SEARCH · PubMed Health

Results for “Microcystins”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

PubMed2003

Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains.

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.

Amino Acid Sequence↗

PubMed2006

Attenuating effects of natural organic matter on microcystin toxicity in zebra fish (Danio rerio) embryos -- benefits and costs of microcystin detoxication.

To contribute to the understanding of joined factors in the environment, impact of pure microcystins (-RR and -LF) on zebra fish (Danio rerio) embryos were investigated individually and in combination with a natural organic matter (NOM). The applied NOM was a reverse osmosis isolate from Lake Schwarzer See (i.e., Black Lake, BL-NOM). Teratogenic effects were evaluated through changes in embryonic development within 48 h of exposure. Detoxication activities were assessed by the activities of phase II biotransformation enzymes, soluble and microsomal glutathione S-transferase (s, mGST). Oxidative stress was assessed by determining both the production of hydrogen peroxide and by analyzing the activities of the antioxidative enzymes, guajacol peroxidase (POD), catalase (CAT), glutathione peroxidase (GPx), and the glutathione restoring enzyme glutathione reductase (GR). Energetic costs were evaluated by determining contents of fat, carbohydrates, and proteins in both exposed and control embryos. BL-NOM attenuated toxic effects of MC-LF and MC-RR verified by less pronounced teratological effects within 24 h, in particular, as well as less rise in the activity of s-GST, when compared with embryos exposed to either pure toxins or in combination with organic matter. BL-NOM also diminished oxidative effects caused by MC-LF; however, it failed to attenuate oxidative stress caused by MC-RR. Content of lipids was significantly reduced in exposed embryos following a trend similar to that obtained with teratological and enzymatic assays confirming the attenuating effect of BL-NOM. Physiological responses to microcystins and NOM required energetic costs, which were compensated to the expense of the energy resources of the yolk, which in turn might affect the normal development of embryos.

Animals↗

PubMed2006

Methods for determining microcystins (peptide hepatotoxins) and microcystin-producing cyanobacteria.

Episodes of cyanobacterial toxic blooms and fatalities to animals and humans due to cyanobacterial toxins (CBT) are known worldwide. The hepatotoxins and neurotoxins (cyanotoxins) produced by bloom-forming cyanobacteria have been the cause of human and animal health hazards and even death. Prevailing concentration of cell bound endotoxin, exotoxin and the toxin variants depend on developmental stages of the bloom and the cyanobacterial (CB) species involved. Toxic and non-toxic strains do not show any predictable morphological difference. The current instrumental, immunological and molecular methods applied for determining microcystins (peptide hepatotoxins) and microcystin-producing cyanobacteria are reviewed.

Animals↗

PubMed1997

14C-labeled microcystin-LR administered to Atlantic salmon via intraperitoneal injection provides in vivo evidence for covalent binding of microcystin-LR in salmon livers.

The tissue distribution and clearance of radiolabeled microcystin-LR administered to Atlantic salmon via i.p. injection has been re-examined using uniformly 14C-labeled toxin. Significant differences were found to exist between these results and those obtained when fish received an i.p. injection of tritium-labeled dihydromicrocystin-LR. In addition, MeOH liver extracts were assayed by both phosphatase assay and 14C counts and the results compared with the total levels of incorporation determined by digestion and subsequent 14C counting of the same live tissues. An attempt to investigate the metabolism and to document the putative products was also undertaken. It was found that microcystin-LR was extensively metabolized to compounds that are more polar than the parent compound.

Animals↗

PubMed2006

Detection of microcystin-producing cyanobacteria in Finnish lakes with genus-specific microcystin synthetase gene E (mcyE) PCR and associations with environmental factors.

We studied the frequency and composition of potential microcystin (MC) producers in 70 Finnish lakes with general and genus-specific microcystin synthetase gene E (mcyE) PCR. Potential MC-producing Microcystis, Planktothrixand Anabaena spp. existed in 70%, 63%, and 37% of the lake samples, respectively. Approximately two-thirds of the lake samples contained one or two potential MC producers, while all three genera existed in 24% of the samples. In oligotrophic lakes, the occurrence of only one MC producer was most common. The combination of Microcystis and Planktothrix was slightly more prevalent than others in mesotrophic lakes, and the cooccurrence of all three MC producers was most widespread in both eutrophic and hypertrophic lakes. The proportion of the three-producer lakes increased with the trophic status of the lakes. In correlation analysis, the presence of multiple MC-producing genera was associated with higher cyanobacterial and phytoplankton biomass, pH, chlorophyll a, total nitrogen, and MC concentrations. Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability of mcyE genes, whereas total phosphorus alone accounted for MC concentrations in the samples by logistic and linear regression analyses. In conclusion, the results suggested that eutrophication increased the cooccurrence of potentially MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation.

Anabaena↗

PubMed2004

Proteomics approach on microcystin binding proteins in mouse liver for investigation of microcystin toxicity.

Microcystins (MC) produced by freshwater cyanobacteria are potent hepatotoxins. MC inhibit protein phosphatases (PP) 1 and 2A. MC and okadaic acid (OA), which is a similar PP inhibitor whereas it has a less affinity to PP1 than PP2A, behave similarly to primary culture hepatocytes, with inducements of phosphorylations of cytoskeleton, morphological changes and apoptosis. Although the distribution of OA in mouse liver was observed immunohistochemically, no OA injury was found. The purpose of this study was therefore to determine why only MC has specific toxicities on the liver. A systematic process of MC affinity chromatography and proteomics, using two-dimensional gel electrophoresis and MALDI-TOFMS, indicated the existence of some MC-binding proteins including the complexes of PP1, PP2A, and PP4 with their own regulatory subunits in mouse liver extracts. The competitive inhibition experiments using affinity chromatography with OA showed that two of the three protein complexes strongly interacted with OA, whereas only the complex of PP1 with the inhibitory subunit NIPP1 did not strongly interacted with OA. These results suggest that the PP1 complex is not related to the common behavior of MC and OA of primary culture hepatocytes, and is related to the specific hepatotoxicities of MC.

Animals↗

PubMed2006

Production of antibodies against microcystin-RR for the assessment of purified microcystins and cyanobacterial environmental samples.

Microcystins (MC) are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. Immunoassays provide a sensitive means to detect these toxins, although cross-reactivity characteristics of different antibodies are variable, and most antibodies have been produced against MC-LR. Here, we have produced the first polyclonal antibodies against the commonly occurring variant, MC-RR, and compared them with MC-LR antibodies for the analysis of purified MCs and cyanobacterial environmental samples. Both antisera cross-reacted with all MCs tested, and with the related cyanobacterial hepatotoxin nodularin-R, but not with non-toxic cyanobacterial peptides. In general, better cross-reactivity characteristics were observed with the MC-RR antisera and limits of quantification were lower for most variants, with all MCs tested and nodularin-R having limits of quantification of 0.31 nM or below. The antisera had different affinities to mixtures containing pooled MC-LR and MC-RR, with MC-LR antisera underestimating total MC concentration when MC-RR represented over 70% of the total MC pool. Both antisera correlated well with HPLC-UV data when incorporated into ELISAs to screen previously characterised environmental samples from Aland, Finland. MC-RR antisera are useful for screening samples containing multiple MCs, and particularly for samples primarily containing MC-RR variants.

Antibodies, Bacterial↗

PubMedDate not supplied

Immunohistochemical localization of microcystin-LR in the liver of mice: a study on the pathogenesis of microcystin-LR-induced hepatotoxicity.

The relationship between the intralobular sites of hepatotoxic injury and the distribution of microcystin-LR (MCLR), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), was examined using an immunohistochemical method with a monoclonal antibody specific to MCLR on the livers of mice receiving a single i.p. injection of the MCLR. Immunoblotting and high-performance liquid chromatography analyses of liver extracts were also performed to determine the binding form of MCLR to PP1 and PP2A (MCLR-PP1/PP2A adducts) and free MCLR. Immunohistochemistry revealed a discernible intensity of staining in the centrilobular regions where hemorrhage and apoptosis occurred. In these regions, immunopositivity was evident in the cytoplasm and nuclei of the hepatocytes; some apoptotic cells were also immunopositive. In contrast, coagulative necrosis, which was mainly evident in the midlobular regions, was completely negative. Analysis of liver extracts demonstrated MCLR-PP1/PP2A adducts, but free MCLR was below detection limit. These results suggest that the immunohistochemical localization of MCLR in centrilobular hepatocytes is closely associated with the onset of hemorrhage and apoptosis and is related to adduct formation. The occurrence of coagulative necrosis however might also be related to other factors such as ischemia/hypoxia.

Animals↗

PubMed2006

Microcystin analysis in human sera and liver from human fatalities in Caruaru, Brazil 1996.

In 1996, an extensive exposure of Brazilian hemodialysis patients at a dialysis center, using a municipal water supply water contaminated with cyanotoxins, provided the first evidence for acute lethal human poisoning from the cyclic peptide hepatotoxins called microcystins. During this outbreak, 100 of 131 patients developed acute liver failure and 52 of these victims were confirmed to have been exposed to lethal levels of microcystins. Detection and quantitation of microcystins in these biological samples posed some analytical challenges since there were no well-established and routine analytic methods to measure total microcystins in tissue or sera samples. At the time of the 1996 exposure we used analytic methods that combined the use of enzyme linked immunosorbant assay (ELISA), analytical high performance liquid chromatography (HPLC), electrospray ionization ion-trap mass spectroscopy (ES-ITMS) and matrix assisted laser desorption ionization-time of flight spectroscopy (MALDI-TOF). In the intervening years these methods have been improved and others developed that allow a more quantitative and critical analysis of microcystin contaminated tissue and sera. For these reasons, and to see how storage with time might effect the detection and stability of microcystins in these matrices, we reanalyzed selected liver tissues and sera from the Caruaru victims in Brazil. We developed and validated a procedure to measure total microcystins in Caruaru human sera and liver tissue using a combination of ELISA, liquid chromatography and liquid chromatography-mass spectrometry (LC/MS), GC/MS and MS/MS techniques. GC/MS and LC/MS were followed by MS/MS to obtain a fingerprint fragment spectra for the microcystins. The validity of the extraction procedure for free microcystins was confirmed by recovery experiments with blood sera spiked with microcystin-LR. We removed proteins with the Microcon Centrifugal Filter prior to LC/MS and ELISA analysis. A solid phase extraction (SPE) procedure was used for analysis of protein bound microcystins by conversion of ADDA to erythro-2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) combined with GC/MS. We found that the GC/MS method yielded a higher concentration of microcystin than that obtained by ELISA and LC/MS. We hypothesize that this difference is due to better GC/MS detection of the covalently bound form of microcystins in human liver tissue. We also concluded that microcystins are very stable when stored under these conditions for periods of almost 10 years.

Bacterial Toxins↗

PubMed2005

Distribution of microcystins in a lake foodweb: no evidence for biomagnification.

Microcystins, toxins produced by cyanobacteria, may play a role in fish kills, although their specific contribution remains unclear. A better understanding of the eco-toxicological effects of microcystins is hampered by a lack of analyses at different trophic levels in lake foodwebs. We present 3 years of monitoring data, and directly compare the transfer of microcystin in the foodweb starting with the uptake of (toxic) cyanobacteria by two different filter feeders: the cladoceran Daphnia galeata and the zebra mussel Dreissena polymorpha. Furthermore foodwebs are compared in years in which the colonial cyanobacterium Microcystis aeruginosa or the filamentous cyanobacterium Planktothrix agardhii dominated; there are implications in terms of the types and amount of microcystins produced and in the ingestion of cyanobacteria. Microcystin concentrations in the seston commonly reached levels where harmful effects on zooplankton are to be expected. Likewise, concentrations in zooplankton reached levels where intoxication of fish is likely. The food chain starting with Dreissena (consumed by roach and diving ducks) remained relatively free from microcystins. Liver damage, typical for exposure to microcystins, was observed in a large fraction of the populations of different fish species, although no relation with the amount of microcystin could be established. Microcystin levels were especially high in the livers of planktivorous fish, mainly smelt. This puts piscivorous birds at risk. We found no evidence for biomagnification of microcystins. Concentrations in filter feeders were always much below those in the seston, and yet vectorial transport to higher trophic levels took place. Concentrations of microcystin in smelt liver exceeded those in the diet of these fish, but it is incorrect to compare levels in a selected organ to those in a whole organism (zooplankton). The discussion focuses on the implications of detoxication and covalent binding of microcystin for the transfer of the toxin in the foodweb. It seems likely that microcystins are one, but not the sole, factor involved in fish kills during blooms of cyanobacteria.

Animals↗

PubMed2001

Effects of cell-bound microcystins on survival and feeding of Daphnia spp.

The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones and Daphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to all Daphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among Daphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.

Animals↗

PubMed2004

Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp. strain IO-102-I.

The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda(5)]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda(5)]microcystin-LR and [d-Asp(3),ADMAdda(5)]microcystin-LR and a partial structure of three new [ADMAdda(5)]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.

Bacterial Toxins↗

PubMed1995

Isolation and characterization of microcystins from laboratory cultures and environmental samples of Microcystis aeruginosa and from an associated animal toxicosis.

Six microcystins were identified in a laboratory culture of the cyanobacterium (blue-green alga) Microcystis aeruginosa PCC 7813 using high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) and mass spectrometry (LC-MS). The toxins were purified and further characterized by amino acid analysis and tandem mass spectrometry (MS-MS). The presence of the previously reported microcystin-LR and microcystin-LY was confirmed. Two further microcystins were characterized as microcystin-LW and microcystin-LF. Another two toxins were partially characterized and are believed to be an analog of microcystin-LR (molecular weight 1008) and microcystin-LM (molecular weight 969). Natural bloom material of M. aeruginosa collected from 2 reservoirs was found to have similar microcystin profiles using HPLC-DAD and LC-MS, indicating the widespread occurrence of these microcystin variants. In addition, the presence of 5 of the microcystins was confirmed in the rumen contents of a lamb by LC-MS and LC-MS-MS, providing the first report of microcystins identified in an animal suspected of being poisoned by cyanobacterial hepatotoxins.

Amino Acid Sequence↗

PubMed2001

Diversity of microcystin genes within a population of the toxic cyanobacterium Microcystis spp. in Lake Wannsee (Berlin, Germany).

In order to find out how many genotypes determine microcystin production of Microcystis spp. in field populations, single colonies (clones) were sampled from Lake Wannsee (Berlin, Germany), characterized morphologically, and subsequently analyzed by PCR for a region within the mcyB gene encoding the activation of one amino acid during microcystin biosynthesis. The different morphospecies varied considerably in the proportion of microcystin-producing genotypes. Most colonies (73%) of M. aeruginosa contained this gene whereas only 16% of the colonies assigned to M. ichthyoblabe and no colonies of M. wesenbergii gave a PCR product of the mcyB gene. Restriction fragment length polymorphism revealed seven restriction profiles showing low variability in nucleotide sequence within each restriction type (0.4-4%) and a low to high variability (1.6-38%) between restriction types. In addition, the sequences of amino acids within the mcyB gene were analyzed to compare the specificity of the amino acid activation during microcystin biosynthesis between restriction types and with the occurrence of amino acids in microcystin variants as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Most of the microcystin-producing colonies showed high similarity in the sequence of amino acids and contained microcystin-LR (LR refers to leucine and arginine in the variable positions of the heptapeptide), microcystin-RR, and microcystin-YR, as well as other variants in minor concentrations. It is concluded that the gene product found for most of the microcystin-producing colonies in the lake is rather unspecific and the diversity of microcystin variants in the lake results from activation of various amino acids during microcystin biosynthesis in the same genotypes.

Amino Acid Sequence↗

PubMed2005

Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin.

Microcystins are toxins produced by freshwater cyanobacteria. They are cyclic heptapeptides that exhibit hepato- and neurotoxicity. However, the transport systems that mediate uptake of microcystins into hepatocytes and across the blood-brain barrier have not yet been identified. Using the Xenopus laevis oocyte expression system we tested whether members of the organic anion transporting polypeptide superfamily (rodent: Oatps; human: OATPs) are involved in transport of the most common microcystin variant microcystin-LR by measuring uptake of a radiolabeled derivative dihydromicrocystin-LR. Among the tested Oatps/OATPs, rat Oatp1b2, human OATP1B1, human OATP1B3, and human OATP1A2 transported microcystin-LR 2- to 5-fold above water-injected control oocytes. This microcystin-LR transport was inhibited by co-incubation with the known Oatp/OATP substrates taurocholate (TC) and bromosulfophthalein (BSP). Microcystin-LR transport mediated by the human OATPs was further characterized and showed saturability with increasing microcystin-LR concentrations. The apparent K(m) values amounted to 7 +/- 3 microM for OATP1B1, 9 +/- 3 microM for OATP1B3, and 20 +/- 8 microM for OATP1A2. No microcystin-LR transport was observed in oocytes expressing Oatp1a1, Oatp1a4, and OATP2B1. These results may explain some of the observed organ-specific toxicity of microcystin-LR. Oatp1b2, OATP1B1, and OATP1B3 are responsible for microcystin transport into hepatocytes, whereas OATP1A2 mediates microcystin-LR transport across the blood-brain barrier.

Animals↗

PubMed1998

Determination of some physicochemical parameters of microcystins (cyanobacterial toxins) and trace level analysis in environmental samples using liquid chromatography.

Some physicochemical parameters of three microcystin standards, known to be potent hepatotoxins produced by cyanobacteria, were determined using well defined chromatographic measurements. The logarithm of their retention factor on octadecylsilica (ODS) with water as the eluent, which is an estimation of the hydrophobicity of a molecule, was assessed at pH 7 at 3.9, 4.2 and 4.4 for microcystins-YR, -LR and -RR, respectively. Though being rather hydrophobic, microcystins also possess polar functions, namely carboxylic acids, amino and amido groups. The ionization of carboxylic groups occurs at pH values of 3.3-3.4. In environmental waters, microcystins are neutral or anionic. They are readily soluble in water, the solubility of microcystin-LR being higher than 1 g/l. Owing to their hydrophobicity and their polar functions, microcystins remain in the aqueous phase rather than being adsorbed on sediments or on suspended particulate matter. In a river water spiked with microcystins at 5 micrograms/l, only 10% was adsorbed on particles and 7% on the sandy sediment after three days. A method using solid-phase extraction on ODS followed by high-performance--or micro--liquid chromatography was optimized to detect microcystins at trace level in water. A clean-up was introduced to eliminate part of the interfering compounds coextracted during the sample percolation. Good recoveries (75-80%) were obtained. The method was linear, reproducible (with relative standard deviations ranging from 5 to 8%) and enabled the determination of microcystins at levels as low as 30 ng/l in drinking water and 100-200 ng/l in surface waters. The production of toxins by two strains of cyanobacteria was evaluated. Variations in the microcystin-LR content in the cells and in the medium of Microcystis aeruginosa PCC7806 were recorded over a five-week period. Toxin production was not correlated to the biomass but depended on the growth stage and was maximal at the end of the exponential growth phase. The release of toxin in water occurred essentially in old cultures where microcystin-LR was determined at concentrations of 170 and 280 micrograms/l in the media of M. aeruginosa PCC7806 and M. aeruginosa PCC7813, respectively. Other microcystins are likely to be synthesized by these strains. But owing to the lack of standards, mass spectrometric detection is required for further identification. This study points out the need of having other standards for water quality monitoring.

Chromatography, High Pressure Liquid↗

PubMed1997

A molecular basis for different interactions of marine toxins with protein phosphatase-1. Molecular models for bound motuporin, microcystins, okadaic acid, and calyculin A.

The hepatotoxic cyclic heptapeptide microcystins and cyclic pentapeptide nodularins are powerful liver tumor promoters and potent inhibitors of the catalytic subunits of protein phosphatase-1 and -2A (PP-1c and PP-2Ac). In marked contrast to microcystins, which interact covalently with PP-1 and PP-2A, the nodularins do not bind covalently to PP-1 and PP-2A and may additionally possess unique carcinogenic properties. The conformation of microcystin-LR has been determined in solution and bound to PP-1c. We show here that the free NMR solution structures of two distinct microcystin structural congeners (microcystin-LR and -LL) are remarkably similar to the bound crystal structure of microcystin-LR. We have exploited this finding by using Metropolis Monte Carlo modeling to dock the solution structures of microcystin-LL and the marine toxin motuporin (nodularin-V) onto the crystal structure of PP-1c. Both of these toxins occupy a position similar to that of microcystin-LR when bound to PP-1c. However, although there are relatively minor differences in the structural orientation of microcystin-LL compared with microcystin-LR, there is a striking difference in the position of the N-methyldehydrobutyrine residue in motuporin relative to the comparable N-methyldehydroalanine residue in microcystin-LR. We propose that this difference in orientation provides a molecular explanation for why nodularins are incapable of forming a covalent linkage with PP-1c. Furthermore, the predicted position of N-methyldehydrobutyrine in motuporin is at the surface of the PP-1c-toxin complex, which may thus facilitate chemical interaction with a further macromolecule(s) possibly relating to its carcinogenic properties. PP-1c and PP-2Ac are also targets for other marine toxins such as okadaic acid and calyculin A. It was therefore of interest to use Metropolis Monte Carlo modeling to dock the known free crystal structures of okadaic acid and calyculin A to the crystal structure of PP-1c. These experiments predict that both okadaic acid and calyculin A are strikingly similar to microcystins and motuporin in their tertiary structure and relative PP-1c binding position.

Animals↗

PubMed1992

Separation and identification of microcystins in cyanobacteria by frit-fast atom bombardment liquid chromatography/mass spectrometry.

In order to separate and identify microcystins, a new analytical method was developed using a frit probe as an interface for fast atom bombardment mass spectral analysis of high performance liquid chromatographic (HPLC) effluents. Two types of HPLC conditions were designed for separation of standard microcystins RR, YR and LR. The HPLC conditions, for example, methanol:0.01% trifluoroacetic acid = 61:39 (containing 0.8% glycerol) as a mobile phase and 0.5 ml/min as a flow rate, provided a base line separation of standard microcystins RR, YR and LR. The HPLC conditions were also effective for separation of the non-toxic geometrical isomers of microcystins RR and LR. The total ion chromatogram of a mixture of standard microcystins showed excellent correlation with the HPLC separation using a u.v. detector. The method was subsequently applied to analysis of microcystins contained in both a culture strain and a field sample, and the procedure from toxin extraction to identification of microcystins was performed within 1 day. The mass chromatogram monitored at m/z 135 that is always observed with abundance in the FAB mass spectra of the purified microcystins, differentiated between microcystins and other types of compounds. This technique allowed the rapid identification of unknown microcystins without standard samples. Additionally, compounds other than microcystins were also found, which would not be seen by u.v. detection at 238 nm.

Chromatography, High Pressure Liquid↗