Effects of diatoms on reproduction and growth of marine copepods

Two hot spots in marine ecology, deleterious effects of diatoms and feeding selectivity of copepods, as well as new progress on these two issues achieved in the recent ten years, are reviewed. These two issues are considered correlated closely. Diatoms and their metabolites can induce deleterious effects on growth, reproduction and development of copepods, including increase of mortality and decrease of egg production, hatching and growth rates. Such negative effects, resulting from either chemical toxin or nutritional deficiency, can be conquered in natural environments by diverse feeding. It is therefore concluded that deleterious effects of diatoms observed in laboratory or during blooming period are only a special case that accommodation of feeding strategy of copepods is disabled. To understand their feeding strategy in natural environments is a prerequisite to explaining the mechanisms of deleterious effects caused by diatoms, and makes it possible to re-evaluate the energy flow in marine ecosystems.

Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense

We report an apparently novel toxic effect of the dinoflagellate Alexandrium tamarense, manifested by inhibition of the egg hatching success of the scallop, Chlamys farreri. The hatching rate of C. farreri approached only 30% of controls when its fertilised eggs were exposed for 36 h to A. tamarense cells or cellular fragments at a concentration of 100 cells/ml, and the hatching rate was just 5% after exposure to A. tamarense of 500 cells/ml. Similar exposures of the fertilised scallop eggs to two other algal species, the diatom Phaeodactylum tricornutum and the raphidophyte Heterosigma carterae, resulted in no such toxicity or inhibitory effects.. Likewise, exposure of eggs to standard STX toxin. as well as to A. tamarense cell contents (supernant of re-suspended algal cells following ultrasonication and centrifugation), did not elicit this inhibitory response. However, exposure of the scallop eggs to cell cultures, intact algal cells, or cell fragments of A. tamarense produced marked toxicity. The alga also influenced larvae at early D-shape stage of scallop. The survival rates began to decrease significantly after exposed for 6 days at concentration of 3000 cells/ml and above: no larvae could survive after 14-day exposure to A. tamarense at 10,000 cells/ml or 20-day at 5000 cells/ml. The results indicated the production of novel substances from A. tamarense which can cause adverse effects on egg hatching and survival of the scallop larvae, The experiment also found that the developmental stages before blastula was the developmental period most sensitive to the A. tamarense toxin(s) and the alga at early exponential stage had the strongest effect on egg hatching comparing with other growth phases. The adverse effect of A. tamarense on early development of scallops may cause decline of shellfish population and may have further impact on marine ecosystem. (C) 2001 Elsevier Science Ltd. All rights reserved.

A modified HPLC method for analysis of PSP toxins in algae and shellfish from China

Improvements to an established HPLC method are introduced. The modified method is more efficient for separation and detection of the toxins responsible for paralytic shellfish poisoning (PSP). The PSP toxin content of two strains of Alexandrium tamarense and approximately forty shellfish samples collected from different locations in China have been analyzed with this HPLC method. Only one shellfish sample, collected from Lianyungang, China, contained PSP toxins.

Investigation of Extration Method for Paralytic Shellfish Poisoning Toxins in Shellfish

Me optimal conditions were established for the extraction of paralytic shellfish poisoning toxins from gonad of Chlamys nobills using acetic acid and hydrochloric acid in the concentration range of 0.04-1.0 mol/L. A 10-g portion of gonad of Chlamys nobilis was extracted by boiling for 5 min with 1.0 mL acetic acid and hydrochloric acid in a 50-mL beaker. Meanwhile, a portion of gonad of Chlamys nobilis was extracted by sonication in the solution of 0.3 mol/L HAc + 0.2 mol/L HCl for a total period of 5-30 min. The raw extract was centrifuged at 3500 r/min for 5 min and the pH of supernatant was adjusted from 2.0 to 4.0 by 0.1 mol/L NaOH or 5 mol/L HCL After passing through a Millipore ultrafiltration membrane (10000 MW cut-off), ultrafiltrate was then analyzed by HPLC. The results showed that hydrochloric acid in the concentration range of 0.25-1.0 mol/L caused a significant decrease of N-sulfocarbarnoyl-11-hydroxysulfate toxin C1 (C1), C2 and gonyautoxin 5 (GTX5) and the concomitant increase of GTX2,3. However, the amount of the three unstable toxins did not show any change using the extraction with acetic acid. Under the same concentration of acetic acid (0.3 mol/L) and hydrochloric acid (0.2 mol/L), the amount of C1 in the ultrasonic extraction was obviously lower than the boiling one, while C2 showed slightly higher than the latter.

Transfer of paralytic shellfish toxins via marine food chains: A simulated experiment

Objective To study the transfer of paralytic shellfish toxins (PST) using four simulated marine food chains: dinoflagellate Alexandrium tamarense -> Arterriia Artemia salina -> Mysid shrimp Neomysis awatschensis; A. tamarense-N. awatschensis: A. taniarense A. salina -> Perch Lateolabrax japonicus; and A. tamarense -> L. japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels iii the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly ibrough the vector of A. salina was then studied. The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense (2 000 cells(.)mL(-1)) for 70 minutes, the content of ChLa in A. salina and N. awatschensis reached 0.87 and 0.024 mu g-mg(-1), respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU(.)g(-1), respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in arternia sample collected on the 1st day was estimated to be 1.65x10(-5) pg STX equa Vindividual. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly froin the vector of A. salina was also studied. The mice injected with extracts from L. japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. taniarense directly or indirectly via the food chains.

Effects of azadirachtin on six inorganic cation distributions in Ostrinia furnacalis (G.)

Azadirachtin (Az), as a botanical insecticide, is relatively safe and biodegradable. It affects a wide vaariety of biological processes, including the reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using ion chromatography, we analyzed changes in six inorganic cation (Li+, Na+, NH4+, K+, Mg2+, and Ca2+) distributions of the whole body and hemolymph in Ostrinia furnacalis (G.) after exposure to sublethal doses of Az. The results showed that Az dramatically interfered with Na+, NH4+, K+, Mg2+, and Ca2+ distributions in hemolymph of O. furnacalis (G.) and concentrations of these five cations dramatically increased. However, in the whole body, the levels of K+, Mg2+, and Ca2+ significantly, decreased after exposure to Az, except that Na+ and NH4+ remained constant. Li+ was undetected in both the control and treated groups in the whole body and hemolymph. It is suggested that Az exerts its insecticidal effects on O. furnacalis (G.) by interfering with the inorganic cation distributions related to ion channels.

Síndrome Dolorosa Miofascial no contexto da Medicina Dentária

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

Ecotoxicology of marine biotoxins in bivalve shellfish

A small proportion of harmful algae produce toxins which are harmful to human health. Strict monitoring programmes are in place within Ireland and the EU to effectively manage risk to human consumers of shellfish species that have accumulated marine biotoxins in their tissues. However, little is known about the impacts of HABs on shellfish health. This study used Solid Phase Adsorption and Toxin Tracking (SPATT) for the passive sampling of algal biotoxins at Lough Hyne Marine Nature Reserve in West Cork, Ireland. Spatial and temporal monitoring of the incidence of a wide range of lipophilic toxins was assessed over a 4-month period. Active sampling accumulated sufficient quantities of toxin for use in subsequent experimentation. In addition to commonly occurring Diarrhetic Shellfish Poisoning (DSP) toxins, Dinophysis toxin-1 and Pinnatoxin-G were both detected in the samples. This is the first identification of these latter two toxins in Irish waters. The effects of the DSP toxin okadaic acid (OA) were investigated on three shellfish species: Mytilus edulis, Ruditapes philippinarum and Crassostrea gigas. Histological examination of the gill, mantle and hepatopancreas tissues revealed varying intensity of damage depending both on the tissue type and the species involved. At the cellular level, flow cytometric analysis of the differential cell population distribution was assessed. No change in cell population distribution was observed in Mytilus edulis or Ruditapes philippinarum, however significant changes were observed in Crassostrea gigas granulocytes at the lower levels of toxin exposure. This indicated a chemically-induced response to OA. DNA fragmentation was measured in the haemolymph and hepatopancreas cells post OA-exposure in Mytilus edulis and Crassostrea gigas. A significant increase in DNA fragmentation was observed in both species over time, even at the lowest OA concentrations. DNA fragmentation could be due to genotoxicity of OA and/or to the induction of cell apoptosis.

Epidemiology and comparative analysis of Yersinia in Ireland

Yersiniosis is an acute or chronic enteric zoonosis caused by enteropathogenic Yersinia species. Although yersiniosis is predominantly associated with gastroenteric forms of infection, extraintestinal forms are often reported from the elderly or patients with predisposing factors. Yersiniosis is often reported in countries with cold and mild climates (Northern and Central Europe, New Zealand and North of Russian Federation). The Irish Health Protection Surveillance Centre (HPSC) currently records only 3-7 notified cases of yersiniosis per year. At the same time pathogenic Yersinia enterocolitica is recovered from pigs (main source of pathogenic Y. enterocolitica) at the levels similar to that observed in Yersinia endemic countries. Introduction of Yersinia selective culture procedures may increase Yersinia isolation rates. To establish whether the small number of notifications of human disease was an underestimate due to lack of specific selective culture for Yersinia we carried out a prospective culture study of faecal samples from outpatients with diarrhoea, with additional culture of appendix and throat swabs. Higher levels of anti-Yersinia seroprevalence than yersiniosis notification rates in endemic countries suggests that most yersiniosis cases are unrecognised by culture. Subsequently, in addition to a prospective culture study of clinical specimens, we carried out serological screening of Irish blood donors and environmental screening of human sewage. Pathogenic Yersinia strains were not isolated from 1,189 faeces samples, nor from 297 throat swabs, or 23 appendix swabs. This suggested that current low notification rates in Ireland are not due to the lack of specific Yersinia culture procedures. Molecular screening detected a wider variety of Y. enterocolitica-specific targets in pig slurry than in human sewage. A serological survey for antibodies against Yersinia YOP (Yersinia Outer Proteins) proteins in Irish blood donors found antibodies in 25%, with an age-related trend to increased seropositivity, compatible with the hypothesis that yersiniosis may have been more prevalent in Ireland in the recent past. Y. enterocolitica is a heterogeneous group of microorganisms that comprises strains with different degree of pathogenicity. Although non-pathogenic Y. enterocolitica lack conventional virulence factors, these strains can be isolated from patients with diarrhoea. Insecticidal Toxin Complex (ITC) and Cytolethal Distending Toxins can potentially contribute to the virulence of non-pathogenic Y. enterocolitica in the absence of other virulence factors. We compared distribution of ITC and CDT loci among pathogenic and non-pathogenic Y. enterocolitica. Additionally, to demonstrate potential pathogenicity of non-pathogenic Y. enterocolitica we compared their virulence towards Galleria mellonella larvae (a non-mammalian model of human bacterial infections) with the virulence of highly and mildly pathogenic Y. enterocolitica strains. Surprisingly, virulence of pathogenic and non-pathogenic Y. enterocolitica in Galleria mellonella larvae observed at 37°C did not correlate with their pathogenic potential towards humans. Comparative phylogenomic analysis detects predicted coding sequences (CDSs) that define host-pathogen interactions and hence providing insights into molecular evolution of bacterial virulence. Comparative phylogenomic analysis of microarray data generated in Y. enterocolitica strains isolated in the Great Britain from humans with diarrhoea and domestic animals revealed high genetic heterogeneity of these species. Because of the extensive human, animal and food exchanges between the UK and Ireland the objective of this study was to gain further insight into genetic heterogeneity and relationships among clinical and non-clinical Y. enterocolitica strains of various pathogenic potential isolated in Ireland and Great Britain. No evidence of direct transfer of strains between the two countries was found.

Development and characterization of novel transgenic techniques for the study of neuronal circuitry

Modern neuroscience relies heavily on sophisticated tools that allow us to visualize and manipulate cells with precise spatial and temporal control. Transgenic mouse models, for example, can be used to manipulate cellular activity in order to draw conclusions about the molecular events responsible for the development, maintenance and refinement of healthy and/or diseased neuronal circuits. Although it is fairly well established that circuits respond to activity-dependent competition between neurons, we have yet to understand either the mechanisms underlying these events or the higher-order plasticity that synchronizes entire circuits. In this thesis we aimed to develop and characterize transgenic mouse models that can be used to directly address these outstanding biological questions in different ways. We present SLICK-H, a Cre-expressing mouse line that can achieve drug-inducible, widespread, neuron-specific manipulations in vivo. This model is a clear improvement over existing models because of its particularly strong, widespread, and even distribution pattern that can be tightly controlled in the absence of drug induction. We also present SLICK-V::Ptox, a mouse line that, through expression of the tetanus toxin light chain, allows long-term inhibition of neurotransmission in a small subset (<1%) of fluorescently labeled pyramidal cells. This model, which can be used to study how a silenced cell performs in a wildtype environment, greatly facilitates the in vivo study of activity-dependent competition in the mammalian brain. As an initial application we used this model to show that tetanus toxin-expressing CA1 neurons experience a 15% - 19% decrease in apical dendritic spine density. Finally, we also describe the attempt to create additional Cre-driven mouse lines that would allow conditional alteration of neuronal activity either by hyperpolarization or inhibition of neurotransmission. Overall, the models characterized in this thesis expand upon the wealth of tools available that aim to dissect neuronal circuitry by genetically manipulating neurons in vivo.

Electrochemical biosensor based on microfabricated electrode arrays for life sciences applications

In developing a biosensor, the utmost important aspects that need to be emphasized are the specificity and selectivity of the transducer. These two vital prerequisites are of paramount in ensuring a robust and reliable biosensor. Improvements in electrochemical sensors can be achieved by using microelectrodes and to modify the electrode surface (using chemical or biological recognition layers to improve the sensitivity and selectivity). The fabrication and characterisations of silicon-based and glass-based gold microelectrode arrays with various geometries (band and disc) and dimension (ranging from 10 μm-100 nm) were reported. It was found that silicon-based transducers of 10 μm gold microelectrode array exhibited the most stable and reproducible electrochemical measurements hence this dimension was selected for further study. Chemical electrodeposition on both 10 μm microband and microdisc were found viable by electro-assisted self-assembled sol-gel silica film and nanoporous-gold electrodeposition respectively. The fabrication and characterisations of on-chip electrochemical cell was also reported with a fixed diameter/width dimension and interspacing variation. With this regard, the 10 μm microelectrode array with interspacing distance of 100 μm exhibited the best electrochemical response. Surface functionalisations on single chip of planar gold macroelectrodes were also studied for the immobilisation of histidine-tagged protein and antibody. Imaging techniques such as atomic force microscopy, fluorescent microscopy or scanning electron microscope were employed to complement the electrochemical characterisations. The long-chain thiol of self-assembled monolayer with NTA-metal ligand coordination was selected for the histidine-tagged protein while silanisation technique was selected for the antibody immobilisation. The final part of the thesis described the development of a T-2 labelless immunosensor using impedimetric approach. Good antibody calibration curve was obtained for both 10 μm microband and 10 μm microdisc array. For the establishment of the T-2/HT-2 toxin calibration curve, it was found that larger microdisc array dimension was required to produce better calibration curve. The calibration curves established in buffer solution show that the microelectrode arrays were sensitive and able to detect levels of T-2/HT-2 toxin as low as 25 ppb (25 μg kg-1) with a limit of quantitation of 4.89 ppb for a 10 μm microband array and 1.53 ppb for the 40 μm microdisc array.

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway.

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.

Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans.

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.

Adenovirus F protein as a delivery vehicle for botulinum B.

BACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.

BACKGROUND: Stimulation of beta(1)- and beta(2)-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the beta(3)AR is also expressed in the human heart and that its stimulation leads to negative inotropic effects. METHODS AND RESULTS: To better understand the role of beta(3)ARs in cardiac function, we generated transgenic mice with cardiac-specific overexpression of 330 fmol/mg protein of the human beta(3)AR (TGbeta(3) mice). Hemodynamic characterization was performed by cardiac catheterization in closed-chest anesthetized mice, by pressure-volume-loop analysis, and by echocardiography in conscious mice. After propranolol blockade of endogenous beta(1)- and beta(2)ARs, isoproterenol resulted in an increase in contractility in the TGbeta(3) mice (30%), with no effect in wild-type mice. Similarly, stimulation with the selective human beta(3)AR agonist L-755,507 significantly increased contractility in the TGbeta(3) mice (160%), with no effect in wild-type mice, as determined by hemodynamic measurements and by end-systolic pressure-volume relations. The underlying mechanism of the positive inotropy incurred with L-755,507 in the TGbeta(3) mice was investigated in terms of beta(3)AR-G-protein coupling and adenylyl cyclase activation. Stimulation of cardiac membranes from TGbeta(3) mice with L-755,507 resulted in a pertussis toxin-insensitive 1.33-fold increase in [(35)S]GTPgammaS loading and a 1.6-fold increase in adenylyl cyclase activity. CONCLUSIONS: Cardiac overexpression of human beta(3)ARs results in positive inotropy only on stimulation with a beta(3)AR agonist. Overexpressed beta(3)ARs couple to G(s) and activate adenylyl cyclase on agonist stimulation.