一种快速提取分析微囊藻毒素的方法

以五种群体和单细胞微囊藻及自然微囊藻水华为材料,在沸水浴中经不同时间处理,过滤后直接进行HPLC—UV检测,发现12min的沸水浴处理就足以达到抽提目的。研究中发现,去离子水比蒸馏水是更有效的抽提溶剂。传统的甲醇抽提结果与沸水浴处理的相对误差主要在0．2％—16．59％之间。结果还显示,群体微囊藻需要比单细胞微囊藻抽提更长时间。本研究提供了一种经过改进的高效、廉价和快速的微囊藻毒素抽提分析方法。     

群体和单细胞微囊藻对短期高光胁迫的生理响应

为了探讨不同形态的微囊藻（Microcystis）对光的耐受能力及其应对机制,研究比较了短期高光强条件下群体微囊藻和单细胞微囊藻的生理响应,结果表明,在高光强胁迫下,群体和单细胞微囊藻的叶绿素含量、最大电子传递速率（ETR_max）均降低,但与单细胞微囊藻相比,群体微囊藻的下降幅度较小;在高光强胁迫下,群体微囊藻的过氧化氢酶（CAT）与超氧化物歧化酶（SOD）的活性均显著增加,而单细胞微囊藻只有CAT活性增加;在短期高光胁迫下,群体微囊藻的死亡率没有显著变化。这些结果表明群体微囊藻比单细胞微囊藻能耐受更高的光强,也暗示了群体微囊藻在野外高光强条件下更具竞争优势。     

微囊藻毒素LR对大鼠肝细胞Caspase-3酶活性的影响

水体富营养化的加剧,导致藻类水华频频发生。在全球由于藻类水华所产生的毒素,对人和动物都产生了极为严重的影响,使之成为日益突出的环境问题。其中危害最严重的 是微囊藻毒素(Micnocystin,MCYST)。而微囊藻毒素LR(LR)是微囊藻毒素中存在最为普遍且毒性作用最明显的一种,是MCYST的主要代表物。       

光强对微囊藻群体形态的影响及其生理机制研究

为了探讨光照对微囊藻形态的影响,研究了6株不同种的群体微囊藻在不同光强下群体形态的变化及其响应机制。研究发现,随着光强的增加,6株群体微囊藻的群体尺寸变大。当光强为80200 mol/（m2s）时,群体微囊藻DH-M1和DC-M2的比生长速率显著增大,而另4株在高光强下比生长速率无显著性差异;对多糖含量分析发现,高光强对群体微囊藻TH-M2、DC-M1、FACHB1174和FACHB1027胞外及胶被多糖的分泌与释放有显著的促进效果,而DH-M1和DC-M2多糖含量增加不明显。对于不同的微囊藻株,高光强促进群体形态变化的作用机理不同:光饱和点低的微囊藻是通过分泌大量的胞外及胶被多糖使群体尺寸变大,而光饱和点高的微囊藻是通过生长来促进群体尺寸的增大。此外,对产毒藻株在不同光强下的毒素基因表达及胞内毒素测定发现,高光强组的群体微囊藻mcyB和mcyD表达量升高,且胞内微囊藻毒素含量增加显著,推测微囊藻毒素也可能是影响微囊藻群体形态及大小的作用因子之一。       

群体和单细胞微囊藻对短期温度变化的生理响应

为了探索短期温度变化对群体微囊藻和单细胞微囊藻的影响, 在室内受控模拟条件下研究了在10℃、25℃和35℃三个温度梯度下, 群体和单细胞微囊藻对短期温度变化的生理响应。研究表明: 与对照组25℃相比, 在10℃培养下, 微囊藻叶绿素浓度显著降低, SOD活性和死亡率均显著增加。与群体微囊藻相比, 在10℃下单细胞微囊藻叶绿素浓度显著下降, F_v/F_m下降, SOD活性显著增加。在35℃培养下, 单细胞微囊藻叶绿素浓度上升, 死亡率和SOD活性增加, 而群体微囊藻则呈现出叶绿素浓度和死亡率降低, CAT活性增加。结果表明短期的温度变化影响了群体和单细胞微囊藻生理机制, 与单细胞微囊藻相比, 群体更能适应短期的温度胁迫, 导致其更具优势。     

光强对微囊藻群体形态的影响及其生理机制研究简

为了探讨光照对微囊藻形态的影响,研究了6株不同种的群体微囊藻在不同光强下群体形态的变化及其响应机制。研究发现,随着光强的增加,6株群体微囊藻的群体尺寸变大。当光强为80—200μmol/(m2·s)时,群体微囊藻DH-M1和DC-M2的比生长速率显著增大,而另4株在高光强下比生长速率无显著性差异;对多糖含量分析发现,高光强对群体微囊藻TH-M2、DC-M1、FACHB1174和FACHB1027胞外及胶被多糖的分泌与释放有显著的促进效果,而DH-M1和DC-M2多糖含量增加不明显。对于不同的微囊藻株,高光强促进群体形态变化的作用机理不同:光饱和点低的微囊藻是通过分泌大量的胞外及胶被多糖使群体尺寸变大,而光饱和点高的微囊藻是通过生长来促进群体尺寸的增大。此外,对产毒藻株在不同光强下的毒素基因表达及胞内毒素测定发现,高光强组的群体微囊藻mcyB和mcyD表达量升高,且胞内微囊藻毒素含量增加显著,推测微囊藻毒素也可能是影响微囊藻群体形态及大小的作用因子之一。     

微囊藻毒素LR诱导细胞毒性机制研究的细胞模型建立

以18种细胞系为材料,研究微囊藻毒素LR(20μg/mL和50μg/mL)所诱导的细胞毒性。形态观察表明,在经过30h以上的微囊藻毒素处理后,PC-3,J82,786-0,5637,VERO-E6等5种细胞出现了明显的细胞形态改变,毒素浓度越高,形态改变越厉害。微囊藻毒素LR的细胞毒性用LDH泄漏来表示。结果显示,5种毒素处理细胞的LDH泄漏呈剂量依赖性增加,其中5637和PC-3的LDH泄漏在同样的处理后较为厉害;同对照比较,SOD活力在20μg/mL MCLR处理下呈增加趋势,但在50μg/mL浓度下则下降;GSH含量在两种浓度处理下呈总体下降趋势。鉴于对微囊藻毒素敏感性差异分析,作者选择以5637细胞为基础,建立微囊藻毒素的毒理机制研究模型。       

微囊藻毒素LR影响人肝细胞HL7702的ERK及JNK的蛋白磷酸化

微囊藻毒素(Microcystin,MCYST)是蓝藻的一些属产生的次级代谢产物,在发生水华的水体中普遍存在。微囊藻毒素LR(MCLR)是微囊藻毒素中存在最为普遍且毒性作用最强的一种。已有研究表明,微囊藻毒素可以诱发肝毒性并且与人群中的肝癌发生密切相关1,2,因此进一步阐明其致毒机理具有重要的意义。       

滇池水华蓝藻铜锈微囊藻和绿色微囊藻的生长生理特性及毒素分析

从滇池分离纯化了两种常见水华微囊藻即铜锈微囊藻和绿色微囊藻。在常规培养条件下,两种藻类在对数生长期的生长速率μ值分别为0.61和0.63;早期生长的抑制光强不大于100μmol m~(-2)s~(-1)。铜锈微囊藻主要产生3种微囊藻毒素:MYCST-RR,MYCST-YR和MYCST-LR,绿色微囊藻产生的主要微囊藻毒素为[Dha~7]-MYCST-RR,和[Dha~7]-MYCST-LR,另含有少量的[Dha~7]-MYCST-YR。在低光强15μE m~(-2)s~(-1)时,毒素含量每毫克干重细胞达到3.127μg微囊藻毒素,当光强达到100μE m~(-2)s~(-1)时,毒素含量降低到每毫克干重细胞1.971μg;光强对毒素形成的影响受到温度的调节,而温度对毒素形成的影响不大。探讨了两种微囊藻细胞在不同光照强度下叶绿素荧光比值Fv/Fm的变化,此比值的变化可以间接反映细胞受外界光照强度抑制程度。     

微囊藻毒素对水环境的影响研究进展

微囊藻毒素是富营养化淡水水体中最常见的藻类毒素,是湖泊蓝藻产生的一类肽类毒素,它的产生受到藻类的遗传和环境因素的共同影响。由于其毒性大,分布广,结构稳定,从而成为水环境中的潜在危害物质。有关微囊藻毒素性质、毒理毒性、在环境中的迁移、转化以及控制预防已成为关注热点。在总结国内外研究的基础上,综述了微囊藻毒素的性质、产生机理以及其与水环境、水生生物(水生植物、鱼类、无脊椎动物)间的相互作用,讨论了微囊藻毒素对水生生物的影响以及水生生物对微囊藻毒素的降解作用,为水体中微囊藻毒素的防治提供科学的依据。     

Dissociation in Pseudomonas aeruginosa

Zierdt, C. H. (National Institutes of Health, Bethesda, Md.), and P. J. Schmidt. Dissociation in Pseudomonas aeruginosa. J. Bacteriol. 87:1003-1010. 1964.-Evidence is presented that dissociation of Pseudomonas aeruginosa occurs in vivo as well as in vitro, although it is suppressed in the blood stream. Of 116 primary cultures on blood agar, 77 (66%) had more than one colony type, with a range of 2 to 6 types per culture. Dissociation was studied in 14 primary cultures during 30 serial blood agar passages. Six of these did not dissociate. Of the six, three were originally primary monocolony strains, and three were strains with two colonial types. Seven of the remaining eight cultures had more than one colony type on the primary culture plate. These eight cultures were observed to dissociate at varying rates; 25 morphological and biochemical tests failed to reveal important differences in the colonial dissociants. However, they may be differentiated by bacteriophage action. Colonial morphology in a given strain of P. aeruginosa can be correlated with its bacteriophage lytic pattern, but patterns frequently undergo drastic change during subculture of the organism. The frequently seen different colonial forms in a specific primary culture are usually related, as proven by bacteriophage typing. Phenotypic colony changes after lysogenization were observed. Mucoid colonial variants are markedly more sensitive than are the nonmucoid to streptomycin, tetracycline, and chloramphenicol.     

Composition of cyclic peptide toxins among strains ofMicrocystis aeruginosa (blue-green algae,cyanobacteria)

The distribution of the cyclic heptapeptide toxin, microcystin, was studied among 31 strains of Microcystis aeruginosa. Microcystins-RR, -YR and -LR were detected in fifteen strains and microcystin-LR was in two strains, but no toxin was found in fourteen strains. All three toxins were detected in all 12 strains belonging to the large cell-size group recognized by cell size and allozyme genotype. This pattern of toxin composition is compatible with that of M. viridis. The small cell-size group showed variation in the toxin composition as in the case of allozyme genotype.     

GENETIC VARIABILITY OF BRAZILIAN STRAINS OF THE MICROCYSTIS AERUGINOSA COMPLEX (CYANOBACTERIA/CYANOPHYCEAE) USING THE PHYCOCYANIN INTERGENIC SPACER AND FLANKING REGIONS (cpcBA)

The genetic and morphological variability among 15 Brazilian strains of Microcystis aeruginosa (Kütz.) Kütz. collected from four locations was examined and compared with several reference strains of M. aeruginosa , M. viridis (A. Br.) Lemm. and M. wesenbergii (Kom.) Kom. in Kondr. Brazilian strains were classified by morphological features and by comparison of the nucleotide sequences of the cpc BA intergenic spacer and flanking regions. Our results indicate that Brazilian strains classified as M. aeruginosa are phylogenetically diverse compared with reference strains of M. aeruginosa and that the current taxonomy underestimates genetic diversity within M. aeruginosa. The data also demonstrate that morphological criteria alone are inadequate to characterize Microcystis species. Although colonial characters were shown to vary considerably in culture, some genetic lineages demonstrated consistent cellular diameter ranges, indicating that cell size has value as a taxonomic character. The detection of six M. aeruginosa genotypes in a single water body indicates that morphological approaches can also seriously underestimate the diversity of Microcystis bloom populations.     

Rosette Colonies of Choanoflagellates (Salpingoeca rosetta) Show Increased Food Vacuole Formation Compared with Single Swimming Cells

Choanoflagellates exist as both single‐celled and colonial forms and filter‐feed by generating water currents using a single apical flagellum. Hydrodynamic modeling studies have differed in predictions of whether single cells or colonies produce greater fluid flow to enhance feeding, and a recent study reported no increase in feeding efficiency of stalked colonies of choanoflagellates compared with single cells. We report that rosette colonies of Salpingoeca rosetta demonstrate higher rates of food vacuole formation compared with unicellular, slow swimmers.     

Regions of toxin A involved in toxin A excretion in Pseudomonas aeruginosa.

Toxin A is excreted by Pseudomonas aeruginosa as a mature 66,583-dalton protein. In this study, we used molecular cloning and deletion analysis to define specific regions of the toxin molecule involved in its excretion. Subclones that express either the amino terminus, the carboxy terminus, or toxin A molecules with internal deletions were constructed. The hypotoxigenic mutant PAO-T1 was used as a host for the expression of the toxin constructs. When overexpressed (by the presence of extra copies of the toxin A-positive regulatory gene, regA, in trans), toxin A-cross-reactive materials produced by most of these constructs were detected in the supernatant of PAO-T1. The supernatant of P. aeruginosa PAO-T1 contained proteolytic activity that degraded toxin A-derived products but not the intact toxin molecule. A single SalI intragenic deletion (coding for the leader peptide, the first 30 amino acids, and the last 305 amino acids of the toxin) resulted in a relatively stable product in the supernatant of PAO-T1. The product of the carboxy terminus construct (which codes for the last 305 amino acids of the toxin) was detected in the lysate of PAO-T1 only. The data suggest that the amino terminus region of toxin A (the leader peptide plus the first 30 amino acid of the mature protein) is sufficient for its excretion, and that a second region, amino acids 309 through 413, protects an internally truncated toxin A molecule from the proteolytic activity in the supernatant of P. aeruginosa PAO-T1.     

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).     

Comparison of reagent-impregnated paper strips and conventional tests for distinguishing Escherichia from Aerobacter: correlation with colonial morphology

The means for distinguishing Escherichia from Aerobacter (Enterobacter) differ in laboratories and range from complete dependence on colonial reactions on typical gram-negative media to reliance on one or more of the classical indole, methyl red, Voges-Proskauer, citrate (IMViC) parameters. Three colonial types (one prejudged as Escherichia) of lactose-positive rods were catalogued on each of the most commonly used selective media, MacConkey Agar, Endo Agar, and E M B Agar. Each cultural type was presumptively diagnosed and then compared with the expected outcome of individual IMViC tests. The distribution of preliminary identifications was similar from growth patterns on MacConkey Agar and E M B Agar, but it differed markedly from Endo Agar. When organisms initially diagnosed by cultural methods were compared by single IMViC tests, it was found that for each colonial type one of the biochemical parameters was best suited. Thus, for those types initially considered Escherichia, the methyl red or Voges-Proskauer test results agreed most consistently; for other types, the citrate reaction was most satisfactory. In addition, when newly formulated reagent-impregnated paper strip methods for indole, Voges-Proskauer, and citrate were evaluated and compared to the standard methods, agreement was 97% for indole, 90% for Voges-Proskauer, and 95% for Simmons' citrate.     

Iron-stimulated toxin production in Microcystis aeruginosa.

Nitrate- and phosphate-limited conditions had no effect on toxin production by Microcystis aeruginosa. In contrast, iron-limited conditions influenced toxin production by M. aeruginosa, and iron uptake was light dependent. A model for production of toxin by M. aeruginosa is proposed.     

Biochemical,morphological, and genetic variations in <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> due to colony disaggregation

Microcystis aeruginosa commonly occurs as large colonial morph under natural conditions, but disaggregates and exists as single cells in laboratory cultures. To demonstrate the adaptive changes, differentiation of carbohydrates, pigments, and protein between colonial and disaggregated M. aeruginosa were examined. Their morphological and ultrastructural characteristics were subsequently observed by scanning electron microscopy and transmission electron microscopy. Results showed that chlorophyll a and phycocyanin in cells, soluble carbohydrate produced in the culture medium, and total carbohydrate in cells and sheath of colonial M. aeruginosa are significantly higher (p < 0.05) compared with those in disaggregated cells. No significant change was found in protein concentration per cell (p > 0.05) between them. Their morphological and ultrastructural characteristics were evidently different, and by morphological criteria they could be separated into two morphotypes. In addition, the genetic diversity of 16S–23S internal transcribed spacer of them were examined and compared with reference strains of M. aeruginosa. The alignment of two sequences revealed that genetic identity level was extremely high (96.94%) and no significant difference was found in the nucleotide diversity (0.014 ± 0.008). This suggested that similar genotypes could present distinct morphotypes in M. aeruginosa. The tree topologies and analysis of molecular variance of the two sequences and reference sequences from GenBank database indicated that the genotypes of M. aeruginosa strains were not always related to their localities and exhibit heterogeneity within a species.