Abstract: Resolving the genetic basis of complex diseases like rheumatoid arthritis will require knowledge of the corresponding diseases in experimental animals to enable translational functional studies. Mapping of quantitative trait loci in mouse models of arthritis, such as collagen-induced arthritis (CIA), using F(2) crosses has been successful, but can resolve loci only to large chromosomal regions. Using an inbred-outbred cross design, we identified and fine-mapped CIA loci on a genome-wide scale. Heterogeneous stock mice were first intercrossed with an inbred strain, B10.Q, to introduce an arthritis permitting MHCII haplotype. Homozygous H2(q) mice were then selected to set up an F(3) generation with fixed major histocompatibility complex that was used for arthritis experiments. We identified 26 loci, 18 of which are novel, controlling arthritis traits such as incidence of disease, severity and time of onset and fine-mapped a number of previously mapped loci.
Abstract: Karyotype structures of Scenedesmus acuminatus (Lagerch.) Chod. and Scenedesmus pectinatus Meyen are compared. The karyotype of S. acuminatus (n = 5) is described for the first time. It reveals four large metacentric and one large submetacentric chromosomes (4M + 1SM). The established karyotype differences have been helpful in clarifying the taxonomic position of these two species. The cytological analyses of other related clonal cultures suggest an evolutionary transition from S. pectinatus towards S. regularis through S. pectinatus f. regularis, which correlates with the morphological data about their variability. These results are discussed from the cytogenetic, morphological and evolutionary point of view. On the basis of the karyotypic analysis, it was confirmed that from a taxonomic point of view S. pectinatus, S. acuminatus and S. regularis are separate biological species.
Abstract: Molecular characterization of ten marine cyanobacterial isolates belonging to the order Oscillatoriales was carried out using the phycocyanin locus (cpcBA-IGS) and the 16S-23S internally transcribed spacer region. DNA sequences from the phycocyanin operon discriminated ten genotypes, which corresponded to seven morphotypes identified by traditional microscopic analysis. The cpcB coding region revealed 17 % nucleotide variation, while cpcA exhibited 29 % variation across the studied species. Phylogenetic analyses support the conclusion that the Phormidium and Leptolyngbya genera are not monophyletic. The nucleotide variations were heterogeneously distributed with no or minimal informative nucleotides. Our results suggest that the discriminatory power of the phycocyanin region varies across the cyanobacterial species and strains. The DNA sequence analysis of the 16S-23S internally transcribed spacer region also supports the polyphyletic nature of the studied oscillatorian cyanobacteria. This study demonstrated that morphologically very similar strains might differ genotypically. Thus, molecular approaches comprising different gene regions in combination with morphological criteria may provide better taxonomical resolution of the order Oscillatoriales.
Abstract: This review is an attempt to summarize the available information about toxin
producing blue-green algae (Cyanoprokaryota). A detailed description of the best
known cyanotoxins, their mode of action with particular attention to the human health
and ecological risks, distribution as well as their detection and quantification by
different assays (biological, biochemical, immunological, physicochemical) are
presented.
Abstract: The search for sources of new biologically active compounds is important for
the discovery of new drugs relative to the treatment of cancer. Cyanoprokaryota are
known to produce anticancer, antiviral and antifungal compounds and antibiotics.
Two strains of Phormidium molle (Cyanoprokaryota, Oscillatoriales) were
investigated for in vitro cytotoxic and anticancer properties using 5 cancer (HeLa,
Jurkat, U-937, A2058, RD) and 2 normal (3T3, FL) cell lines. Treatment with
Phormidium extracts or Phormidium growth media altered the cytoskeletons and
microtubule network of the adherent cells causing dose-dependent destruction of the
monolayer and morphological changes. An extract concentration of 10 μg/mL
showed significant cytotoxicity on HeLa and A2058 cells 24 h after treatment (41%
and 60% respectively) as determined with the MTT assay, while the viability of the
other cells was not significantly affected. Both, extracts and growth media,
significantly inhibited incorporation of [3H]-thymidine in the adherent cancer cell
lines in a dose-dependent manner. No colony formation was observed over a
concentration of 6 μg/mL Phormidium molle extracts for the cancer adherent cells
(HeLa). Chromatographic analyses of the Phormidium extracts and growth media
showed peaks with retention time between 6 and 15 min, which potentially represent
alkaloids. Thus, Cyanoprokaryota could be a novel appropriate choice for
development of anticancer therapeuticals.
Abstract: The variability of the coenobia as well as of the single cells in the
coenobia of Scenedesmus pectinatus Meyen has been investigated. The reproductive
capacity and variability of one single coenobium during the ontogenesis were
examined by using a drop method. Morphotaxonomic characteristics of the species
were analyzed and compared with similar studies of clonal cultures. This study
confirms the results from tests performed with intensive and synchronized cultures.
Abstract: Among the Cyanoprokaryota (blue-green algae), the genus Phormidium has thus far rarely been studied with respect to toxin production and potentially resulting human and environmental health effects. We here show that five previously unexplored freshwater species of this genus (Ph. bijugatum, Ph. molle, Ph. papyraceum, Ph. uncinatum, Ph. autumnale) are indeed capable of producing bioactive compounds. Phormidium extracts caused weight loss as well as neuro/hepatotoxic symptoms in mice, and in the case of Ph. bijugatum even death. Very low levels of saxitoxins and microcystins, as confirmed by ELISA, were insufficient to explain this toxicity and the differing toxic potencies of the Phormidium species. Qualitative HPLC analyses confirmed different substance patterns and in the future could aid in the separation of fractions for more detailed substance characterisation. The results in vivo were confirmed in vitro using cells of human, mouse and fish. The fish cells responded least sensitive but proved useful in studying the temperature dependence of the toxicity by the Phormidium samples. Further, the human cells were more sensitive than the mouse cells thus suggesting that the former may be a more appropriate choice for studying the impact of Phormidium to man. Among the human cells, two cancer cell lines were more responsive to one of the samples than a normal cell line, thereby indicating a potential anti-tumour activity. Thus, the five freshwater Phormidium species should be considered in environmental risk assessment but as well, as a source of therapeutic agents.
Abstract: The accurate determination of species of Cyanoprokaryota/Cyanophyceae has many important applications. These include the assessment of risk with regard to blooms in water reservoirs as well as the identification of species capable of producing valuable bioactive compounds. Commonly, Cyanoprokaryota are classified based on their morphology. However, morphological criteria are not always reliable because they may change, for example, due to environmental factors. Thus, genetic and molecular analyses are a promising additional approach, but their application has so far been limited to relatively few genera. In light of this, we present here the first characterization of species and strains of the genus Phormidium Kütz. based on the cpcB-IGS-cpcA locus of the phycocyanin operon. In phylogenetic analyses using deduced amino acid sequences of the cpcB-cpcA regions, Phormidium was found to be polyphyletic. This analysis appeared to be dominated by the cpcB region, which is characterized by a relatively high percentage of informative substitutions. The percentage of variable positions within the cpcB-IGS-cpcA locus overall was 16.5%, thereby indicating a level of divergence remarkably higher than that reported for Nodularia and Arthrospira in previous studies relying on cpcB-IGS-cpcA. Further, alignment of informative nucleotide substitutions in the cpcB-IGS-cpcA sequences revealed a mosaic distribution, which may be indicative of genetic recombination events. Finally, the length and sequences of the IGS region alone proved useful as markers to differentiate the cyanobacterial genus Phormidium. However, whether the IGS region per se is sufficiently discriminatory to differentiate between Phormidium species or even strains requires further investigation using newly identified Phormidium sequence data.
Abstract: There is an increasing amount of knowledge on the cytotoxic properties of cyanotoxins, but relatively little is known regarding their fine specificity and mechanisms of action. In this study, we investigated the influence of microcystin-LR and AnTx-a on mouse B- and T-lymphocyte subpopulations in vitro. Cyanotoxins significantly decreased the cell viability after 4 and 24 h, compared to the untreated control. After 24 h exposure to microcystin-LR and anatoxin-a, the viability of splenocytes dropped to 23% and 57%, respectively. Our data demonstrate that microcystin-LR induced apoptosis specifically in mouse B cells, probably via the B-cell antigen receptor and mitochondrial pathway, while the T cells were not affected. AnTx-a showed cytotoxic effects on both lymphocyte subpopulations, but the effects were driven by mechanisms different from apoptosis. These findings demonstrate that the cyanotoxins could cause cytotoxic alterations in a variety of cell types different from the major targets, operating via distinct mechanisms.
Abstract: The variability in the morphology, modal number of chromosomes, TCR expression and functional reactivity of a CII-specific T-cell hybridoma at continuous subcultivation have been investigated. As the number of passages increased, besides the oval semiadherent cells (normal phenotype), fibroblast-like cells (transformed phenotype) were also observed. The two cell subpopulations differed in their karyotype characteristic, as well as in their functional reactivity. The cell population with a normal phenotype was characterized by a tetramodal number of chromosomes (30, 40, 48 and 70) and trisomies of chromosomes 6 and 14, while the cell population with a transformed phenotype was characterized by a trimodal number of chromosomes (11, 68 and 74) and trisomy of chromosome 12. A nullisomy of sex chromosomes was established in both types of cells. In the initial passages of subcultivation, 73.04% of the cells with a normal morphological phenotype expressed TCR-CD3 complexes on their surface and possessed high functional reactivity. After a two-week subcultivation, the values of these indices went down considerably: 46.11% of the cells expressed functional TCR-CD3 complexes, as a result of which their functional reactivity decreased. Only 2.71% of the cells with a transformed morphological phenotype expressed functional TCR-CD3 complexes on their surface. In these cells, a total loss of reactivity towards the specific antigens was established. The achieved results show that at continuous subcultivation the T-cell hybridomas are unstable, and with the increase in the number of passages there appear chromosome rearrangements, leading to loss of their functional reactivity.
Abstract: Despite a growing awareness of the presence of cyanobacterial toxins, knowledge about the ability of specific species to produce toxic compounds is still rather limited. It was the overall goal of the current work to investigate if probes derived from the freshwater species Lyngbya aerugineo-coerulea (Kutz.) Gomont, a cyanobacterium frequently found in southern Europe and not previously investigated for the presence of bioactive compounds, were capable of eliciting in vivo and in vitro toxicity. The cyanobacterial extract revealed signs of neuro- as well as hepatotoxicity in mice, although these signs could not be explained by the well-known respective cyanobacterial neuro- and hepatotoxins saxitoxin and microcystin. Cytotoxicity was elicited by the cyanobacterial extract in all mammalian cell lines tested. As well, the rainbow trout liver cell line, RTL-W1, was found to be susceptible to the cytotoxic effects of the extract, although the cytotoxicity was dependent on temperature. In contrast, the cyanobacterial growth medium elicited cytotoxicity independent of temperature, leading to morphological changes indicative of alterations to the cytoskeleton. Overall, the results suggest that Lyngbya aerugineo-coerulea is an important cyanobacterium to be considered for its potential to cause health risks on environmental exposure of it to mammals and fish. Applying a combination of mammalian and piscine cell line bioassays is a unique approach that, combined with chemical analysis, could be used in the future to identify the structure and cellular mechanisms of the as-yet-unknown toxic Lyngbya aerugineo-coerulea metabolites in particular and to screen cyanobacterial extracts for their toxicity in general.
Abstract: The role of Empis meridionalis Meigen (Diptera: Empididae) as a pollinator of flowering plants has been investigated. It was found that 12 flower plants species are visited by this species. The daily activity of E. meridionalis as well as the correllation between the frequency of visit, colour and type of blossom are described. E. meridionalis visits most commonly actinomorphic blossoms with purple or pink color and it plays an important role in the pollination of a series of flowering plants in early summer.
Abstract: In the present paper, flower-visiting by 15 species of Empidoidea to 23 species of flowering plants is reported. The daily activity and frequency of visits to the blossoms were established. The role of Empidoidea in the pollination of flowering plants is discussed.
