Histological, cytological and biochemical alterations induced by microcystin-LR and cylindrospermopsin in white mustard (Sinapis alba L.) seedlings.

This study compares the histological, cytological and biochemical effects of the cyanobacterial toxins microcystin-LR (MCY-LR) and cylindrospermopsin (CYN) in white mustard (Sinapis alba L.) seedlings, with special regard to the developing root system. Cyanotoxins induced different alterations, indicating their different specific biochemical activities. MCY-LR stimulated mitosis of root tip meristematic cells at lower concentrations (1 µg ml-1) and inhibited it at higher concentrations, while CYN had only inhibitory effects. Low CYN concentrations (0.01 µg ml-1) stimulated lateral root formation, whereas low MCY-LR concentrations increased only the number of lateral root primordia. Both inhibited lateral root development at higher concentrations. They induced lignifications, abnormal cell swelling and inhibited xylem differentiation in roots and shoots. MCY-LR and CYN induced the disruption of metaphase and anaphase spindles, causing altered cell divisions. Similar alterations could be related to decreased protein phosphatase (PP1 and PP2A) activities in shoots and roots. However, in vitro phosphatase assay with purified PP1 catalytic subunit proved that CYN in contrast to MCY-LR, decreased phosphatase activities of mustard in a non-specific way. This study intends to contribute to the understanding of the mechanisms of toxic effects of a protein phosphatase (MCY-LR) and a protein synthesis (CYN) inhibitory cyanotoxin in vascular plants.

Alkaloids from cyanobacteria with diverse powerful bioactivities.

Alkaloid containing plants represent a heterogeneous group both taxonomically and chemically, a basic nitrogen being the unifying factor for the various classes. As most alkaloids are extremely toxic, organisms containing them do not feature strongly in medicine but they have always been important in the allopathic system. Typical alkaloids are derived from plant sources, they are basic, they contain one or more nitrogen, and they usually have marked physiological actions in humans or other mammalian species. This review will present various alkaloids generated by cyanobacteria, highlighting their complex structures, powerful bioactivities, and pharmacological properties. The main groups of cyanobacterial alkaloids include the neuromuscular transmission blocker anatoxins, the ion channel blocker saxitoxins, the degenerated amino acid ß-methylamino-L-alanine, the protein synthesis inhibitor guanidine alkaloid cylindrospermopsins, and cyanobacterial indol alkaloids with antiviral, antifungal, and cytotoxic activity.

Cylindrospermopsin and microcystin-LR alter the growth, development and peroxidase enzyme activity of white mustard (Sinapis alba L.) seedlings, a comparative analysis.

This work focuses on the comparative analysis of the effects of two cyanobacterial toxins of different chemical structure cylindrospermopsin (CYN) and microcystin-LR (MC-LR) on the white mustard (Sinapis alba L.) seedlings. Both cyanotoxins reduced significantly the fresh mass and the length of cotyledons, hypocotyls and main roots of seedlings in a concentration dependent manner. For various mustard organs the 50% inhibitory concentration values (IC50) of growth were between 3-5 µg ml(-1) for MC-LR and between 5-10 µg ml-1 for CYN, respectively. Cyanotoxins altered the development of cotyledons, the accumulation of photosynthetically active pigments and anthocyanins. Low MC-LR concentrations (0.01 and 0.1 µg ml(-1)) stimulated anthocyanin formation in the cotyledons but higher than 1 µg ml(-1) MC-LR concentrations strongly inhibited it. The CYN treated chlorotic cotyledons were violet coloured in consequence of high level of anthocyanins, while MC-LR induced chlorosis was accompanied by the appearance of necrotic patches. Necrosis and increases of peroxidase enzyme activity (POD) are general stress responses but these alterations were characteristic only for MC-LR treated mustard plants. These findings provide experimental evidences of developmental alterations induced by protein synthesis and protein phosphatase inhibitory cyanotoxins (CYN and MC-LR) in a model dicotyledonous plant.

Cylindrospermopsin inhibits growth and modulates protease activity in the aquatic plants Lemna minor L. and Wolffia arrhiza (L.) Horkel.

The toxic effects of cylindrospermopsin (cyanobacterial toxin) on animals have been examined extensively, but little research has focused on their effects on plants. In this study cylindrospermopsin (CYN) caused alterations of growth, soluble protein content and protease enzyme activity were studied on two aquatic plants Lemna minor and Wolffia arrhiza in short-term (5 days) experiments. For the treatments we used CYN containing crude extracts of Aphanizomenon ovalisporum (BGSD-423) and purified CYN as well. The maximal inhibitory effects on fresh weight of L. minor and W. arrhiza caused by crude extract were 60% and 54%, respectively, while the maximum inhibitory effects were 30% and 43% in the case of purified CYN at 20 µg ml(-1) CYN content of culture medium. In CYN-treated plants the concentration of soluble protein showed mild increases, especially in W. arrhiza. Protease isoenzyme activity gels showed significant alterations of enzyme activities under the influence of CYN. Several isoenzymes were far more active and new ones appeared in CYN-treated plants. Treatments with cyanobacterial crude extract caused stronger effects than the purified cyanobacterial toxins used in equivalent CYN concentrations.

Analysis of genetic diversity in crocuses with Carpathian Basin origin using AFLP-markers.

Crocus taxonomy has until now been based primarily on morphology, taking chromosome numbers into consideration. The genetics and genome structure of the genus, the relationships and diversity within the genus are not well known. Amplified fragment length polymorphism (AFLP) is a whole genome approach to study genetic variation that is gaining in popularity for lower-level systematics. The present study employed the AFLP technique for analyzing relationships among taxa of the Crocus genus (particularly the Crocus vernus aggregate) with Carpathian Basin origin. The molecular variance obtained was based on amplification, separation and detection of EcoRI and Tru1I double-digested Crocus spp. genomic DNAs. Our results confirm the relatedness of C. tommasinianus, C. vittatus and C. heuffelianus at the Verni series of the Crocus genus. C. banaticus is taxonomically isolated as the sole member of the subgenus Crociris based on unique morphological features, but the difference is not convincing from AFLP data. The second interesting AFLP analysis result is the position of C. scepusiensis which separated it from the Crocus vernus aggregate.

Investigation of toxin content in Cylindrospermopsis raciborski (Woloszynska) Seenaya and Subba Raju and Aphanizomenon ovalisporum (Forti) strains isolated from shallow lakes of Hungary.

Cylindrospermopsin (CYN) is an alkaloid type cytotoxic metabolite produced by several cyanobacterial species, which caused human illnesses. The occurrence of CYN has been mostly associated with tropical and subtropical cyanobacteria, but recently it is appearing in several countries, all over the world. We analyzed CYN concentration and polyketide synthase/peptide synthetase (PKS /PS) genes, important parts of the gene cluster responsible for the CYN biosynthesis, in 14 isolated/collected Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum strains originated mostly from Hungary. CYN and PKS /PS genes were detected in Aphanizomenon ovalisporum strains isolated from Spain (of our isolation) and isolated in Israel (IL C-164), but the Hungarian isolate from the hyposaline Lake Szelidi had a lack of production capacity. In the Hungarian samples of C. raciborskii, we found no CYN and PKS /PS genes content comparing to CYN producer C. raciborskii AQS originated from Australia.

Signalling inhibitors against Mycobacterium tuberculosis--early days of a new therapeutic concept in tuberculosis.

Tuberculosis causes nearly two million deaths per year world-wide. In addition multidrug-resistant mycobacterial strains rapidly emerge so novel therapeutic approaches are needed. Recently, several promising mycobacterial target molecules were identified, which are involved in bacterial or host cell signalling e.g. the serine/threonine protein kinases, PknB and PknG, NAD kinase and the NAD synthetase. Here we describe some early efforts in the development of novel signal transduction inhibitory anti-mycobacterial drugs using a multiple target approach, with special emphasis on the kinase inhibitory field. Initially, we are using the Nested Chemical Library (NCL) technology and pharmacophore modelling. A hit-finding library, consisting of approximately 19000 small molecules with a bias for prototypic kinase inhibitors from our NCL library and commercial sources was virtually screened against these validated target molecules. Protein structures for the virtual screening were taken from the published three dimensional crystal structures of the enzymes. The hits from the virtual screening were subsequently tested in enzymatic assay systems. Potent hits were then tested for biological activity in macrophages, infected with mycobacteria. The final goal of this exercise is not only to identify potent anti-mycobacterial substances, but also a common pharmacophore for the mycobacterial target PknG in combination with PknB, NAD kinase and/or NAD synthetase. This common pharmacophore still needs to be a unique pharmacophore for the mycobacterial target proteins over human off-targets. Such a pharmacophore might then drive the optimization of a completely new profile of an antibiotic agent with activity against latent mycobacteria and resistance mycobacterial strains.

Alterations in protein synthesis in the cyanobacterium Synechococcus sp. strain PCC 6301 in response to Calendula micrantha extract with molluscicidal activity.

The response to the extract of the Egyptian wild herb Calendula micrantha, with molluscicidal activity, was examined in the unicellular cyanobacterium Synechococcus sp. strain PCC 6301. Growth and chlorophyll a of the cells were only slightly affected by low concentrations but drastically reduced by high concentrations. The rate of protein synthesis progressively decreased by increasing extract concentration. The cells preferentially induced the synthesis of a limited number of polypeptides in response to the treatment. Among the induced polypeptides were those with apparent molecular weights of 161K (161,000), 96.7K, 93.4K, 85K, 69.9K, 59K, 49K, 45K, 35K, 32.4K, 28K, 24K, 21.7K, 18K, and 16K based on their mobilities in gel electrophoresis. These initial studies suggest that the plant extract exerted certain stress which stimulated alteration in the pattern of protein synthesis in Synechococcus sp. Some of the induced polypeptides are similar to that known to occur in other stresses, especially heat-shock stress.

Simple and efficient method for isolation and measurement of cyanobacterial hepatotoxins by plant tests (Sinapis alba L.).

A simple and cost-effective method for isolating and assaying microcystins, cyanobacterial toxins, by C-18 cartridges, DEAE-cellulose (DE-52) chromatography, and a mustard (Sinapis alba L.) plant seedling test is described. The procedure results in a purity of up to 95-97% microcystin without the need for an HPLC system and justifies the use of the S. alba L. seedling test in the quantitative assessment of the toxin with an IC50 of 3 micrograms ml-1 instead of the mouse intraperitoneal test.

Alterations in the accumulation of adenylylated nucleotides in heavy-metal-ion-stressed and heat-stressed Synechococcus sp. strain PCC 6301, a cyanobacterium, in light and dark.

Heavy-metal-ion- (Cd2+, Cu2+, Pb2+, Hg2+ and Zn2+) or heat (50 degrees C)-stress treatments of the unicellular cyanobacterium Synechococcus sp., strain PCC 6301, under both light and dark conditions led to the accumulation of bis(5'-nucleosidyl)oligophosphates: Ap4A, Ap4G, Ap3A, Ap3G and Ap3Gp2. Under light regimens, the accumulation of Ap4A and Ap4G is more characteristic of heavy-metal-ion-stressed cells, whereas the accumulation of Ap3A, Ap3G and Ap3Gp2 is the dominant feature of heavy-metal-ion or heat-shock treatment during energy deprivation (i.e. in the dark). This accumulation of bisnucleoside oligophosphates supports a model whereby the adenylylated nucleotides are synthesized by the backward reaction of tRNA-aminoacyl synthetases. These nucleotides may also act to switch or modulate cyanobacterial responses under various environmental stress conditions.

AS-1 cyanophage infection inhibits the photosynthetic electron flow of photosystem II in Synechococcus sp. PCC 6301, a cyanobacterium.

In Synechococcus sp. cells AS-1 cyanophage infection gradually inhibits the photosystem II mediated photosynthetic electron flow whereas the activity of photosystem I is apparently unaffected by the cyanophage infection. Transient fluorescence induction and flash-induced delayed luminescence decay studies revealed that the inhibition may occur at the level of the secondary acceptor, QB of photosystem II. In addition, the breakdown of D1-protein is inhibited, comparable to DCMU-induced protection of D1-protein turnover, in AS-1-infected cells.

Purification and characterization of RNA polymerase from the cyanobacterium Anabaena 7120.

A procedure for the purification of RNA polymerase from vegetative cells of the filamentous cyanobacterium Anabaena 7120 is described. Polyethyleneimine precipitation followed by gel filtration and affinity chromatography steps results in greater than 99% purification with 46% yield. The enzyme has a novel core component of Mr = 66,000, designated gamma, in addition to the typical prokaryotic beta'beta alpha 2 core enzyme. The sigma subunit has been identified by reconstitution of specific transcriptional activity from core enzyme and gel-purified sigma. In transcription assays, this RNA polymerase initiates at a number of Anabaena vegetative cell promoters, as well as from a bacteriophage T4 early promoter, but does not initiate at nitrogen fixation (nif) promoters used in heterocysts. The promoter specificity of Anabaena RNA polymerase is compared with that of Escherichia coli RNA polymerase.

Effects of light deprivation on RNA synthesis, accumulation of guanosine 3'(2')-diphosphate 5'-diphosphate, and protein synthesis in heat-shocked Synechococcus sp. strain PCC 6301, a cyanobacterium.

The rate of total RNA synthesis, the extent of guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) accumulation, and the pattern of protein synthesis were studied in light-deprived and heat-shocked Synechococcus sp. strain PCC 6301 cells. There was an inverse correlation between the rate of total RNA synthesis and the pool of ppGpp, except immediately after a temperature shift up, when a parallel increase in the rate of RNA synthesis and accumulation of ppGpp was observed. The inverse correlation between RNA synthesis and ppGpp accumulation was more pronounced when cells were grown in the dark. Heat shock treatment (47 degrees C) had an unexpected effect on ppGpp accumulation; there was a fairly stable level of ppGpp under heat shock conditions, which coincided with a stable steady-state rate of RNA synthesis even in the dark. We found that the pattern of dark-specific proteins was altered in response to heat shock. The transient synthesis of several dark-specific proteins was abolished by an elevated temperature (47 degrees C) in the dark; moreover, the main heat shock proteins were synthesized even in the dark. This phenomenon might be of aid in the study of cyanobacterial gene expression.

Effect of heat shock on protein synthesis in the cyanobacterium Synechococcus sp. strain PCC 6301.

The response to heat shock at 47 degrees C was examined in the cyanobacterium (blue-green alga) Synechococcus sp. strain PCC 6301. On heat shock, the growth of the cells decreased and they preferentially synthesized a limited number of polypeptides. The rate of synthesis of these proteins increased markedly in the early period of temperature shift up and gradually decreased afterwards. Among the proteins greatly affected by temperature shift up were those with apparent molecular weights of 91,000 (91K), 79K, 78K, 74K, 65K, 64K, 61K, 49K, 45K, 24K, 22K, 18K, 16K, 14K, 12K, and 11.4K, based on their mobilities in sodium dodecyl sulfate-polyacrylamide gels. From these initial studies on Synechococcus sp. strain PCC 6301 we conclude that in cyanobacteria a heat shock response similar to that known to occur in other eucaryotes and procaryotes might exist.

Thioredoxins and the redox modulation of glucose-6-phosphate dehydrogenase in Anabaena sp. strain PCC 7120 vegetative cells and heterocysts.

Glucose-6-phosphate dehydrogenase (G6PDH) was isolated from heterocysts and vegetative cells of Anabaena sp. strain PCC 7120. Both enzyme preparations proved to be more active in their oxidized than in their reduced forms. At least one protein with thioredoxin activity was found in Anabaena sp. which, if reduced with dithiothreitol, deactivated the G6PDH preparations. The deactivated heterocyst G6PDH could be reactivated neither by O2 nor by oxidized thioredoxin. Reactivation of the enzyme was, however, achieved by oxidized glutathione or H2O2. The active form of Anabaena G6PDH was readily deactivated by heterologous thioredoxin(s). The Anabaena thioredoxin(s) modulated heterologous enzymes.

Chiroptical properties of chlorophyll-protein complexes separated on Deriphat/polyacrylamide gel.

Circular dichroism (c.d.) was measured for four chlorophyll-protein complexes, resolved from sodium dodecyl sulphate extracts of chloroplasts by electrophoresis in polyacrylamide gel containing Deriphat 160 (disodium N-dodecyl beta-imidopropionate), a zwitterionic detergent. The slowest-band (1) complex was found to be identical with the complex CP1 as found on electrophoresis in the presence of anion detergent, but it was in a much higher yield (30% of the chlorophyll a). In band-2 and -3 protein complexes a c.d. pattern described for the complex CP2 could be recognized. Another c.d. component of a split-exciton type with extrema at 680 (-) and 669 (+)nm, together with evidence of disorganized chlorophyll, was found in band-2, -3 and -4 complexes. When a barley (Hordeum vulgare) mutant lacking chlorophyll b was examined, only bands 1 and 4 were obtained, and the c.d. of the band-4 complex was much less affected by disorganized chlorophyll. C.D. spectra resembling that of this band-4 complex could be generated by subtracting the c.d. of complex CP1 from the c.d. of photochemically active mutant chloroplast fragments, or by subtracting the c.d. of complexes CP1 and CP2 from pea (Pisum sativum) chloroplast fragments. The Deriphat appears to have preserved at least to some extent a new type of chlorophyll a-protein complex.

Accumulation of guanosine tetraphosphate (ppGpp) under nitrogen starvation in Anacystis nidulans, a cyanobacterium.

The effect of nitrate deprivation on cell growth and nucleotide level was studied in Anacystis nidulans. A 10-fold reduction in nitrate level resulted in a drastic slowdown of growth. Upon addition of nitrate to the starving cultures, after a lag period, the cells resumed growth. Nutritional shift-down induced a transitory expansion of the guanosine tetraphosphate (ppGpp) pool, preceded by a transitory increase in GTP and ATP concentrations. After having reached peak values, the concentration of ppGpp, GTP and ATP dropped to the respective base levels. The expansion of the ppGpp pool was found to be due to an increase in ppGpp synthesis, rather than to a decrease in ppGpp breakdown. After nutritional shift-up, no decrease in the ppGpp level was found. In starving cells, a decrease in free amino acids was observed to occur concomitantly with the expansion of the ppGpp pool. The level of free amino acids started to increase simultaneously with the contraction of the ppGpp pool.

Bacteriophage infection interferes with guanosine 3'-diphosphate-5'-diphosphate accumulation induced by energy and nitrogen starvation in the cyanobacterium Anacystis nidulans.

Anacystis nidulans accumulates large amounts of guanosine 3'-diphosphate-5'-diphosphate (ppGpp) upon nutritional or energy starvation induced by light-to-dark shift, treatment with carbonylcyanide-m-chlorophenylhydrazone (an uncoupler), or treatment with L-methionine-DL-sulfoximine (an inducer of nitrogen starvation). In contrast to healthy A. nidulans cells, those infected by AS-1 cyanophage do not respond with ppGpp accumulation when starved after about one-third of the complete infection cycle, except, to some extent, under extreme conditions when both nitrogen deprivation and energy deprivation are induced simultaneously (darkening plus L-methionine-DL-sulfoximine treatment). In contrast to cyanophage infection in Anacystis, infection with T4 phage of Escherichia coli CP 78 cells does not affect their accumulation of ppGpp under treatments identical with or similar to those applied in the experiments with Anacystis. This difference in response of phage-infected heterotrophic and photoautotrophic cells to starvation seems to reflect differences in control of nutritional or energy metabolism rather than differences in ability to synthesize ppGpp.