Kinetic analysis of interaction between lipopolysaccharide and biomolecules

Lipopolysaccharide ( LPS) is a major component of the outer membrane of all gram-negative bacteria. It is a heat-resistant toxin which can cause toxic shock in animals. LPS interacts with some biomolecules and triggers its toxic reaction. In this study, the interaction between LPS from Salmonella Minnesota and some biomolecules using syrface okasnib resibabce ( SPR) biosensor. biomolecules were imobilized on CM5 sensor-chip suing amion coupling method and LPS was injected over the immobilized surfaces.

Direct toxicity assessment of toxic chemicals with electrochemical method

Electrochemical measurement of respiratory chain activity is a rapid and reliable screening for the toxicity on microorganisms. Here, we investigated in-vitro effects of toxin on Escherichia coli (E. coli) that was taken as a model microorganism incubated with ferricyanide. The current signal of ferrocyanide effectively amplified by ultramicroelectrode array (UMEA), which was proven to be directly related to the toxicity. Accordingly, a direct toxicity assessment (DTA) based on chronoamperometry was proposed to detect the effect of toxic chemicals on microorganisms. The electrochemical responses to 3,5-dichlorophenol (DCP) under the incubation times revealed that the toxicity reached a stable level at 60 min, and its 50% inhibiting concentration (IC50) was estimated to be 8.0 mg L-1. At 60 min incubation, the IC50 values for KCN and As2O3 in water samples were 4.9 mg L-1 and 18.3 mg L-1, respectively. But the heavy metal ions, such as Cu2+ Pb2+ and Ni2+, showed no obvious toxicity on E. coli.

The interaction of recombinant Pseudomonas aeruginosa exotoxin A with DNA and mimic biomembrane investigated by surface plasmon resonance and electrochemical methods

Based on the multidomain structure of Pseudomonas aeruginosa exotoxin A, a fusion protein termed rPEA has been constructed, which is expected to serve as a gene carrier in vitro. The expression and purification of rPEA are described. The basal properties of rPEA as a gene carrier are evaluated by investigating its interaction with plasmid DNA and mimic biomembrane by surface plasmon resonance (SPR) and electrochemical methods. rPEA is proved to be able to bind with plasmid DNA with high affinity. It can also interact with lipid membrane and increase permeability of the membrane, so the probe molecules can easily reach the gold surface and exhibit the electrochemical response.

Self-assembled monolayer growth of phospholipids on hydrophobic surface toward mimetic biomembranes: Scanning probe microscopy study

Atomic force microscopy (AFM) and lateral force microscopy (LFM) were used simultaneously to analyze a model membrane bilayer structure consisting of a phospholipid outer monolayer deposited onto organosilane-derivatized mica surfaces, which were constructed by using painting and self-assembly methods. The phospholipid used as outer monolayer was dimyristoylphosphatidylcholine (DMPC). The hydrocarbon-covered substrate that formed the inner half bilayer was composed of a self-assembly monolayer (SAM) of octadecyltrichloroorganosilane (OTS) on mica. SAMs of DMPC were formed by exposing hydrophobic mica to a solution of DMPC in decane/isobutanol and subsequently immersing into pure water. AFM images of samples immersed in solution for varying exposure times showed that before forming a complete monolayer the molecules aggregated into dense islands (2.2-2.6 nm high) on the surface. The islands had a compact and rounded morphology. LFM, coupled with topographic data obtained with the atomic force mode, had made possible the distinction between DMPC and OTS. The rate constant of DMPC growth was calculated. This is the first systematic study of the SAM formation of DMPC by AFM and LFM imaging. It reveals more direct information about the film morphology than previous studies with conventional surface analytical techniques such as infrared spectroscopy, X-ray, or fluorescence microscopy.

氮、磷营养盐对链状亚历山大藻（东海株）生长和产毒的影响研究

近年来，东海长江口邻近海域春季常常发生大规模链状亚历山大藻（Alexandrium catenella）赤潮。通常认为，由长江携带入海的大量营养盐导致的富营养化是该海域大规模赤潮频繁发生的重要原因。为阐明该海域富营养化特征与链状亚历山大藻赤潮的发生及其危害效应之间的关系，本文根据东海氮、磷营养盐的浓度与结构特征，设计实验研究了营养盐对链状亚历山大藻（东海株ACDH）的生长和产毒的影响。 实验结果表明，ACDH 藻株的比增长率在硝酸盐浓度大于 28.0 µM 时接近最高水平，长江口邻近海域海水中硝酸盐的浓度可以使 ACDH 藻株的比增长率接近最高值；藻株的比增长率在磷酸盐浓度大于 2.5 µM 时也接近最高水平，现场海水中磷酸盐的浓度可能对ACDH藻株的生长构成限制。ACDH藻株生长的最适氮磷比在9到11之间 ，表明链状亚历山大藻的生长需要较多的磷。ACDH 藻株对磷酸盐的最大吸收速率 (Vm) 和半饱和常数(Ks) 分别为 0.22 pmol/cell•h 和 0.75 µM，与中肋骨条藻等东海常见赤潮藻种相比并不利于其对低浓度磷酸盐的竞争吸收，但处于磷限制的链状亚历山大藻细胞有明显的碱性磷酸酶表达，这将有助于其吸收利用有机态的磷营养物质，获取所需的磷维持生长。链状亚历山大藻可以利用尿素、硝酸盐和铵盐这三种氮源进行生长，有相近的比增长率和最大细胞密度。综合本实验得到的结果与以往的文献资料可以认为，东海链状亚历山大藻的适应策略利于其在长江口邻近海域形成赤潮，但东海长江口邻近海域的富营养化并非链状亚历山大藻赤潮形成的唯一原因，链状亚历山大藻赤潮的形成还应与其生活史和垂直迁移等行为特征有关。而高浓度的营养盐对于亚历山大藻赤潮规模的增加可能具有直接作用。 在常见的铵盐、硝酸盐和尿素这三种氮源中，铵盐和硝酸盐的加入会导致细胞毒素含量的迅速增加，但是，添加尿素却不能显著提高藻细胞的毒素含量。长期以尿素作为氮源甚至会使藻细胞的毒素含量低于氮限制条件下的细胞毒素含量。磷限制能显著提高其细胞毒素含量，对野外采集的浮游植物样品分析结果显示，其细胞毒素含量在 34.7-88.4 fmol/cell 之间，显著高于室内培养的营养盐充足条件下的链状亚历山大藻的细胞毒素含量，而与磷限制条件下培养的藻细胞毒素含量接近，说明东海海水中磷酸盐的限制有可能提高藻细胞的毒素含量，从而增加其潜在的危害效应。 总之，通过我们的研究可以看出，东海的富营养化特征直接影响链状亚历山大藻赤潮的形成与潜在的危害效应。东海链状亚历山大藻的适应策略利于其在长江口邻近海域形成赤潮，同时，该海域高氮磷比和磷酸盐浓度限制会提高藻细胞的毒素含量，增加其危害效应。以上研究将有助于了解链状亚历山大藻赤潮的爆发机理，并对赤潮危害的预测和防范提供科学依据。

中国沿海典型区域织纹螺毒性及毒素成分研究

织纹螺（Nassarius spp.）味道鲜美，是中国及其它一些亚洲国家沿海地区居民习惯食用的一种水产品。但是，近几十年来，中国沿海频繁发生食用织纹螺中毒事件，严重威胁着人们的身体健康和生命安全。加之人们对织纹螺体内的毒素成分、来源及其毒性变化规律还没有清晰的认识，因此难以有效预防和控制食用织纹螺引起的中毒事件。本文根据文献报道，在中国沿海食用织纹螺中毒事件多发的典型区域，包括江苏省的连云港市和盐城市、浙江省的舟山市和宁波市、福建省的宁德市、厦门市和莆田市设立了监测点，于2006年和2007年间进行了连续采样，应用小鼠生物测试法调查了织纹螺毒性的消长情况，并利用高效液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry，LC-MS）和高效液相色谱技术（High Performance Liquid Chromatography，HPLC）对织纹螺体内的毒素成分进行了分析。 实验结果表明，2006年于江苏省盐城市射阳海域采集的织纹螺样品中，阳性样品检出率为56%，毒性在2-5 MU/g组织（湿重）之间变化，在2007年于同地采集的8个样品中，除一个样品毒性为3.14 MU/g组织（湿重）以外，其余样品均表现为阴性；而2007年采集自连云港市赣榆海域的织纹螺样品，在采样期间则呈现出极高的毒性，最高达到846.52 MU/g 组织（湿重），毒性在监测期间呈“M”状波动，在5月和7月下旬出现两个毒性高峰。2006年于浙江省宁波市象山港采集的织纹螺样品中，阳性样品检出率为25%，毒性均在2.5 MU/g组织（湿重）左右；而同年采集自舟山市定海的织纹螺样品中，阳性样品检出率为100%，最高毒性达18.40 MU/g组织（湿重），毒性在监测期间也呈“M”状波动，高峰期出现在6月初和7月底。2006年3-9月采集自福建省宁德霞浦、厦门同安和莆田涵江采集的织纹螺样品中，阳性样品检出率分别为20%、43%和14%，除7月中旬采集自宁德霞浦的一个样品毒性达到16.19 MU/g组织（湿重）之外，其余样品毒性均在2-5 MU/g组织（湿重）间波动。从阳性样品的时间分布规律来看，3月份和6、7月份是阳性样品集中出现的时期。根据以上调查结果可以看出，织纹螺的毒性消长呈现出较明显的地域性和季节性特征，不同地区的织纹螺毒性存在差异，而同一区域织纹螺毒性的消长则表现出明显的季节性集中趋势。除了2007年采集自连云港赣榆的织纹螺样品毒性与其平均个体组织重量有相似的变化趋势以外，其余地区的织纹螺样品毒性和个体大小无明显相关性。 利用LC-MS和HPLC技术对织纹螺样品中的毒素成分进行了分析，确定河豚毒素（tetrodotoxin, TTX）及其同系物（trideoxyTTX，4-epi-TTX，anhydroTTX，oxoTTX）是所采集织纹螺中的主要致毒成分，样品中没有检测到麻痹性贝毒毒素（Paralytic Shellfish Poison, PSP）。自不同地区采集的织纹螺中毒素成分基本一致，但组成存在一定差异。其中，采自江苏省连云港赣榆和浙江省舟山定海的织纹螺样品中，trideoxyTTX是主要的成分，其次是TTX；而从其它采样地点采集的织纹螺中，TTX都是主要的毒素成分，其次才是trideoxyTTX及其它同系物。对采集自江苏省连云港赣榆和浙江舟山定海的织纹螺体内毒素的解剖学分布进行了分析，结果表明肌肉、消化腺和剩余部分中的毒素组成基本一致，其中trideoxyTTX是主要的毒素成分，其次为TTX，但采自浙江舟山的织纹螺剩余部分中的TTX是主要的毒素成分。在监测期间，各组织中的毒素组成没有明显变化，但毒素含量随季节变化表现出了一定的差异。 综上所述，在中国沿海典型区域开展的织纹螺毒性调查结果表明其毒性消长具有一定的地域性和季节性特征。分析结果显示织纹螺体内的毒素成分是河豚毒素及其同系物，采自不同区域的织纹螺体内毒素成分基本一致，但毒素组成稍有差异。对织纹螺中毒素的解剖学分布研究显示，各组织中的毒素含量随季节变化而表现出一定差异，但毒素组成没有明显的季节性变化。这些结果显示中国沿海的织纹螺应具有相似的毒素来源，研究结果将为相关部门有效监测、预防和控制食用织纹螺中毒事件提供有力的科学依据。

Environmental and health effects associated with Harmful Algal Bloom and marine algal toxins in China

The frequency and scale of Harmful Algal Bloom (HAB) and marine algal toxin incidents have been increasing and spreading in the past two decades, causing damages to the marine environment and threatening human life through contaminated seafood. To better understand the effect of HAB and marine algal toxins on marine environment and human health in China, this paper overviews HAB occurrence and marine algal toxin incidents, as well as their environmental and health effects in this country. HAB has been increasing rapidly along the Chinese coast since the 1970s, and at least 512 documented HAB events have occurred from 1952 to 2002 in the Chinese mainland. It has been found that PSP and DSP toxins are distributed widely along both the northern and southern Chinese coasts. The HAB and marine algal toxin events during the 1990s in China were summarized, showing that the HAB and algal toxins resulted in great damages to local fisheries, marine culture, quality of marine environment, and human health. Therefore, to protect the coastal environment and human health, attention to HAB and marine algal toxins is urgently needed from the environmental and epidemiological view.

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.

As neurotoxinas de clostridium sp.

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

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.