Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay （dot-ELISA）, indirect ELISA and Westem blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Westem blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5-6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards （＂marker＂） so that the alterations ofprotease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10^4 cell/mL in the indirect ELISA, while 10^5 cell/mL in the dot-ELISA.     

Symbiotic Relations of Sediment-Agglutinating Nematodes and Bacteria in Detrital Habitats: The Enzyme-Sharing Concept

Abstract. A new concept (‘enzyme sharing’) concerning the interaction of marine nematodes and microbes in the degradation of sedimentary detritus is presented. Elements of this concept are (1) the notorious tendency of many aquatic nematodes to agglutinate detrital particles by mucus secretions, (2) new observations on the stimulation of microbial growth by nematodes in agar plates, and (3) literature data on limited endogenous proteolytic capacities of aquatic nematodes.
Observations on nematode‐microbe associations in agar plates prompted the conceptual synthesis. In agar medium without the addition of any nutrients a spectacular growth of bacteria was visible on the sinusoidal crawling trails of nematodes only 2 – 3 days after introduction of the worms (species of Adoncholaimus, Anoplostoma and Sabatieria). Juveniles of Anoplostoma that hatched in the agar cultures left their minute trails in the medium and these were rapidly occupied by bacteria. The nematodes repeatedly visited their bacterial trails, which persisted as a peculiar biotic structure for more than one year and survived the nematodes.
In sterile agar preparations containing the fluorogenic methylumbelliferyl‐β‐glucoside in the presence of the nematode Adoncholaimus, an enhanced fluorescence of the medium was visible, indicating β‐glucosidase activity. We therefore assume that oncholaimid nematodes discharge enzymes that alone, or in concert with microbial activities, contribute to the hydrolytic cleavage of refractory polysaccharides containing β‐glucosidic bonds such as agar components and cellulose. The sugars thus produced may then be taken up by the nematodes and concomitantly support the conspicuous growth of microbes. Since we did not observe any feeding of the nematodes on the associated microbes in agar plates, we question the nutritive potential of intact microbial cells for a number of nematodes abounding in detrital habitats, and call attention to the significance of ambient dissolved or adsorbed organic monomeric nutrients.
Consequently, we perceive the puzzling perpetual accretion of detrital organic particles to sediment agglutinations by nematodes as an adaptation for operating an ‘enzymatic reactor’ for the production of dissolved nutrients. We hypothesise a relationship of mutual commensalism of nematodes and heterotrophic microbes in detrital habitats and propose the term ‘enzyme sharing’ for this relationship. Both parties invest in a common enzyme pool that decomposes organic detritus for their nutrition. We present here evidence that nematodes contribute β‐glucosidase, which is involved in the cellulase system. Data from the literature suggest that microbial enzymatic processing of detrital proteins yields amino acids available to nematodes, which apparently have no efficient proteolytic enzyme system in their intestines.     

营养盐水平对四种海洋浮游硅藻胞外多糖产量的影响

4种海洋浮游硅藻(牟勒氏角毛藻、海链藻、三角褐指藻和新月菱形藻)培养在改进的f／2培养基中，研究了不同氮、磷和硅营养水平对它们胞外多糖产量的影响．结果表明，硅藻胞外多糖的生产和释放具有种间特异性，角毛藻和海链藻胞外多糖的生产和释放主要在静止期，而三角褐指藻和新月菱形藻在指数生长期前期和静止期都能生产和分泌较高的胞外多糖；培养液中低浓度磷减少了4种硅藻在静止期胞外多糖的产量，但增加了角毛藻在生长期前期胞外多糖的产量；氮浓度的降低增加了三角褐指藻在指数生长前期胞外多糖的产量，但减少了其他3种藻类胞外多糖的产量；硅浓度的降低对4种硅藻胞外多糖的产量影响不大，在一定程度上还促进了静止期胞外多糖的生产．本研究表明，营养盐水平对硅藻胞外多糖生产的影响因种类和细胞所处生长期不同而存在着很大的差异．     

南极菌Pseudoalteromonas sp．S-15-13产胞外多糖的研究及其分子鉴定

本文对影响南极细菌S-15-13生长和胞外多糖产量的主要环境因子进行了研究,同时采用16S rDNA序列分析及系统发育分析方法对其进行了分子鉴定。结果表明：菌株S-15-13最佳产糖条件为：培养时间,56h；培养温度,8℃；碳源,1．0%葡萄糖；NaCl浓度,3．0%；pH,6．0-7．0。16S rDNA序列分析和系统发育分析表明,菌株S-15-13属于假交替单胞菌属(Pseudoaltero- monas)。     

绿藻水溶性多糖的研究概况和进展

对近年来绿藻多糖的提纯方法、组成结构及应用的研究进展进行了综述。绿藻多糖的提纯需要经过样品的前处理、提取和精制三个步骤,提取方法包括酸提、酶提、加入钙螯合剂提取、微波及超声波辅助提取。绿藻多糖是水溶性的硫酸化杂多糖。从石莼(Ulva)、浒苔(Enteromorpha)、礁膜(Monostroma)、小球藻(Chlorella)、蕨菜(Bracken)及刚毛藻(Bristles)中提取的多糖的化学组成及结构特征各不相同,而且多糖的化学组成和结构特征受提取方式的影响。绿藻多糖的生物相容性、生物可降解性及抗氧化、抗肿瘤、抗凝血、抗炎、免疫调节等多种生物活性使其在食品、医药、化妆品及农业中具有广泛的应用。有关绿藻多糖的精确结构、提纯方法及化学修饰的进一步研究将使绿藻多糖的应用更加广泛。     

南海沉积物细菌胞外蛋白酶在家族水平上的多样性

海洋产蛋白酶细菌及其分泌的胞外蛋白酶在降解有机氮以推动海洋氮循环进行方面发挥着重要作用,但目前对这二者多样性的认识非常有限。本研究自南海沉积物中筛选得到90株产蛋白酶细菌,并通过N-端氨基酸序列,分析了其胞外蛋白酶在家族水平上的多样性。16S rRNA基因序列分析的结果表明,筛选的产蛋白酶细菌均属于Gammaproteobacteria纲,且大多数属于Alteromonadales目和Vibrionales目的不同属。对其中14株菌株的14个胞外蛋白酶的N-端氨基酸序列分析表明,所有这些蛋白酶属于金属蛋白酶的M4家族或丝氨酸蛋白酶的S8家族。本研究提供了海洋沉积物产蛋白酶细菌在类群及其胞外蛋白酶在类型上的新细节,这将有助于全面了解微生物酶促降解海洋沉积物有机氮的过程和机理。     

Effect of polysaccharide from Enteromorpha prolifera on maize seedlings under NaCl stress

In this study, a polysaccharide from Enteromorpha prolifera (EP) was extracted and its effect on maize seedlings under NaCl stress was investigated. Firstly, the components and structure of the EP were determined. We found that EP is a sulfated polysaccharide of high-molecular weight (Mw, 1 840 KDa) heteropolysaccharides and the main monosaccharide is rhamnose. The polysaccharide was applied to explore its effect on the growth of maize seedlings and its defense response under a salt stress. The results show that EP could promote the growth of maize seedlings under the salt stress. In addition, EP was shown able to significantly regulate membrane permeability and adjustment of osmotic substances such as soluble protein, soluble sugar, and proline, antioxidant enzymes containing superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase. Therefore, EP is an effective salt-resistant substance for the growth of maize seedlings under NaCl stress.     

NaHSO3对两种微藻生长及胞外多糖和藻胆蛋白含量的影响

本文研究了0.25~3 mmol/L浓度的NaHSO3对紫球藻(Porphyridium violaceum)和0.06~0.96 mmol/L浓度的NaHSO3对蓝隐藻(Chroomonas placoidea)的叶绿素荧光参数(PSII最大光能转化效率Fv/Fm、相对电子传递速率rETR、光化学淬灭qP、非光化学淬灭NPQ)、细胞密度、干重、胞外多糖和藻胆蛋白含量的影响。研究表明,较低浓度的NaHSO3对紫球藻和蓝隐藻的生长及活性物质积累有促进作用,2种微藻进行生长和活性物质积累的最适NaHSO3浓度分别为0.5和0.12 mmol/L,此条件下,实验结束时,2种微藻的Fv/Fm、rETR、qP、细胞密度、干重、胞外多糖和藻胆蛋白含量均达到最大值,显著高于对照组。其中,紫球藻的最终细胞密度、干重、胞外多糖和藻胆蛋白含量分别比对照组增加了36.0%、19.1%、71.8%和69.0%。蓝隐藻的上述参数在NaHSO3浓度为0.12 mmol/L时则比对照组增加了60.8%、45.4%、60.0%和53.1%。另一方面,NaHSO3浓度为2~3 mmol/L时显著抑制紫球藻生长及活性物质积累,NaHSO3浓度为0.48~0.96 mmol/L则不利于蓝隐藻生长及活性物质积累。研究结果表明,适合紫球藻和蓝隐藻生长及胞外多糖和藻胆蛋白积累的最佳NaHSO3浓度分别为0.5和0.12 mmol/L,该研究为2种微藻的开发利用提供了理论依据。     

Cu2+对序批式反应器中活性污泥胞外聚合物产量及其组分的影响

研究了长期暴露条件下Cu2+对序批式反应器(SBR)性能及其活性污泥胞外聚合物(EPS)特性的影响。结果表明,进水中加入10 mg·L-1的Cu2+后,在SBR运行的第16~55天,COD和NH+4-N的去除率保持稳定;在第56~75天,COD和NH+4-N的平均去除率与进水Cu2+浓度为0 mg·L-1时相比分别下降了3.88%和6.41%。浓度为10 mg·L-1的Cu2+长期作用下,活性污泥中EPS、松散附着EPS(LB-EPS)和紧密附着EPS(TB-EPS)产量及LB-EPS和TB-EPS中蛋白质(PN)含量增加。傅里叶变换红外光谱分析表明10 mg·L-1 Cu2+的长期暴露导致TB-EPS中PN的C=O键、N-H键和C-O键相对含量降低。X射线光电子能谱(XPS)测试结果显示在10 mg·L-1 Cu2+长期暴露条件下,LB-EPS和TB-EPS中元素Cu和O百分含量增加。     

钝顶螺旋藻藻蓝蛋白和多糖的抗肿瘤免疫活性研究

对钝顶螺旋藻藻蓝蛋白 (PC)和多糖 (PSP)的抗肿瘤免疫活性进行了研究。采用免疫酶标技术、MTT法及定量溶血分光光度测定法等免疫分析实验 ,从免疫器官、免疫细胞、免疫分子 3个水平上进行藻蓝蛋白和多糖的抗肿瘤免疫活性检测。结果显示 ,藻蓝蛋白和多糖处理组小鼠瘤块直径及瘤体重量均小于对照组 ,T细胞及 B细胞活性明显增强 ,体液抗体量显著提高 ,螺旋藻藻蓝蛋白比多糖抑制肿瘤细胞生长和提高机体免疫力的作用更明显。