Conversion of the BASE prion strain into the BSE strain: the origin of BSE?

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine "amyloidotic" spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrP(Sc), and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans.     

Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA

Ensemble and single-molecule fluorescence measurements of 2′-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)– calf thymus (CT) DNA complexes at various [H-258]/[DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a [H-258]/[DNA bp] ratio of 0.05 the relative fluorescence quantum yield, Φ_f, of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of ~3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low [H-258]/[DNA bp] ratios. With increasing [H-258]/[DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher [H-258]/[DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5–2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher [H-258]/[DNA bp] ratios reflected by a lifetime component of 1.5–2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher [H-258]/[DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of ~500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.     

Precise derivatization of structurally distinct chitosans with rhodamine B isothiocyanate

Work to date shows that structurally distinct chitosans have reacted inefficiently and unpredictably with fluorescein isothiocyanate (FITC) in an acid–methanol solvent that maintains both chitosan and fluorophore solubility. Since isothiocyanate preferentially reacts with neutral amine groups, and chitosan solubility typically depends upon a minimal degree of protonation, we tested the hypothesis that precise derivatization of chitosan with rhodamine isothiocyanate (RITC) can be achieved by controlling the reaction time and the degree of protonation. Addition of 50% v/v methanol reduced the chitosan degree of protonation in acetic acid but not HCl solutions. At various degrees of protonation, chitosan reacted inefficiently with RITC as previously observed with FITC. Nevertheless, precise derivatization was achieved by allowing the reaction to proceed overnight at a given degree of protonation (p < 0.0001, n = 18) and fixed initial fluorophore concentration. A reproducible 2% to 4% fraction of neutral amines reacted with RITC in proportion to the initial fluorophore concentration (p < 0.005). Using our optimized protocol, chitosans with different degree of deacetylation and molecular weight were derivatized to either 1% or 0.5% mol/mol RITC/chitosan-monomer with a precision of 0.1% mol/mol. The average molecular weight of fluorescent RITC-chitosan was similar to the unlabeled parent chitosan. Precise molar derivatization of structurally distinct chitosans with RITC can be achieved by controlling chitosan degree of protonation, initial fluorophore concentration, and reaction time.     

CULTIVATION STUDIES OF GIGARTINA SKOTTSBERGII IN SOUTHERN CHILE

Increases in population and agriculture in coastal areas can result in increased nutrient inputs and alterations in the ratios of organic to inorganic nutrients in coastal waters. Such changes in coastal nutrient regimes can affect phytoplankton community structure by creating conditions favorable for growth and dominance of algae that were not dominant before. The effect that changes in ratios and concentrations of nutrients have on toxicity of harmful algal species is not well known. There seems to be a relationship; however, between nutrient stress and toxin production among harmful phytoplankton producing low-N toxins, e.g. Diarrhetic Shellfish Poisoning (DSP) toxins. Even less is known about the relationship between organic nutrient uptake and toxin production. Benthic species and species in coastal areas are probably exposed to greater fluxes of dissolved organic nitrogen (DON). In this study, benthic and planktonic species of Prorocentrum were grown on L1 media with the sole N-source varying among treatments as nitrate, ammonium, urea, L-glutamic acid, and high molecular weight natural DON. An ELISA specific to the DSP toxins, okadaic acid and 35-methylokadaic acid, was used to determine toxin production by each species when grown on the different N sources. Preliminary results indicate that some organic forms of N support growth as well as inorganic forms for Prorocentrum minimum , P. mexicanum , and P. hoffmannianum.     

INTEGRATING SEAWEEDS INTO MARINE AQUACULTURE SYSTEMS: A KEY TOWARD SUSTAINABILITY

The rapid development of intensive fed aquaculture (e.g. finfish and shrimp) throughout the world is associated with concerns about the environmental impacts of such often monospecific practices, especially where activities are highly geographically concentrated or located in suboptimal sites whose assimilative capacity is poorly understood and, consequently, prone to being exceeded. One of the main environmental issues is the direct discharge of significant nutrient loads into coastal waters from open-water systems and with the effluents from land-based systems. In its search for best management practices, the aquaculture industry should develop innovative and responsible practices that optimize its efficiency and create diversification, while ensuring the remediation of the consequences of its activities to maintain the health of coastal waters. To avoid pronounced shifts in coastal processes, conversion, not dilution, is a common-sense solution, used for centuries in Asian countries. By integrating fed aquaculture (finfish, shrimp) with inorganic and organic extractive aquaculture (seaweed and shellfish), the wastes of one resource user become a resource (fertilizer or food) for the others. Such a balanced ecosystem approach provides nutrient bioremediation capability, mutual benefits to the cocultured organisms, economic diversification by producing other value-added marine crops, and increased profitability per cultivation unit for the aquaculture industry. Moreover, as guidelines and regulations on aquaculture effluents are forthcoming in several countries, using appropriately selected seaweeds as renewable biological nutrient scrubbers represents a cost-effective means for reaching compliance by reducing the internalization of the total environmental costs. By adopting integrated polytrophic practices, the aquaculture industry should find increasing environmental, economic, and social acceptability and become a full and sustainable partner within the development of integrated coastal management frameworks.     

The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin, and vimentin filaments.

We investigated the structure of the chondrocyte cytoskeleton in intact tissue sections of mature bovine articular cartilage using confocal fluorescence microscopy complemented by protein extraction and immunoblotting analysis. Actin microfilaments were present inside the cell membrane as a predominantly cortical structure. Vimentin and tubulin spanned the cytoplasm from cell to nuclear membrane, the vimentin network appearing finer compared to tubulin. These cytoskeletal structures were present in chondrocytes from all depth zones of the articular cartilage. However, staining intensity varied from zone to zone, usually showing more intense staining for the filament systems at the articular surface compared to the deeper zones. These results obtained on fluorescently labeled sections were also corroborated by protein contents extracted and observed by immunoblotting. The observed cytoskeletal structures are compatible with some of the proposed cellular functions of these systems and support possible microenvironmental regulation of the cytoskeleton, including that due to physical forces from load-bearing, which are known to vary through the depth layers of articular cartilage.     

Ecological engineering in aquaculture: use of seaweeds for removing nutrients from intensive mariculture

Rapid scale growth of intensive mariculture systems can often lead to adverse impacts on the environment. Intensive fish and shrimp farming, being defined as throughput-based systems, have a continuous or pulse release of nutrients that adds to coastal eutrophication. As an alternative treatment solution, seaweeds can be used to clean the dissolved part of this effluent. Two examples of successfully using seaweeds as biofilters in intensive mariculture systems are discussed in this paper. The first example shows that Gracilaria co-cultivated with salmon in a tank system reached production rates as high as 48.9 kg m⁻² a⁻¹, and could remove 50% of the dissolved ammonium released by the fish in winter, increasing to 90–95% in spring. In the second example, Gracilaria cultivated on ropes near a 22-t fish cage farm, had up to 40% higher growth rate (specific growth rate of 7% d⁻¹) compared to controls. Extrapolation of the results showed that a 1 ha Gracilaria culture gave an annual harvest of 34 t (d. wt), and assimilated 6.5% of the released dissolved nitrogen. This production and assimilation was more than twice that of a Gracilaria monoculture. By integrating seaweeds with fish farming the nutrient assimilating capacity of an area increases. With increased carrying capacity it will be possible to increase salmon cage densities before risking negative environmental effects like eutrophication and toxic algal blooms sometimes associated with the release of dissolved nutrients. The potential for using mangroves and/or seaweeds as filters for wastes from intensive shrimp pond farming is also discussed. It is concluded that such techniques, based on ecological engineering, seems promising for mitigating environmental impacts from intensive mariculture; however, continued research on this type of solution is required. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Southern Ocean ecosystem under multiple climate change stresses ‐ an integrated circumpolar assessment

A quantitative assessment of observed and projected environmental changes in the Southern Ocean (SO) with a potential impact on the marine ecosystem shows: (i) large proportions of the SO are and will be affected by one or more climate change processes; areas projected to be affected in the future are larger than areas that are already under environmental stress, (ii) areas affected by changes in sea‐ice in the past and likely in the future are much larger than areas affected by ocean warming. The smallest areas (<1% area of the SO) are affected by glacier retreat and warming in the deeper euphotic layer. In the future, decrease in the sea‐ice is expected to be widespread. Changes in iceberg impact resulting from further collapse of ice‐shelves can potentially affect large parts of shelf and ephemerally in the off‐shore regions. However, aragonite undersaturation (acidification) might become one of the biggest problems for the Antarctic marine ecosystem by affecting almost the entire SO. Direct and indirect impacts of various environmental changes to the three major habitats, sea‐ice, pelagic and benthos and their biota are complex. The areas affected by environmental stressors range from 33% of the SO for a single stressor, 11% for two and 2% for three, to <1% for four and five overlapping factors. In the future, areas expected to be affected by 2 and 3 overlapping factors are equally large, including potential iceberg changes, and together cover almost 86% of the SO ecosystem.     

Opportunities and challenges for the development of an integrated seaweed-based aquaculture activity in Chile: determining the physiological capabilities of <Emphasis Type="Italic">Macrocystis</Emphasis> and <Emphasis Type="Italic">Gracilaria</Emphasis> as biofilters

Seaweed production is a reality in Chile. More than ten species are commercially used to produce phycocolloids, fertilizers, plant growth control products, human food or animal fodder and feed additives. These multiple uses of algae offer a number of possibilities for coupling this activity to salmon, abalone and filter-feeder farming. In this context, different experiments carried out in Chile have demonstrated that Gracilaria chilensis and Macrocystis pyrifera have great potential in the development of an integrated aquaculture strategy. The present Integrated Multi-Trophic Aquaculture (IMTA) approach study showed that Gracilaria can be cultured best at 1 m depth whereas Macrocystis has an especially good growth response at 3 m depth. Both species use available nitrogen efficiently. On the other hand, high intensities of solar radiation (UV and PAR) can be critical at low depths of cultivation, and our results indicate that both species show photosynthetic susceptibility mainly at noon during the summer. The demand of Macrocystis for abalone feeding is increasing, thus improving the opportunity for developing an integrated nutrient waste recycling activity in Chile. Although Gracilaria shows a higher nitrogen uptake capacity than Macrocystis, its market value does not yet allow a massive commercial scaling.