Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copperATPase

Wilson disease is an autosomal recessive copper transport disorder resulting from defective biliary excretion of copper and subsequent hepatic copper accumulation and liver failure if not treated. The disease is caused by mutations in the ATP7B (WND) gene, which is expressed predominantly in the liver and encodes a copper-transporting P-type ATPase that is structurally and functionally similar to the Menkes protein (MNK), which is defective in the X-linked copper transport disorder Menkes disease. The toxic milk (tx) mouse has a clinical phenotype similar to Wilson disease patients and, recently, the tx mutation within the murine WND homologue (Wnd) of this mouse was identified, establishing it as an animal model for Wilson disease. In this study, cDNA constructs encoding the wild-type (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) were generated and expressed in Chinese hamster ovary (CHO) cells. The fx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, co-localization experiments demonstrated that while Wnd and MNK are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell, Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multivesicular structures resembling late endosomes that may represent a novel compartment for copper transport. The data presented provide further support for a relationship between copper transport activity and the copper-induced relocalization response of mammalian copper ATPases, and an explanation at a molecular level for the observed phenotype of fx mice.

The effect of volatile fatty acids in acidified fermented piggery effluent on shiga-toxigenic and non-toxic resident strains of Escherichia coli

Aims: To examine the effects of acidified acidogenically fermented piggery effluent containing Volatile Fatty Acids (VFA) on shiga-toxigenic and resident strains of Escherichia coli (E. coli) as part of the development of a waste treatment process. Methods and Results: Four shiga-toxigenic E. coli strains (O157:H7, 091.H-, 0111.H-, and 0123.H-) and four non-toxic resident enzootic strains were all killed by 3 h treatment with fermented piggery effluent liquor (153 mmol l(-1) total VFA) at pH 4.3. The shiga-toxigenic strains showed greater sensitivity after 1 h of treatment. The fermented liquor at pH 6.8 was not inhibitory. Conclusions: The shiga-toxigenic strains were no more resistant to the toxic effects of VFA than the non-toxic strains tested. Significance and Impact of the Study: Shiga-toxigenic strains and resident enzootic non-toxigenic strains are equally susceptible to inactivation by this waste treatment process and by acidified VFA in general.

The DMSO reductase family of microbial molybdenum enzymes; Molecular properties and role in the dissimilatory reduction of toxic elements

The dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes is a large and diverse group that is found in bacteria and archaea. These enzymes are characterised by a bis(molybdopterin guanine dinucleotide)Mo form of the molybdenum cofactor, and they are particularly important in anaerobic respiration including the dissimilatory reduction of certain toxic oxoanions. The structural and phylogenetic relationship between the proteins of this family is discussed. High-resolution crystal structures of enzymes of the DMSO reductase family have revealed a high degree of similarity in tertiary structure. However, there is considerable variation in the structure of the molybdenum active site and it seems likely that these subtle but important differences lead to the great diversity of function seen in this family of enzymes. This diversity of catalytic capability is associated with several distinct pathways of electron transport.

Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants

In Australian freshwaters, Anabaena circinalis, Microcystis spp. and Cylindrospermopsis raciborskii are the dominant toxic cyanobacteria. Many of these Surface waters are used as drinking water resources. Therefore, the National Health and Medical Research Council of Australia set a guideline for MC-LR toxicity equivalents of 1.3 mug/l drinking, water. However, due to lack of adequate data, no guideline values for paralytic shellfish poisons (PSPs) (e.g. saxitoxins) or cylindrospermopsin (CYN) have been set. In this spot check. the concentration of microcystins (MCs), PSPs and CYN were determined by ADDA-ELISA, cPPA, HPLC-DAD and/or HPLC-MS/MS, respectively, in two water treatment plants in Queensland/Australia and compared to phytoplankton data collected by Queensland Health, Brisbane. Depending on the predominant cyanobacterial species in a bloom, concentrations of up to 8.0, 17.0 and 1.3 mug/l were found for MCs, PSPs and CYN, respectively. However, only traces (< 1.0 mug/l) of these toxins were detected in final water (final product of the drinking water treatment plant) and tap water (household sample). Despite the low concentrations of toxins detected in drinking water, a further reduction of cyanobacterial toxins is recommended to guarantee public safety. (C) 2004 Elsevier Ltd. All rights reserved.

Factors affecting akinete differentiation in Cylindrospermopsis raciborskii (Nostocals, Cyanobacteria)

1. Cylindrospermopsis raciborskii is a potentially toxic freshwater cyanobacterium which can produce akinetes (reproductive spores) that on germinating can contribute to future populations. To further understand factors controlling the formation of these specialised cells, the effects of diurnal temperature fluctuations (magnitude and frequency), in combination with different light intensities and phosphorus concentrations were investigated under laboratory conditions. 2. Akinete differentiation was affected by the frequency of temperature fluctuations. Maximum akinete concentrations were observed in cultures that experienced multiple diurnal temperature fluctuations. 3. Akinete concentrations increased with increasing magnitude of temperature fluctuation. A maximum akinete concentration was achieved under multiple diurnal temperature fluctuations with a magnitude of 10degreesC (25degreesC to 15degreesC). 4. A fourfold increase in light intensity (25-100 mumol m(-2) s(-1)) resulted in an approximate 14-fold increase in akinete concentration. 5. High filterable reactive phosphorus (FRP) concentrations (> 70 mug L-1) in the medium, combined with a multiple diurnal temperature fluctuation of 10degreesC, supported the development of the highest akinete concentration.

Blooms of the cyanobacterium Lyngbya majuscula in coastal Queensland, Australia: disparate sites, common factors

During the last decade there has been a significant rise in observations of blooms of the toxic cyanobacterium, Lyngbya majuscula along the east coast of Queensland, Australia. Whether the increase in cyanobacterial abundance is a biological indicator of widespread water quality degradation or also a function of other environmental change is unknown. A bioassay approach was used to assesses the potential for runoff from various land uses to stimulate productivity of L. majuscula. In Moreton Bay, L. majuscula productivity was significantly (p < 0.05) stimulated by soil extracts, which were high in phosphorus, iron and organic carbon. Productivity of L. majuscula from the Great Barrier Reef was also significantly (p < 0.05) elevated by iron and phosphorus rich extracts, in this case seabird guano adjacent to the bloom site. Hence, it is possible that other L. majuscula blooms are a result of similar stimulating factors (iron, phosphorus and organic carbon), delivered through different mechanisms. (c) 2004 Elsevier Ltd. All rights reserved.

Living in a potentially toxic environment: comparisons of endofauna in two congeneric sponges from the Great Barrier Reef

The abundance and community composition of the endofauna in 2 species of sponge, Haliclona sp. 1 and Haliclona sp. 2 (phylum Porifera: order Haplosclerida), were examined at different sites on the slope at Heron Island Reef, in the southern Great Barrier Reef, on 2 separate occasions. Both species of Haliclona Occupy Similar habitats on the reef slope and are often found living adjacent to each other, but the major groups of secondary metabolites and the gross external morphology in the 2 species of sponge are different. The 2 species of sponge supported significantly different endofaunal communities, with Haliclona sp. 2 Supporting 3 to 4 times more individuals than Haliclona sp. 1. Fewer demersal zooplankton (copepods), nematodes and some peracarid crustaceans were found in Haliclona sp. I compared with Haliclona sp. 2. There were also differences in the numbers of spionid, nereidid and syllid. polychaetes living in the 2 species of sponge. The only taxon that was more abundant in Haliclona sp. 1 than Haliclona sp. 2 was the spionid Polydorella prolifera, and this difference was only evident on 1. of the 2 occasions. The amount of free space (pores, channels, cavities) for a given weight of sponge was only 19% greater in Haliclona sp. 2 than in Haliclona sp. 1, suggesting other factors, such as the differences in the allelochemicals, may have a role in determining the numbers and types of animals living in these 2 species of sponge.

Effects of physical disturbance on infaunal and epifaunal assemblages in subtropical, intertidal seagrass beds

We assessed the impact of large-scale commercial and recreational harvesting of polychaete worms Marphysa spp. on macrobenthic assemblages in a subtropical estuary in Queensland, Australia, by examining: (1) the spatial extent of harvesting activities and the rate of recovery of the seagrass habitat over an 18 to 20 mo period; (2) the recovery of infauna in and around commercial pits of known age; (3) the indirect effects of physical disturbance from trampling and deposition of sediments during harvesting on epibenthos in areas adjacent to commercial and recreational pits; (4) impacts of potential indirect effects through manipulative experimentation. Harvesting caused a loss of seagrass, changes to the topography and compaction of the sediments associated with the creation of walls around commercial pits, and the deposition of rubble dug from within the pit. The walls and rubble were still evident after 1.8 to 20 mo, but comprised only a small proportion of the total area on the intertidal banks. There was a shift from an intertidal area dominated by Zostera capricorni to one with a mixture of Z. capricorni, Halophila spp. and Halodule uninervis, but there was no overall decline in the biomass of seagrass in these areas. There were distinct impacts from harvesting on the abundance of benthic infauna, especially amphipods, polychaetes and gastropods, and these effects were still detectable after 4 mo of potential recovery. After 12 me, there were no detectable differences in the abundances of these infauna between dug areas and reference areas, which suggested that infauna had recovered from impacts of harvesting; however, an extensive bloom of toxic fireweed Lyngbya majsucula may have masked any remaining impacts. There were no detectable impacts of harvesting on epifauna living in the seagrass immediately around commercial or recreational pits.

Feeding preference and deterrence in rabbitfish Siganus fuscescens for the cyanobacterium Lyngbya majuscula in Moreton Bay, south-east Queensland, Australia

Rabbitfish Siganus fuscescens preferences for Lyngbya majuscula collected from three bloom locations in Moreton Bay, Queensland, Australia, were tested along with a range of local plant species in the laboratory. Consumption of L. majuscula by fish did not differ between wild and captive-bred fish (P = 0.152) but did differ between bloom location (P = 0.039). No relationship was found between consumption rates and lyngbyatoxin-a concentration (r(2) = 0.035, P = 0.814). No correlation existed between C : N and proportion of food consumed when all food types were analysed statistically, whereas a clear correlation was observed when L. majuscula was removed from the calculations. In simulated bloom conditions, fish avoided ingestion of L. majuscula by feeding through gaps in the L. majuscula coverage. Both wild and captive-bred S. fuscescens showed a distinct feeding pattern in 10 day no-choice feeding assays, with less L. majuscula being consumed than the preferred red alga Acanthophora spicifera. Lyngbya majuscula however, was consumed in equal quantities to A. spicifera by wild S. fuscescens when lyngbyatoxin-a was not detectable. Wild fish probably do not preferentially feed on L. majuscula when secondary metabolites are present and are not severely impacted by large L. majuscula blooms in Moreton Bay. Furthermore, poor feeding performance in both captive-bred and wild S. fuscescens suggests that they would exert little pressure as a top-down control agent of toxic L. majuscula blooms within Moreton Bay. (c) 2006 The Fisheries Society of the British Isles.

Phototrophic microendoliths bloom during coral "white syndrome"

Following rapid lesion progression of white syndrome in tabular Acropora spp., the white bare skeleton gradually changes to green, a result of endolithic algae blooms (primarily Ostreobium spp.). Endolithic algal biomass and chlorophyll concentration were found to be an order of magnitude higher in the green zone compared with healthy appearing parts of each colony. Chl b to Chl a ratio increased from 1:1.6 in the healthy area to 1:2 and 1:3.5 in the white exposed skeleton and green zones, respectively. These observations together with pulse amplitude modulated (PAM) fluorometry suggest photoacclimation of the endoliths in the green zone. Histopathological microscopy revealed that the endolithic algal filaments penetrate the coral tissue. This study highlights the interaction of endolithic algae with both the skeleton and host tissue. This may have a critical role in the processes that accompany the post-disease state in reef-building corals.

Dietary selectivity for the toxic cyanobacterium Lyngbya majuscula and resultant growth rates in two species of opisthobranch mollusc

Trophodynamics of blooms of the toxic marine cyanobacterium Lyngkya majuscula were investigated to determine dietary specificity in two putative grazers: the opisthobranch molluscs, Stylocheilus striatus and Bursatella leachii. S. striatus is associated with L. majuscula blooms and is known to sequester L. majuscula metabolites. The dietary specificity and toxicodynamics of B. leachii in relation to L. majuscula is less well documented. In this study we found diet history had no significant effect upon dietary selectivity of S. striatus when offered a range of plant species. However, L. majuscula chemotype may alter S. striatus' selectivity for this cyanobacterium. Daily biomass increases between small and large size groups of both species were recorded in no-choice consumption trials using L. majuscula. Both S. striatus and B. leachii preferentially consumed L. majuscula containing lyngbyatoxin-a. Increase in mass over a 10-day period in B. leachii (915%) was significantly greater than S. striatus (150%), yet S. striatus consumed greater quantities of L. majuscula (g day(-1)) and thus had a lower conversion efficiency (0.038) than B. leachii (0.081) based on sea hare weight per gram of L. majuscula consumed day(-1). Our findings suggest that growth rates and conversion efficiencies may be influenced by sea hare maximum growth potential, acquisition of secondary metabolites or diet type. (C) 2005 Elsevier B.V. All rights reserved.

Responses of four Australian tree species to toxic concentrations of copper in solution culture

In Australia, metal-contaminated sites, including those with elevated levels of copper (Cu), are frequently revegetated with endemic plants. Little is known about the responses of Australian plants to excess Cu. Acacia holosericea, Eucalyptus crebra, Eucalyptus camaldulensis, and Melaleuca leucadendra were grown in solution culture with six Cu treatments (0.1 to 40 mu M). While A. holosericea was the most tolerant to excess Cu, all of the species tested were sensitive to excess Cu when compared with exotic tree and agricultural species. The critical external concentrations for toxicity were < 0.7 mu M for all species tested. There was little differentiation between shoot-tissue Cu concentrations in normal versus treated plants, thus, the derivation of critical shoot concentrations was possible only for the most tolerant species, A. holosericea. Critical root Cu concentrations were approximately 210 mu g g(-1) (A. holosericea), 150 mu g g(-1) (E. crebra), 25 mu g g(-1) (E. camaldulensis), and 165 mu g g(-1) (M. leucadendra). These results provide the first comprehensive combination of growth responses, critical concentrations, and toxicity symptoms for three important Australian genera for use in the management of Cu-contaminated sites.