10 were measured by ELISA. Coefficients of variation for the day-to-day variation in the blood sampling, transport and stimulation as well as in the whole blood stimulation per se ranged from 7.5% to 12.3%. The intra-individual variation was 15% (TNF-alpha) and 19% (IL10) in contrast to the inter-individual variation of, on average, 35%. No interchanging of ranks between high and low producers was observed after repeating the whole blood stimulation on distinct days. The whole blood stimulation system is able to distinguish high and low producers of TNF-alpha and IL10.     

Comparison of ELISA and HPLC for the determination of desmosine or isodesmosine in aortic tissue elastin

We developed a rapid and simple method for estimating tissue elastin content by measuring desmosine (D) in tissue hydrolysates by competitive ELISA. We compared the ELISA previously reported HPLC methods. When D or isodesmosine (ID) in hydrolysate of the same elastin preparation were measured by the two different methods, a good linear relationship was obtained (r = 0.854 for human aorta or r = 0.938 for rabbit aorta, respectively). The ELISA method can detect as little as 6 pmol/ml and it may be useful in monitoring elastin metabolism in patients with various connective tissue diseases.     

Clinical benefits of diagnosing incipient AA amyloidosis in pediatric rheumatic diseases as estimated from a retrospective study

We describe here a newly established cell line from an eccrine carcinoma which produced an abundant amount of granulocyte colony-stimulating factor (G-CSF). An eccrine carcinoma of the scalp of a 69 year-old-Japanese female had metastasized to the pleura. Clinically, she had marked neutrophilia (up to 60,000/mm3), and a high level of G-CSF (38.7 x 10(3) pg/ml) was detected in the pleural effusion, as determined by enzyme-linked immunosorbent assay (ELISA). We established a cell line in vitro and maintained the cells in culture for 30 months in 90 subcultures. We investigated whether these tumor cells were able to produce G-CSF in culture and found that they were. We also found that the amount of G-CSF produced paralleled the rise in cell number (26.5 x 10(3) pg/ml at confluency). When culture media were administered to rabbits (25 ml/rabbit), the amount of circulating neutrophils increased until the number was equal to or greater than that resulting from injection of recombinant human G-CSF (rhG-CSF)(75 micrograms). This effect persisted for 7 days. When tumors were induced in SCID and nude mice by injecting cultured cells (1 x 10(7) cells/mouse), the number of circulating neutrophils also correlated well with tumor size in these mice (200,000/mm3, 3 cm tumor). After tumor removal, the neutrophil number returned to normal within 30 days. G-CSFmRNA in cultured, cells was detected by RT-PCR. Based on these results, it was confirmed that the marked neutrophilia observed in the patient was caused by the tumor-generated G-CSF. This is the first G-CSF-producing cell line developed from a cancer of the skin.     

Distinction of CYP1A1 and CYP1A2 activity by selective inhibition using fluvoxamine and isosafrole

A one step competitive Enzyme-Linked Immunosorbent assay (ELISA) method was developed to detect mycobacterial antigen in cerebrospinal fluid (CSF) for the diagnosis of tuberculous meningitis and compared with a standard competitive ELISA method. Indigenously prepared soluble extract of Mycobacterium tuberculosis H37 Rv was used as antigen. The study was conducted using CSF of 230 clinically diagnosed cases of tuberculous meningit is and 208 control subjects. A cutoff value of 0.57 ng/ml by the one step ELISA and 0.5 ng/ml by the standard ELISA method were determined. The specificity of both methods were 100% and positivity was 68.26% and 70.43% respectively. A follow up study was conducted in 63 cases at various interval of time after starting anti-tubercular therapy i.e. at 3 weeks (63 cases), 6 weeks (27 cases) and > or = 4-12 months (13 cases). It was observed that antigen levels decreased gradually, but were much above the cutoff range. Indigenously prepared antigen was compared with antigen prepared in other laboratories and standard molecular weight markers using SDS PAGE (Sodium Do-decyl Sulphate Polycrylamide Gel Electrophoresis).     

Microcystin uptake inhibits growth and protein phosphatase activity in mustard (Sinapis alba L.) seedlings

Mustard (Sinapis alba L.) seeds were cultivated for seven days on a solid nutrient medium supplemented with 040 microg microcystin-RR per ml. Microcystin-RR affected seedling growth (IC50 0.8 microg/ml) and microcystin concentrations > or =5.0 microg/ml produced malformed plants. The inhibition of protein phosphatase 1 and 2A activity correlated with the growth inhibition. The seedlings were also shown to take up 3H-dihydromicrocystin-LR derived radioactivity up to a level corresponding to ca. 80 ng toxin per mg plant protein.     

Heparin and low-dose aspirin restore placental human chorionic gonadotrophin secretion abolished by antiphospholipid antibody-containing sera

This study was conducted to determine whether drugs used for conventional treatments of pregnant women with antiphosholipid syndrome might be able to restore the gonadotrophin-releasing hormone (GnRH)-induced secretion of placental human chorionic gonadotrophin (HCG) in vitro. We tested this hypothesis using a modified enzyme-linked immunosorbent assay (ELISA) and an in-vitro placental culture system. Pharmacological dose of low molecular weight heparin (20 IU/ml) significantly (P < 0.02) reduced the antiphospholipid antibody (aPL) binding in the ELISA and was able to restore GnRH-induced HCG secretion (P < 0.05) in presence of aPL-containing sera. Low-dose aspirin (0.03 M) did not modify aPL binding in the ELISA, but partially restored HCG secretion (P < 0.05). These observations may help to explain the role of these treatments in antiphospholipid syndrome.     

Color Doppler ultrasound used for screening varicocele: the clinical significance as compared to physical examination

Here we present an enzyme-linked immunosorbent assay (ELISA) on microtiter plates for the quantitative determination of anti-D. This method is based on the solubilization of red blood cells sensitized with anti-D and the subsequent measurement of immunoglobulin G by ELISA. The measuring range of the assay is 40-5,000 IU/ml and the lowest quantifiable concentration in plasma is 0.5 IU/ml. The interassay relative standard deviation for concentrations above 130 IU/ml ranges from 3.2 to 8.1% and below 50 IU/ml from 10.5 to 19.7%. Comparison of ELISA with automated hemagglutination shows that the results of the two assays correlate well: r = 0.992, n = 26. The assay was validated for donor plasma samples and anti-D immunoglobulin preparations and it can also be used in assessing the severity of Rh (D) hemolytic disease during pregnancy.     

Effects of phencyclidine on immediate early gene expression in the brain

An enzyme-linked immunosorbent assay (ELISA) was developed for sensitive and specific determination of pravastatin (PS) sodium, a HMG-CoA reductase inhibitor. Preparation of immunogens to obtain antisera was carried out using chemically modified PS; beta-alanine derivative of PS (for ELISA-1) and 5-deoxy- PS (for ELISA-2) were linked to bovine serum albumin via its terminal carboxylic acid by the N-succinimidyl ester method, to avoid intramolecular lactonization of PS. Enzyme-labeled antigens were prepared similarly by coupling with horseradish peroxidase, and were used by homogeneous combination of antisera. The enzymic activity was determined using a microtiter plate coated with second antibody and tetramethylbenzidine as a chromogenic substrate. Both of the ELISA systems enabled the determination of PS in a range of 5 to 500 pg/well, with an IC50 of 36 to 130 pg/well. Cross-reactivties with main metabolites in plasma, which differed from PS in decaline moiety, were less than a few percent. When ELISA-1 was applied to the determination of PS in human plasma directly after dilution with the ELISA buffer, the detection limit and the intra-assay coefficient (5 ng/ml of PS) were 500 pg/ml and 4.5%, respectively. Further, ELISA-1 was validated by gas chromatography-mass spectrometry with the determination of PS in human plasma after oral administration at a dose of 10 mg/body.     

First identification of microcystins in Irish lakes aided by a new derivatisation procedure for electrospray mass spectrometric analysis

Recent animal and bird deaths at several lakes in Ireland were indicative of possible cyanobacterial poisoning. Using protein phosphatase inhibition assays, microcystins (MCs) were identified in extracts of cyanobacteria from several lakes at concentrations ranging from 1.6 to 168 micrograms/g. This is the first report of MCs in Irish freshwaters. The protein phosphatase inhibition assay was used to screen fractions during HPLC purification of the MCs in cyanobacteria (Anabaena and Oscillatoria) and water samples from Corbally and Caragh Lakes. MC-LR, MC-HtyR, MC-FR, and MC-YR and 3 unidentified MCs of m/z values 1028.5, 981, 1042.7 were isolated from the Corbally sample; while the Caragh Lake sample contained largely MC-LR and MC-YR. A new microanalytical technique was developed for the confirmation of MCs which involved the derivatisation of the methyldehydroalanine group of MCs with 2-aminoethanethiol. Electrospray mass spectrometry of these products showed characteristic double-charged ions, and this novel technique was useful for differentiating MCs from co-eluting impurities in HPLC fractions of cyanobacterial extracts.