Gene expression and hormone secretion profile of urotensin I associated with osmotic challenge in caudal neurosecretory system of the euryhaline flounder, Platichthys flesus.

The caudal neurosecretory system (CNSS) is a part of stress response system, a neuroendocrine structure unique to fish. To gain a better understanding of the physiological roles of CNSS in fluid homeostasis, we characterized the tissue distribution of urotensin I (UI) expression in European flounder (Platichthys flesus), analyzed the effect chronic exposure to seawater (SW) or freshwater (FW), transfer from SW to FW, and reverse transfer on mRNA levels of UI, L-type Ca2+ channels and Ca-activated K+ channels transcripts in CNSS. The tissue distribution demonstrated that the CNSS is dominant sites of UI expression, and UI mRNA level in fore brain appeared greater than other non-CNSS tissues. There were no consistent differences in CNSS UI expression or urophysis UI content between SW- and FW-adapted fish in July and September. After transfer from SW to FW, at 8 h CNSS UI expression was significantly increased, but urophysis UI content was no significantly changes. At 24 h transfer from SW to FW, expression of CNSS UI was no apparent change and urophysis UI content was reduced. At 8 h and 24 h after transfer from FW to SW UI expression and urophysis UI content was no significantly effect. The expression of bursting dependent L-type Ca2+ channels and Ca-activated K+ channels in SW-adapted fish significantly decreased compared to those in FW-adapted. However, there were no differences in transfer from SW to FW or from FW to SW at 8 h and 24 h. Thus, these results suggest CNSS UI acts as a modulator in response to osmotic stress and plays important roles in the body fluid homeostasis.

Aluminium exposure disrupts elemental homeostasis in Caenorhabditis elegans.

Aluminium (Al) is highly abundant in the environment and can elicit a variety of toxic responses in biological systems. Here we characterize the effects of Al on Caenorhabditis elegans by identifying phenotypic abnormalities and disruption in whole-body metal homeostasis (metallostasis) following Al exposure in food. Widespread changes to the elemental content of adult nematodes were observed when chronically exposed to Al from the first larval stage (L1). Specifically, we saw increased barium, chromium, copper and iron content, and a reduction in calcium levels. Lifespan was decreased in worms exposed to low levels of Al, but unexpectedly increased when the Al concentration reached higher levels (4.8 mM). This bi-phasic phenotype was only observed when Al exposure occurred during development, as lifespan was unaffected by Al exposure during adulthood. Lower levels of Al slowed C. elegans developmental progression, and reduced hermaphrodite self-fertility and adult body size. Significant developmental delay was observed even when Al exposure was restricted to embryogenesis. Similar changes in Al have been noted in association with Al toxicity in humans and other mammals, suggesting that C. elegans may be of use as a model for understanding the mechanisms of Al toxicity in mammalian systems.

Characterisation of chemosensory trigeminal receptors in the rainbow trout, Oncorhynchus mykiss: responses to chemical irritants and carbon dioxide.

Trigeminally innervated, mechanically sensitive chemoreceptors (M) were previously identified in rainbow trout, Oncorhynchus mykiss, but it is not known whether these receptors are responsive only to noxious, chemical irritants or have a general chemosensory function. This study aimed to characterise the stimulus-response properties of these receptors in comparison with polymodal nociceptors (P). Both P and M gave similar response profiles to acetic acid concentrations. The electrophysiological properties were similar between the two different afferent types. To determine whether the receptors have a nociceptive function, a range of chemical stimulants was applied to these receptors, including non-noxious stimuli such as ammonium chloride, bile, sodium bicarbonate and alarm pheromone, and potentially noxious chemical irritants such as acetic acid, carbon dioxide, low pH, citric acid, citric acid phosphate buffer and sodium chloride. Only irritant stimuli evoked a response, confirming their nociceptive function. All receptor afferents tested responded to carbon dioxide (CO(2)) in the form of mineral water or soda water. The majority responded to 1% acetic acid, 2% citric acid, citric acid phosphate buffer (pH 3) and 5.0 mol l(-1) NaCl. CO(2) receptors have been characterised in the orobranchial cavity and gill arches in fish; however, this is the first time that external CO(2) receptors have been identified on the head of a fish. Because the fish skin is in constant contact with the aqueous environment, contaminants with a low pH or hypercapnia may stimulate the nociceptive system in fish.

Nitric oxide potentiates cAMP-gated cation current by intracellular acidification in feeding neurons of pleurobranchaea.

A pH-sensitive cAMP-gated cation current (I(Na,cAMP)) is widely distributed in neurons of the feeding motor networks of gastropods. In the sea slug Pleurobranchaea this current is potentiated by nitric oxide (NO), which itself is produced by many feeding neurons. The action of NO is not dependent on either cGMP or cAMP signaling pathways. However, we found that NO potentiation of I(Na,cAMP) in the serotonergic metacerebral cells could be blocked by intracellular injection of MOPS buffer (pH 7.2). In neurons injected with the pH indicator BCECF, NO induced rapid intracellular acidification to several tenths of a pH unit. Intracellular pH has not previously been identified as a specific target of NO, but in this system NO modulation of I(Na,cAMP) via pH(i) may be an important regulator of the excitability of the feeding motor network.

Trophic transfer of aluminium through an aquatic grazer-omnivore food chain.

The potential for trophic transfer of aluminium (Al) was investigated using a grazing detritivore, the freshwater snail Lymnaea stagnalis, and a predator, the signal crayfish Pacifastacus leniusculus. Snails were exposed to either aqueous Al (500 microg l(-1)) in the presence or absence of an inorganic ligand (phosphate (+P); 500 microg l(-1)) for 30 days, or kept as unexposed controls. Subcellular partitioning of Al in the snail tissues was characterised using ultracentrifugation. Al content in the soft tissues and the subcellular fractions was measured using inductively coupled plasma atomic emission spectroscopy. Exposed and control snails were fed to individually housed crayfish (n=6 per group) over 40 days. Water samples, uneaten snail tissue and faeces were collected throughout the experiment in order to assess the fate of Al. Behavioural toxicity to the crayfish was assessed at four time points, and tissue accumulation of Al in soft tissues was measured following a 2-day depuration period. Snails exposed to Al+P accumulated more Al per snail than those exposed to Al only (291 microg vs 206 microg), and also contained a higher proportion of detoxified Al (in inorganic granules and associated with heat stable proteins) (39% vs 26%). There were no significant differences in behavioural activity between the different groups of crayfish at any time point. Crayfish fed snails exposed to only Al accumulated significant levels of Al in their total soft tissues, compared to controls; crayfish fed Al+P-exposed snails did not, even though concentrations of Al in these snails were higher. The highest concentrations of Al were found in the green gland in both crayfish feeding groups, and the gut and hepatopancreas in crayfish fed Al only exposed snails; all of these were significantly higher than in crayfish fed control snails. There was no significant accumulation of Al in the gills or flexor muscle in any group. At least 17% of trophically available Al in the snail tissues was accumulated by the crayfish. This proportion was similar in both feeding groups but, as the proportion of trophically available Al in the snails exposed to Al+P was lower, this led to lower accumulation in the Al+P crayfish feeding group. This study indicates that in comparison to vertebrates, aquatic invertebrates accumulate a higher proportion of Al via oral ingestion but it does not accumulate in tissues that may pose a threat to human consumers.

The suitability of gallium as a substitute for aluminum in tracing experiments.

Aluminum is a toxic metal whose complex aquatic chemistry, mechanisms of toxicity and trophic transfer are not fully understood. The only isotope of Al suitable for tracing experiments in organisms-(26)Al-is a rare, costly radioisotope with a low emission energy, making its use difficult. Gallium shares a similar chemistry with Al and was therefore investigated as a potential substitute for Al for use in aquatic organisms. The freshwater snail, Lymnaea stagnalis was exposed to either Al or Ga (0.0135 mM) under identical conditions for up to 40 days. Behavioural toxicity, metal accumulation in the tissues, and sub-cellular partitioning of the metals were determined. Al was more toxic than Ga and accumulated to significantly higher levels in the soft tissues (P < 0.05). The proportion of Al in the digestive gland (DG; detoxificatory organ) relative to other tissues was significantly lower than that of Ga (P < 0.05) from day 14 onwards. There were also differences in the proportions of Al and Ga associated with heat stable proteins (HSPs) in the digestive gland, with significantly more HSP present in the DGs of snails exposed to Al, but significantly less Al than Ga associated with the HSP per unit mass protein present. From this evidence, we conclude that Ga may be of limited use as a tracer for Al in animal systems.

Tissue accumulation of aluminium is not a predictor of toxicity in the freshwater snail, Lymnaea stagnalis.

The amount of toxic metal accumulated by an organism is often taken as an indicator of potential toxicity. We investigated this relationship in the freshwater snail, Lymnaea stagnalis, exposed to 500 microg l(-1) Al over 30 days, either alone or in the presence of phosphate (500 microg l(-1) P) or a fulvic acid surrogate (FAS; 10 mg l(-1) C). Behavioural activity was assessed and tissue accumulation of Al quantified. Lability of Al within the water column was a good predictor of toxicity. FAS increased both Al lability and behavioural dysfunction, whereas phosphate reduced Al lability, and completely abolished Al-induced behavioural toxicity. Tissue accumulation of Al was not linked to toxicity. Higher levels of Al were accumulated in snails exposed to Al + P, compared to those exposed to Al alone, whereas FAS reduced Al accumulation. These findings demonstrate that the degree of tissue accumulation of a metal can be independent of toxicity.

Precise and fuzzy coding by olfactory sensory neurons.

The exact nature of the olfactory signals that arrive in the brain from the periphery, and their reproducibility, remain essentially unknown. In most organisms, the sheer number of olfactory sensory neurons (OSNs) makes it impossible to measure the individual responses of the entire population. We measured the individual in situ electrophysiological activity of OSNs in Drosophila larvae, in response to stimulation with 10 aliphatic odors (alcohols and esters). We studied control larvae (a total of 296 OSNs) and larvae with a single functional OSN, created using the Gal4-upstream activator sequence system. Most OSNs showed consistent, precise responses (either excitation or inhibition) in response to a given odor. Some OSNs also showed qualitatively variable responses ("fuzzy coding"). This robust variability was an intrinsic property of these neurons: it was not attributable to odor type, concentration, stimulus duration, genotype, or interindividual differences, and was seen in control larvae and in larvae with one and two functional OSNs. We conclude that in Drosophila larvae the peripheral code combines precise coding with fuzzy, stochastic responses in which neurons show qualitative variability in their responses to a given odor. We hypothesize that fuzzy coding occurs in other organisms, is translated into differing degrees of activation of the glomeruli, and forms a key component of response variability in the first stages of olfactory processing.

Cortisol and prolactin modulation of caudal neurosecretory system activity in the euryhaline flounder Platichthys flesus.

Previous studies have shown roles for cortisol and prolactin in osmoregulatory adaptation to seawater and freshwater, respectively, in euryhaline fish. This study of the European flounder investigated the potential for these hormones to modulate activity of the caudal neurosecretory system (CNSS), which is thought to be involved in physiological adaptation to changing external salinity. Superfusion of isolated CNSS with either cortisol or prolactin (10 microM; 15 min) led to changes in firing activity in neuroendocrine Dahlgren cells, recorded extracellularly. Cortisol evoked a modest increase in overall firing activity, with the response delayed by 4 h after treatment. The response to prolactin was short latency, continued to build up over the subsequent 4-h wash period, and comprised increased firing activity together with recruitment of previously silent Dahlgren cells. Immunoreactivity for glucocorticoid and prolactin receptors was localised to Dahlgren cells. The CNSS expression level for glucocorticoid-2 receptor mRNA, measured by Q-PCR, was significantly lower in fish fully acclimated to freshwater, compared to seawater. No differences were seen between these two states for prolactin receptor mRNA expression. These results provide evidence for a modulatory action of both hormones on the neurosecretory function of the CNSS.

Avoidance of aluminum toxicity in freshwater snails involves intracellular silicon-aluminum biointeraction.

Silicon (Si) ameliorates aluminum (Al) toxicity to a range of organisms, but in almost all cases this is due to ex vivo Si-Al interactions forming inert hydroxyaluminosilicates (HAS). We hypothesized a Si-specific intracellular mechanism for Al detoxification in aquatic snails, involving regulation of orthosilicic acid [Si(OH)4]. However, the possibility of ex vivo formation and uptake of soluble HAS could not be ruled out Here we provide unequivocal evidence for Si-Al interaction in vivo, including their intracellular colocalization. In snails preloaded with Si(0H)4, behavioral toxicity in response to subsequent exposure to Al was abolished. Similarly, recovery from Al-induced toxicity was faster when Si(OH)4 was provided, together with rapid loss of Al from the major detoxificatory organ (digestive gland). Temporal separation of Al and Si exposure excluded the possibility of their interaction ex vivo. Elemental mapping using analytical transmission electron microscopy revealed nanometre-scale colocalization of Si and Al within excretory granules in the digestive gland, consistent with recruitment of Si(OH)4, followed by high-affinity Al binding to form particles similarto allophane, an amorphous HAS. Given the environmental abundance of both elements, we anticipate this to be a widespread phenomenon, providing a cellular defense against the profoundly toxic Al(III) ion.

Nociception in fish: stimulus-response properties of receptors on the head of trout Oncorhynchus mykiss.

This study examined stimulus-response properties of somatosensory receptors on the head of rainbow trout, Oncorhynchus mykiss, using extracellular recording from single cells in the trigeminal ganglion. Of 121 receptors recorded from 39 fish, 17 were polymodal nociceptors, 22 were mechanothermal nociceptors, 18 were mechanochemical receptors, 33 were fast adapting mechanical receptors and 31 were slowly adapting mechanical receptors. Mechanical thresholds were higher in polymodal nociceptors than in either slowly adapting or fast adapting mechanical receptors, whereas thermal thresholds of mechanothermal nociceptors were higher than those of polymodal nociceptors. Polymodal nociceptors and mechanochemical receptors gave similar responses to topical applications of acid. All receptor types except mechanothermal nociceptors showed an increase in peak firing frequency with increased strength of mechanical stimulation, with evidence of response saturation at higher intensities. Mechanothermal, but not polymodal, nociceptors showed an increase in firing response to increased temperature. None out of 120 receptors tested gave any response to the temperature range +4 degrees C to -7 degrees C, indicating an absence of cold nociceptors. Attempts to evoke sensitization of receptors using chemical or heat stimuli were unsuccessful, with receptors showing either a return to control responses or irreversible damage. Comparisons are made between somatosensory receptors characterized here in a fish and those of higher vertebrates.

Seasonal changes in peptide, receptor and ion channel mRNA expression in the caudal neurosecretory system of the European flounder (Platichthys flesus).

The caudal neurosecretory system (CNSS) of the euryhaline flounder Platichthys flesus has suggested roles in osmoregulatory, reproductive and nutritional adaptation, as fish migrate between seawater (winter) and brackish/freshwater (summer) environments. This study examined seasonal changes in mRNA expression profile of functionally important genes in the CNSS. cDNAs encoding neuropeptides, receptors and ion channels were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and screening of a flounder CNSS cDNA library. The expression profile of cloned genes was determined by real-time RT-PCR at 2-month intervals throughout the year in CNSS from seawater-adapted fish. Plasma cortisol (measured by radioimmunoassay) showed a peak in April, the time of spawning. Expression levels of mRNA for peptides urotensins I and II (UI, UII) and corticotropin releasing factor (CRF) all showed a seasonal cycle, with lowest expression in April and highest in August-October. The expression of CRF2(UI), UT(UII) and CRF1 receptors was not correlated with corresponding peptide expression. Receptors for potential neuromodulators of CNSS activity also displayed a seasonal mRNA expression profile. Glucocorticoid, 5-hydroxytryptamine, kappa-opioid and glutamate receptor expression peaked around April, suggesting that modulation of electrical activity of the neurosecretory Dahlgren cells is of particular importance at this time. Expression of mRNA for L-type Ca(2+) and Ca-activated K(+) channels was lower during the summer months. These channels underlie electrical bursting activity in Dahlgren cells. Ion channel mRNA expression was also lower in CNSS from flounder fully adapted to freshwater as opposed to seawater, consistent with previously reported observations of reduced bursting activity in Dahlgren cells from freshwater-adapted CNSS. These findings support the hypothesis that the CNSS is functionally reprogrammed to cope with changes in physiological challenge as fish migrate between sea and estuaries in winter and spring.

Evidence for nitric oxide role in the caudal neurosecretory system of the European flounder, Platichthys flesus.

A neuromodulatory role for nitric oxide has been reported for magnocellular neuroendocrine cells in mammalian hypothalamus. We examined its potential as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the euryhaline flounder. Immunocytochemistry using an antibody raised against human neuronal nitric oxide synthase (NOS) indicated the presence of NOS in the Dahlgren cells. Quantitative RT-PCR, using a flounder-specific probe, revealed NOS mRNA expression in the CNSS. In July, though not in September, NOS mRNA expression was significantly higher in fish fully adapted to seawater, compared to freshwater-adapted fish. Following acute transfer of fish from freshwater to seawater, NOS mRNA expression was elevated at 8h and then recovered by 24h. In pharmacological experiments in vitro, application of NO donors (SNAP, SNP) caused an increase in electrical activity (firing frequency) of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The NOS substrate, l-arginine, led to increased firing frequency, cell recruitment and enhanced bursting activity. However, this effect was not blocked by the NOS inhibitor L-NAME. These findings suggest that NO acts as a modulator within the CNSS, potentially enhancing electrical activity and hence secretory output. A role in supporting adaptation to hyperosmotic conditions is also indicated.

Fish caudal neurosecretory system: a model for the study of neuroendocrine secretion.

The caudal neurosecretory system (CNSS) is unique to fish and has suggested homeostatic roles in osmoregulation and reproduction. Magnocellular neuroendocrine Dahlgren cells, located in the terminal segments of the spinal cord, project to a neurohaemal organ, the urophysis, from which neuropeptides are released. In the euryhaline flounder Platichthys flesus Dahlgren cells synthesise at least four peptides, including urotensins I and II and CRF. These peptides are differentially expressed with co-localisation of up to three in a single cell. Dahlgren cells display a range of electrical firing patterns, including characteristic bursting activity, which is dependent on L-type Ca(2+) and Ca-activated K(+)channels. Activity is modulated by a range of extrinsic and intrinsic neuromodulators. This includes autoregulation by the secreted peptides themselves, leading to enhanced bursting. Electrophysiological and mRNA expression studies have examined changes in response to altered physiological demands. Bursting activity is more robust and more Dahlgren cells are recruited in seawater compared to freshwater adapted fish and this is mirrored by a reduction in mRNA expression for L-type Ca(2+) and Ca-activated K(+) channels. Acute seawater/freshwater transfer experiments support a role for UII in adaptation to hyperosmotic conditions. Responses to stress suggest a shared role for CRF and UI, released from the CNSS. We hypothesise that the Dahlgren cell population is reprogrammed, both in anticipation of and in response to changed physiological demands, and this is seen as changes in gene expression profile and electrical activity. The CNSS shows striking parallels with the hypothalamic-neurohypophysial system, providing a highly accessible system for studies of neuroendocrine mechanisms. Furthermore, the presence of homologues of urotensins throughout the vertebrates has sparked new interest in these peptides and their functional evolution.

Properties of corneal receptors in a teleost fish.

Corneal receptors have not previously been identified in lower vertebrates. The present study describes the properties of trigeminal ganglion corneal receptors in a teleost fish, the rainbow trout (Oncoryhnchus mykiss). Out of 27 receptors, 7 were polymodal nociceptors, 6 were mechanothermal nociceptors, 2 were mechanochemical receptors and the largest group, 12, were only responsive to mechanical stimulation. No cold responsive receptors were found on the trout cornea. Mechanical and thermal thresholds were lower and receptive field diameters smaller than those of cutaneous trigeminal receptors in the trout, demonstrating greater sensitivity in the cornea. The lack of cold sensitive neurons may provide evidence for the evolution of cold nociceptors in vertebrates that is related to the transition from poikilothermy to homeothermy.

Molecular characterization and expression of urotensin II and its receptor in the flounder (Platichthys flesus): a hormone system supporting body fluid homeostasis in euryhaline fish.

Urotensin II (UII) is a potent vasoconstrictor in mammals, but the source of circulating UII remains unclear. Investigations of the caudal neurosecretory system (CNSS), considered the major source of UII in fish, alongside target tissue expression of UII receptor (UT), can provide valuable insights into this highly conserved regulatory system. We report UII gene characterization, expression of the first fish UT, and responses to salinity challenge in flounder. The 12-aa UII peptide shares 73% sequence identity with pig and human UII. Flounder UT receptor shares 56.7% identity with rat. Although the CNSS is the major site of UII expression, RT-PCR revealed expression of UII and UT in all tissues tested. Around 30-40% of large CNSS Dahlgren cells expressed UII, alone or in combination with urotensin I and/or corticotrophin releasing hormone. Immunolocalization of UT in osmoregulatory tissues (gill, kidney) was associated with vascular elements. There were no consistent differences in CNSS UII expression or plasma UII between seawater (SW)- and freshwater (FW)-adapted fish, although gill and kidney UT expression was lower in FW animals. After acute transfer from SW to FW, plasma UII and kidney and gill UT expression were reduced, whereas UT expression in kidney was increased after reverse transfer. UII appears to be more important to combat dehydration and salt-loading in SW than the hemodilution faced in FW. Potentially, altered target tissue sensitivity through changes in UT expression, is an important physiological controlling mechanism, not only relevant for migratory fish but also likely conserved in mammals.

Influence of aqueous aluminium on the immune system of the freshwater crayfish Pacifasticus leniusculus.

Little is known of the effects of aluminium (Al) on invertebrate immunity despite the ubiquitous nature of the metal and its toxicity to aquatic organisms. Here we examine the effect of Al at neutral pH on the immune system of the freshwater crayfish Pacifasticus leniusculus. Heat-killed bacteria were injected at intervals into the haemolymph of crayfish continuously exposed to a sub-lethal concentration (500 microg l(-1)) of Al over 40 days. Circulating haemocyte and bacterial numbers were monitored for 16 days post-injection at each time interval. In the absence of bacterial challenge, the number of circulating haemocytes increased in Al-exposed crayfish compared to unexposed controls. Aluminium exposure initially reduced the crayfish's ability to clear bacteria from the circulation and decreased the rate of recovery in haemocyte numbers following bacterial challenge. These effects on bacterial and haemocyte numbers were abolished after prolonged exposure (>10 days) to Al, indicating adaptation to the metal. Aqueous Al impairs gill function in P. leniusculus by inducing hypersecretion of mucus and we suggest that the decrease in immunocompetence is due to haemolymph hypoxia. We conclude that exposure to episodic pulses of aqueous Al over the short term (<10 days) increases the risk of infection in the crayfish by impairing the ability of haemocytes to recognise and/or remove bacteria from the circulation.

Coexpression of corticotropin-releasing hormone and urotensin i precursor genes in the caudal neurosecretory system of the euryhaline flounder (Platichthys flesus): a possible shared role in peripheral regulation.

CRH and urotensin I (UI) are neuroendocrine peptides that belong to the superfamily of corticotropin-releasing factors. In mammals, these peptides regulate the stress response and other central nervous system functions, whereas in fish an involvement for UI in osmoregulation has also been suggested. We have identified, characterized, and localized the genes encoding these peptides in a unique fish neuroendocrine organ, the caudal neurosecretory system (CNSS). The CRH and UI precursors, isolated from a European flounder CNSS library, consist of 168 and 147 amino acid residues, respectively, with an overall homology of approximately 50%. Both precursors contain a signal peptide, a divergent cryptic region and a 41-amino acid mature peptide with cleavage and amidation sites. Genomic organization showed that whole CRH and UI coding sequences are contained in a single exon. Northern blot analysis and quantitative PCR of a range of tissues confirmed the CNSS as a major site of expression of both CRH and UI and thus serves as a likely source of circulating peptides. In situ hybridization demonstrated that CRH and UI colocalize to the same cells of the CNSS. Our findings suggest that, in euryhaline fish, the CNSS is a major site of production of CRH and probably contributes to the high circulating levels observed in response to specific environmental challenges. Furthermore, the localization of CRH and UI within the same cell population suggests an early, possibly shared role for these peptides in controlling stress-mediated adaptive plasticity.

Partial characterisation of high-molecular weight glycoconjugates in the trail mucus of the freshwater pond snail Lymnaea stagnalis.

We have studied the glycoconjugates in trail mucus of the pond snail Lymnaea stagnalis. The mucus was dissolved with 6 M guanidinium hydrochloride (GuHCl) and the major component was comprised of very high-M(r) glycoconjugates that were eluted in the void volume of a Sepharose CL-4B gel-filtration column. This high-M(r) material was pooled and thereafter subjected to density gradient centrifugation first in 4 M GuHCl/CsCl and subsequently 0.2 M GuHCl/CsCl to further remove non-glycosylated proteins and DNA. The harvested glycoconjugate pool chromatographed in the void volume of Sepharose CL-2B. However, reduction of disulfide bonds lowered the molecular size of approximately 80% of the void material yielding a major fragment and some minor smaller fragments in gel chromatography. The reduced glycoconjugates were digested with papain and yielded high molecular weight, proteinase-resistant glycopeptides. This fragmentation pattern is similar to that found for oligomeric gel-forming mucins in mammals and the amino acid composition (60% Ser/Thr) and sugar analysis of the glycopeptides is consistent with mucin-like molecules, there being no significant amounts of xylose or uronic acids. The residual 20% of the preparation, which apparently resisted reduction and protease digestion, had a similar amino acid composition to the bulk, but was somewhat different in sugar composition, containing some xylose and a significant amount of glucuronic acid. The two groups of molecules had very different morphologies in the electron microscope. Taken together, these data suggest that trail mucus is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though we cannot rule out that polysaccharides may also be present.

Effect of orthosilicic acid on the accumulation of trace metals by the pond snail Lymnaea stagnalis.

Silicon (Si) has a marked affinity for aluminium (Al(III)), but not other trace metals such as cadmium (Cd(II)) and zinc (Zn(II)). Exogenous orthosilicic acid (Si(OH)(4)) ameliorates the toxicity of Al(III) to the pond snail Lymnaea stagnalis, but its mechanism of action is unclear. Here, studies were conducted to ascertain whether interaction between orthosilicic acid and Al(III) occurs in the water column to prevent Al(III) uptake, or in the tissues to reduce the toxicity of accumulated metal. Silicon did not reduce the accumulation of Al(III) by the digestive gland (the main "sink" for trace metals in L. stagnalis) following exposure of the snail for 30 days to 500 microg l(-1) added Al(III) and 13-fold molar excess of orthosilicic acid. However, Si concentrations correlated well with Al(III) levels in the digestive gland (R(2)=0.77), giving a ratio of 2.5:1 (Al(III):Si). Exposure to Zn(II) or Cd(II) and 13-fold molar excess of orthosilicic acid did not prevent uptake of these metals, or result in a correlation between metal and Si concentrations of the snail digestive gland. These data show that aquated orthosilicic acid does not prevent Al(III) accumulation by L. stagnalis. However, following exposure, the ratio of Al(III) to Si in the digestive gland is suggestive of the early formation of hydroxyaluminosilicates, probably proto-imogolites (2-3:1 Al(III):Si). Whether hydroxyaluminates are formed ex vivo in the water column and taken up by snails into the digestive gland, or formed in situ within the digestive gland remains to be established. Either way, orthosilicic acid clearly prevents the in vivo toxicity of Al(III) rather than reducing its uptake. Silicon appears to have an important role in the handling Al(III) by the pond snail which may also have wider relevance in understanding the role of Si in ameliorating Al(III) toxicity.