Cytogenetic index and functional genome alterations in Chironomus piger Strenzke (Diptera, Chironomidae) in the assessment of sediment pollution: a case study of Bulgarian and UK rivers.

The genotoxicity of trace metals in the sediments from a number of polluted sites on UK and Bulgarian rivers to Chironomus piger was assessed by an examination of genome instability as demonstrated by structural and functional changes to the salivary glands chromosomes. Based on the metal assays, the sediments were characterized to range from 'extremely' to 'strongly contaminated'. The cytogenetic index calculated on the basis of somatic structural chromosome alterations in the polytene chromosomes indicates a high level of pollution (0.07-0.06 in Bulgarian and 0.10-0.13 in UK stations). Exposure of C. piger to contaminated sediments resulted in a high level of chromosome damage as indicated by a somatic index of between 1.96 and 4.0. The transcription mechanism of the Balbiani rings and nucleolar organizer was damaged as their activity was either partially or completely suppressed. We have demonstrated that the C. piger genome is a sensitive sublethal indicator of sediment contamination, and is a highly suitable candidate for ecotoxicological monitoring of running waters.

Effect of humic acid on water chemistry, bioavailability and toxicity of aluminium in the freshwater snail, Lymnaea stagnalis, at neutral pH.

The influence of humic acid on the water chemistry of environmentally relevant concentrations of Al at neutral pH was studied, together with its effect on the bioavailability and toxicity of Al in Lymnaea stagnalis. Humic acid significantly reduced the loss of Al from the water and increased the fraction of filterable Al, although this was a relatively small fraction of total Al. Filterable Al concentration in the presence or absence of humic acid was independent of initial Al concentration. Humic acid only partly reduced toxicity, as observed by a reduction in behavioural suppression, and had no effect on the level of Al accumulated in tissues. These results suggest that humic acid maintains Al in a colloidal form that is bioavailable to L. stagnalis. However, these colloidal Al-humic acid species were less toxic since behavioural toxicity was reduced. Humic acid may play an important role in limiting the toxicity of Al to freshwater organisms.

Characterization of surface sugars on algal cells with fluorescein isothiocyanate-conjugated lectins.

We used qualitative and quantitative fluorescence microscopy of the fluorescein isothiocyanate-conjugated lectins Concanavalin A, phytohaemagglutinin-erythroagglutinin, pokeweed mitogen, and peanut agglutinin to examine sugar composition on the cell surface and cell-associated mucilage (where present) in a number of cultured and environmental algae. Lectin-binding activity was markedly different between laboratory-cultured and environmental samples of the same species. Sugar composition of the cyanobacterium Anabaena cylindrica varied with growth cycle, although no clear pattern of change was observed. Akinetes typically showed lectin-binding activity higher than that of the vegetative cells or heterocysts throughout the growth cycle. Algae with mucilage showed greater lectin binding, indicating that mucilage contained more surface sugars accessible to the lectin probe compared with the cell wall surface. A low level of galactose and N-acetyl galactosamine (detected by peanut agglutinin) was associated with the surface mucilage of most algal species. Relatively high amounts of mannose, glucose, and N-acetyl glucosamine (detected by Concanavalin A, phytohaemagglutinin, and pokeweed mitogen) were also present. Lectin binding was shown to be a highly specific and sensitive approach to the examination of cell surface chemistry of both cultured and environmental algae and to the study of biodiversity in phytoplankton.

Role of exogenous and endogenous silicon in ameliorating behavioural responses to aluminium in a freshwater snail.

Aluminium accumulation by the freshwater snail Lymnaea stagnalis is correlated with behavioural depression which is ameliorated by addition of orthosilicic acid. We hypothesised that Si is relocated to the digestive gland in response to Al, leading to the formation of non-toxic hydroxyaluminosilicates (HAS). Exposure to 500 microg l(-1) Al for 30 days was associated with an initial period of behavioural depression, followed by apparent tolerance and subsequent depression, suggesting saturation of the cellular detoxification pathway during prolonged exposure. Exogenous Si (7.77 mg l(-1)) completely ameliorated all behavioural effects of Al but did not prevent its accumulation. In the presence of added Al, significantly more of this Si was accumulated by the tissues, compared to controls and snails exposed to Si alone. In snails exposed to Al plus Si, Al and Si concentrations were significantly correlated, with a ratio around 3:1 Al:Si, consistent with the presence of the non-toxic HAS protoimogolite.

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors.

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g(-1) astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l(-1), astragaloside IV 0.9 mg g(-1)). Polysaccharide yields were similar amongst the different bioreactors (range 25-32 mg g(-1)). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.

Functional and structural rearrangements of salivary gland polytene chromosomes of Chironomus riparius Mg. (Diptera,Chironomidae) in response to freshly neutralized aluminium.

Although recent work has shown that environmentally relevant concentrations of freshly neutralized aluminium (AI) are bioavailable and toxic to freshwater invertebrates, the genotoxicity of Al has not been examined. Here we show that freshly neutralized Al affects structure and function of the salivary gland polytene chromosomes of the ubiquitous chironomid larva Chironomus riparius over three generations. Exposure to 500 microg l-1 added Al for 24-25 days resulted in a significantly higher frequency of numerous somatic aberrations, while no structural aberrations were found in F1 controls and few in the second and third generation. Aberrations also included deletions of sections of chromosome G of C. riparius larvae as well as deletions of one or more Balbiani rings. Changes in functional activity included decreased activity of the Balbiani rings (BR), and an increase in the number of decondensed centromeres. The activity of the nucleolar organizer (NOR) significantly decreased in F1 chironomids exposed to Al, while in the F2 and F3 generations the NOR showed normal (high) activity. First generation chironomids were generally more susceptible to Al although no clear evidence of tolerance was apparent over three generations. The possible use of alterations in chironomid polytene chromosomes as biomarkers of trace metal pollution is discussed.

Bioavailability and toxicity of freshly neutralized aluminium to the freshwater crayfish Pacifastacus leniusculus.

Freshly neutralized aluminium (Al) is toxic to a variety of freshwater organisms despite its insolubility at circumneutral pH. Insoluble Al acts exogenously--for example, on the fish gill--thereby impairing respiratory function, and endogenously in grazing and filter-feeding invertebrates following ingestion during drinking and feeding. This paper examines the bioavailability and behavioral toxicity of freshly neutralized Al to the freshwater crayfish Pacifastacus leniusculus exposed to 500 microg L(-1) added Al for 20 days under controlled conditions. We test the hypothesis that aqueous Al is toxic to the crayfish and that this is largely due to the metal's association with the gill rather than following accumulation in the body. Little Al was accumulated in the digestive gland (hepatopancreas) or flexor muscle, but large amounts were associated with the gills, resulting in concentration factors of up to 1 x 10(4). Histochemistry showed that much of this metal was extracellular to the gill epithelium and associated with the mucus layer. Behavioral dysfunction was observed following exposure to Al for five days. Reduction in the amount of Al in the water column, due to binding to snail trail mucus attached to the substrate, reduced the amount of Al associated with the gill and delayed the onset of behavioral dysfunction. We conclude that freshly neutralized Al is toxic to the crayfish and that main site of Al action is the gill.

Influence of oligomeric silicic and humic acids on aluminum accumulation in a freshwater grazing invertebrate.

This study examined the influence of oligomeric silicic acid and humic acid on aluminum in the water column and its accumulation in the freshwater snail Lymnaea stagnalis. Forty-eight hours after addition of Al (500 microg L(-1)), 83% of the metal was lost from the water column. This loss was reduced by oligomeric silica (20 mg L(-1)) and by humic acid (10 mg L(-1)). Aluminum accumulated in the digestive gland and, to a lesser extent, in the remaining soft tissues, and this accumulation was reduced by oligomeric silica. In the presence of humic acid, Al accumulation in the digestive gland was unaffected, though less was accumulated in the remaining tissues. Snails accumulated Si preferentially in the digestive gland and this accumulation was increased in the presence of added Al. Thus, both oligomeric silica and humic acid influence Al bioavailability and Si is upregulated in the digestive gland in the presence of Al.

Effect of sub-lethal concentrations of aluminium on the filtration activity of the freshwater mussel Anodonta cygnea L. at neutral pH.

Significant amounts of aluminium (Al) are commonly present in rivers and lakes, largely in particulate form in neutral waters. Freshwater bivalves, as filter feeders are therefore exposed to both particulate and dissolved metal and are potentially vulnerable to Al. The effect of Al on filtering behaviour of the freshwater mussel Anodonta cygnea L. was investigated during short (1 hour) and long-term (15 days) exposure to environmentally relevant concentrations (250 and 500 microg l(-1)) at neutral pH. Water flow through the outflow siphon was monitored as an indicator of pumping capacity. Short-term (1 hour) exposure to 500 microg l(-1) added Al produced an irreversible decrease in the duration of filtering periods, presumably as an avoidance response to the toxicant. One-hour exposure 250 microg l(-1) Al had no detectable effect. When mussels were exposed to 250 or 500 microg l(-1) added Al for 15 days, siphon activity measured in days 11-15 of exposure was inhibited by 50% and 65%, respectively, compared to pre-exposure levels. Recovery occurred following transfer of mussels to uncontaminated water. Interaction between Al and freshwater bivalves at neutral pH may affect both the performance of the mussels and the chemical speciation of the metal in the natural environment.

Down-regulation of liver iron-regulatory protein 1 in haemochromatosis.

Cellular iron homoeostasis is maintained by iron sensor proteins known as iron-regulatory proteins (IRPs), which act post-transcriptionally by binding RNA stem-loop structures, termed iron-responsive elements (IREs), present on the mRNAs of proteins involved in iron storage, utilization and transport. IRP1 is a bifunctional protein that can act either as a cytoplasmic aconitase or as an IRE-binding protein. The RNA-binding activity of IRP1 is regulated post-translationally by the insertion or extrusion of a 4Fe-4S cluster, without changes in the levels of protein. In hereditary haemochromatosis (HH) accumulation of iron in parenchymal tissues, including the liver, occurs, possibly through dysfunctional IRP1. Investigation of IRP1 expression in liver biopsies from HH patients showed that the protein is completely absent or markedly reduced in heavily iron-loaded HH patients. Real-time PCR was then conducted in an attempt to investigate the mRNA levels and establish the underlying mechanism behind the disappearing act of IRP1. The two possibilities are: transcriptional regulation (through the inhibition of transcription) or post-transcriptional regulation (either through increased turnover of protein or inhibition of translation) of IRP1. Preliminary data suggest that transcription of IRP1 is not affected by chronic iron overload, and down-regulation may be attributable instead to degradation of the protein.

Rapid non-equilibrium aluminium-ligand interactions: studies on the precipitation of aluminium by laser light scattering, ultrafiltration and centrifugation.

The study aimed to develop simple assays to study aluminium-ligand interactions in natural/biological systems where equilibrium is rarely reached and thus where the initial seconds or hours of interactions are important. The immediate and non-equilibrium precipitation of aluminium hydroxide, in aqueous solution at neutral pH, was therefore studied by laser light scattering (diffraction), ultrafiltration and centrifugation. The interaction of weak ligands, present in the gastrointestinal lumen, on the precipitation of aluminium hydroxide was also investigated. The initial kinetics and particle sizes of precipitated aluminium hydroxide were sensitive to a number of external factors, including the presence of weak ligand (bicarbonate), sheer force (stirring), electrolyte concentration and initial (i.e. added) aluminium concentration. However, after a few seconds (no weak ligand), or several hundred seconds (with weak ligand), the subsequent observed changes to the solid phase were of small magnitude and occurred slowly. Thus, a 25-min window, within 5 and 30 min of pH adjustment, can be used to study the interactions of aluminium-ligand. This may approximate better to most natural systems where unperturbed aluminium-ligand equilibrium must rarely exist.

Avoidance responses to aluminium in the freshwater bivalve Anodonta cygnea.

This study examined the effect of aluminium (Al) on the filtering behaviour (shell opening or gape) of the freshwater bivalve Anodonta cygnea L in neutral fresh water. Parallel measurements of Al concentration in the soft tissues were made to examine the relationship between changes in behaviour and accumulation of Al. The number of lysosomal granules in the gill, kidney and digestive gland were counted, as lysosomes are known to be involved in the excretion and detoxification of trace metals. The bivalves were exposed to two environmentally relevant concentrations of added Al i.e. 250 and 500 microg l(-1) (9.25 and 18.5 microM l(-1)) at neutral pH for 15 days and shell movement monitored continuously. Aluminium affected the mussels' filtering activity, producing an avoidance reaction whose magnitude was concentration-dependent; 250 microg l(-1) added Al produced no detectable change, while 500 microg l(-1) Al reduced mean duration of shell opening by 50%. This effect was irreversible over a 15 day recovery period. Tissue levels of Al after 15 days exposure were an order of magnitude higher in animals exposed to 250 microg l(-1) added Al than in those exposed to 500 microg l(-1). This was consistent with the inhibition of filtering activity due to valve closure at the higher concentration, which may have prevented uptake of Al. In addition, probable different chemical speciation of Al in the water column (soluble for 250 and colloidal for 500 microg l(-1)) may lead to marked differences in tissue uptake. The kidney and digestive gland were the main sites of accumulation of Al and concentrations remained significantly elevated 15 days after transfer of animals to clean water. It is suggested that mucus plays a role in the exclusion of Al as elevated concentrations were measured in the pseudofaeces of animals during and after exposure. Lysosomal granules may be involved in the intracellular handling and detoxification of Al as numbers increased significantly in all organs during exposure and continued to increase after the animals were transferred to clean water. The present study provides evidence for the bioavailability and toxicity of Al to mussels at neutral pH and at concentrations which are known to enter neutral freshwaters when mobilised by natural or anthropogenic acidity. The changes in behaviour and uptake of Al in the mussel observed in this investigation are, therefore, likely to be reflected in the natural environment and the degree to which Al affects the 'fitness' of the mussel populations and the transfer of Al through the food chain merit investigation.

Characterization of CD44 splicing patterns in normal keratinocytes, dysplastic and squamous carcinoma cell lines.

The CD44 glycoprotein is spliced from a complex gene of 10 constitutive and 10 variant exons. In this study, CD44 splicing patterns and intron 9 retention were investigated by exon-specific RT-PCR for variant exons v1-v10 and intron 9 in normal, immortalized, dysplastic and malignant keratinocytes. Expression of product was determined immunohistochemically for some of the exons. Normal keratinocytes showed one major transcript including exons v2-v10 and 3 minor transcripts. No lines showed a normal CD44 splicing pattern but rather a variety of truncated transcripts of contiguous variant exons which overall correlated with expression. Squamous cell carcinoma (SCC)-4 and SCC-9 lines showed relatively normal transcripts although protein was expressed only by SCC-9. SCC-12B2, SCC-15, SCC-25 and SCC-27 showed a series of shorter overlapping transcripts, with loss of exons v8-v10 in the major transcripts. Intron 9 was not retained in normal keratinocytes or cell lines. Despite the fact that keratinocytes constitutively express all variant exons, splicing patterns are distinctly abnormal and merit investigation as potential markers for epidermal and oral squamous malignancy.

Functional mobility discriminates nonfallers from one-time and frequent fallers.

BACKGROUND: Given that 90% of hip fractures result from a fall, individuals who fall frequently are more likely to be at greater risk for fracture than one-time fallers. Our aim was to determine whether performance variables associated with injurious falls could be used to distinguish frequent fallers from both one-time fallers and nonfallers. METHODS: A total of 157 men and women (77.4-5.4 years) were recruited and categorized into one of the following three groups based on falls status over the previous 12 months: nonfallers (n = 48), one-time fallers (n = 56), and frequent fallers (more than one fall) (n = 53). All subjects were evaluated on functional mobility and lower extremity strength and power. RESULTS: Using multivariate analysis of covariance with height as a covariate, nonfallers were significantly faster than both one-time and frequent fallers during the Get Up and Go (a test involving lower extremity strength and power, and mobility) and faster than one-time fallers on the Tandem Gait (p < .01). There were no significant differences between groups for other mobility variables or for laboratory measures of strength and power. Because one-time and frequent fallers were similar on all measures. they were grouped as "fallers" in discriminant analysis. The Get Up and Go discriminated between the fallers and nonfallers with a final Wilks's Lambda of .900 (p < .001) and correctly classified 72.4% of fallers and nonfallers before crossvalidation and 71.2% of the cases after validation. CONCLUSIONS: Given that the Get Up and Go discriminates between fallers and nonfallers and is associated with lower extremity strength and power, fall prevention strategies should focus on improving both functional mobility and lower extremity strength and power.

Bioaccumulation and toxicity of aluminium in the pond snail at neutral pH.

The low solubility of aluminium (Al) at neutral pH means that it largely exists as colloidal particulates in aquatic systems. However, the pond snail Lymnaea stagnalis accumulates significant amounts of Al following exposure to water containing added Al (up to 500 microg l(-1)) at pH 7. This is accompanied by depression of behavioural activity (locomotion, feeding) which subsequently recovers, suggesting tolerance to the metal. The presence of silica ameliorates behavioural toxicity of Al, but does not prevent uptake of the metal. In vitro studies using the isolated central nervous system demonstrate toxicity at the cellular level. Extracellular application of Al (100 microM) led to membrane depolarisation, bursts of action potentials and action potential broadening. The chemical form in which Al is applied influences the extent of bioaccumulation and toxicity. Detailed knowledge of its solution chemistry is therefore essential.

The state, the market, and general practice: the Australian case.

This article examines the development of general practice in the latter half of the 20th century, documenting the issues of concern to both the profession and the state. General practice developed hand in hand with the welfare state in Australia. As the structural changes associated with restructuring of the welfare state have advanced, so have the fortunes of general practice declined, despite significant attempts in the 1970s and 1980s to "save" general practice by both the profession and the state. These structural changes have operated on two fronts, the economic and the cultural. On the economic, changes to the employment of general practitioners clearly indicate ongoing proletarianization, particularly in a changing environment of labor-capital relations. At the cultural level, development of the self-help and the women's movements and the elective affinity of these groups with the individualism of the new right are leading to deprofessionalization. The author advances this argument in a review of general practice over the last 40 years and in a case study of community health services. Theoretically he argues for a combination of the proletarianization and the deprofessionalization theses.

Localization and fate of aluminium in the digestive gland of the freshwater snail Lymnaea stagnalis.

The digestive gland of the freshwater snail Lymnaea stagnalis, exposed to water containing an elevated concentration of aluminium at neutral pH for up to 30 days, followed by a 20 day recovery period, was examined by light and electron microscopy and X-ray microanalysis. Aluminium was localized in the yellow granules present in the digestive and excretory cells and in the green and small granules present in the digestive cells. More aluminium, silicon, phosphorus and sulphur were present in all three granule types from aluminium exposed snails. The number of yellow and green granules from the digestive gland of aluminium exposed snails showed a progressive increase over the experimental period compared to controls. The number and aluminium content of the granules is likely to reflect the role of the digestive gland as a 'sink' for accumulated aluminium. We propose that intracellular monomeric silica is involved in the detoxification of aqueous aluminium which at neutral pH is largely in the form of an insoluble polyhydroxide. The increased amounts of sulphur and phosphorus in the granules are likely to be part of a broad response to metal loading but probably do not play a significant role in the storage and detoxification of aluminium.

Aluminum toxicity in a molluscan neuron: effects of counterions.

Previous studies using the freshwater snail Lymnaea stagnalis have indicated significant accumulation of aluminum (Al) from simple salts (chloride or nitrate) or Al lactate [Al(lactate)3 preparations, but not from the Al maltol complex [Al(maltol)3]. This is in contrast to findings in mammalian systems, where uptake and neurotoxicity are greatest for the soluble and lipophilic Al(maltol)3 complex. This study was undertaken to investigate the direct effects of extracellular Al (100 microM) from three Al preparations [AlCl3, Al(lactate)3 and Al(maltol)3] on electrophysiological parameters of an identified neuron, the right parietal dorsal 1 (RPD1) neuron, of L. stagnalis in vitro. The effects of the corresponding counterion/ligand on the solubility and availability of Al in solution were also examined. Significant effects of Al on electrical properties, including membrane depolarization, increased firing activity, and abnormal firing patterns, were seen in the presence of AlCl3 and Al(lactate)3, which formed polyhydroxy and labile Al species in aqueous solution, but not with Al(maltol)3, which remained as the soluble monomeric complex. Qualitative differences were also observed between the response to AlCl3 and Al(lactate)3, despite their similar chemistry. The extent of action potential broadening was greater with Al(lactate)3, suggesting some interaction between Al and lactate in their cellular uptake and/or toxicity. It is suggested that polyhydroxy Al species are toxic to molluscan neurons, possibly via disruption of intracellular Ca2+ homeostasis.

Cloning and characterization of metallothionein cDNAs in the mussel Mytilus edulis L. digestive gland.

Metallothioneins are small metal-binding proteins found in all species of animals and are transcriptionally-induced by heavy metal ions, oxidative stresses, and inflammation. In the blue sea mussel, Mytilus edulis, several apparent subtypes of each isoform have been purified and biochemically sequenced. To determine whether the high number of metallothionein forms present in M. edulis were specific to the digestive gland, and to understand how these proteins evolved, we cloned five variants of metallothionein from M. edulis. MT10 and MT20 isoform fragments were amplified by PCR, and used as radiolabelled probes to screen digestive gland cDNA libraries. The MT10 transcripts were 321-353 nucleotides long and the MT20 transcripts, 513-555 nucleotides. Previously identified primary structures of MT10 subtypes were confirmed and, in addition, a novel subtype was identified. Expression of MT10 and MT20 isoforms shown by clonal representation and Northern blot analysis indicated that the MT10 message was more prevalent than the MT20 message. Only the MT20 II transcript could be identified among the MT20 clones. The high degree of untranslated region similarity between each isoform indicates that these additional forms are recent gene duplication events in the Mytilus lineage. Exposure of 0.4 mg l-1 of cadmium to the mussels resulted in a marked increase in both mRNAs suggesting that the MT20 isoform represents a primarily inducible metallothionein not highly expressed under basal conditions.