Biomedical subjects

Jutta Fastner

Publications and source records attributed to Jutta Fastner.

18 recordsLinked to original sources

PubMed2005

First report on cylindrospermopsin producing Aphanizomenon flos-aquae (Cyanobacteria) isolated from two German lakes.

Three single-filament isolates of Aphanizomenon flos-aquae from two German lakes were found to produce remarkable amounts of the cyanobacterial hepatotoxin cylindrospermopsin (CYN). CYN-synthesis of the strains were evidenced both by LC-MS/MS analysis and detection of PCR products of gene fragments which are implicated in the biosynthesis of the toxin. The strains contain CYN in the range of 2.3-6.6 mg g(-1) of cellular dry weight. To our knowledge this is the first report of CYN in A. flos-aquae.

Alkaloids↗

PubMed2005

Toxicity of culturable cyanobacteria strains isolated from the Portuguese coast.

Cyanobacteria are common inhabitants of terrestrial and aquatic ecosystems. In the aquatic environments their success is particularly well documented in fresh and brackish waters. References related to the occurrence of these organisms in marine habitats and especially in what concerns to toxicology are scarce. Reports of cyanotoxins in marine habitats are restricted to the warmer temperate and tropical regions. In this study, cyanobacteria strains belonging to the genera Cyanobacterium, Oscillatoria, Synechocystis and Synechococcus were isolated from rocky beaches along the Portuguese coast and were cultured and screened for toxicological properties. The toxicity to mice was observed after intraperitoneal injection of centrifuged and non-centrifuged extracts. The results showed a wide range of responses with strains showing no signs of poisoning and strains producing protracted or slightly protracted toxic effects. Neurotoxic symptoms and effects in liver, kidney, small intestine and lungs are reported. The identity of the toxic compounds is thus far unknown but the implication drawn from the present data is that more than one toxic compound is produced. There was a clear discrepancy between the toxicity of centrifuged and non-centrifuged extracts. In most cases the pellet was toxic indicating that toxicity can be associated with compounds tightly bound to the cell walls.

Animals↗

PubMed2005

Variability of the microcystin synthetase gene cluster in the genus Planktothrix (Oscillatoriales, Cyanobacteria).

In populations of Planktothrix, microcystin-producers and non-producers, which are morphologically identical, coexist. In order to develop a basis for the reliable detection of microcystin producers in field samples with polymerase chain reaction (PCR) based methods, we studied the presence and variability of eight regions of the mcy gene cluster in 46 Planktothrix strains, including both microcystin-producing and non-producing ones. PCR-amplification products for two mcy gene regions were also found in non-microcystin-producing strains, indicating the existence of natural mutants. PCR-products of the other regions studied were only detected in microcystin-producing strains. Two of these mcy-amplicons were variable in sequence and length. Four gene regions remained that were conserved and specific for microcystin-producing Planktothrix strains, and thus qualified to detect the respective chemotypes in environmental samples.

Bacterial Proteins↗

PubMed2005

Genetic identification of microcystin ecotypes in toxic cyanobacteria of the genus Planktothrix.

Microcystins (MCs) are toxic heptapeptides which are synthesized by the filamentous cyanobacterium Planktothrix and other genera via non-ribosomal peptide synthesis. MCs share the common structure cyclo(-D-ala1-L-X2-D-erythro-beta-iso-aspartic acid3-L-Z4-adda5-D-Glu6-N-methyl-dehydroalanine7) [Adda; (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid], in which numerous MC variants have been reported. In general, the variation in structure is due to different amino acid residues in positions 7, 2 and 4 within the MC molecule, which are thought to be activated by the adenylation domains mcyAAd1, mcyBAd1 and mcyCAd, respectively. It was the aim of the study (i) to identify MC ecotypes that differed in the production of specific MC variants and (ii) to correlate the genetic variation within adenylation domains with the observed MC variants among 17 Planktothrix strains. Comparison of the sequences of mcyAAd1 revealed two distinctive Ad-genotypes differing in base pair composition and the insertion of an N-methyl transferase (NMT) domain. The mcyAAd1 genotype with NMT (2854 bp) correlated with N-methyl-dehydroalanine and the mcyAAd1 genotype without NMT (1692 bp) correlated with dehydrobutyrine in position 7. Within mcyBAd1, a lower genetic variation (0-4 %) and an exclusive correlation between one Ad-genotype and homotyrosine as well as another Ad-genotype and arginine in position 2 was found. The sequences of mcyCAd were found to be highly similar (0-1 % dissimilarity) and all strains contained arginine in position 4. The results on adenylation domain polymorphism do provide insights into the evolutionary origin of adenylation domains in Planktothrix and may be combined with ecological research in order to provide clues about the abundance of genetically defined MC ecotypes in nature.

Adenosine Monophosphate↗

PubMed2005

Microcystins (cyanobacterial toxins) in surface waters of rural Bangladesh: pilot study.

In Bangladesh the exposure of millions of inhabitants to water from (shallow) tube wells contaminated with high geogenic loads of arsenic is a major concern. As an alternative to the costly drilling of deep wells, the return to the use of surface water as a source of drinking water is considered. In addition to the well-known hazards of water borne infectious diseases associated with the use of surface water, recently the potential public health implications of toxic cyanobacteria have been recognized. As a first step towards a risk assessment for cyanotoxins in Bangladesh surface waters, seston samples of 79 ponds were analysed in late summer 2002 for the presence of cyanobacteria and microcystins (MCYST), the most frequently detected cyanobacterial toxins worldwide. Microcystins could be detected in 39 ponds, mostly together with varying abundance of potentially microcystin-producing genera such as Microcystis, Planktothrix and Anabaena. Total microcystin concentrations ranged between <0.1 and > 1,000 microg l(-1), and more than half of the positive samples contained high concentrations of more than 10 microg l(-1). The results clearly show that concentrations of microcystins well above the provisional WHO guideline value of 1 microg l(-1) MCYST-LR can be frequently detected in Bangladesh ponds. Thus, an increasing use of surface water for human consumption introduces a risk of replacing one health hazard by another and therefore needs to be accompanied by cyanotoxin hazard assessments.

Bacterial Toxins↗

PubMed2004

Interlaboratory comparison trial on cylindrospermopsin measurement.

The hepatotoxin cylindrospermopsin (CYN) is a potent inhibitor of protein synthesis in mammalian cells. It is produced by freshwater cyanobacterial blooms in countries such as Australia, the United States, Israel, Thailand, and Brazil. An interlaboratory comparison was organized as a first step to evaluate the measurement of CYN in lyophilized cyanobacterial cells. Six laboratories from Europe, Israel, and Australia participated in the trial. All of the methods used for extraction of the toxin and the high-performance liquid chromatography (HPLC) analysis were satisfactory on the basis of statistical evaluation, according to ISO standards 5725-1 and -2. Further comparison of all the extraction methods by the organizer indicated that the most effective extraction procedure used 5% formic acid to prevent interference in chromatograms by contaminant compounds when analyzed using HPLC employing isocratic conditions of 5% (v/v) aqueous methanol plus 0.1% (v/v) trifluoroacetic acid as the mobile phase.

Alkaloids↗

PubMed2004

First report of a microcystin-containing bloom of the cyanobacteria Microcystis spp. in Lake Oubeira, eastern Algeria.

Lake Oubeira has been used as the main source of drinking water for many communities in the East of Algeria. In this lake, nutrient loading coupled with year-round warm weather favors the growth of cyanobacteria, several of which can produce cyanotoxins, especially the potent liver toxins called microcystins (MCYSTs). The present study evaluated microcystin levels and characterized the different microcystin variants present in the raw water during a 17-month period (April 2000-September 2001), as measured by protein phosphatase inhibition assays and by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, respectively. The results showed that microcystin concentrations in the lake water varied between 3 and 29,163 microg microcystin-LR equivalent per liter. The microscopic examination of the phytoplankton samples showed the dominance of the Microcystis genus in the cyanobacterial bloom. The highest MCYST concentration was observed in August 2001, at 29,163 microg/l. Therefore, the highest total MCYST content per phytoplankton biomass was found in August 2001, with 4,590 microg MCYST-LR equivalents/g dried bloom material. Analysis of the field bloom extract by MALDI-TOF mass spectrometry demonstrated the presence of four variants of microcystins: microcystin-LR (MCYST-LR), microcystin-YR (MCYST-YR), microcystin-RR (MCYST-RR), and a demethylated variant of MCYST-LR (D-MCYST-LR).

Algeria↗

PubMed2004

Distribution of microcystin-producing and non-microcystin-producing Microcystis sp. in European freshwater bodies: detection of microcystins and microcystin genes in individual colonies.

Microcystis is a well-known cyanobacterial genus frequently producing hepatotoxins named microcystins. Toxin production is encoded by microcystin genes (mcy). This study aims (i) to relate the mcy occurrence in individual colonies to the presence of microcystin, (ii) to assess whether morphological characteristics (morphospecies) are related to the occurrence of mcy genes, and (iii) to test whether there are geographical variations in morphospecies specificity and abundance of mcy genes. Individual colonies of nine different European countries were analysed by (1) morphological characteristics, (2) PCR to amplify a gene region within mcyA and mcyB indicative for microcystin biosynthesis, (3) matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to detect microcystins. Almost one hundred percent of the colonies predicted to produce microcystins by PCR analysis were found to contain microcystins. A high similarity in microcystin variants in the different colonies selected from lakes across Europe was demonstrated. The different morphospecies varied in the frequency with which they contained mcy genes. Most colonies (>75%) of M. aeruginosa and M. botrys contained the mcy genes, whereas < or = 20% of the colonies identified as M. ichthyoblabe and M. viridis gave a PCR product of the mcy genes. No colonies of M. wesenbergii gave a PCR product of either mcy gene. In addition, a positive relationship was found between the size of the colony and the frequency of those containing the mcy genes. It is concluded that the analysis of morphospecies is indicative for microcystin production, although the quantitative analysis of microcystin concentrations in water remains indispensable for hazard control.

Bacterial Proteins↗

PubMed2004

Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp.

To investigate the abundance of active and inactive microcystin genotypes in populations of the filamentous cyanobacterium Planktothrix spp., individual filaments were grown as clonal strains in the laboratory and analysed for microcystin synthetase (mcy) genes and microcystin. Twenty-three green-pigmented strains of P. agardhii originating mostly from shallow water bodies fell into two groups, those possessing mcyA and those lacking mcyA. In contrast, all of the 49 strains that were assigned to the red-pigmented P. rubescens contained mcyA. One strain of P. agardhii and eight strains of P. rubescens contained the total microcystin synthetase gene cluster but were found inactive in microcystin synthesis. To investigate the natural abundance of inactive mcy genotypes in P. rubescens individual filaments sampled from Lake Irrsee and Lake Mondsee (Austria) were analysed directly for the presence of mcyA and microcystin by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. All filaments assigned to P. rubescens contained mcyA. The proportion of inactive microcystin genotypes in populations with a low (Irrsee) or high density (Mondsee) of P. rubescens was 5% and 21%, each. The results of this study demonstrate that P. rubescens typically contain mcy genes whereas P. agardhii have a patchy distribution of mcy genes. In both species microcystin producers co-occur with non-microcystin producers due to the absence/inactivation of mcy genes.

Bacterial Proteins↗

PubMed2004

Toxic and nontoxic microcystis colonies in natural populations can be differentiated on the basis of rRNA gene internal transcribed spacer diversity.

Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.

Base Sequence↗

PubMed2003

Isolation, characterization, and quantitative analysis of Microviridin J, a new Microcystis metabolite toxic to Daphnia.

This paper describes the purification and characterization of microviridin J. a newly discovered metabolite of Microcystis that causes a lethal molting disruption in Daphnia spp., upon ingestion of living cyanobacterial cells. Microviridin J consists of an acetylated chain of 13 amino acids arranged in three rings and two side chains. Unlike other known isoforms of microviridin, microviridin J contains arginine that imparts a unique solution conformation characterized by proximal hydrophobic interactions between Arg and other regions of the molecule. This eventually results in the formation and stabilization of an additional ring system. Microviridin J potently inhibits porcine trypsin, bovine chymotrypsin, and daphnid trypsin-like proteases. The activity against trypsin is most likely due to Arg and its distinctive conformational interactions. Overall, the data presented for microviridin J emphasize once again the ability of cyanobacteria to produce numerous and potent environmental toxins.

Animals↗

PubMed2003

Effects of light on the microcystin content of Microcystis strain PCC 7806.

Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 micro mol of photons m(-2) s(-1) on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell(-1) and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 micro mol of photons m(-2) s(-1), the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 micro mol of photons m(-2) s(-1) than at 10 micro mol of photons m(-2) s(-1) and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.

Bacterial Toxins↗

PubMed2003

Microcystin biosynthesis in planktothrix: genes, evolution, and manipulation.

Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.

Amino Acid Sequence↗

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↗

PubMed2002

An international intercomparison exercise for the determination of purified microcystin-LR and microcystins in cyanobacterial field material.

The comparability of current microcystin analysis methods has been evaluated in an international intercomparison exercise. The focus was on the analysis of microcystins by high-performance liquid chromatography coupled with ultraviolet or photodiode-array detection (HPLC-PDA/UV), currently the most widespread method for microcystin analysis, but the exercise was open for other methods such as enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay (PPA) and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS).Thirty-one laboratories from 13 countries participated in the study. For a microcystin-LR (MC-LR) standard solution (S1) of undisclosed quantity, and for a field sample (S3) from a natural cyanobacterial bloom, repeatabilities between 4 and 15% and reproducibilities between 24 and 49% were obtained. No significant differences between single methods were found for S1 and S3, except for a significantly higher repeatability value of ELISA for S1. However, the analysis of microcystins in the field sample (S3) by HPLC-PDA/UV was significantly more variable than for the standard solution (S1). Both the extraction and the analysis of the microcystins appeared to contribute to this variability. It is concluded that standard MC-LR (S1) can be measured with adequate precision by all participating laboratories independently of the method used. With respect to the different methods used the results for the field sample can also be regarded as satisfactory, but clearly showed the need for improvement by standardisation between laboratories. Furthermore, quantification with in-house standards compared to quantification using the supplied MC-LR standard indicated that routine microcystin analysis in laboratories may be also influenced by the variability of available standards, emphasising the need for the production of certified reference materials (CRM).

Chromatography, High Pressure Liquid↗

PubMed2002

Applications of MALDI-TOF MS analysis in cyanotoxin research.

Recent advances in analytical chemistry have provided an array of techniques to analyze cyanotoxins in a variety of samples. Mass spectral analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a reliable and rapid tool to detect and identify microcystin variants in very small samples such as single Microcystis colonies. Mass signals presumptive of microcystin variants can be characterized further and identified by postsource-decay fragmentation and comparison of observed fragment spectra with theoretical ones or with those of pure reference compounds. By analyzing Microcystis colonies from water blooms, the high metabolic diversity among individual colonies in the communities could be established. For monitoring microcystins in environmental samples, MALDI-TOF MS can provide considerable support to HPLC by identifying microcystin variants not available as purified standards, especially in Planktothrix-dominated blooms in which demethylated variants are predominant. A short description of the principles of MALDI-TOF MS is provided, followed by examples of its application and a discussion of its potential use in cyanotoxin research.

Cyanobacteria↗

PubMed2002

HPLC-PDA detection of cylindrospermopsin--opportunities and limits.

The cyanobacterial hepatotoxic alkaloid cylindrospermopsin (CYL) is of increased concern to public health due to the spreading of its main producer, Cylindrospermopsis raciborskii, around the globe. Here we present results of an evaluation of the possibility to analyse environmental samples for their content of CYL based on HPLC with photo diode array detection as an alternative to costly LC-MS approaches. A gradient from 0% to 50% aqueous methanol (+0.05% trifluoroacetic acid) in 20 min proved to be highly reproducible with respect to peak height, peak area, and retention time of purified CYL. Good linearity of peak area response was found for 1-300 ng CYL on column. For a good performance the duration of equilibration prior to individual runs was crucial. Extraction from cell material (culture and bloom) was efficiently done with pure water in one extraction step and CYL contents determined matched well with results previously obtained by LC-MS. When different seston matrices were added to cultured cells to mimic realistic environmental samples, however, peaks eluting close to CYL in chromatograms restrained the performance. The data presented show a limitation of HPLC-PDA analysis for trace amounts of CYL in environmental samples but also underline the potential of an inexpensive and fast analysis for various purposes.

Alkaloids↗

PubMed2002

Microcystins in natural blooms and laboratory cultured Microcystis aeruginosa from Laguna de Bay, Philippines.

Laguna de Bay, the largest freshwater lake in the Philippines, experiences periodic blooms of the cyanobacteria Microcystis aeruginosa. Blooms of these cyanobacteria in 1996, 1998 and 1999 were sampled. HPLC and MALDI-TOF mass spectrometry were used to analyze for microcystins. A total of 16 structural variants of the toxin were isolated from the samples with microcystin LR (MC-LR) as the most abundant variant in the samples from 1996 and 1999 making up 77 to 85% of the total, respectively. MC-RR was the dominant variant in the 1998 bloom making up 38%. The samples from 1996 had the highest total toxin concentration (4049 microg g(-1)) followed by those from 1998 (1577 microg g(-1)) and 1999 (649 microg g(-1)). A strain of M. aeruginosa previously isolated from the lake was also cultured in the laboratory under different nitrogen concentrations (1, 3 and 6 mg L(-1)) and elevated phosphorus concentration (0.5 mg L(-1)) to determine the influence of these factors on toxin production. A total of 9 different structural variants of microcystin were isolated from the laboratory cultures with MC-LR consisting more than 75% of the total in all treatments. No significant differences in the total toxin concentration as well as the % distribution of the different variants among treatments were observed. However, the strain of M. aeruginosa cultured in the laboratory had from 3 to 20 times higher total microcystin than those harvested from the lake.

Bacterial Toxins↗