Characterization of fluorophores released from three kinds of lake phytoplankton using gel chromatography and fluorescence spectrophotometry.
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Three kinds of phytoplankton were cultivated, and the contribution of dissolved organic matter (DOM) released from the phytoplankton was examined to clarify the cause of organic pollution of Lake Biwa. Microcystis aeruginosa, Staurastrum dorcidentiferum, and Cryptomonas ovata were evaluated with regard to cultivation. A significant peak (M(w): <3000 Da) was mainly detected in the algal DOM released from plankton during cultivation by gel chromatography with a fluorescence detector (E(x) = 340 nm, E(m) = 435 nm). Since this peak corresponds to a peak with lower molecular weight in three peaks detected in the surface water of Lake Biwa, it can be concluded that the algal DOM released from the plankton during cultivation makes a considerable contribution to the refractory organic matter in Lake Biwa. Three fluorescence maxima were observed in the cultivation of three kinds of phytoplankton: two fulvic-like fluorescence peaks (A and B) and a protein-like fluorescence peak (C). These peaks became larger as their cell counts of plankton increased. As for the fractionations of algal DOM using DAX-8, the ratio of hydrophilic DOM is fairly high in DOM produced by three kinds of phytoplankton. The order of the amount of algal DOM per cell volume during cultivation was Cryptomonas ovata > Microcystis aeruginosa > Staurastrum dorcidentiferum. These results suggest that the increase of the refractory organic matter in Lake Biwa may be attributed to a change of the predominant phytoplankton. (+info)
Toxicity and toxins of natural blooms and isolated strains of Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures.
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All samples of cyanobacterial blooms collected from 1986 to 1989 from Lake Kasumigaura, Ibaraki Prefecture, Japan, were hepatotoxic. The 50% lethal doses (LD50s) of the blooms to mice ranged from 76 to 556 mg/kg of body weight. Sixty-eight Microcystis cell clones (67 Microcystis aeruginosa and 1 M. viridis) were isolated from the blooms. Twenty-three strains (including the M. viridis strain) were toxic. However, the ratio of toxic to nontoxic strains among the blooms varied (6 to 86%). Microcystins were examined in six toxic strains. Five toxic strains produced microcystin-RR, -YR, and -LR, with RR being the dominant toxin in these strains. Another strain produced 7-desmethylmicrocystin-LR and an unknown microcystin. This strain showed the highest toxicity. Establishment of axenic strains from the Microcystis cells exhibiting extracellularly mucilaginous materials was successful by using a combination of the agar plate technique and two-step centrifugation. (+info)
Plasticity and evolution of aeruginosin biosynthesis in cyanobacteria.
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Genetic analysis of the microcystin biosynthesis gene cluster in Microcystis strains from four bodies of eutrophic water in Japan.
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The highly conserved organization of microcystin biosynthesis (mcy) gene clusters, which includes nonribosomal peptide synthetase (NRPS) genes, polyketide synthase (PKS) genes, and fused NRPS-PKS genes, has been characterized in the genus Microcystis. In this study, a total of 135 cyanobacterial strains from four different geographical locations in Japan were isolated. Fourteen mcy-possessing (mcy+) strains were identified according to PCR amplification between two genes from domestic mcy+ strains and the mcy gene's organization was classified into five types. Phylogenetic relationships of the 16S-23S internal transcribed spacer region indicated that the five types of mcy gene cluster structure classified into two groups of the genus Microcystis. HPLC of the isolated mcy+ strain containing a partial deletion of mcyI (DeltamcyI) revealed that microcystin production disappeared. A transcriptional analysis of the Delta mcyI-strain and an assay of recombinant McyI dehydrogenase activity showed that McyI is responsible for microcystin biosynthesis. Based on patterns of the PCR amplicons and analyses of nucleotide sequences in the mcy gene cluster of Microcystis, we confirmed the presence of inserts at three specific loci, between mcyA and mcyD, and downstream of mcyC and mcyJ. Our study is the first investigation of the mcy gene cluster structure in the genus Microcystis from environmental samples. (+info)
Recombination, cryptic clades and neutral molecular divergence of the microcystin synthetase (mcy) genes of toxic cyanobacterium Microcystis aeruginosa.
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Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue-green alga, Microcystis aeruginosa.
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The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes. Male Sprague Dawley rats were used for all studies. For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation. Cell suspensions and and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 micrograms/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 micrograms/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg. Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin. In cell suspensions, MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes. In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments. These alterations were dose and time dependent. Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR. In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing. These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments. (+info)