Microcystin interferes with defense against high oxidative stress in harmful cyanobacteria.

Harmful cyanobacteria producing toxic microcystins are a major concern in water quality management. In recent years, hydrogen peroxide (H2O2) has been successfully applied to suppress cyanobacterial blooms in lakes. Physiological studies, however, indicate that microcystin protects cyanobacteria against oxidative stress, suggesting that H2O2 addition might provide a selective advantage for microcystin-producing (toxic) strains. This study compares the response of a toxic Microcystis strain, its non-toxic mutant, and a naturally non-toxic Microcystis strain to H2O2 addition representative of lake treatments. All three strains initially ceased growth upon H2O2 addition. Contrary to expectation, the non-toxic strain and non-toxic mutant rapidly degraded the added H2O2 and subsequently recovered, whereas the toxic strain did not degrade H2O2 and did not recover. Experimental catalase addition enabled recovery of the toxic strain, demonstrating that rapid H2O2 degradation is indeed essential for cyanobacterial survival. Interestingly, prior to H2O2 addition, gene expression of a thioredoxin and peroxiredoxin was much lower in the toxic strain than in its non-toxic mutant. Thioredoxin and peroxiredoxin are both involved in H2O2 degradation, and microcystin may potentially suppress their activity. These results show that microcystin-producing strains are less prepared for high levels of oxidative stress, and are therefore hit harder by H2O2 addition than non-toxic strains.

Draft Genome Sequences of Two Uncultured Armatimonadetes Associated with a Microcystis sp. (Cyanobacteria) Isolate.

Two genome sequences of the phylum Armatimonadetes, derived from terrestrial environments, have been previously described. Here, two additional Armatimonadetes genome sequences were obtained via single-molecule real-time (SMRT) sequencing of an enrichment culture of the bloom-forming cyanobacterium Microcystis sp. isolated from a eutrophic lake (Brandenburg, Germany). The genomes are most closely affiliated with the class Fimbriimonadales, although they are smaller than the 5.6-Mbp type strain genome.

Advances in genomics, transcriptomics and proteomics of toxin-producing cyanobacteria.

A common misconception persists that the genomes of toxic and non-toxic cyanobacterial strains are largely conserved with the exception of the presence or absence of the genes responsible for toxin production. Implementation of -omics era technologies has challenged this paradigm, with comparative analyses providing increased insight into the differences between strains of the same species. The implementation of genomic, transcriptomic and proteomic approaches has revealed distinct profiles between toxin-producing and non-toxic strains. Further, metagenomics and metaproteomics highlight the genomic potential and functional state of toxic bloom events over time. In this review, we highlight how these technologies have shaped our understanding of the complex relationship between these molecules, their producers and the environment at large within which they persist.

Transcriptomics-aided dissection of the intracellular and extracellular roles of microcystin in Microcystis aeruginosa PCC 7806.

Recent studies have provided evidence for both intracellular and extracellular roles of the potent hepatotoxin microcystin (MC) in the bloom-forming cyanobacterium Microcystis. Here, we surveyed transcriptomes of the wild-type strain M. aeruginosa PCC 7806 and the microcystin-deficient ΔmcyB mutant under low light conditions with and without the addition of external MC of the LR variant (MC-LR). Transcriptomic data acquired by microarray and quantitative PCR revealed substantial differences in the relative expression of genes of the central intermediary metabolism, photosynthesis, and energy metabolism. In particular, the data provide evidence for a lower photosystem I (PSI)-to-photosystem II (PSII) ratio and a more pronounced carbon limitation in the microcystin-deficient mutant. Interestingly, only 6% of the transcriptional differences could be complemented by external microcystin-LR addition. This MC signaling effect was seen exclusively for genes of the secondary metabolism category. The orphan polyketide synthase gene cluster IPF38-51 was specifically downregulated in response to external MC-LR under low light. Our data suggest a hierarchical and light-dependent cross talk of secondary metabolites and support both an intracellular and an extracellular role of MC in Microcystis.

Sensitive detection of organophosphates in river water by means of a piezoelectric biosensor.

A highly sensitive piezoelectric biosensor has been developed for detection of cholinesterase inhibitors. The inhibitor benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on a monolayer of 11-mercaptomonoundecanoic acid (MUA) self-assembled on the gold surface of the sensor. The binding of high-molecular-weight cholinesterase to the immobilized cocaine derivative was monitored with a mass sensitive piezoelectric quartz crystal (quartz crystal nanobalance; QCN). In the presence of an inhibiting substance in the sample, the binding of cholinesterase to the immobilized inhibitor was reduced. The decrease of the rate of mass change was proportional to the concentration of free inhibitor in the sample. This way the affinity sensor followed anti-cholinesterase toxicity and the enzyme activity of ChE was not addressed. A assay for detection of organophosphates (OP) was optimized. Regeneration of the sensor surface was achieved with 1 mol L(-1) formic acid, which enabled 40 measurements with one sensor. All assays were carried out in a flow-through arrangement. The total measurement time (binding+regeneration) was 25 min and the detection limit for different OP (paraoxon, diisopropylfluorophosphate, chlorpyriphos, and chlorfenvinphos) was down to 10(-10) mol L(-1) (0.02 microg L(-1)). This sensor was used for determination of organophosphate (diisopropylfluorophosphate) levels in river water samples.

[Comparative stability assessment of laccases from the basidiomycetes Coriolus hirsutus and Coriolus zonatus in the presence of effectors]. / Sravnitel'noe izuchenie stabil'nosti lakkaz iz bazidiomitsetov Coriolus hirsutus i Coriolus zonatus v prisutstvii éffectorov.

Stability characteristics of the laccases of the basidiomycetes Coriolus hirsutus and Coriolus zonatus were measured comparatively at temperatures 25 and 40 degrees C in the presence of various effectors (proteins, salts, polyalcohols, polyacids, and polyelectrolytes). Stabilization effects of cations on the laccases from C. hirsutus and C. zonatus decreased in the descending series Cu2+ > Mg2+ > Ca2+ and Ca2+ > Mg2+ > Mn2+, respectively. Tween 20 caused insignificant stabilization of the two enzymes. The C. zonatus laccase was also insignificantly stabilized as a result of treatment with bovine serum albumin. The enzymatic activity of the laccase preparations from C. hirsutus and C. zonatus was conserved virtually completely after vacuum drying (84 and 93%, respectively). The most effective stabilizer of the C. hirsutus laccase was found to be dextran (17 kD). Dry preparations treated with this agent conserved up to 95% of the enzymatic activity. The most effective stabilizer of the C. zonatus was polyacrylic acid (102% of the initial activity).

Bioelectrochemical analysis of neuropathy target esterase activity in blood.

Bioelectrochemical analysis of neuropathy target esterase (NTE) and its inhibitors is based on the combination of the NTE-catalyzed hydrolysis of phenyl valerate and phenol detection by a tyrosinase carbon-paste electrode. The use of the tyrosinase electrode improves 10-fold the sensitivity of NTE detection in comparison with a spectrophotometric method. The tyrosinase electrode was found to be suitable for measurements in whole human blood where spectrophotometric detection is considerably restricted. The specificity of NTE in blood for mipafox and di-2-propyl phosphorofluoridate was close to that for neuronal NTE. The NTE-like activity in blood was determined to be 0.19 +/- 0.02 nmol/min/mg of protein.

A new approach for determination of neuropathy target esterase activity.

Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE.

Laccase of Coriolus zonatus: isolation, purification, and some physicochemical properties.

Laccase is one of the lignolytic enzymes found in liquid cultures of the fungus Coriolus zonatus in defined medium. The enzyme was isolated from culture liquid and characterized. Laccase from C. zonatus is a single-chain protein with a molecular mass of 60 kDa. Carbohydrate moiety of enzyme consisted of mannose, galactose and N-acetyl-glucosamine in a ratio of 6:2:0.6, respectively, and comprised 10% of the entire molecule. Isoelectric point was detected at pH 4.6. Laccase was found to have a pH optimum of 4.9 and temperature optimum of 55 degrees C. Substrate specificity studies were conducted with catechol, K-ferrocyanide, hydroquinone, and sinapinic acid as substrates. The highest efficiency of catalysis was observed with sinapic acid as the substrate. The kinetic constants kcat and Km of this reaction were 624 s-1 and 7 microM, respectively.

Automated amplified flow immunoassay for cocaine.

An amplified flow immunoassay (AFIA) was developed for cocaine, which combines a noncompetitive immunoenzymometric assay (IEMA) with an on-line detection of the enzyme label alkaline phosphatase (ALP) by a substrate-recycling biosensor. In the IEMA, the analyte cocaine first binds to a labeled polyclonal anti-cocaine antibody. Then, the excess labeled antibody is separated on an affinity column that contains a perfusion chromatography carrier modified by immobilized cocaine. The unbound complexes of the analyte cocaine with the ALP-labeled antibody are detected postcolumn. The detector senses phenol produced by ALP from phenyl phosphate. As detector, an amperometric substrate-recycling biosensor was used, which consists of a Clark-type oxygen electrode covered by tyrosinase and pyrroloquinoline quinone-dependent glucose dehydrogenase. The lower limit of detection is 380 pM (38 fmol) for cocaine. The sampling rate is 26/h. Cocaine could be detected from "real samples" with an imprecision of +/- 10% (n = 3) and with a recovery of 49 +/- 3% for various concentrations. AFIA is generally important as a new approach for the fast detection of picomolar concentrations of haptens.

Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis.

An isolate of Coriolus hirsutus constitutively expresses substantial amounts of extracellular laccase on a defined growth medium. The most efficient inducer of extracellular laccase synthesis was syringaldazine, which increased the enzyme yield by 1000% at a concentration of 0.11 microM. The constitutive form of the enzyme was purified 312-fold. Laccase from C. hirsutus, with an estimated molecular mass of 55 kDa and pI of 4.0, is a monomeric glycoprotein containing 12% carbohydrate consisting of mannose and N-acetylglucosamine. The laccase was found to contain 3.9-4.1 copper atoms per molecule. The absorption spectrum shows a maximum at 610 nm and a shoulder at 330 nm, which is typical of laccase possessing type 1 and type 3 copper atoms. The parameters of the first type of copper were determined by EPR as g perpendicular=2.046 and g parallel=2.200, A parallel=8.103 x 10(-3) cm-1. Laccase was found to be a pH-stable and thermostable enzyme. With organic substrates it exhibits a pH optimum of 4.5, but with the inorganic substrate K4[Fe(CN)6] this decreased to 3.5. The highest efficiency of catalysis was observed with sinapinic acid as the substrate. The kinetic constants kcat and Km of this reaction were 578 s-1 and 24 microM respectively. It was established that the kinetics of the assayed reaction shows a Ping Pong mechanism.

Ultrasensitive bienzyme sensor for adrenaline.

A biosensor consisting of an analyte-recycling two-enzyme system using laccase (Coriolus hirsutus) and PQQ-dependent glucose dehydrogenase in combination with the electrochemical detection of oxygen depletion at a platinum electrode was used for adrenaline determination in the nano- and subnanomolar concentration range. Measurements were performed in a flow cell providing excellent baseline stability and fast recovery of the sensor. Improved design of the polymer matrix resulted in a lower detection limit of 200 pmol/l for adrenaline. The sensor has successfully been applied to the analysis of adrenaline in effluate of isolated rabbit hearts.

Catecholamine detection using enzymatic amplification.

Different amplification sensors based on the substrate recycling principle were investigated with respect to their applicability to catecholamine detection. In the bioelectrocatalytic approach, glassy carbon electrodes were modified by laccase or a PQQ-dependent glucose dehydrogenase. Substrate recycling occurs and the detection limit is in the lower nanomolar concentration range (e.g. 10 nM dopamine and 1 nM noradrenaline for the laccase- and glucose dehydrogenase-modified electrodes, respectively). Combinations of glucose dehydrogenase with laccase or tyrosinase were investigated as bienzymatic probes. Among the systems we studied, the laccase/glucose dehydrogenase sensor is the most sensitive (detection limit: 0.5 nM adrenaline). The selectivities of the different sensor systems are discussed. Application of the laccase/glucose dehydrogenase electrode in different media (i.e. brain homogenate, heart effluate) was successfully shown. For samples with high concentrations of interfering substances (uric and ascorbic acid), the interferences can be effectively removed using enzymatic methods.

Zeptomole-detecting biosensor for alkaline phosphatase in an electrochemical immunoassay for 2,4-dichlorophenoxyacetic acid.

A bienzyme substrate-recycling biosensor in a flow injection analysis system is described for the sensitive measurement of alkaline phosphatase (ALP) and applied to the fast readout of a competitive immunoassay for the widely used pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The phenol-indicating biosensor consists of a Clark-type electrode covered by a membrane with coentrapped tyrosinase and quinoprotein glucose dehydrogenase. ALP dephosphorylates phenyl phosphate to phenol (K(m) = 36 microM) outside the flow system. Phenol is oxidized in the sensor membrane by the oxygen-consuming tyrosinase via catechol to o-quinone. The quinone is reconverted to catechol by glucose dehydrogenase. This substrate cycling results in a 350-fold amplified sensor response to phenol. The oxygen consumption of the enzyme couple in the presence of phenol is monitored as a decrease in current. A total of 3.2 fM ALP (320 zmol/ 100 microL) has been detected after a 57.5 min incubation with phenyl phosphate. All involved reagents are stable over the time of measurement. The sensor does not produce any measurable blank signals. The immunoassay detects 0.1 microgram/L 2,4-D, the maximum concentration for pesticides allowed in drinking water by European Community regulations. The applicability of this biosensor for fast immunoassay readout is demonstrated by a 2 min incubation. By comparison, a standard photometric method (p-nitrophenyl phosphate) requires overnight incubation.

A new sensitive and simple method for detection of catecholamines from adrenal chromaffin cells.

A biosensor was used for the analysis of catecholamines in media and lysates of cultured bovine adrenal chromaffin cells. The sensor is composed of coimmobilised laccase and glucose dehydrogenase coupled with an oxygen electrode, using the catalytic effect of cate cholamines for glucose oxidation in this system. The analysis time is almost 5 min. The correlation between the biosensor and HPLC determination is 0.99.

Biosensor based on an enzyme modified electrode for highly-sensitive measurement of polyphenols.

The use of glucose dehydrogenase from Acinetobacter calcoaceticus for highly sensitive measurement of polyphenols, based on bioelectrocatalytic analyte recycling, has been demonstrated. A polyphenol (analyte) is oxidized on the surface of a glassy carbon electrode at an anodic potential and is regenerated by immobilized glucose dehydrogenase (GDH) in the presence of glucose, resulting in an amplified response. The dynamic properties of the enzyme-modified glassy carbon electrode allow the convenient monitoring of subnanomolar analyte concentration. The detection limits for p-aminophenol and norepinephrine are 0.2 nM and 0.5 nM, respectively.

Regulation of growth-plate chondrocytes by insulin-like growth-factor I and basic fibroblast growth factor.

A study was performed in order to investigate the possible functional roles of insulin-like growth-factor I (IGFI) and basic fibroblast growth factor (bFGF) in the regulation of mitotic and metabolic activity of growth-plate chondrocytes. Chondrocytes from the distal radial growth plates of calves and the costal physeal cartilage of rats were exposed to these factors, individually and in combination, in primary monolayer culture, to assess their effects. The data showed that bFGF had both a greater potency and a greater efficacy as a mitogen for bovine growth-plate chondrocytes than did IGF-I. The maximum incorporation of 3H-thymidine by bFGF was 8.3 times that in serum-free (control) cultures; the maximum stimulation of incorporation by IGF-I was 2.5 times that in the control medium. In contrast, IGF-I stimulated a maximum incorporation of 35S-sulfate into glycosaminoglycans that was 2.6 times that in the IGF-I serum-free control cultures, while bFGF had no effect or was mildly inhibitory. When used together, these two factors acted synergistically. Incorporation of 3H-thymidine was more than two times greater than the sum of the effects of the growth factors when used alone and 20.5 times greater than that of the growth factor-free control cultures. Physeal chondrocytes from six-day-old rats were mitotically more responsive to bFGF than to IGF-I, but they were more responsive to IGF-I when they had been derived from twenty-eight-day-old rats. Interaction between bFGF and factors in the serum enhanced the mitotic activity of the rat chondrocytes, but bFGF did not interact with IGF-I under the same experimental conditions. In the presence of bFGF, there was a reduction in the stimulation by IGF-I of incorporation of 35S-sulfate and a decrease in the percentage of chondrocytes containing alkaline phosphatase. These growth factors also influenced cellular morphology in culture. In the presence of IGF-I or serum, the rat chondrocytes manifested the polygonal morphology typical of chondrocytes in culture, while bFGF promoted a more elongated spindle shape. Removal of bFGF and replacement by IGF-I restored the polygonal morphology, indicating that this transition is reversible.

Amperometric bi-enzyme based biosensor for the detection of lactose--characterization and application.

Based on the glucose oxidase-beta-galactosidase sequence an enzyme probe for the specific determination of lactose has been developed. beta-Galactosidases from different sources have been compared, the sensor containing beta-galactosidase from Curvularia inaequalis has been characterized in respect of optimal pH, enzyme loading, apparent activity and functional stability. The response of the bi-enzyme probe depends linearly on lactose concentration between 0.02 and 3.00 mmol dm-3. The application to different milk and foodstuff samples resulted in good correlations toward enzymatic photometric (y = (0.956x-1.67) mmol dm-3) and infrared detection (y = (1.0772x-0.3909)%). Using a measuring frequency of 100 h-1 the serial imprecision is about 2% for diluted milk, urine, or foodstuff samples.

Binding of insulin-like growth factor-I (IGF-I) to primary cultures of chondrocytes from rat rib growth cartilage.

Binding of insulin-like growth factor I (IGF-I) to cultured resting, proliferative and hypertrophic growth plate chondrocytes was investigated. The optimal binding conditions and the extent of degradation of the 125I-IGF-I at 20 degrees C were analyzed in a time-course study. The maximal binding without noticeable degradation was observed after 3 h. The binding of IGF-I to the proliferative cells was 2-fold higher than to the resting and the hypertrophic cells. On the proliferative chondrocytes two classes of receptors with different affinities were found. 125I-IGF-I could be displaced from the proliferative cells by unlabelled IGF-I, IGF-II and insulin, respectively. Half maximal binding was observed at 0.3 nmol/l (= 2.2 micrograms/l) of IGF-I, 4.3 nmol/l (= 32 micrograms/l) of IGF-II and 350 nmol/l (= 2000 micrograms/l) of insulin. No specific binding of human growth hormone (hGH) could be demonstrated. When binding of epidermal growth factor (EGF) to the proliferative cells was assessed, little, but specific binding was observed.

Effects of IGF-I, rGH, FGF, EGF and NCS on DNA-synthesis, cell proliferation and morphology of chondrocytes isolated from rat rib growth cartilage.

The effects of IGF-I, rGH, FGF, EGF and NCS on DNA-synthesis were analyzed in resting, proliferative and hypertrophic chondrocytes obtained by fractionation. Proliferation and morphology were studied on non-fractionated cells. The highest stimulation of DNA-synthesis was induced by NCS followed by IGF-I at all stages of chondrocyte differentiation. DNA-synthesis was also stimulated by a low concentration of FGF (1 microgram/1) in proliferative and hypertrophic chondrocytes, while FGF in a higher concentration (10 micrograms/1) had no significant mitogenic effect. Cell proliferation was stimulated by both NCS and IGF-I, whereas FGF and EGF only caused morphological changes. Our data indicate that IGF-I is the main serum growth factor regulating growth and proliferation by interacting with chondrocytes at all stages of differentiation.