Crystal structures of protein phosphatase-1 bound to motuporin and dihydromicrocystin-LA: elucidation of the mechanism of enzyme inhibition by cyanobacterial toxins

The microcystins and nodularins are tumour promoting hepatotoxins that are responsible for global adverse human health effects and wildlife fatalities in countries where drinking water supplies contain cyanobacteria. The toxins function by inhibiting broad specificity Ser/Thr protein phosphatases in the host cells, thereby disrupting signal transduction pathways. A previous crystal structure of a microcystin bound to the catalytic subunit of protein phosphatase-1 (PP-1c) showed distinct changes in the active site region when compared with protein phosphatase-1 structures bound to other toxins. We have elucidated the crystal structures of the cyanotoxins, motuporin (nodularin-V) and dihydromicrocystin-LA bound to human protein phosphatase-1c (gamma isoform). The atomic structures of these complexes reveal the structural basis for inhibition of protein phosphatases by these toxins. Comparisons of the structures of the cyanobacterial toxin:phosphatase complexes explain the biochemical mechanism by which microcystins but not nodularins permanently modify their protein phosphatase targets by covalent addition to an active site cysteine residue.     

Oval cell proliferation in p16INK4a expressing mouse liver is triggered by chronic growth stimuli.

Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a).Like other organs, the adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes. These cells retain the ability to proliferate in response to liver injury or reduction of liver mass. However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells.For therapeutic application a non-toxic activation of this stem cell compartment is required. We have established transgenic mice with conditional overexpression of the cell cycle inhibitor p16(INK4a) in hepatocytes and have provoked and examined oval cell activation in adult liver in response to a range of proliferative stimuli.We could show that the liver specific expression of p16(INK4a) leads to a faster differentiation of hepatocytes and an activation of oval cells already in postnatal mice without negative consequences on liver function.     

Effect of total dissolved solids and irradiance on growth and toxin production by Nodularia spumigena

The objective of this work was to determine the influence of total dissolved solids/salinity (TDS mgL^-1) on growth and biomass specific rates of nodularin (hepatotoxin) production by Nodularia spumigena 001E isolated from Lake Alexandrina, South Australia. Maximum biomass yield (dry matter, chlorophyll a and particulate organic carbon/POC) at 80 μmol photon m^-2 s^-1 was recorded at 3300 mg TDS L^-1 and decreased at salinities above or below this value (p < 0.05). The maximum biomass yield (dry matter and chlorophyll a) at 30 μmol m^-2 s^-1 occurred at a higher salinity of 9900 mg TDS L^-1. Cultures grown at 80 μmol m^-2 s^-1, at a TDS> 6600 mg L^-1, had significantly (p < 0.05) lower nodularin content (ml^-1 medium) than cultures grown at the same salinities at 30 μmolm^-2 s^-1. The maximum total toxin concentration (mL^-1 medium) occurred at 9900 and 3300 mg TDS L^-1 at 30 μmol m^-2 s^-1and 80 μmol m^-2 s^-1 respectively. Toxin per unit biomass, expressed as dry matter, chlorophyll a and POC was similar for cultures grown at 30 μmol m^-2 s^-1 or 80 μmol m^-2s^-1 at salinities < 6600 mg TDS L^-1. At salinities > 9900 mg TDS L^-1 the toxin content per unit biomass decreased at both irradiances, however, cultures grown at 30 μmol m^-2s^-1 had a higher toxin content than those grown at 80 μmol m^-2 s^-1. The results indicate that not only do changes in irradiance and salinity directly influence growth and toxin production but that changes in irradiance affected the influence of salinity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Physiological effects in juvenile three-spined sticklebacks feeding on toxic cyanobacterium Nodularia spumigena-exposed zooplankton

Feeding rate, growth and nutritional condition as well as nodularin concentration of juvenile three-spined sticklebacks Gasterosteus aculeatus were assessed in an experimental study where field-collected fish were given a diet of zooplankton fed with toxic Nodularia spumigena for 15 days. Food consumption was higher in N. spumigena bloom conditions compared with the cyanobacterium-free control, but despite this the growth rate of exposed fish did not improve. Control fish and fish fed N. spumigena -exposed zooplankton had higher RNA:DNA ratios and protein content than fish grown in cyanobacterial bloom conditions indicating good nutritional condition and recent growth of fish, whereas in bloom conditions metabolic transformation of nodularin to less toxic compounds may cause an energetic cost to the fish affecting the growth rate of the whole organism. Juvenile three-spined sticklebacks collected from the field contained higher concentrations of nodularin at the beginning of the experiment (mean 503·1 μg kg⁻¹). After 15 days, the lowest nodularin concentrations in fish were measured in the control treatment, suggesting that fish fed with non-toxic food are able to detoxify nodularin from their tissues more effectively than fish in continuing exposure.     

Chloroplast structure and starch grain production as phylogenetic indicators in the lower Rhodophyceae

The position of starch grain production, the shape of the starch grains and the depth to which the pyrenoid is embedded in the chloroplast are used as indicators of evolution in the lower Rhodophyceae. A cell with cytoplasmic allantoid starch grains encasing the pyrenoid and no thylakoids between the chloroplast envelope and the pyrenoid is considered to be evolutionarily primitive. A cell with oval starch grains not associated with the pyrenoid and with a pyrenoid deeply embedded in the chloroplast is thought to be evolutionarily advanced. A polyphyletic origin of the Porphyridiales is discussed.     

Effects of Cyanobacteria on Survival and Reproduction of the Littoral Crustacean Gammarus zaddachi (Amphipoda)

Toxic cyanobacteria can have harmful or fatal impacts on aquatic organisms. In the archipelagos of the northern Baltic Sea, the open sea blooms often drift into littoral areas, where they decompose and release toxins and other chemical compounds in the water. However, the effects of cyanobacteria on the littoral organisms have not previously been investigated. We studied the effects of three cyanobacteria species (toxic Nodularia spumigena, non-toxic N. sphaerocarpa and non-toxic Aphanizomenon flos-aquae) and purified dissolved nodularin (produced by N. spumigena) on a common littoral amphipod Gammarus zaddachi. Nodularin was transferred to eggs, juveniles and adults of G. zaddachi, but no significant negative effects of dissolved nodularin were detected on adults, eggs or juveniles. However, survival of adults decreased by the exposure to toxic N. spumigena cells. The egg hatching rate and juvenile survival were not affected when exposed to the three cyanobacteria species. In contrast, a weak decrease in the egg production and an increased abortion of embryos from the brood pouch of females was observed, the later indicating a failure in parental care. Further, a decrease in grazing rate on the filamentous green alga Enteromorpha intestinalis was observed. The results suggest that toxic cyanobacteria blooms are not extremely fatal, but may have, in high concentrations, negative effects on the adult survival, fecundity, and feeding behaviour of gammarids inhabiting the littoral zone.     

Nodularin,a cyanobacterial toxin,is synthesized in planta by symbiotic Nostoc sp.

The nitrogen-fixing bacterium, Nostoc, is a commonly occurring cyanobacterium often found in symbiotic associations. We investigated the potential of cycad cyanobacterial endosymbionts to synthesize microcystin/nodularin. Endosymbiont DNA was screened for the aminotransferase domain of the toxin biosynthesis gene clusters. Five endosymbionts carrying the gene were screened for bioactivity. Extracts of two isolates inhibited protein phosphatase 2A and were further analyzed using electrospray ionization mass spectrometry (ESI-MS)/MS. Nostoc sp. ‘Macrozamia riedlei 65.1'' and Nostoc sp. ‘Macrozamia serpentina 73.1'' both contained nodularin. High performance liquid chromatography (HPLC) HESI-MS/MS analysis confirmed the presence of nodularin at 9.55±2.4 ng μg−1 chlorophyll a in Nostoc sp. ‘Macrozamia riedlei 65.1'' and 12.5±8.4 ng μg−1 Chl a in Nostoc sp. ‘Macrozamia serpentina 73.1'' extracts. Further scans indicated the presence of the rare isoform [L-Har²] nodularin, which contains ℒ-homoarginine instead of ℒ-arginine. Nodularin was also present at 1.34±0.74 ng ml⁻¹ (approximately 3 pmol per g plant ww) in the methanol root extracts of M. riedlei MZ65, while the presence of [L-Har²] nodularin in the roots of M. serpentina MZ73 was suggested by HPLC HESI-MS/MS analysis. The ndaA-B and ndaF genomic regions were sequenced to confirm the presence of the hybrid polyketide/non-ribosomal gene cluster. A seven amino-acid insertion into the NdaA-C1 domain of N. spumigena NSOR10 protein was observed in all endosymbiont-derived sequences, suggesting the transfer of the nda cluster from N. spumigena to terrestrial Nostoc species. This study demonstrates the synthesis of nodularin and [L-Har²] nodularin in a non-Nodularia species and the production of cyanobacterial hepatotoxin by a symbiont in planta.     

PROTEIN PHOSPHATASE INHIBITORY ACTIVITY IN EXTRACTS OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

A large-scale screening program was initiated to evaluate laboratory-cultured blue-green algae (cyanobacteria) as a source of novel compounds with inhibitory activity against certain serine/threonine protein phosphatases. Over 1600 extracts, representing 816 cyanophyte strains with broad habitat and taxonomic diversity, were screened. Inhibitors were identified in extracts produced from all orders tested except Chamaesiphonales, and the family Stigonemataceae was found to contain proportionally more inhibitors as compared to the total screen.