Development, calibration, and evaluation of a model of Pseudo-nitzschia and domoic acid production for regional ocean modeling studies.

Pseudo-nitzschia species are one of the leading causes of harmful algal blooms (HABs) along the western coast of the United States. Approximately half of known Pseudo-nitzschia strains can produce domoic acid (DA), a neurotoxin that can negatively impact wildlife and fisheries and put human life at risk through amnesic shellfish poisoning. Production and accumulation of DA, a secondary metabolite synthesized during periods of low primary metabolism, is triggered by environmental stressors such as nutrient limitation. To quantify and estimate the feedbacks between DA production and environmental conditions, we designed a simple mechanistic model of Pseudo-nitzschia and domoic acid dynamics, which we validate against batch and chemostat experiments. Our results suggest that, as nutrients other than nitrogen (i.e., silicon, phosphorus, and potentially iron) become limiting, DA production increases. Under Si limitation, we found an approximate doubling in DA production relative to N limitation. Additionally, our model indicates a positive relationship between light and DA production. These results support the idea that the relationship with nutrient limitation and light is based on direct impacts on Pseudo-nitzschia biosynthesis and biomass accumulation. Because it can easily be embedded within existing coupled physical-ecosystem models, our model represents a step forward toward modeling the occurrence of Pseudo-nitzschia HABs and DA across the U.S. West Coast.

Adaptogenic potential of ginsenosides against domoic acid-induced toxicity by regulating neuronal stress and kinate receptors: Ex vivo and in silico studies.

This study is focused on potential effects of ginsenosides from Panax ginseng (PG) against amnesic shell fish poison, that is, domoic acid-induced excitotoxicity. Mice received PG at two different dosages by oral feeding for a period of 28 days (50 and 100 mg kg-1 bwt.-1 ). Domoic acid was injected to the mice to induce excitotoxicity (DA; 3 mg kg-1  bwt.-1 ) and piracetam-injected animals (PIR; 100 mg kg-1  bwt.-1 ) were treated as positive control. DA-induced cognitive impairment was reverted by PG supplementation, which was observed in Morris water maze and novel object task. Moreover, PG supplementation restored levels of GABA and antioxidant enzymes. Our results further elucidated ameliorative effects of PG supplementation on DA-induced changes in the expression of synaptic plasticity (BDNF), inflammation (NFkB), and apoptotic (Bcl2, Bax, and Caspase 3) markers. Hence, this study elucidates potential nootropic effects of ginsenosides from P. ginseng extract against DA-induced neuronal impairments via, modulation of behavioral and biochemical mechanisms involved in excitotoxicity, oxidative stress, neuro-inflammation, and apoptosis. PRACTICAL APPLICATIONS: This study highlights potential effects of ginsenosides from Panax ginseng against amnesic shell fish poison, that is, domoic acid-induced excitotoxicity for the first time. This study confirms that ginsenosides have the beneficial effects against amelioration of DA-induced toxicity. This study elucidates the potential nootropic effects of P. ginseng extract against DA-induced neuronal impairments via, modulation of synaptic plasticity markers and oxido-inflammatory responses leading to apoptosis. This study will be helpful in offering various mechanisms involved in pharmacological applications of P. ginseng in the management of DA-induced excitotoxicity.

A Generic LC-HRMS Screening Method for Marine and Freshwater Phycotoxins in Fish, Shellfish, Water, and Supplements.

Phycotoxins occur in various marine and freshwater environments, and can accumulate in edible species such as fish, crabs, and shellfish. Human exposure to these toxins can take place, for instance, through consumption of contaminated species or supplements and through the ingestion of contaminated water. Symptoms of phycotoxin intoxication include paralysis, diarrhea, and amnesia. When the cause of an intoxication cannot directly be found, a screening method is required to identify the causative toxin. In this work, such a screening method was developed and validated for marine and freshwater phycotoxins in different matrices: fish, shellfish, water, and food supplements. Two LC methods were developed: one for hydrophilic and one for lipophilic phycotoxins. Sample extracts were measured in full scan mode with an Orbitrap high resolution mass spectrometer. Additionally, a database was created to process the data. The method was successfully validated for most matrices, and in addition, regulated lipophilic phycotoxins, domoic acid, and some paralytic shellfish poisoning toxins could be quantified in shellfish. The method showed limitations for hydrophilic phycotoxins in sea water and for lipophilic phycotoxins in food supplements. The developed method is a screening method; in order to confirm suspected compounds, comparison with a standard or an additional analysis such as NMR is required.

Determination of Cyanotoxins and Phycotoxins in Seawater and Algae-Based Food Supplements Using Ionic Liquids and Liquid Chromatography with Time-Of-Flight Mass Spectrometry.

An analytical procedure is proposed for determining three cyanotoxins (microcystin RR, microcystin LR, and nodularin) and two phycotoxins (domoic and okadaic acids) in seawater and algae-based food supplements. The toxins were first isolated by a salting out liquid extraction procedure. Since the concentration expected in the samples was very low, a dispersive liquid-liquid microextraction procedure was included for preconcentration. The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate (80 mg) was used as green extractant solvent and acetonitrile as disperser solvent (0.5 mL) for a 10 mL sample volume at pH 1.5, following the principles of green analytical chemistry. Liquid chromatography with electrospray ionization and quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) was used. The selectivity of the detection system, based on accurate mass measurements, allowed the toxins to be unequivocally identified. Mass spectra for quadrupole time of flight-mass spectrometry (Q-TOF-MS) and Q-TOF-MS/MS were recorded in the positive ion mode and quantification was based on the protonated molecule. Retention times ranged between 6.2 and 17.9 min using a mobile phase composed by a mixture of methanol and formic acid (0.1%). None of the target toxins were detected in any of the seawater samples analyzed, above their corresponding detection limits. However, microcystin LR was detected in the blue green alga sample.

Neonatal domoic acid alters in vivo binding of [11C]yohimbine to α2-adrenoceptors in adult rat brain.

RATIONALE: Epilepsy is a debilitating seizure disorder that affects approximately 50 million people. Noradrenaline reduces neuronal excitability, has anticonvulsant effects and is protective against seizure onset. OBJECTIVE: We investigated the role of α2-adrenoceptors in vivo in a neonatal domoic acid (DOM) rat model of epilepsy. METHODS: We injected male Sprague-Dawley rats daily from postnatal day 8-14 with saline or one of two sub-convulsive doses, 20 µg/kg (DOM20) or 60 µg/kg (DOM60) DOM, an AMPA/kainate receptor agonist. The rats were observed in open field, social interaction and forced swim tests at day 50, 75 and 98, respectively. At ~120 days of age, four rats per group were injected and scanned with [11C]yohimbine, an α2-adrenoceptor antagonist, and scanned in a Mediso micro positron emission tomography (PET) scanner to measure α2-adrenoceptor binding. RESULTS: DOM60-treated rats spent more time in the periphery during the open field test and had a significant 26-33 % reduction in [11C]yohimbine binding in the hypothalamus, hippocampus and orbital prefrontal cortex compared to saline-treated rats. On the other hand, DOM20 rats had a significant 34-40 % increase in [11C]yohimbine binding in the hypothalamus, amygdala and entorhinal cortex compared to saline-treated rats, with no obvious behavioural differences. CONCLUSIONS: The current data clearly indicate that low concentrations of DOM given to rats in their second week of life induces long-term changes in α2-adrenoceptor binding in rat brain that may have relevance to the progression of an epilepsy phenotype.

Fetal domoic acid exposure affects lateral amygdala neurons, diminishes social investigation and alters sensory-motor gating.

Domoic acid (DA) is an algal neurotoxin that accumulates in marine fish and shellfish. DA can move across the placenta and concentrate in amniotic fluid, which can be swallowed during late gestation. DA also transfers to infants via milk. Preclinical studies to determine effects of developmental DA expose have primarily involved DA exposure during the postnatal period and little is known about late CNS effects following prenatal DA. In the present study, we tested the hypothesis that prenatal exposure of FVB mice to low levels of DA would result in diminished social interaction and sensory motor gating associated with alterations in parvalbumin immunoreactivity in relevant brain regions undergoing development during and following DA exposure. In addition to parvalbumin, we stained with NeuN for a neuronal specific nuclear protein to determine if neuronal loss followed prenatal DA exposure. A single moderate dose of DA administered during gestation produces diminishes social investigation and alters sensorimotor gating, behavioral effects more pronounced in males than females. These behavioral changes were associated with discrete alterations in the parvalbumin-positive subtype of GABAergic neurons in the dentate gyrus and lateral amygdala.

Algal toxin impairs sea lion memory and hippocampal connectivity, with implications for strandings.

Domoic acid (DA) is a naturally occurring neurotoxin known to harm marine animals. DA-producing algal blooms are increasing in size and frequency. Although chronic exposure is known to produce brain lesions, the influence of DA toxicosis on behavior in wild animals is unknown. We showed, in a large sample of wild sea lions, that spatial memory deficits are predicted by the extent of right dorsal hippocampal lesions related to natural exposure to DA and that exposure also disrupts hippocampal-thalamic brain networks. Because sea lions are dynamic foragers that rely on flexible navigation, impaired spatial memory may affect survival in the wild.

Ceftriaxone modulates uptake activity of glial glutamate transporter-1 against global brain ischemia in rats.

Ceftriaxone(Cef) selectively increases the expression of glial glutamate transporter-1 (GLT-1), which was thought to be neuroprotective in some circumstances. However, the effect of Cef on glutamate uptake of GLT-1 was mostly assayed using in vitro studies such as primary neuron/astrocyte cultures or brain slices. In addition, the effect of Cef on neurons in different ischemic models was still discrepant. Therefore, this study was undertaken to observe the effect of Cef on neurons in global brain ischemia in rats, and especially to provide direct evidence of the up-regulation of GLT-1 uptake for glutamate contributing to the neuronal protection of Cef against brain ischemia. Neuropathological evaluation indicated that administration of Cef, especially pre-treatment protocols, significantly prevented delayed neuronal death in hippocampal CA1 subregion normally induced by global brain ischemia. Simultaneously, pre-administration of Cef significantly up-regulated the expression of GLT-1. Particularly, GLT-1 uptake assay with (3) H-glutamate in living cells from adult rats showed that up-regulation in glutamate uptake accompanied up-regulated GLT-1 expression. Inhibition of GLT-1 by antisense oligodeoxynucleotides or dihydrokainate significantly inhibited the Cef-induced up-regulation in GLT-1 uptake and the neuroprotective effect against global ischemia. Thus, we may conclude that Cef protects neurons against global brain ischemia via up-regulation of the expression and glutamate uptake of GLT-1. Glutamate uptake by glial glutamate transporter-1 (GLT-1) is the principal way to regulate extracellular glutamate homeostasis in central nervous system. Over-accumulation of glutamate results in excitotoxicity and injures neurons after cerebral ischemia. Ceftriaxone up-regulates GLT-1 expression and uptake of glutamate, diminishes the excitotoxicity of glutamate and then protects neurons against global brain ischemia.

Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice.

Recent findings suggest that endoplasmic reticulum stress may be involved in the pathogenesis of domoic acid-induced neurodegeneration. Purple sweet potato color, a class of naturally occurring anthocyanins, has beneficial health and biological effects. Recent studies have also shown that anthocyanins have estrogenic activity and can enhance estrogen receptor-α expression. In this study, we evaluated the effect of purple sweet potato color on cognitive deficits induced by hippocampal mitochondrial dysfunction in domoic acid-treated mice and explored the potential mechanisms underlying this effect. Our results showed that the oral administration of purple sweet potato color to domoic acid-treated mice significantly improved their behavioral performance in a step-through passive avoidance task and a Morris water maze task. These improvements were mediated, at least in part, by a stimulation of estrogen receptor-α-mediated mitochondrial biogenesis signaling and by decreases in the expression of p47phox and gp91phox. Decreases in reactive oxygen species and protein carbonylation were also observed, along with a blockade of the endoplasmic reticulum stress pathway. Furthermore, purple sweet potato color significantly suppressed endoplasmic reticulum stress-induced apoptosis, which prevented neuron loss and restored the expression of memory-related proteins. However, knockdown of estrogen receptor-α using short hairpin RNA only partially blocked the neuroprotective effects of purple sweet potato color in the hippocampus of mice cotreated with purple sweet potato color and domoic acid, indicating that purple sweet potato color acts through multiple pathways. These results suggest that purple sweet potato color could be a possible candidate for the prevention and treatment of cognitive deficits in excitotoxic and other brain disorders.

Effect of inhibition of spinal cord glutamate transporters on inflammatory pain induced by formalin and complete Freund's adjuvant.

BACKGROUND: Spinal cord glutamate transporters clear synaptically released glutamate and maintain normal sensory transmission. However, their ultrastructural localization is unknown. Moreover, whether and how they participate in inflammatory pain has not been carefully studied. METHODS: Immunogold labeling with electron microscopy was carried out to characterize synaptic and nonsynaptic localization of glutamate transporters in the superficial dorsal horn. Their expression and uptake activity after formalin- and complete Freund's adjuvant (CFA)-induced inflammation were evaluated by Western blot analysis and glutamate uptake assay. Effects of intrathecal glutamate transporter activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline and inhibitors (DL-threo-ß-benzyloxyaspartate [TBOA], dihydrokainate, and DL-threo-ß-hydroxyaspartate), or TBOA plus group III metabotropic glutamate receptor antagonist (RS)-α-methylserine-O-phosphate, on formalin- and CFA-induced inflammatory pain were examined. RESULTS: In the superficial dorsal horn, excitatory amino acid carrier 1 is localized in presynaptic membrane, postsynaptic membrane, and axonal and dendritic membranes at nonsynaptic sites, whereas glutamate transporter-1 and glutamate/aspartate transporter are prominent in glial membranes. Although expression of these three spinal glutamate transporters was not altered 1 h after formalin injection or 6 h after CFA injection, glutamate uptake activity was decreased at these time points. Intrathecal (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline had no effect on formalin-induced pain behaviors. In contrast, intrathecal TBOA, dihydrokainate, and DL-threo-ß-hydroxyaspartate reduced formalin-evoked pain behaviors in the second phase. Intrathecal TBOA also attenuated CFA-induced thermal hyperalgesia at 6 h after CFA injection. The antinociceptive effects of TBOA were blocked by coadministration of (RS)-α-methylserine-O-phosphate. CONCLUSION: Our findings suggest that spinal glutamate transporter inhibition relieves inflammatory pain through activation of inhibitory presynaptic group III metabotropic glutamate receptors.

Application of micro-electrode arrays (MEAs) as an emerging technology for developmental neurotoxicity: evaluation of domoic acid-induced effects in primary cultures of rat cortical neurons.

Due to lack of knowledge only a few industrial chemicals have been identified as developmental neurotoxicants. Current developmental neurotoxicity (DNT) guidelines (OECD and EPA) are based entirely on in vivo studies that are both time consuming and costly. Consequently, there is a high demand to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. One of the most promising tools for neurotoxicity assessment is the measurement of neuronal electrical activity using micro-electrode arrays (MEAs) that provides a functional and neuronal specific endpoint that until now has been used mainly to detect acute neurotoxicity. Here, electrical activity measurements were evaluated to be a suitable endpoint for the detection of potential developmental neurotoxicants. Initially, primary cortical neurons grown on MEA chips were characterized for different cell markers over time, using immunocytochemistry. Our results show that primary cortical neurons could be a promising in vitro model for DNT testing since some of the most critical neurodevelopment processes such as progenitor cell commitment, proliferation and differentiation of astrocytes and maturation of neurons are present. To evaluate if electrical activity could be a suitable endpoint to detect chemicals with DNT effects, our model was exposed to domoic acid (DomA), a potential developmental neurotoxicant for up to 4 weeks. Long-term exposure to a low concentration (50nM) of DomA increased the basal spontaneous electrical activity as measured by spike and burst rates. Moreover, the effect induced by the GABA(A) receptor antagonist bicuculline was significantly lower in the DomA treated cultures than in the untreated ones. The MEA measurements indicate that chronic exposure to DomA changed the spontaneous electrical activity leading to the possible neuronal mal functioning. The obtained results suggest that the MEAs could be a useful tool to identify compounds with DNT potential.

Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy).

Twenty-six Pseudo-nitzschia multistriata cultures were tested for intracellular domoic acid production and fourteen were found to be toxic. Four suboptimal growth conditions were compared with conditions observed to be optimal to explore possible triggers for intracellular domoic acid production. Silica- and phosphate-limitation and low light treatment induced elevated toxin concentrations whereas high temperature appeared to suppress it. Inheritance of the toxin-production ability was investigated by measuring intracellular toxin content in a total of thirty-nine F(1) strains from two different crosses. Results showed radical differences in domoic acid levels among the F(1) offspring from the same parents.

Cortical inhibition during burst suppression induced with isoflurane anesthesia.

Isoflurane is a widely used anesthetic which safely and reversibly induces deep coma and associated burst suppression (BS) electroencephalographic patterns. Here we investigate possible underlying causes for the state of cortical hyperexcitability which was recently shown to be one of the characteristics of BS. Our hypothesis was that cortical inhibition is diminished during isoflurane-induced BS. Experiments were performed in vivo using intracellular recordings of cortical neurons to assess their responsiveness to stimulations of connected thalamic nuclei. We demonstrate that during BS EPSPs were diminished by 44%, whereas inhibitory potentials were completely suppressed. This finding was supported by additional results indicating that a decrease in neuronal input resistance normally found during inhibitory responses under low isoflurane conditions was abolished in the BS condition. Moreover, removal of inhibition occasionally revealed excitatory components which were absent during recordings before the induction of BS. We also show that the absence of inhibition during BS is not caused by a blockage of GABA receptors, since iontophoretically applied GABA shows receptor availability. Moreover, the concentration of extracellular chloride was increased during BS, as would be expected after reduced flow of chloride through GABA(A) receptors. Also inhibitory responses were reinstated by selective blockage of glial glutamate transporters with dihydrokainate. These results suggest that the lack of inhibition during BS is caused by reduced excitation, probably resulting from increased glial uptake of glutamate stimulated by isoflurane, which creates a diminished activation of cortical interneurons. Thus cortical hyperexcitability during BS is favored by suppressed inhibition.

GLT-1 upregulation impairs prepulse inhibition of the startle reflex in adult rats.

We tested the hypothesis that glutamate transporter GLT-1 (also known as EAAT2) plays a role in the regulation of prepulse inhibition (PPI) of the acoustic startle reflex, a simple form of information processing which is reduced in schizophrenia. To do this, we studied PPI in rats treated with ceftriaxone (200 mg/kg/day for 8 days), an antibiotic that selectively enhances GLT-1 expression and activity. We showed that ceftriaxone-induced GLT-1 upregulation is associated with impaired PPI of the startle, that this effect is reversed by dihydrokainate, a GLT-1 antagonist, that GLT-1 expression correlates negatively with PPI, and that PPI normalizes when GLT-1a levels return to baseline. Our data indicate that GLT-1 regulates PPI of the startle reflex.

Transcriptional responses of xenobiotic metabolizing enzymes, HSP70 and Na+/K+ -ATPase in the liver of rabbitfish (Siganus oramin) intracoelomically injected with amnesic shellfish poisoning toxin.

Amnesic shellfish poisoning toxin domoic acid (DA) is a marine neurotoxin that accumulates in fish and shellfish, and has been implicated to be involved in human and marine wildlife mortality. The transcriptional responses of cytochrome P-450 1A (CYP1A), glutathione S-transferase alpha (GSTA), glutathione S-transferase rho (GSTR), heat shock protein 70 (HSP70), and Na(+)/K(+)-ATPase alpha 1 (ATP1A1) in the liver of rabbitfish (Siganus oramin) intracoelomically injected with DA, were investigated. Experimental fish were administered with one injection of DA (2 microg/g wet weight) or PBS as control. After 24 h, fish were killed and hepatic RNA was isolated. Partial cDNA of rabbitfish CYP1A, GSTA, GSTR, HSP70, ATP1A1, and beta-actin were obtained by PCR using degenerate primers. Using beta-actin as an external control, the relative liver CYP1A, GSTA, GSTR, HSP70, and ATP1A1 mRNA abundance of rabbitfish were determined by semi-quantitative RT-PCR within the exponential phase. The ratio CYP1A/beta-actin mRNA (%) of exposure group was determined to be 148.92+/-12.69, whereas the ratio of control group was 82.3+/-8.35, indicating that CYP1A was induced significantly in rabbitfish following DA exposure (P<0.05). Although the expressions of GSTA, HSP70, and ATP1A1 tended to increase and GSTR tended to decrease, no significant changes were found (P>0.05). The induction of hepatic CYP1A in response to DA suggests a potential role for fish phase I xenobiotic metabolizing enzyme in DA metabolism.

A synthetic kainoid, (2S,3R,4R)-3-carboxymethyl-4-(phenylthio)pyrrolidine-2-carboxylic acid (PSPA-1) serves as a novel anti-allodynic agent for neuropathic pain.

In spite of prominent progress in basic pain research, neuropathic pain remains a significant medical problem, because it is often poorly relieved by conventional analgesics. Thus this situation encourages us to make more sophisticated efforts toward the discovery of new analgesics. We previously showed that i.t. administration of acromelic acid-A (ACRO-A), a Japanese mushroom poison, provoked prominent tactile pain (allodynia) at an extremely low dose of 1 fg/mouse. In the present study we synthesized ACRO-A analogues (2S,3R,4R)-3-carboxymethyl-4-phenoxypyrrolidine-2-carboxylic acid (POPA-2) and (2S,3R,4R)-3-carboxymethyl-4-(phenylthio)pyrrolidine-2-carboxylic acid (PSPA-1) chemically and examined their ability to induce allodynia in conscious mice. Whereas POPA-2 induced allodynia at extremely low doses from 1 to 100 fg/mouse, similar to ACRO-A, PSPA-1 did not induce allodynia; rather, it inhibited the ACRO-A-induced allodynia with an ID(50) value (95% confidence limits) of 2.19 fg/mouse (0.04-31.8 fg/mouse). Furthermore, PSPA-1 relieved neuropathic pain produced by L5 spinal nerve transection on day 7 after the operation in a dose-dependent manner from 1 to 100 pg/mouse. In contrast, it did not affect thermal or mechanical nociception or inflammatory pain. PSPA-1 reduced the increase in neuronal nitric oxide synthase activity in the spinal cord of neuropathic pain mice assessed by NADPH-diaphorase histochemistry and blocked the allodynia induced by N-methyl-d-aspartate. These results demonstrate that PSPA-1 may represent a novel class of anti-allodynic agents for neuropathic pain acting by blocking the glutamate-nitric oxide pathway.

Acute and late effects on induction of allodynia by acromelic acid, a mushroom poison related structurally to kainic acid.

1. Ingestion of a poisonous mushroom Clitocybe acromelalga is known to cause severe tactile pain (allodynia) in the extremities for a month and acromelic acid (ACRO), a kainate analogue isolated from the mushroom, produces selective damage of interneurons of the rat lower spinal cord when injected either systemically or intrathecally. Since ACRO has two isomers, ACRO-A and ACRO-B, here we examined their acute and late effects on induction of allodynia. 2. Intrathecal administration of ACRO-A and ACRO-B provoked marked allodynia by the first stimulus 5 min after injection, which lasted over the 50-min experimental period. Dose-dependency of the acute effect of ACRO-A on induction of allodynia showed a bell-shaped pattern from 50 ag x kg(-1) to 0.5 pg x kg(-1) and the maximum effect was observed at 50 fg x kg(-1). On the other hand, ACRO-B induced allodynia in a dose-dependent manner from 50 pg x kg(-1) to 50 ng x kg(-1). 3. N-methyl-d-aspartate (NMDA) receptor antagonists and Joro spider toxin, a Ca(2+)-permeable AMPA receptor antagonist, inhibited the allodynia induced by ACRO-A, but not by ACRO-B. However, other AMPA/kainate antagonists did not affect the allodynia induced by ACRO. 4. Whereas no neuronal damage was observed in the spinal cord in ACRO-A-treated mice, induction of allodynia by ACRO-A (50 fg x kg(-1)) and ACRO-B (50 ng x kg(-1)) was selectively lost 1 week after i.t. injection of a sublethal dose of ACRO-A (50 ng x kg(-1)) or ACRO-B (250 ng x kg(-1)). Higher doses of ACRO-A, however, could evoke allodynia dose-dependently from 50 pg x kg(-1) to 500 ng x kg(-1) in the ACRO-A-treated mice. The allodynia induced by ACRO-A (500 ng x kg(-1)) was not inhibited by Joro spider toxin or NMDA receptor antagonists. These properties of the late allodynia induced by ACRO-A were quite similar to those of the acute allodynia induced by ACRO-B. 5. ACRO-A could increase [Ca(2+)](i) in the deeper laminae, rather than in the superficial laminae, of the spinal cord. This increase was not blocked by the AMPA-preferring antagonist GYKI52466 and Joro spider toxin. 6. Taken together, these results demonstrate the stereospecificity of ACRO for the induction of allodynia and suggest the presence of a receptor specific to ACRO.

Diatom cultivation and biotechnologically relevant products. Part II: current and putative products.

While diatoms are widely present in terms of diversity and abundance in nature, few species are currently used for biotechnologically applications. Most studies have focussed on intracellularly synthesised eicosapentaenoic acid (EPA), a polyunsaturated fatty acid (PUFA) used for pharmaceutical applications. Applications for other intracellular molecules, such as total lipids for biodiesel, amino acids for cosmetic, antibiotics and antiproliferative agents, are at the early stage of development. In addition, the active principle component must be identified amongst the many compounds of biotechnological interest. Biomass from diatom culture may be applied to: (1). aquaculture diets, due to the lipid- and amino-acid-rich cell contents of these microorganisms, and (2). the treatment of water contaminated by phosphorus and nitrogen in aquaculture effluent, or heavy metal (bioremediation). The most original application of microalgal biomass, and specifically diatoms, is the use of silicon derived from frustules in nanotechnology. The competitiveness of biotechnologically relevant products from diatoms will depend on their cost of production. Apart from EPA, which is less expensive when obtained from Phaeodactylum tricornutum than from cod liver, comparative economic studies of other diatom-derived products as well as optimisation of culture conditions are needed. Extraction of intracellular metabolites should be also optimised to reduce production costs, as has already been shown for EPA. Using cell immobilisation techniques, benthic diatoms can be cultivated more efficiently allowing new, biotechnologically relevant products to be investigated.

Kinetics and activation of postsynaptic kainate receptors at thalamocortical synapses: role of glutamate clearance.

Kainate (KA) receptor-mediated excitatory postsynaptic currents (EPSCs) exhibit slow kinetics at the great majority of synapses. However, native or heterologously expressed KA receptors exhibit rapid kinetics in response to agonist application. One possibility to explain this discrepancy is that KA receptors are extrasynaptic and sense glutamate diffusing from the synaptic cleft. We investigated this by studying the effect of three manipulations that change glutamate clearance on evoked KA EPSCs at thalamocortical synapses. First, we used high-frequency stimulation to increase extrasynaptic glutamate levels. This caused an apparent increase in the relative contribution of the KA EPSC to transmission and slowed the decay kinetics. However, scaling and summing the EPSC evoked at low frequency reproduced this, demonstrating that the effect was due to postsynaptic summation of KA EPSCs. Second, we applied inhibitors of high-affinity glutamate transport. This caused a depression in both AMPA and KA EPSC amplitude due to the activation of a presynaptic glutamatergic autoreceptor. However, transport inhibitors had no selective effect on the amplitude or kinetics of the KA EPSC. Third, to increase glutamate clearance, we raised temperature during recordings. This shortened the decay of both the AMPA and KA components and increased their amplitudes, but this effect was the same for both. Therefore these data provide evidence against glutamate diffusion out of the synaptic cleft as the mechanism for the slow kinetics of KA EPSCs. Other possibilities such as interactions of KA receptors with other proteins or novel properties of native synaptic heteromeric receptors are required to explain the slow kinetics.

Inhibition of glutamate transporter by theanine enhances the therapeutic efficacy of doxorubicin.

Theanine, a major amino acid existing in green tea, enhanced the antitumor activity of doxorubicin (DOX) due to inhibition of DOX efflux from tumor cells. In order to clarify the mechanism, we have investigated the contribution of glutamate transporters to the action of theanine, because theanine is a glutamate analogue. In M5076 ovarian sarcoma cells, glutamate transport inhibitors reduced the efflux of DOX, as well as theanine. Incidentally, theanine significantly inhibited the glutamate uptake by M5076 cells in a concentration-dependent manner similar to specific inhibitors. These results suggested that the inhibition of DOX efflux was induced by the inhibition of glutamate transport by theanine. In addition, RT-PCR and Western blot analysis revealed the expression of GLAST and GLT-1, astrocytic high-affinity glutamate transporters, in M5076 cells. Thus, theanine was shown to competitively inhibit the glutamate uptake by acting on these glutamate transporters. This action suggested the contribution of glutamate transporters to the inhibition of DOX efflux by theanine. We revealed the novel mechanism of enhancement of the antitumor efficacy of DOX via the inhibition of glutamate transporters by theanine.