76 resultados para killer toxin
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
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.
Resumo:
目前多数学者认为树鼩(Tree shrews)是灵长目攀居目树鼩科的一种小型哺乳动物。因其具有体型小、易饲养、价格低、新陈代谢和解剖结构与人类非常接近等优点,树鼩已经成为多种人类疾病研究的良好动物模型。树鼩作为乙型肝炎病毒(HBV)感染的动物模型已经被广泛应用。 自然杀伤性T细胞(Natrual killer T cells, NKT cells)可同时表达NK细胞和T细胞的表面标记,是CD1d限制性的特殊的淋巴细胞亚群,在机体的免疫反应中起重要作用。研究表明,在HBV感染及乙型肝炎的发病过程中,NKT细胞既有抑制和清除病毒的作用,也有加重肝炎及相关病症的负作用。因此,对NKT细胞的研究及恰当地调节将有助于理解和解决在研究乙肝防治中遇到的问题,但在树鼩中,至今尚无NKT细胞的相关报道。 目前公认的检测NKT细胞的手段是用加载了α-GalCer的CD1d四聚体检测。但至今尚无树鼩特异的相关抗体,给树鼩NKT细胞的研究造成很大障碍。因此,获得树鼩特异的CD1d四聚体是研究其NKT细胞特征和功能的前提。本论文作为其中一部分,目的是克隆树鼩CD1d编码区全长序列,为今后抗体的制备奠定基础。 本论文中,我们完成了相关工作并得到了以下结果:1、首次克隆得到了树鼩CD1d基因编码区的全长序列(1002bp),并预测了其氨基酸序列;2、通过比对分析得知其与灵长类的CD1d同源性较高,达到80%以上;同时,通过系统进化树分析,得知树鼩与灵长类亲缘关系较其它物种更近;3、首次分析了树鼩的CD1d mRNA在不同组织中的表达情况,发现树鼩的肝,肺中CD1d表达量较高,外周血和脾脏中也有少量表达。 总之,我们首次克隆得到了树鼩CD1d的编码区的全长序列,为今后制备CD1d四聚体进而研究树鼩的NKT细胞奠定了基础;同时首次分析了CD1d mRNA在树鼩的部分组织中的表达情况,结合以往其它物种中CD1d组织分布情况的检测结果,为树鼩中CD1d分子的研究提供了材料和数据基础。
Resumo:
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。
Resumo:
Purpose: To estimate the biological risks to the immune system of the type of space radiation, 12C6+, encountered by cosmonauts during long-term travel in space. Materials and methods: The Kun-Ming strain mice were whole-body irradiated by 12C6+ ion with 0, 0.01, 0.05, 0.075, 0.2, 0.3, 0.5, 0.75, 1 or 2 Gy, at a dose rate of 1 Gy/min. At 35 days after irradiation, the thymus and spleen weights were measured, the natural killer (NK) cells activity of spleen was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), and the interferon-gamma (IFN-gamma) levels in serum and thymus were detected with enzyme-linked immunosorbent assays (ELISA). Results: The results showed that the thymus weight, IFN-gamma levels in serum and the activity of splenic NK-cells had significantly increased at a dose of 0.05 Gy. With further dose increase, the weight of spleen continued to increase but the weight of thymus, IFN-gamma level and NK-cells activity declined. Conclusions: These results suggest that the dose of 0.05 Gy irradiation has a stimulatory effect on mouse immunity; this effect declined with increasing dose.
Resumo:
实验目的:随着科技的发展,人类活动范围已经逐渐向外太空扩展,对于人类太空探索的最大威胁是太空中的各种粒子辐射。这些辐射包括太阳辐射(质子和电子)和银河辐射(质子占85%,氦离子占14%,重离子占1%)。众所周知,重离子与常规X和γ射线相比有较高的传能线密度(linear energy transfer, LET)和相对生物学效应(relative biological effectiveness, RBE),对机体组织和器官有较强的影响。放射治疗是肿瘤治疗的重要手段之一,由于肿瘤细胞的异质性,其对放、化疗的反应相差悬殊。本研究的目的是: 1评估辐射对健康机体产生的生物学风险; 2研究抗氧化剂氮乙酰半胱氨酸(NAC)对机体辐射损伤的保护作用 3不同肿瘤细胞辐射敏感性的差异。实验方法: 1 X射线或12C6+离子对小鼠进行不同剂量的全身辐射。NAC处理组小鼠在照射前1小时腹腔注射200mg/kg的NAC,对照组注射等体积的生理盐水。照射后不同时间点取样,利用流式细胞仪检测小鼠免疫细胞周期和凋亡情况,单细胞电泳检测淋巴细胞DNA损伤,MTT法(3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)检测脾脏NK(natural killer,NK)细胞活性,微核法检测淋巴细胞染色体损伤情况,小鼠体内干扰素-γ(Interferon-γ,IFN-γ)由ELISA方法得到,小鼠血清中超氧化物岐化酶(Surperoxide dismutase SOD)由分光光度法测定,并观察胸腺和脾脏指数变化。 2 不同剂量X射线和12C6+离子辐射人肺腺癌细胞H1299和A549,用细胞克隆法检测照射后细胞存活曲线,流式细胞仪检测细胞周期和凋亡,Western-blot 检测A549 细胞P53蛋白表达。 结果: 1小鼠外周血淋巴细胞、胸腺细胞和脾脏淋巴细胞周期随着X射线照射剂量的增大而被阻滞在了G0/G1期,相同剂量的12C6+离子辐射时外周血淋巴细胞周期被阻滞在S期,分次连续X射线照射时,外周血淋巴细胞周期随着累积剂量的增加被阻滞在G2/M期;细胞凋亡比例随着照射剂量的增加而增加。小鼠血清中IFN-γ水平和脾脏中NK细胞活性在重离子照射剂量为0.05Gy时有显著增加,脾脏NK细胞活性随着照射剂量的增加而减弱。 2重离子照射后,小鼠淋巴细胞DNA和染色体的损伤随辐射剂量和照射后时间的延长而加剧。脾脏NK细胞活性在照射后各个时间点减弱,血清中IFN-γ水平和SOD酶活性随着重离子照射剂量的增加而降低。预防性给予NAC,12C6+离子辐射对淋巴细胞DNA和染色体所致损伤,胸腺细胞周期和凋亡,脾脏NK细胞活性,血清中IFN-γ的水平和SOD酶的活性的损伤与盐水组比较均有显著改善。 3 X射线照射对肺腺癌H1299细胞周期和凋亡率未产生明显影响,重离子照射后随着照射剂量的增加细胞周期被阻滞在G2/M期,细胞凋亡率也呈剂量依赖性;X射线和12C6+离子照射A549细胞后,细胞周期均被阻滞在G2/M期,凋亡率剂量依赖性增加。A549细胞P53蛋白的表达水平随着重离子照射剂量的增加而增加。结论: 1重离子辐射造成细胞DNA和染色体损伤随着照射剂量的增加和照射后时间的延长而增加,比X射线辐射损伤复杂和难以修复,产生这种现象的机理为辐射导致活性氧分子簇的产生,细胞因子和与细胞氧化反应有关的酶活性的变化,同时这种损伤对胸腺细胞周期、凋亡和胸腺、脾脏指数以及机体免疫系统都有影响;低剂量重离子辐射(0.05Gy)对小鼠机体的免疫力有刺激作用,机体免疫能力随着照射剂量增加和照射后时间的推移而减弱,不同的免疫器官对辐射的敏感性也不同; 2 200mg/kg 的NAC对辐射所致小鼠免疫系统损伤有很好的保护作用; 3 肺腺癌细胞H1299比同系A549具有较强的辐射敏感性,A549细胞凋亡的增加与P53蛋白表达水平升高有关
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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.