Suppressed apoptosis in the inflamed gastric mucosa of Helicobacter pylori-colonized iNOS-knockout mice

Deregulated cell turnover in Helicobacter pylori (H. pylori)-colonized gastric mucosa has been suggested to be linked to the gastric carcinogenesis pathway. We previously reported attenuation of apoptosis and enhancement of cellular proliferation in the H. pylori-colonized gastric mucosa of Mongolian gerbils as compared to that in mice, which might reflect a specific link between H. pylori colonization and carcinogenesis in the Mongolian gerbils; the difference between the two strains could be attributable to differences in the host genetic background. Inducible-type nitric oxide synthase (iNOS) is thought to participate in not only the inflammatory response, but also in the regulation of gastric mucosal cell turnover in H. pylori-colonized gastric mucosa. Thus, the present study was designed to examine gastric leukocyte activation and epithelial cell apoptosis in the gastric mucosa following H. pylori inoculation in iNOS-knockout mice. Methods: iNOS-knockout mice (iNOS^−/−) and their iNOS^+/+ littermates were orally inoculated with the Sydney strain of H. pylori (SS1, 10⁸ colony-forming units [CFU]). H. pylori infection was confirmed by microaerobic bacterial culture. The stomach of each mouse was evaluated 14 weeks and 30 weeks after the inoculation. Gastric mucosal accumulation of polymorphonuclear leukocytes (PMN) was assessed by determining the myeloperoxidase (MPO) activity and histological score based on the updated Sydney system. The level of apoptosis was determined by estimation of the cytoplasmic levels of mono- and oligonucleosomes and by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. Results: The SS1-inoculated mice showed persistent H. pylori colonization for 12 weeks. While gastric mucosal PMN infiltration increased following SS1 inoculation in both iNOS^+/+ and iNOS^−/−strains, enhanced DNA fragmentation was observed in only SS1-colonized iNOS^+/+ mice, and not in the iNOS^−/− mice. In conclusion, although the recruitment of PMN in response to H. pylori was evoked even in the gastric mucosa of iNOS^−/− mice, epithelial cell apoptosis induced by H. pylori was attenuated in this strain. These data suggest that iNOS may play an important role in promoting apoptosis in the H. pylori-infected inflamed gastric mucosa, and that persistent inflammation without apoptosis in iNOS^−/− mice with H. pylori infection may be linked to preneoplastic transformation.     

The role of neutrophils and inflammation in gastric mucosal injury

Gastric inflammation is a highly complex biochemical protective response to cellular/tissue injury. When this process occurs in an uncontrolled manner, the result is excessive cellular/tissue damage that results chronic inflammation and destruction of normal tissue. Current evidence suggests that Helicobacter pylori (H. pylori) infection and nonsteroidal anti-inflammatory drug (NSAID) ingestion are major causative factors in the pathogenesis of gastric mucosal injury in humans. In response to H. pylori infection or NSAID, neutrophils are recruited to the site of inflammation and generate reactive oxygen and nitrogen species and proteases. However, neutrophils are not able to kill the bacteria that live in the gastric mucus, and compounds produced by activated neutrophils themselves may be potentially harmful for normal tissue. It has been shown that leukocyte-vascular endothelial cell interaction is regulated by various cell adhesion molecules, and that this interaction is directly or indirectly modified by many factors, the origin of which is H. pylori and NSAIDs. This review describes the potential role of neutrophils and neutrophil-associated inflammation for gastric oxidative stress and injury induced by H. pylori and/or NSAID.     

Antimicrobial activity of aqueous extracts of Larrea divaricata Cav (jarilla) against Helicobacter pylori

We studied here the effect of aqueous extracts of Larrea divaricata Cav on the growth of Helicobacter pylori. Results show that cold extract, infusion, decoction and simulated digestion had inhibitory activity at 0.04–0.1 mg/l against clarithromycin and metronidazole susceptible and resistant H. pylori strains. These results support the popular use of L. divaricata Cav in gastric disturbances and prompt further research to characterize these compounds with a therapeutic potential against gastric ulcers and gastric cancer associated with H. pylori.     

Mutation as an origin of genetic variability in Helicobacter pylori

The availability of two complete Helicobacter pylori genome sequences and recent studies of its population genetics have provided a detailed picture of genetic diversity in this important human gastric pathogen. It is believed that, in addition to genetic recombination, de novo mutation could have a role in generating the high level of genetic variation in H. pylori.     

The distribution of 4-hydroxynonenal-modified proteins in gastric mucosa of duodenal peptic ulcer patients

This study used monoclonal antibody specific for 4-hydroxynonenal (HNE)-histidine to evaluate immunohistochemical distribution of HNE-protein adducts in gastric mucosa biopsies of 52 peptic ulcer patients (all positive for H. pylori) and of 20 healthy volunteers (eight positive and 12 negative for H. pylori). HNE-modified proteins were present in glandular epithelium in all subjects, both patients with duodenal peptic ulcer and healthy subjects. Hence, the presence of HNE did not appear to be related to the presence of H. pylori. However, in patients with duodenal peptic ulcer accumulation of HNE-protein adducts was frequently observed also in nuclei, while in the control group such subcellular distribution of HNE was not observed at all. This study shows physiological presence of HNE in human gastric mucosa, but also suggests its role in pathology of gastric dysfunction in duodenal peptic ulcer patients manifested by accumulation of HNE-protein adducts in particular in nuclei of gastric glandular epithelium.     

Why Helicobacter pylori has Lewis antigens

In mimicry with human gastric epithelial cells, the lipopolysaccharide of Helicobacter pylori expresses Lewis blood group antigens. Recent data suggest that molecular mimicry does not promote immune evasion, nor does it lead to induction of autoantibodies, but that H. pylori Lewis X mediates adhesion to gastric epithelial cells and is essential for colonization.     

Oxidative stress defense mechanisms to counter iron-promoted DNA damage in Helicobacter pylori

Iron, a key element in Fenton chemistry, causes oxygen-related toxicity to cells of most living organisms. Helicobacter pylori is a microaerophilic bacterium that infects human gastric mucosa and causes a series of gastric diseases. Exposure of H. pylori cells to air for 2 h elevated the level of free iron by about 4-fold as measured by electron paramagnetic resonance spectroscopy. H. pylori cells accumulated more free iron as they approached stationary phase growth, and they concomitantly suffered more DNA damage as indicated by DNA fragmentation analysis. Relationships between the intracellular free iron level, specific oxidative stress enzymes, and DNA damage were identified, and new roles for three oxidative stress-combating enzymes in H. pylori are proposed. Mutant cells defective in either catalase (KatA), in superoxide dismutase (SodB) or in alkyl hydroperoxide reductase (AhpC) were more sensitive to oxidative stress conditions; and they accumulated more free (toxic) iron; and they suffered more DNA fragmentation compared to wild type cells. A significant proportion of cells of sodB, ahpC, or katA mutant strains developed into the stress-induced coccoid form or lysed; they also contained significantly higher amounts of 8-oxo-guanine associated with their DNA, compared to wild type cells.     

幽门螺杆菌多价表位疫苗CWAE的表达及其免疫学性质的研究 *

目的 利用生物信息学软件评价幽门螺杆菌(Helicobacter pylori)多价表位疫苗CWAE的抗原结构,经原核表达获得高纯度CWAE蛋白,进而鉴定多价表位疫苗CWAE的免疫学性质。方法 通过生物信息学软件分析H. pylori多价表位疫苗CWAE的抗原结构;用人工合成的H. pylori多价表位肽融合基因WAE替换重组质粒pET28a-CUE中的UE基因,构建重组质粒pET28a-CWAE。然后,将pET28a-CWAE转入大肠杆菌BL21(DE3)中,经IPTG诱导表达,并通过Ni-NTP镍离子亲和层析纯化抗原蛋白CWAE;利用GM1-ELISA鉴定CWAE中CTB组分的黏膜免疫佐剂活性。最后,通过ELISA和小鼠脾脏淋巴细胞增殖实验检测CWAE激发BALB/c小鼠产生抗H. pylori抗体体液免疫和淋巴细胞免疫应答的能力。结果 通过生物信息学软件证实H. pylori多价表位疫苗CWAE具有科学合理的结构;重组表达质粒pET28a-CWAE经PCR、双酶切和基因测序鉴定,融合基因CWAE与设计序列完全一致;重组基因工程菌株pET28a-CWAE/BL21(DE3)经IPTG诱导表达,抗原蛋白CWAE主要以包涵体形式存在,经Ni-NTP镍离子亲和层析纯化,纯度约达93.2%;GM1-ELISA实验证实,CWAE中CTB组分依旧保持有较好的黏膜佐剂活性;ELISA结果证实CWAE能够激发BALB/c小鼠产生H. pylori特异性抗体,而小鼠脾脏淋巴细胞增殖实验进一步证实CWAE能够激发针对H. pylori多种致病因子的淋巴细胞免疫反应。结论 H. pylori多价表位疫苗CWAE具有科学合理的抗原结构,经原核表达可获得高纯度抗原蛋白,能够激发BALB/c小鼠产生H. pylori特异性抗体体液免疫和淋巴细胞免疫应答。为研发防治H. pylori感染的多价表位疫苗奠定实验基础。     

Contribution of ferrous iron to maintenance of the gastric colonization of Helicobacter pylori in miniature pigs

Our previous study showed that the colonization levels of Helicobacter pylori were higher in the stomachs of 5-day-old miniature pigs than in 2-week-old ones. As dietary factors can cause these differences, we compared two diets, i.e., Weanymilk and a similar formula with a higher concentration of Fe(II), Weanylobulin. The colonization levels in the fundic mucosa were significantly higher in 2-week-old pigs fed Weanylobulin than in those fed Weanymilk. Supplementing Weanylobulin with an iron chelator, deferoxamine mesylate, significantly lowered the bacteria counts in the gastric mucosa. Normal diets supplemented with Fe(II) in 2-month-old pigs caused significantly more sites of bacteria in the antrum compared with normal diets alone. In addition, ranitidine, an inhibitor of gastric acid secretion that reduces Fe(III) to Fe(II) in the stomach, decreased the bacteria counts in 10-month-old pigs. These results suggested that Fe(II) maintained the colonization levels of H. pylori in the stomach of the miniature pigs.     

Cross-talk of NO, superoxide and molecular oxygen, a majesty of aerobic life

Because nitric oxide (NO) reacts with various molecules, such as hemeproteins, superoxide and thiols including glutathione (GSH) and cysteine residues in proteins, biological effects and metabolic fate of this gaseous radical are affected by these reactants. Although the lifetime of NO is short particularly under air atmospheric conditions (where the oxygen tension is unphysiologically high), it increases significantly under physiologically low oxygen concentrations. Because oxygen tensions in human body differ from one tissue to another and change depending on their metabolism, biological activity of NO in various tissues might be affected by local oxygen tensions. To elucidate the role of NO and related radicals in the regulation of circulation and energy metabolism, their effects on arterial resistance and energy metabolism in mitochondria, mammalian cells and enteric bacteria were studied under different oxygen tensions. Kinetic analysis revealed that NO-dependent generation of cGMP in resistance arteries and their relaxation were strongly enhanced by lowering oxygen tensions in the medium. NO reversibly suppressed the respiration and ATP synthesis of isolated mitochondria and intact cells particularly under low oxygen tensions. Kinetic analysis revealed that cross-talk between NO and superoxide generated in and around endothelial cells regulates arterial resistance particularly under physiologically low oxygen tensions. NO also inhibited the respiration and ATP synthesis of E. coli particularly under low oxygen tensions. Because concentrations of NO and H⁺ in gastric juice are high, most ingested bacteria are effectively killed in the stomach. However, the inhibitory effects of NO on the respiration and ATP synthesis of H. pylori are extremely small. Kinetic analysis revealed that H. pylori generates the superoxide radical thereby inhibiting the bactericidal action of NO in gastric juice. Based on such observations, critical roles of the cross-talk of NO, superoxide and molecular oxygen in the regulation of energy metabolism and survival of aerobic and microaerophilic organisms are discussed.     

幽门螺杆菌致病岛CagL重组抗原的可溶性表达及其多克隆抗体的制备和分析*

目的:利用原核表达和蛋白质纯化技术获得高纯度的幽门螺杆菌致病岛CagL重组抗原(rCagL),利用其制备anti-CagL多克隆抗体,并分析抗体的特异性。方法:通过生物信息学软件分析rCagL的抗原结构;利用PCR长片段DNA合成技术合成不含有信号肽序列的幽门螺杆菌致病岛CagL基因,将其插入表达质粒pCzn1中,构建重组质粒pCzn1-rCagL。然后,将pCzn1-rCagL转入大肠杆菌Arctic Express中,经IPTG诱导表达后,通过Ni-IDA镍离子亲和层析纯化重组抗原rCagL,利用Western blot鉴定rCagL与His标签抗体和Anti-H. pylori抗体的免疫反应性;最后,通过rCagL辅以弗氏佐剂免疫BALB/c小鼠,制备anti-CagL多克隆抗血清,通过ELISA方法分析抗血清的特异性。结果:生物信息学软件表明重组抗原rCagL具有较好的抗原性质;重组质粒pCzn1-rCagL经双酶切和基因测序等技术鉴定,证实rCagL核苷酸序列与理论序列完全一致;基因工程菌株pCzn1-rCagL/Arctic Express在低温11℃条件经IPTG诱导表达。 SDS-PAGE实验结果证实:rCagL可实现相对高效地可溶性蛋白表达,可溶性蛋白约占包涵体的62.07%。经Ni-IDA亲和层析柱纯化,可获得高纯度rCagL,纯度约为96.6%。Western blot结果证实:重组抗原rCagL可特异性与His标签抗体和Anti-H. pylori抗体结合。ELISA结果证实:经rCagL免疫小鼠制备的多克隆抗体anti-CagL可特异性识别rCagL和H. pylori裂解物,具有较高的抗体特异性。结论:重组抗原rCagL在低温条件下可实现可溶性表达,经纯化可获得高纯度抗原蛋白;rCagL具有较好的抗原性,制备的多克隆抗体具有较好的免疫特异性,为发展H. pylori相关诊断试剂奠定了实验基础。     

Up-expression of NapA and other oxidative stress proteins is a compensatory response to loss of major Helicobacter pylori stress resistance factors

Twenty-six Helicobacter pylori targeted mutant strains with deficiencies in oxidative stress combating proteins, including 12 double mutant strains were analyzed via physiological and proteomic approaches to distinguish the major expression changes caused by the mutations. Mutations were introduced into both a Mtz^S and a Mtz^R strain background. Most of the mutations caused increased growth sensitivity of the strains to oxygen, and they all exhibited clear compensatory up-expression of oxidative stress resistance proteins enabling survival of the bacterium. The most frequent up-expressed oxidative stress resistance factor (observed in 16 of the mutants) was the iron-sequestering protein NapA, linking iron sequestration with oxidative stress resistance. The up-expression of individual proteins in mutants ranged from 2 to 10 fold that of the wild type strain, even when incubated in a low O₂ environment. For example, a considerably higher level of catalase expression (4 fold of that in the wild-type strain) was observed in ahpC napA and ahpC sodB double mutants. A Fur mutant up-expressed ferritin (Pfr) protein 20-fold. In some mutant strains the bacterial DNA is protected from oxidative stress damage apparently via overexpression of oxidative stress-combating proteins such as NapA, catalase or MdaB (an NADPH quinone reductase). Our results show that H. pylori has a variety of ways to compensate for loss of major oxidative stress combating factors.     

Isolation and characterization of Histophilus somni (Haemophilus somnus) in semen samples of rams with epididymitis

Histophilus somni (Haemophilus somnus) has been reported as the cause of epididymitis in rams. This bacterium has also been found in the preputial mucosa of rams without epididymitis lesions. H. somni is a bacterium that is difficult to characterize, since it is a pleomorphic Gram-negative bacilli of characteristics similar to Actinobacillus seminis, which is also found in ram epididymitis lesions. The objective of this work was to determine if H. somni (H. somnus) is involved in cases of sheep epididymitis. A clinical examination was performed in 160 rams, extracting semen by electro-ejaculation of 28 of them, which had epididymal lesions. The penis was exteriorized in order to avoid prepuce contamination. The semen samples were cultivated in chocolate agar in a 10% CO₂ environment. Two strains were isolated in pure culture with a colony morphology and microscopy similar to H. somni (H. somnus). These were identified using the API 20 E system, using as a control the reference strain of H. somnus (2336ATCC). One of the isolates (129H) resulted identical to the reference strain and the other (827) presented differences in the arginine decarboxylase, H₂S, catalase and inositol reactions, although these differences have been reported (in strains isolated from different geographic origins, animal species and anatomical region). To characterize the isolates, an electrophoretic analysis of total proteins was performed (PAGE–SDS) finding identical profiles between the reference strain of H. somnus and isolate 129H and similar in relation to isolate 827. The amplification of a fragment of approximately 407 bp was observed in the 129H isolate and the ATCC strain, but not in 827. In other samples, isolations were made of Brucella ovis, Corynebacterium spp., Staphylococcus and other pleomorphic Gram-negative bacilli similar to A. seminis. Therefore, it has been confirmed that H. somni is present in the reproductive tract of rams and it could be involved in the presentation of ovine epididymitis. It is important that we underline that this is the first report of H. somni isolation in Mexico from ram semen samples.     

NBA1/MERIT40在3种沙棘中的适应性分化研究——兼论二代测序技术在杂种鉴定中的应用

随着高通量测序技术的发展，从"组学"水平研究适应性进化分子机制成为进化生物学的研究热点。本研究以沙棘属的中国沙棘、肋果沙棘及二者的同倍化杂交物种棱果沙棘为材料，通过转录组测序，筛选出了受到正选择（w>1）的BRCA1-A&BRISC复合体亚基NBA1基因。生物信息学分析表明，NBA1基因在3种沙棘中编码区全长为771 bp，共编码256个氨基酸，为核定位的亲水性蛋白。中国沙棘与肋果沙棘中NBA1的氨基酸序列在218与236位存在差异，中国沙棘中靠近结合位点的218位突变为甲硫氨酸，不同于肋果沙棘及其他陆生植物中NBA1亚基VWA结构域中保守的亮氨酸，导致二者的蛋白质三级结构发生明显变化，且使中国沙棘与肋果沙棘中该蛋白结合位点的空间排布产生显著差异。综上，推测二者的NBA1亚基在结合其他亚基方面有所不同，主要导致了BRCA1-A复合体修复紫外辐射引起的DNA损伤修复功能方面出现差异，这可能与中国沙棘与肋果沙棘对不同海拔环境的适应有关。此外，Sanger测序验证了二代测序结果的准确性，证明该基因在亲本种中国沙棘与肋果沙棘中是纯合的单拷贝，在杂种棱果沙棘中以共显性的杂合方式存在。进一步分析发现第二代测序数据不适用于在个体水平鉴定杂种，但群体内多个个体的平行测序一定程度上可随机反映杂种的亲本来源。本研究为进一步揭示沙棘属植物对不同海拔的适应分子机制提供了参考数据，同时为利用转录组测序方法鉴定杂交物种提出了建议。     

中国西北地区萱草属花粉形态研究

本文借助光学显微镜和扫描电镜,对我国西北地区萱草属6种1变种的花粉形态进行了比较研究,不同的种类,其花粉形态或多或少有所不同,说明花粉形态在种间具有分类意义,而且6种、1变种可自然地分为两大类群,第一类群包括小黄花菜、北萱草、黄花菜,第二类群包括北黄花菜、折叶萱草、萱草、重瓣萱草。#br#北黄花菜、小黄花菜的花粉形态差别较大,证明把小黄花菜做为北黄花菜的变种是不妥当的。     

Correlation of self- and interspecific incompatibility among sympatric Hedyotis species (Rubiaceae) and consequences for hybridization

Breakdown of self-incompatibility increases opportunities for both self-fertilization and interspecific hybridization, although the latter is dependent on the extent of competition between heterospecific and conspecific pollen. We investigate the mating system and pollination biology of five phylogenetically closely related species within a distylous species complex in Hedyotis L. (Rubiaceae) in southern China. The complex comprises Hedyotis acutangula Champ. ex Benth., Hedyotis shiuyingiae T.Chen, Hedyotis vachellii (Hook. & Arn.) Kuntze, and two putative hybrid species, Hedyotis bodinieri (H.Lév.) Chun and Hedyotis loganioides Benth., hypothesized to result from interbreeding between these species. We test the hypothesis that the breakdown of self- and interspecific incompatibilities in sympatric Hedyotis species might allow interspecific hybridization in natural populations. We assessed the extent of self- and interspecific incompatibility in sympatric populations, including investigations of spontaneous and artificial self-pollination, geitonogamy, inter- and intramorph xenogamy. Artificial interspecific crosses were undertaken between H. acutangula, H. shiuyingiae, and H. bodinieri, between H. acutangula and H. vachellii, and between H. acutangula and H. loganioides. Hedyotis acutangula is demonstrated to be self- and interspecific compatible, whereas H. vachellii, H. bodinieri, and H. loganioides are self-compatible and interspecific incompatible; H. shiuyingiae, in contrast, is strictly self- and interspecific incompatible. Comparisons of pollen tube growth rates in hybridizing species-pairs revealed that heterospecific pollen of H. shiuyingiae, H. vachellii, and H. bodinieri can compete with conspecific self-pollen of H. acutangula. Our study therefore indicates that the breakdown of self-incompatibility directly and indirectly facilitates interspecific hybridization and provides a platform for better understanding evolutionary directionality in Hedyotis.     

Lumen phase specific cross immunity between Hymenolepis microstoma and H. nana in mice

, and 1988. Lumen phase specific cross immunity between Hymenolepis microstoma and H. nana in mice. International Journal for Parasitology 18: 1019–1027. When mice inoculated with five cysticercoids of Hymenolepis microstoma were challenged with H. nana, they showed strong resistance to challenges with both eggs and cysticercoids of H. nana from day 20. The immunity became complete from day 30 onward: no tissue cysticercoids or lumenal adults of H. nana were established in these mice. However, when mice were challenged with H. nana 10 or 20 days after 10-day old immature H. microstoma were removed by an anthelmintic, the immunity evoked was directed exclusively against the lumenal phase of the cysticercoid challenge but not the tissue cysticercoids of the egg challenge. When mice experienced the prepatent infection with H. microstoma twice, the immunity evoked was also against the lumenal phase of the egg challenge: the oncospheres developed into tissue cysticercoids but thereafter completely failed to develop into lumenal adult tapeworms. Infection with a single cysticercoid of H. microstoma was shown to be sufficient to evoke immunity against H. nana cysticercoid infections in two strains of mice. Sera from mice which experienced a patent infection with H. microstoma revealed that IgG, IgA, IgM isotypes reacted against oncospheres and cysticercoids of both species, while sera from mice which experienced two prepatent infections reacted with cysticercoids only. Sera from H. microstoma infected mice resistant to H. nana caused precipitations on 4-day-old H. nana in vitro. A correlation exists between the presence of stage specific antibodies and resistance to the different stages.     

Differential detection of Hammondia hammondi from Toxoplasma gondii using polymerase chain reaction

Hammondia hammondi and Toxoplasma gondii are two related coccidian parasites, with cats as definitive hosts and warm-blooded animals as intermediate hosts. It is difficult to differentiate them by morphological and serological parameters. In the present study, primers were designed to specifically amplify the ITS-1 region of H. hammondi to differentiate it from T. gondii. Attempts were made to detect the presence of H. hammondi DNA in the tissues of mice infected with H. hammondi alone, as well as from mixed infections with T. gondii, using the newly designed primers. The de novo primers effectively amplified the H. hammondi-specific target fragment from all samples containing H. hammondi, including those with concomitant T. gondii infection. Further, the primers did not amplify any fragment from the related parasites like T. gondii, Neospora caninum and Hammondia heydorni. The new primers provide simple and efficient means to differentially diagnose H. hammondi from T. gondii even in samples containing both parasites, thus obviating the need for other labourious techniques like mouse bioassay and in vitro cultivation.     

Enhanced liver cell mutations in trematode-infected Big Blue transgenic mice

Parasite infections in humans have long been associated with specific types of cancers. Schistosoma hematobium is a known inducer of urinary bladder cancer, Helicobacter pylori is a gastric carcinogen, and hepatitis B virus and Opisthorchis viverrini are causative agents of liver cell cancers. Another liver fluke, Fasciola hepatica, has also been identified as a neoplastic risk agent, primarily in animals. We used F. hepatica as a model agent to determine if the presence of an aggressive liver fluke could induce mutagenic events in mammalian tissue. Using the Big Blue^® transgenic mouse assay, we found a two-fold increase in lacI mutations in cells harvested from mice harboring F. hepatica worms when compared to uninfected control animals. These data indicate that biological infections can cause increased genetic damage in surrounding host tissue.     

A new simple screening method for the detection of cytotoxic substances produced by harmful red tide phytoplankton

A highly sensitive, but simple and quantitative, cytotoxic assay method for the detection of toxic substances produced by red tide phytoplankton was developed by utilizing Vero cells which were the most resistant to seawater among the six cell lines tested. Heterocapsa circularisquama, which is known to be highly toxic to shellfish, showed cytotoxicity to Vero cells in a cell-density dependent manner when Vero cells were directly exposed to the cell suspension of H. circularisquama in seawater-based plankton culture medium, whereas Heterocapsa triquetra, which is morphologically similar to H. circularisquama but non-toxic to shellfish, showed no cytotoxic effect. Since the potent cytotoxicity was also detected in the cell-free culture supernatant of H. circularisquama, it was suggested that a certain cytotoxic substance is extracellularly secreted by H. circularisquama. Furthermore, by this direct exposure method, we found that Alexandrium fraterculus, Alexandrium tamiyavanichii, Alexandrium tamarense, and Alexandrium affine but not Alexandrium taylorii and Alexandrium catenella cause toxic effect on Vero cells with different extent depending on species. By gel-filtration and subsequent two cytotoxicity assays using Vero and mouse neuroblastoma cell line (Neuro-2a), we found that high molecular weight cytotoxic substance distinct from paralytic shellfish poisoning toxins is present in the aqueous extract of A. tamarense. These results suggest that our 96-well microplate cytotoxicity assay using Vero cells is useful not only as a primary screening assay for the detection of potential toxic activity of harmful phytoplankton but also as a quantitative routine toxicity assay for following the active substances during the extraction and purification processes.