Salinity effect on growth and toxin production of four tropical Alexandrium species (Dinophyceae).

Four tropical PSP toxins-producing dinoflagellates, Alexandrium minutum, Alexandrium tamiyavanichii, Alexandrium tamarense and Alexandrium peruvianum from Malaysian waters were studied to investigate the influences of salinity on growth and toxin production. Experiments were conducted on constant temperature 25 degrees C, 140 microE mol m(-2) s(-1) and under 14:10 light:dark photo-cycle with salinity ranged from 2 to 30 psu. The PSP-toxin congeners, GTX 1-6, STX, dcSTX, NEO and C1-C2 were analysed by high performance liquid chromatography. Salinity tolerance of the four species in decreasing order is A. minutum>A. peruvianum>A. tamarense>A. tamiyavanichii. Specific growth rates and maximum densities varied among these species with A. minutum recorded as the highest, 0.5 day(-1) and 6 x 10(4) cells L(-1). Toxin content decreased with elevated salinities in A. minutum, the highest toxin content was about 12 fmole cell(-1) at 5 psu. In A. tamiyavanichii, toxin content peaked at optimal growth salinity (20 and 25 psu). Toxin content of A. tamarense, somehow peaked at sub-optimal growth salinity (15 and 30 psu). Results of this study implied that salinity fluctuation not only influenced the growth physiology but also toxin production of these species.     

Localization of epitopes in the toxins of Tityus serrulatus scorpions and neutralizing potential of therapeutic antivenoms.

Overlapping pentadecapeptides covering the complete amino acid sequence of TsII, TsVII and TsIV toxins from the venom of scorpion Tityus serrulatus (Ts), were prepared by use of the Spot method of multiple peptide synthesis. Horse anti-Ts antisera for therapeutic use were tested for their binding to peptides. All nine antisera tested showed reactivity with several peptides from the three toxins. Three antigenic regions, one in the very N-terminal, the second in the central part and the other in the C-terminal part of the three toxins were frequently, but not constantly recognized, with an intensity that seemed to be related to the neutralizing potency of the tested antivenom. Thus the corresponding peptides (residues 1-15 and 48-62 of TsII; residues 1-15, 16-30 and 48-62 of TsIV and residues 1-15 and 47-61 of TsVII) were synthesized, coupled to KLH and used as antigens to coat the microtitration plates to determine any relationship between their ELISA reactivity with therapeutic horse antivenoms and the neutralizing potential of these antivenoms. The mixture of the N-terminal peptide of TsII, of the N-terminal TsVII peptide and of the C-terminal of TsIV was found to give a linear relationship with the neutralizing titer of horse serum of low neutralizing potency (< or =1 mg/ml). However, high neutralizing antivenoms did not show the expected response in peptide ELISA. This observation is discussed in the context of the occurrence of continuous and discontinuous epitopes on toxins.     

Biomonitoring of ciguatoxin exposure in mice using blood collection cards.

Ciguatera is a human food poisoning caused by consumption of tropical and subtropical fish that have, through their diet, accumulated ciguatoxins in their tissues. This study used laboratory mice to investigate the potential to apply blood collection cards to biomonitor ciguatoxin exposure. Quantitation by the neuroblastoma cytotoxicity assay of Caribbean ciguatoxin (C-CTX-1) spiked into mice blood was made with good precision and recovery. The blood collected from mice exposed to a sublethal dose of Caribbean ciguatoxic extract (0.59 ng/g C-CTX-1 equivalents) was analyzed and found to contain detectable toxin levels at least 12 h post-exposure. Calculated concentration varied from 0.25 ng/ml at 30 min post-exposure to 0.12 ng/ml at 12 h. A dose response mice exposure revealed a linear dose-dependent increase of ciguatoxin activity in mice blood, with more polar ciguatoxin congeners contributing to 89% of the total toxicity. Finally, the toxin measurement in mice blood exposed to toxic extracts from the Indian Ocean or from the Pacific Ocean showed that the blood collection card method could be extended to each of the three known ciguatoxin families (C-CTX, I-CTX and P-CTX). The low matrix effect of extracted dried-blood samples (used at 1:10 or 1:20 dilution) and the high sensitivity of the neuroblastoma assay (limit of detection 0.006 ng/ml C-CTX-1), determined that the blood collection card method is suitable to monitor ciguatoxin at sublethal doses in mice and opens the potential to be a useful procedure for fish screening, environmental risk assessment or clinical diagnosis of ciguatera fish poisoning in humans or marine mammals.     

青岛沿海海洋附着生物抗肿瘤活性的筛选

对青岛沿海海洋附着生物资源进行调查，采用海虾细胞毒性法及流式细胞术对已鉴定的18种附着生物进行抗肿瘤活性筛选。结果表明，星座美洲海鞘Amaroucium ecostellatum Verril、大菊海鞘Botryllus magnicoecus Hartmeyer、山形海绵Mycale macilenta、海黍子Sargassum kjellmanianum Yendo、裙带菜Undaria pinnatifida(Harv．)Sur．甲醇提取物具有较强的抗肿瘤活性。并进一步进行活性成分追踪研究，确定星座美洲海鞘、大菊海鞘、山形海绵、海黍子、裙带菜的活性部位。     

一株抗肿瘤放线菌的筛选及其初步研究

在经过MTT法初筛得到的具有细胞毒的海洋微生物中 ,我们又采用荧光显微观测法 ,发现放线菌AK 1 1处理的细胞发生凋亡。在琼脂糖电泳上呈现DNA梯度带 ,进一步验证了其具有使细胞凋亡的作用。AK 1 1活性化合物纯品的裸鼠试验结果表明 :其试验浓度为 0 .0 1 6 μg·kg- 1,0 .1mL/次和 0 .0 0 8μg·kg- 1,0 .1mL/次时抑瘤率分别为 5 2 .0 %和 33.6 %。对人胃癌细胞MGC 80 3及人胃肝细胞SMMC772 1的IC50 的测定值略小于阿霉素 ,显示了AK 1 1具有较高的抗肿瘤活性。FCM对细胞周期的分析表明 :经AK 1 1处理后细胞在S期发生阻滞。DNA合成受阻     

DNA提取方法进展

DNA的提取是分子生物学研究的基础技术，提取的DNA的纯度及结构完整性是进行基因工程各项研究所必需的条件。近年来一些新的或改进的DNA提取纯化方法不断出现，本文对从陆生动物、植物、微生物以及海洋生物提取DNA的方法进行综述。     

海洋贝类提取物的药理作用研究

目的 通过观察海洋贝类提取物(EMS)抗血小板聚集和抗氧化的药理作用,探讨EMS抗动脉粥样硬化(AS)作用机理。方法1.采用二磷酸腺苷(ADP)和胶原作诱导剂进行体内及体外血小板聚集实验,观察EMS对大鼠血小板聚集功能的影响。2.检测EMS对血清总超氧化物歧化酶(T-SOD)及脂质过氧化物丙二醛(MDA)含量的影响。结果 1.EMS体外用药(9.76,29.27,48.78 mg·mL~(-1))可显著降低胶原诱导的家兔血小板最大聚集率;在体内实验中,EMS(5,10,20g·kg~(-1))ig对ADP诱导的大鼠血小板聚集率无明显影响。2.EMS(5,10,20g·kg~(-1),ig,qd)可显著升高大鼠血清T-SOD的活性,并降低MDA含量。结论EMS具有抑制血小板聚集功能和抗氧化作用。     

海洋微生物抗菌活性的初步研究

从南海、东海、黄海等海域的海洋底质、生物样品中分离到1003个海洋微生物菌株．包括520株放线菌、111株真菌、222株常温细菌和150株耐热细菌。通过纸片法对其抗菌活性进行筛选．结果发现321株海洋微生物具有抑菌活性，占总分离菌株的32．0％。放线菌、真菌、常温细菌、耐热细菌的活性菌株比例分别为39．6％，32．4％。17．1％，27．3％。具有抑菌活性的菌株比例与样品来源密切相关。生物样品来源的细菌抑菌活性较高的菌株比例较底质样品来源的要高，而海洋放线菌与海洋真菌结果相反。各类海洋微生物菌株的抗菌谱有很大的不同。     

Comparative study on differential accumulation of PSP toxins between cockle (Acanthocardia tuberculatum) and sweet clam (Callista chione).

At the western Mediterranean coast of Morocco, the cockle (Acanthocardia tuberculatum) contained persistent high levels of paralytic shellfish toxins for several years, while other bivalve molluscs such as sweet clam (Callista chione) from the same vicinity were contaminated seasonally to a much lesser extent. In order to understand the causes of this prolonged contamination, a comparative study on PSP decontamination between sweet clam and cockle was conducted from November 2001 until June 2002. PSP toxicity was analysed by automated pre-column oxidation (Prechromatographic oxidation and LC-FD) in several organs of both species, namely digestive gland, foot, gill, mantle, muscle and siphon for sweet clams. The results showed that cockle sequester PSP toxins preferably in non-visceral organs (Foot, gill and mantle) contrary to sweet clam that sequester them in visceral tissues (digestive gland). The toxin profile of cockle organs indicated dominance of dcSTX, whereas sweet clam tissues contained especially C-toxins. Substantial differences in toxin profile between cockle and sweet clam, from the same area as well as from the composition of PSP toxin producer, Gymnodinium catenatum, confirm the bioconversion of PSP toxins in cockle.     

Toxicity and mAChRs binding activity of Cassiopea xamachana venom from Puerto Rican coasts.

A separation of toxic components from the upside down jellyfish Cassiopea xamachana (Cx) was carried out to study their cytotoxic effects and examine whether these effects are combined with a binding activity to cell membrane receptors. Nematocysts containing toxins were isolated from the autolysed tentacles, ruptured by sonication, and the crude venom (CxTX) was separated from the pellets by ultracentrifugation. For identifying its bioactive components, CxTX was fractionated by gel filtration chromatography into six fractions (named fraction I-VI). The toxicity of CxTX and fractions was tested on mice; however, the hemolytic activity was tested on saline washed human erythrocytes. The LD50 of CxTX was 0.75 microg/g of mouse body and for fraction III, IV and VI were 0.28, 0.25 and 0.12 microg/g, respectively. Fractions I, II and V were not lethal at doses equivalent to LD50 1 microg/g. The hemolytic and phospholipase A2 (PLA2) activities of most fractions were well correlated with their mice toxicity. However, fraction VI, which contains the low molecular mass protein components (< or =10 kDa), has shown no PLA2 activity but highest toxicity to mice, highest hemolytic activity, and bound significantly to the acetylcholine muscarinic receptors (mAChRs) isolated from rat brain. The results suggested that fraction VI contains proteinaceous components contributing to most of cytolysis as well as membrane binding events. Meanwhile, fraction IV has shown high PLA2 that may contribute to the venom lethality and paralytic effects.