Analysis of paralytic shellfish toxins in Aphanizomenon DC-1 from Lake Dianchi, China

Lake Dianchi is in Yunnan Province in southwestern China. In recent years, significant cyanobacterial blooms have occurred in this lake nearly every year because of eutrophication. Monitoring data for the past 5 years acquired by our research group showed that phytoplankton composition alternated between species of Microcystis sp. during warm seasons and those of Aphanizomenon sp. during cool seasons. In March 2003, when phytoplankton composition was highly dominated by Aphanizomenon sp., samples were taken from the lake for toxin detection and immediate strain isolation. A mouse bioassay with extracts from the lyophilized field material showed obvious intoxication from paralytic shellfish poisons (PSPs), and all mice died within 30 min. Further analysis of both field and isolated algal strain Aphanizomenon DC-1 by the postcolumn HPLC-FLD method confirmed its PSP-producing ability The analogues found in the extracts from the field material were neoSTX, dcSTX, and dcGTX3, with contents of 2.279, 1.135, and 0.547 ng/mg DW, respectively. Under laboratory culture condition, toxin content in the Aphanizomenon strain DC-1 varied greatly during different growth phases, with two peaks: in the early-exponential and late-stationary growth phases. When the culture grew at a relatively high rate during the mid- to late-exponential growth phase, toxin content declined gradually. Moreover, the types of toxin in the DC-1 strain varied greatly during a single culture cycle. The HPLC results showed that dcSTX was the only toxin isomer detected throughout the culture period, and its level remained stable. On the other hand, dcGTX2 and GTX4 were the major toxins during the early-exponential and stationary phases, respectively. This article presents the first data on the identification and detection of paralytic shellfish toxins from cyanobacteria in Lake Dianchi. As far as we know, this is also the first report of this type of toxin in inland water bodies in China. Our study indicates the threat associated with PSP toxins in Lake Dianchi and suggests that necessary measures and programs for control are urgently needed to prevent the spread of toxic cyanobacterial blooms. (c) 2006 Wiley Periodicals, Inc.

Effect of inclusion of blue-green algae meal on growth and accumulation of microcystins in gibel carp (Carassius auratus gibelio)

Six isonitrogenous (crude protein content: 38%) and isoenergetic (gross energy content: 17 kJ g(-1)) diets were formulated to investigate the effects of inclusion of blue-green algae meal on gibel carp (Carassius auratus gibelio). In each diet, 15% of the protein was supplied by fishmeal; the remainder was supplied by soybean meal and blue-green algae meal. Diet 1 was used as control with no blue-green algae meal whereas the content in diets 2-6 was 15.15, 29.79, 44.69, 59.58 and 74.48%, respectively. Each diet was fed to five groups of gibel carp for 12 weeks in a flow-through system. Final body weight and specific growth rate (SGR) of fish fed diet 5 were significantly lower than the control diet (P < 0.05). Mortality of gibel carp increased with increase in algae meal inclusion (P < 0.05), but there was no significant difference between fish fed diets 3-6 (P > 0.05). Feed conversion efficiency (FCE) decreased with the increase in algae meal inclusion (P < 0.05). Fish-fed diet 6 showed the highest feeding rate (P < 0.05), while there were no significant differences among the other groups (P > 0.05). Apparent digestibility coefficient of dry matter, protein, and energy decreased with increasing algae meal inclusion in the diets (P < 0.05). Aspartate aminotransferase (GOT) activity in the liver was not significantly different among groups (P > 0.05). Liver alanine aminotransferase (GPT) activity of fish-fed diets 4, 5 and 6 was significantly lower than the control diet (diet 1; P < 0.05). Microcystins in the muscle, liver, gallbladder, and spleen increased with increasing algae inclusion (P < 0.05).

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in two freshwater shrimps, Palaemon modestus and Macrobrachium nipponensis, from a large shallow, eutrophic lake of the subtropical China

So far no information is available on microcystin (MC) contents in shrimps, prawns or crayfish from natural freshwaters. Tissue distributions and seasonal dynamics of the hepatotoxic MC-LR and -RR in two freshwater shrimps, Palaemon modestus and Macrobrachium nipponensis were studied monthly (during June-November, 2003) in a Chinese lake containing toxic cyanobacterial blooms. The shrimps P. modestus and M. nipponensis accumulated high MCs not only in the hepatopancreas (mean 4.29 and 0.53 mu g g(-1) DW, respectively) but also in the gonad (mean 1.17 and 0.48 mu g g-1 DW, respectively), and the crayfish Procambarus clarkii accumulated as much as 0.93 mu g g(-1) DW in the gonad. This indicates that gonads of these invertebrates are the second important target organ of MCs. P. modestus apparently accumulated more MCs in their organs than M. nipponensis, which might be a reflection of their difference in trophic niche. Eggs of the shrimps accumulated 8.4% (M. nipponensis, 0.27 mu g g(-1) DW) and 29.0% (P. modestus, 2.34 mu g g(-1) DW) of total toxin burden, indicating that MCs had been transferred into offspring from their adults. Among the shrimp muscle samples analyzed, 31% were above the provisional WHO TDI level, suggesting the risk of consuming shrimps in Lake Chaohu. (c) 2005 Elsevier Ltd. All rights reserved.

Growth and antioxidant system of the cyanobacterium Synechococcus elongatus in response to microcystin-RR

Microcystins, one type of the cyanobacterial toxins, show a broad range of hazardous effects on other organisms. Most of the researches on the toxic effects of microcystins have involved in animals and higher plants. Little work, however, has been done on evaluating the mechanisms of microcystin toxicity on algae. In this study, the toxicological effects of microcystin-RR (MC-RR) on the cyanobacterium Synechococcus elongatus were investigated. For this purpose, six physio-biochemical parameters (cell optical density, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST)) were tested in algal cells when exposed to 100 mug(-1) microcystin-RR. The results showed that the growth of Synechococcus elongatus ( expressed as optical density) was significantly inhibited compared with the control. At the same time, the treated algae exhibited a pronounced increase in production of ROS and MDA after 6 days exposure to microcystin-RR. Signi. cant changes in GSH levels and GSH-Px, GSH activities were also detected in algal cells, with higher values being observed in the toxin treated algae after 6 days exposure. GST activities in the treated algae exhibited a decline after exposure and rapid augmentation on day 3, thereafter, they kept at a high level when compared to the control group. GSH contents and GSH-Px activities were also significantly raised in the toxin-treated algae cells from day 3, but they showed a sharp decrease on day 4, which was the onward of cell proliferation. These results suggested that oxidative stress manifested by elevated ROS levels and MDA contents might be responsible for the toxicity of microcystin to Synechococcus elongatus and the algal cells could improve their antioxidant ability through the enhancement of enzymatic and non-enzymatic preventive substances.

Physiological and biochemical microcystin-RR toxicity to the Synechococcus elongatus

Freshwater Microcystis may form dense blooms in eutrophic lakes. It is known to produce a family of related cyclic hepatopeptides (microcystins, MC) that constitute a threat to aquatic ecosystems. Most toxicological studies of microcystins have focused on aquatic animals and plants, with few examining the possible effects of microcystins on phytoplankton. In this study we chose the unicellular Synechococcus elongatus (one of the most studied and geographically most widely distributed cyanobacteria in the picoplankton) as the test material and investigated the biological parameters: growth, pigment (chlorophyll-a, phycocyanin), photosynthetic activity, nitrate reductase activity, and protein and carbohydrate content. The results revealed that microcystin-RR concentrations above 100 mug (.) L-1 significantly inhibited the growth of Synechococcus elongatus. In addition, a change in color of the toxin-treated algae (chlorosis) was observed in the experiments. Furthermore, MC-RR markedly inhibited the synthesis of the pigments chlorophyll-a and phycocyanin. A drastic reduction in photochemical efficiency of PSII (F-v/F-m) was found after a 96-h incubation. Changes in protein and carbohydrate concentrations and in nitrate reductase activity also were observed during the exposure period. This study aimed to evaluate the mechanisms of microcystin toxicity on a cyanobacterium, according to the physiological and biochemical responses of Synechococcus elongatus to different doses of microcystin-RR. The ecological role of microcystins as an allelopathic substance also is discussed in the article. (C) 2004 Wiley Periodicals, Inc.

An ELISA-like time-resolved fluorescence immunoassay for microcystin detection

Background: A time-resolved fluorescence immunoassay (TRFIA), based on anti-microcystin-LR (MCLR) monoclonal antibodies (MAbs) and europium-labeled antimouse IgG conjugate, was first developed for microcystin detection. Methods: Anti-MCLR MAbs were prepared by a standard method, and the attained MAbs showed a good cross reactivity with MCLR, MCRR and MCYR. The TRFIA was performed in an indirect competitive mode. The detection method of TRFIA was compared with indirect competitive enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). Results: The TRFIA exhibited a typical sigmoidal response for MCLR at concentrations of 0.005-50 ng/ml, with a wide quantitative range between 0.01 and 10 ng/ml, indicating the broadest detective range and the most sensitive of all the methods for microcystins (MCs) detection. Additionally, the TRFIA maintained good reliability through its quantitative range, as evidenced by low coefficients of variation (1.6-12.2%). The toxin data of algal samples assayed from TRFIA were in the same range as those with ELISA and HPLC, implying that the method was reliable and practical for the detection of MCs. Conclusions: The TRFIA may offer a valuable alternative or a substitute for conventional ELISA for microcystin detection. (C) 2004 Elsevier B.V. All rights reserved.

Dynamics of microcystins-LR and -RR in the phytoplanktivorous silver carp in a sub-chronic toxicity experiment

A sub-chronic toxicity experiment was conducted to examine tissue distribution and depuration of two microcystins (microcystin-LR and microcystin -RR) in the phytoplanktivorous filter-feeding silver carp during a course of 80 days. Two large tanks (A, B) were used, and in Tank A, the fish were fed naturally with fresh Microcystis viridis cells (collected from a eutrophic pond) throughout the experiment, while in Tank B, the food of the fish were M. viridis cells for the first 40 days and then changed to artificial carp feed. High Performance Liquid Chromatography (HPLC) was used to measure MC-LR and MC-RR in the M. viridis cells, the seston, and the intestine, blood, liver and muscle tissue of silver carp at an interval of 20 days. MC-RR and MC-LR in the collected Microcystis cells varied between 268-580 and 110-292 mug g(-1) DW, respectively. In Tank A, MC-RR and MC-LR varied between 41.5-99.5 and 6.9-15.8 mug g(-1) DW in the seston, respectively. The maximum MC-RR in the blood, liver and muscle of the fish was 49.7, 17.8 and 1.77 mug g(-1) DW, respectively. No MC-LR was detectable in the muscle and blood samples of the silver carp in spite of the abundant presence of this toxin in the intestines (for the liver, there was only one case when a relatively minor quantity was detected). These findings contrast with previous experimental results on rainbow trout. Perhaps silver carp has a mechanism to degrade MC-LR actively and to inhibit MC-LR transportation across the intestines. The depuration of MC-RR concentrations occurred slowly than uptakes in blood, liver and muscle, and the depuration rate was in the order of blood > liver > muscle. The grazing ability of silver carp on toxic cyanobacteria suggests an applicability of using phytoplanktivorous fish to counteract cyanotoxin contamination in eutrophic waters. (C) 2003 Elsevier Ltd. All rights reserved.

Responses of antioxidant systems in the hepatocytes of common carp (Cyprinus carpio L.) to the toxicity of microcystin-LR

The freshwater, bloom-forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin, which causes the damage of animal liver. Recently, toxic Microcystis blooms frequently occur in the eutrophic Dianchi Lake (300 km(2) and located in the South-Westem of China). Microcystin-LR from Microcystis in Dianchi was isolated and purified by high performance liquid chromatography (HPLC) and its toxicity to mouse and fish liver was studied (Li et al., 2001). In this study, six biochemical parameters (reactive oxygen species, glutathione, superoxide dismutase, catalase, glutathione peroxide and glutathione S-transferase) were determined in common carp hepatocytes when the cells were exposed to 10 mug microcystin-LR per litre. The results showed that reactive oxygen species (ROS) contents increased by more than one-time compared with the control after 6 h exposure to the toxin. In contrast, glutathione (GSH) levels in the hepatocytes exposed to microcystin-LR decreased by 47% compared with the control. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GSH-Px) increased significantly after 6 h exposure to microcystin-LR, but glutathione S-transferase (GST) activity showed no difference from the control. These results suggested that the toxicity of microcystin-LR caused the increase of ROS contents and the depletion of GSH in hepatocytes exposed to the toxin and these changes led to oxidant shock in hepatocytes. Increases of SOD, CAT and GSH-Px activities revealed that these three kinds of antioxidant enzymes might play important roles in eliminating the excessive ROS. This paper also examined the possible toxicity mechanism of microcystin-LR on the fish hepatocytes and the results were similar to those with mouse hepatocytes. (C) 2003 Elsevier Science Ltd. All rights reserved.

Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples

Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used.

Changes in culture growth dynamics and cell size in a Scenedesmus quadricauda upon action of potassium dichromate

The effect of potassium dichromate in concentrations of 0.5 to 10 mg/l on a laboratory culture of Sc. quadricauda algae was studied in standard conditions. The total cell numbers decreased at potassium dichromate concentrations over 1 mg/l, and the proportion of living cells decreased at all studied concentrations. A positive correlation was found between changes in cell size and their numbers at toxin concentrations of 1 and 3 mg/l, and a negative correlation was found between the relative size and the cell numbers at 3 and 10 mg/l. This may be due to different intensity of growth inhibition and cell division under the influence of the toxin. The culture sensitivity to the toxin increased in autumn and decreased in the spring.

CYCLIC PEPTIDE HEPATOTOXINS FROM FRESH-WATER CYANOBACTERIAL (BLUE-GREEN-ALGAE) WATERBLOOMS COLLECTED IN CENTRAL CHINA

Toxic cyanobacteria (blue-green algae) waterblooms have been found in several Chinese water bodies since studies began there in 1984. Waterbloom samples for this study contained Anabaena circinalis, Microcystis aeruginosa and Oscillatoria sp. Only those waterblooms dominated by Microcystis aeruginosa were toxic by the intraperitoneal (i.p.) mouse bioassay. Signs of poisoning were the same as with known hepatotoxic cyclic peptide microcystins. One toxic fraction was isolated from each Microcystis aeruginosa sample. Two hepatotoxic peptides were purified from each of the fractions by high-performance liquid chromatography and identified by amino acid analysis followed by low and high resolution fast-atom bombardment mass spectrometry (FAB-MS). LD50 i.p. mouse values for the two toxins were 245-mu-g/kg (Toxin A) and 53-mu-g/g (Toxin B). Toxin content in the cells was 0.03 to 3.95 mg/g (Toxin A) and 0.18 to 3.33 mg/kg (Toxin B). The amino acid composition of Toxin A was alanine [1], arginine [2], glutamic acid [1] and beta-methylaspartic acid [1]; for Toxin B it was the same, except one of the arginines was replaced with a leucine. Low- and high-resolution FAB-MS showed that the molecular weights were 1,037 m/z (Toxin A) and 994 m/z (Toxin B), with formulas of C49H76O12N13 (Toxin A) and C49H75O12N10 (Toxin B). It was concluded that Toxin A is microcystin-RR and Toxin B is microcystin-LR, both known cyclic heptapeptide hepatotoxins isolated from cyanobacteria in other parts of the world. Sodium borohydride reduction of microcystin-RR yielded dihydro-microcystin-RR (m/z = 1,039), an important intermediate in the preparation of tritium-labeled toxin for metabolism and fate studies.

A switch-on mechanism to activate maize ribosome-inactivating protein for targeting HIV-infected cells

Maize ribosome-inactivating protein (RIP) is a plant toxin that inactivates eukaryotic ribosomes by depurinating a specific adenine residue at the a-sarcin/ricin loop of 28S rRNA. Maize RIP is first produced as a proenzyme with a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP with full N-glycosidase activity. This naturally occurring switch-on mechanism provides an opportunity for targeting the cytotoxin to pathogen-infected cells. Here, we report the addition of HIV-1 protease recognition sequences to the internal inactivation region and the activation of the maize RIP variants by HIV-1 protease in vitro and in HIV-infected cells. Among the variants generated, two were cleaved efficiently by HIV-1 protease. The HIV-1 protease-activated variants showed enhanced N-glycosidase activity in vivo as compared to their un-activated counterparts. They also possessed potent inhibitory effect on p24 antigen production in human T cells infected by two HIV-1 strains. This switch-on strategy for activating the enzymatic activity of maize RIP in target cells provides a platform for combating pathogens with a specific protease.

白喉毒素-血管内皮生长因子突变体融合毒素的研究

本研究旨在利用突变后的血管内皮生长因子（VEGF）与白喉毒素（DT）构建靶向毒素，使其特异性地杀伤肿瘤血管，达到阻断血管增生，切断肿瘤的血液供应的目的。首先通过基因工程技术从白喉杆菌中提取基因组DNA，扩增出其中的DTC区、T区基因。并运用不同的突变方法，构建了四个VEGF的突变体。即VEGF R82A，K84A，H86A突变，VEGF D63A，E64A，E67A突变，和截去了肝素结合区和NP-l（neuropilin-l）受体结合区的VEGF R82A，K84A，H86A突变和VEGF D63A，E64A，E67A突变。以这些突变体的编码基因与DT的T区、C区基因制成四个融合基因，并在大肠杆菌中表达、纯化、复性。以相似条件下制备的不加VEGF突变体的DTT区、C区蛋白为阴性对照，以VEGFR-l＋和VEGFR-2＋的Lovo细胞和VEGFR-l＋VEGFR-2-的SMMC-7721细胞做细胞实验。制成的四个融合毒素中有三个通过细胞学检验，具有靶向杀伤作用。特异性结合VEGFR-1受体的蛋白PmV8D、PsmVSD既可以抑制VEGFR-1+、VEGFR-2-的肝癌细胞SMMC-7721又可以抑制VEGFR-1+、VEGFR-2＋的结肠癌细胞Lovo。而不带VEGF突变体的DT391无抑制作用。特异性结合VEGFRZ受体的蛋白PmV6D可以抑制VEGFR-1+、VEGFR-2＋结肠癌细胞Lovo的生长，但不可以抑制VEGFR-1＋，VEGFR-2-的肝癌细胞SMMC-7721。受体结合力过弱的PsmV6D不对细胞的生长产生任何影响。

Spatial variability in water quality and surface sediment diatom assemblages in a complex lake basin,lake of the woods,ontario,canada

Lake of the Woods (LOW) is an international waterbody spanning the Canadian provinces of Ontario and Manitoba, and the U.S. state of Minnesota. In recent years, there has been a perception that water quality has deteriorated in northern regions of the lake, with all increase in the frequency and intensity of toxin-producing cyanobacterial blooms. However, given the lack of long-term data these trends are difficult to verify. As a first step, we examine spatial and seasonal patterns in water quality in this highly complex lake on the Canadian Shield. Further, we examine surface sediment diatom assemblages across multiple sites to determine if they track within-take differences in environmental conditions. Our results show that there are significant spatial patterns in water quality in LOW. Principal Component Analysis divides the lake into three geographic zones based primarily on algal nutrients (i.e., total phosphorus, TP), with the highest concentrations at sites proximal to Rainy River. This variation is closely tracked by sedimentary diatom assemblages, with [TP] explaining 43% of the variation in diatom assemblages across sites. The close correlation between water quality and the surface sediment diatom record indicate that paleoecological models could be used to provide data on the relative importance of natural and anthropogenic sources of nutrients to the lake.

杀伤血管内皮生长因子受体1阳性细胞的靶向毒素

白喉毒素(diphtheria toxin DT) 是棒状白喉杆菌被茁噬菌体感染后分泌的一种外毒素. 它可以阻断真核细胞的蛋白质合成,杀死细胞. 血管内皮生长因子(VEGF) 的R82A,K84A,H86A突变体可以和肿瘤血管上高表达的VEGF受体1 (VEGFR-1) 特异性结合. 首先从白喉杆菌中提取基因组DNA,扩增出白喉毒素C区、T区基因. 并运用点突变技术,制成VEGF的R82A,K84A,H86A突变体. 利用这个可以和肿瘤血管上特异性受体相结合的VEGF的突变体,代替白喉毒素上的受体结合区,制成了针对VEGFR-1的靶向融合毒素——DT391-mVEGF. 以去除了受体结合区的DT391为阴性对照,细胞实验表明,融合毒素对VEGFR-1阳性的肿瘤细胞有特异性杀伤作用.