南海东海岛沉积物分离细菌的鉴定及其抑菌活性

[背景] 海洋微生物在活性物质开发方面具有巨大的应用前景,而目前有关南海东海岛微生物的研究鲜少。[方法] 对从东海岛沉积物中分离纯化的海洋细菌,采用形态学观察、生理生化以及16S rRNA基因序列的系统发育分析方法进行鉴定;以大肠杆菌（Escherichia coli）、枯草芽孢杆菌（Bacillus subtilis）和金黄色葡萄球菌（Staphylococcus aureus）作为指示菌,测定其抑菌活性;对具有抑菌活性的菌株扩增聚酮合酶（Polyketide synthase I,PKSI）基因,并与已知的PKSI氨基酸序列比对;选择具有PKSI基因的代表菌株,检测菌株及其发酵抑菌物的稳定性。[结果] 分离纯化到25株海洋细菌,分属于不动杆菌属（Acinetobacter）、交替单胞菌属（Alteromonas）、芽孢杆菌属（Bacillus）、嗜冷杆菌属（Psychrobacter）、假交替单胞菌属（Pseudo-alteromonas）、海洋单胞菌属（Oceanimonas）、葡萄球菌属（Staphylococcus）、微球菌属（Micrococcus）和海杆菌属（Marinobacter）。12株菌株通过基因筛选检测到PKSI编码基因,其中6株菌株具有抑菌活性和PKSI编码基因,并分属于芽孢杆菌属和交替单胞菌属;PKSI氨基酸序列同源性分析推测菌株DHD-15和DHD-a可能产生新的I型聚酮合酶结构。菌株DHD-15和DHD-L生长温度范围为15-40℃,可耐受10% NaCl高盐以及pH 3和pH 11的酸碱条件,但不耐高温;菌株DHD-15产生的抑菌物质可耐受100℃和pH 11的高温碱性条件,在50℃、pH 9条件下制备和室温保藏条件下抑菌活性较高,其稳定性较好。[结论] 南海东海岛沉积物筛选的细菌种具有抑菌活性,具有产聚酮类活性物质的潜力。     

盐渍海带盐中可培养嗜盐古菌多样性、胞外酶活及拮抗作用探究

盐渍海带盐是一种高盐环境,其中可能生存有嗜盐微生物,如嗜盐古菌。嗜盐古菌是一类生活于高盐环境的极端环境微生物。为了探究盐渍海带盐中嗜盐微生物的物种多样性,筛查分离了菌株的几种常见胞外功能酶活性和拮抗活性。采用纯培养技术,从盐渍海带盐样品中分离培养嗜盐微生物,并对其16S rRNA基因进行扩增和测序;基于16S rRNA基因序列的相似度分析,确定分离菌株在属级水平的分类地位;依据种属信息,挑选代表菌株,进行胞外常见功能酶活性和拮抗作用测定。从盐渍海带盐样品中分离到来自Haloarcula（盐盒菌属）、Halorubrum（盐红菌属）、Halarchaeum、Halobacterium（盐杆菌属）、Halococcus（盐球菌属）、Halolamina（盐尊片形菌属 ）和Haloplanus（盐扁平菌属）等7个属的131株嗜盐古菌;检测到产胞外蛋白酶菌株1株,产酯酶菌2株,产明胶酶菌7株,产氧化酶菌1株和产触菌酶5株;此外,筛选到6株具有拮抗活性的菌株,其中来自盐红菌属菌株Halorubrum sp. ZSA68较其他菌株生长快,产抑菌活性物质快,并显示出较强的抑菌活性和较广的抑菌谱,初步推测Halorubrum sp. ZSA68所产物质为多肽或蛋白类抑菌活性物质,其分子量大小约为30~50 kDa,该抑菌活性物质在60 ℃以上或NaCl浓度低于2%时容易失去活性。通过本研究认识了海带盐中可培养嗜盐古菌的分布,获得多株具有较高胞外酶活性及具拮抗作用的菌株,积累了丰富的嗜盐古菌菌株资源,这在高盐食品的防腐方面具有潜在的应用价值。     

短头熊蜂肠道拮抗菌株的分离筛选鉴定及其生物特性评价

【背景】近年来,由于栖息地减少、农药的大量使用及病原菌侵染等综合因素,导致全世界的熊蜂种类与数量逐年减少,病原菌的侵染可通过微生物在自身生长过程中会产生的抑菌物质进行有效抑制或杀灭。【目的】短头熊蜂(Bombus breviceps)长期生存在野外环境中,其肠道内存在着大量微生物资源。从短头熊蜂肠道内筛选拮抗菌株,并对其抑菌特性进行研究。【方法】采用牛津杯双层法筛选拮抗菌株,测定抑菌活性最佳菌株发酵液的抑菌物质稳定性与抑菌广谱性等抑菌特性,并借助细胞膜通透性、流式细胞仪检测等试验探究其抑菌机制。【结果】得到了5株具有明显抑菌作用的拮抗菌株,其中果杆菌(Fructobacillus tropaeoli)CZ01对金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌(Salmonella choleraesuis)、大肠杆菌(Escherichia coli)、福氏志贺氏菌(Shigella flexneri)和无乳链球菌(Streptococcus agalactiae)这5种病原指示菌都具有高度抑菌效果。菌株CZ01对金黄色葡萄球菌的抑菌效果最佳,抑菌圈直径可达到(21.21±0.25) mm,在121 ℃处理后仍具有67.36%以上的抑菌活性,调整pH值为10.0时仍具有78.16%的抑菌活性。【结论】短头熊蜂肠道微生物资源较丰富,尤其是果杆菌(F.tropaeoli)CZ01具有抑菌活性高、稳定性好、抑菌谱广等特性,对金黄色葡萄球菌具有良好的杀灭效果,显示出良好的应用潜能。     

中药抑制嗜水气单胞菌、副溶血弧菌和溶藻弧菌的分子机制研究进展

嗜水气单胞菌（Aeromonas hydrophila）、副溶血弧菌（Vibrio parahaemolyticus）和溶藻弧菌（Vibrio alginolyticus）是常见的鱼类致病菌,导致鱼类患出血性败血症、溃疡等疾病。大黄、金银花、甘草等中药因具有天然活性、不易产生细菌耐药性而有望替代抗生素抑制这些鱼类致病菌。回顾细菌的致病性和中药的药理活性,从蛋白质组学和基因转录水平阐述了中药抑制嗜水气单胞菌、副溶血弧菌和溶藻弧菌的分子机制。这些研究对人类了解和控制致病菌的感染、提高鱼类免疫力以及推广应用中药作为饲料添加剂具有重要的指导意义。     

产抑菌物质乳酸菌的筛选鉴定及生物学特性研究

[目的]筛选高产抑菌活性物质的乳酸菌菌株,并研究抑菌物质的生物学特性。[方法]采用平板琼脂孔扩散法从发酵制品中筛选出具有抑菌活性的菌株,并对其进行形态学特征、生理生化特征及16S r DNA序列分析鉴定,测定抑菌谱,考察发酵产物对p H、温度、5种常见蛋白酶的敏感性。[结果]该菌株被鉴定为干酪乳杆菌(Lactobacillus casei),其发酵产物对细菌有抑制作用,对霉菌和酵母菌无抑制作用; p H 2. 0时发酵液抑菌圈直径达到21. 24±0. 45 mm,比p H 6. 0时高32. 7%;经沸水浴后原始发酵液抑菌圈直径下降了34. 3%,p H 2. 0时仅下降6. 9%;经蛋白酶处理后抑菌圈直径与对照组相比变化不大。[结论]筛选得到的乳酸菌菌株具有良好的抑菌活性,该抑菌物质在酸性条件下有较高的抑菌活性和热稳定性,对5种蛋白酶不敏感。     

CP7抗菌蛋白对嗜水气单胞菌的抑杀作用机理

[目的]研究多粘类芽孢杆菌(Paenibacillus polymyxa) CP7菌株的抗菌蛋白(CP7ACP)对嗜水气单胞菌的抑杀作用机理,为防治嗜水气单胞菌引起的鱼病提供新的潜在天然药物.[方法]采用抑菌试验、钼锑抗比色法和紫外光谱法研究其对嗜水气单胞菌S12菌株生长、磷泄漏和生物大分子的影响,并利用扫描电镜和透射电镜观察了嗜水气单胞菌细胞结构遭受的破坏作用.[结果]CP7ACP对嗜水气单胞菌的抑菌圈直径约8.1 mm,最小抑菌浓度(MIC)与最小杀菌浓度(MBC)分别为原液浓度的1/8和1/4;嗜水气单胞菌受CP7ACP处理后,电镜观察发现其细胞壁、细胞膜、细胞器以及菌体均受到不同程度的破坏,胞内的生物大分子和磷泄漏明显,基因组DNA发生增色效应.[结论]CP7ACP抑制嗜水气单胞菌生长,可用于防治嗜水气单胞菌引起的鱼病.     

几种中药单体和抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性研究

考察没食子酸等6种中药单体和诺氟沙星等3种抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性及其联合抑菌作用。通过琼脂扩散法测定受试物的抑菌作用,用微量二倍稀释法测定受试物最小抑菌浓度（MIC）和最小杀菌浓度（MBC）,用棋盘法考察受试物的联合抑菌作用。6种中药单体中没食子酸和槲皮素对嗜水气单胞菌及温和气单胞菌抑制作用较强,抑菌圈均达到20 mm以上,没食子酸对嗜水气单胞菌及温和气单胞菌的MIC和MBC均为250 g/mL;槲皮素对嗜水气单胞菌的MIC和MBC均为500 g/mL,对温和气单胞菌的MIC和MBC分别为250和500 g/mL;而大黄素甲醚等4种中药单体无明显的抑菌作用。嗜水气单胞菌及温和气单胞菌对诺氟沙星、恩诺沙星、氟苯尼考等抗生素均极敏感。在联合药敏试验中,没食子酸与恩诺沙星或氟苯尼考、槲皮素与氟苯尼考联合用药对嗜水气单胞菌具有相加抑菌作用;没食子酸与恩诺沙星联合用药对温和气单胞菌具有协同抑菌作用,没食子酸与诺氟沙星或氟苯尼考、槲皮素与氟苯尼考联合用药对温和气单胞菌具有相加抑菌作用。没食子酸和槲皮素对嗜水气单胞菌及温和气单胞菌具有显著的抑制作用,二者与恩诺沙星、诺氟沙星、氟苯尼考等抗生素联合应用具有相加或协同作用,有助于降低抗生素的用量及残留。       

大黄醇提物对嗜水气单胞菌抑菌活性及其机制

研究大黄醇提物对嗜水气单胞菌的抑菌活性及机制。主要采用牛津杯法和液体倍比稀释法;通过扫描电镜、SDS-PAGE、二维电泳及串联质谱等方法探讨大黄醇提物对嗜水气单胞菌的抑菌机制。结果显示,大黄醇提取物对嗜水气单胞菌有明显的抑制效果,抑菌圈为(24.5±0.2)mm,MIC和MBC均为3.91 mg/m L;在药物作用下,菌体表面变粗糙,细胞膜受损并出现破裂;在经药物处理4 h后,细菌蛋白含量明显下降(P0.05),2-DE电泳发现3个差异性蛋白。结果表明,大黄醇提物对嗜水气单胞菌的抑制作用机制是破坏其细胞膜结构及影响特定蛋白的表达。     

宽体金线蛭嗜水气单胞菌感染的病原检验*

对河北某宽体金线蛭(Whitmania pigra Whitm an)养殖场所养殖的宽体金线蛭发生的病害,进行了发病情况、临床表现、病理变化等方面的检验。同时,择代表菌株进行了16S rRNA基因的分子鉴定,测定了16S rRNA基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树。结果表明所检病例为由嗜水气单胞菌(Aerom onas hydrophila)所引起的感染。分离后做纯培养的10株嗜水气单胞菌均为同种血清型菌株;代表菌株对健康宽体金线蛭的人工感染试验表明了相     

嗜水气单胞菌ntrC调控的生理功能及其机制研究

[目的] NtrC是一种与DNA结合的转录调控因子,在激活氮同化基因的转录和维持氮源供应中具有重要作用,本研究拟探究其对嗜水气单胞菌生理功能的影响及其作用机理。[方法] 本研究采用同源重组方法构建了嗜水气单胞菌ATCC 7966 ntrC的缺失株,并以野生株为对照,对缺失株的生理表型进行测定和分析,利用定量蛋白质组学技术比较野生株和ntrC缺失株的蛋白表达差异。[结果] 发现敲除ntrC基因后,嗜水气单胞菌在缺氮、渗透压、重金属离子、氧化以及不同抗生素胁迫下的耐受性都发生显著变化,且这些表型在其补救菌株中均能得到恢复。定量蛋白质组学分析发现,野生株和ntrC缺失株的差异表达蛋白可能参与氨基酸生物合成、抗坏血酸和醛糖酸盐等代谢通路的调控。[结论] 本研究阐明了ntrC在嗜水气单胞菌中的重要作用及其对细菌生物学功能的影响,探讨了ntrC直接或间接调控的蛋白与生理表型之间的联系,研究结果可为未来水产致病菌的防治提供理论支持。     

Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.     

Inhibition of phosphoglucose isomerase allozymes from the wing polymorphic waterstrider,Limnoporus canaliculatus,by pentose shunt metabolites

Inhibition of phosphoglucose isomerase (PGI) allozymes from the wing-polymorphic waterstrider, Limnoporus canaliculatus, by three pentose-shunt metabolites was studied at several different temperatures. This was done to determine if the allozymes exhibited a differential ability to participate in lipid biosynthesis via differential partitioning of carbon flux through the pentose shunt versus glycolysis. 6-Phosphogluconate and erythrose-4-phosphate proved to be strong competitive inhibitors of PGI, while sedoheptulose-7-phosphate was a very weak inhibitor. The PGI allozymes from L. canalicualtus were differentially inhibited by 6-phosphogluconate at two of the three temperatures studied. However, this property does not appear to be an adaptive difference between the allozymes but, rather, a correlated effect resulting from variation in substrate binding. Estimates of reaction rates for the allozymes indicate that the differences in inhibition result in no detectable differences in reaction velocities. Thus, no evidence in support of the hypothesis that PGI allozymes from Limnoporus canaliculatus were adapted to function in different metabolic capacities via differential inhibition was obtained in this study. However, the importance of this characteristic in allozymic adaptation in natural populations remains an open question.Supported by NSF Grant DEB 7908802 and UPHS Grant GM 21133 to R. K. Koehn and an NSF dissertation improvement grant to A. J. Zera.     

Carbonic anhydrase activity of Prochloron sp. isolated from an ascidian host

The prokaryotic algal symbiont of ascidians, Prochloron sp., was found to exhibit carbonic anhydrase activity which is largely associated with the cell surface. This extracellular carbonic anhydrase activity was inhibited, while the intracellular activity was not affected, by chloride or bromide. Acetazolamide and ethoxyzolamide inhibited carbonic anhydrase activity with I₅₀ values of 7×10^-4 and 3×10^-4M, respectively. These I₅₀ values are similar to those observed for intracellular carbonic anhydrases of Synechococcus sp. PCC7942, Chlamydomonas reinhardii and spinach.Abbreviations AZA acetazolamide - CA carbonic anhydrase - chl chlorophyll - EZA ethozyzolamide - I₅₀ concentration of an inhibitor required to cause 50% inhibition - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - U unit     

Acute inhibition of protease and suppression of growth in zooplankter,Moina macrocopa,by Microcystis blooms collected in Central India

Cyanobacterial blooms consisting of Microcystis spp., collected from 14 water-bodies in Central India, and an adapted culture, were studied for likely impact on zooplankton community. When fed with single cells of Microcystis from several locations, in mixtures with Chlorella, population growth of the cladoceran Moina macrocopa was suppressed. Microcystis alone was unsuitable as food. In three cases, bloom extracts enhanced mortality of starved zooplankton. Extracts from several sources inhibited protease activity when trypsin or a crude extract from zooplankton served as enzyme source. Upon fractionation by solid-phase extraction, the C-18 passed extract contained the anti-protease and toxic substances for zooplankton, whereas a methanol eluted fraction retained the trypsin inhibitory substance. The study suggests that production of protease inhibitors by cyanobacteria is a factor responsible for feeding inhibition and mortality in zooplankton, which in turn could regulate the community structure of grazers.     

Suppression of female bagworm, Thyridopteryx ephemeraeformis reproduction potential with its sex pheromone, 1-methylbutyl decanoate

Mating of the female bagworm, Thyridopteryx ephemeraeformis (Haworth) (Lepidoptera: Psychidae) on host trees treated with the racemate of the species' female sex pheromone, 1-methylbutyl decanoate, was suppressed by 87% in the field. The suppressive effect of the pheromone was independent of insect population density when it was evaporated from female-infested trees at 712 (±29; S.E.) mg/tree/3 day from a 488 cm × 0.63 cm polyvinylchloride laminated tape. However, evaporation of pheromone at a lower rate did not suppress mating. Observations of adult male behavior in the vicinity of the pheromone-impregnated tape revealed that males were attracted to the tape and that they made copulatory thrusts toward the tape and any object touching the tape. These observations and analyses of the pheromone titers of individual females showed that males can respond to a wide range of pheromone concentrations. Indications are that the mechanism of mating suppression probably involved male sensory adaptation or central nervous habituation to the synthetic 1-methylbutyl decanoate and that the compound, evaporated from host trees, could be useful as a plant protectant against the bagworm.

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Résumé Les accouplements des femelles de Thyridopteryx ephemeraeformis Haworth (Lep. Psychidae) ont été réduits de 87% sur des arbres hôtes traités dans la nature par des racémates du 1-méthylburyldécanoate, la phéromone sexuelle femelle de cette espèce. Lorsque la phéromone s'évapore à partir d'un ruban de 488 cm sur 0,63 de chlorure de polyvinyle portant 712 (±29 S.E.) mg, par arbre pendant 3 jours, l'efficacité était indépendante de la densité de population de l'insecte. Cependant, l'évaporation de la phéromone à une plus faible concentration était sans effet. L'observation du comportement des mâles au voisinage des rubans imprégnés de phéromone a montré qu'ils étaient attirés par le ruban et qu'ils tentaient de s'accoupler avec le ruban et tout objet à son contact. Ces observations et les analyses des concentrations en phéromones des femelles ont montré que les âles pouvaient répondre à une gamme étendue de concentrations en phéromone. Il y a des indices que le mécanisme de suppression de l'accouplement implique une adaptation sensorielle du mâle ou une accoutumance du système nerveux central au 1-méthylbutyl méthylbutyl décanoate synthétique et que ce composé s'évaporant sur les arbres hôtes, pourrait être utilisable pour protéger efficacemment contre T. ephemeraeformis.

     

Inhibition of photosynthesis ofPopulus euramericana andHelianthus annuus by SO2, NO2 and O3

Populus euramericana cv. I-214 andHelianthus annuus L. cv. Russian Mammoth were exposed to various concentrations of O₃ SO₂ or NO₂ for 2 h in a cylindrical assimilation chamber. The threshold concentrations of air pollutants for inhibition of net photosynthesis differed between the two species and also between the pollutants tested. Furthermore, the lethal concentrations where the net photosynthetic rates were completely inhibited, also differed between species and between pollutants. For SO₂ and NO₂,P. euramericana was more tolerant photosynthetically thanH. annuus when related to the concentration of pollutants used during the experiment. However, when related to the cumulative uptake rate of each pollutant, the photosynthetic tolerance of the two species was similar. In contrast to the effects of SO₂ or NO₂, the influence of O₃ on net photosynthesis was quite different. The relative rates of net photosynthesis in both species showed the same linear relationship with O₃ concentration. However, the relationship between the relative rate of net photosynthesis and the cumulative uptake rate of O₃ differed between the two species, although it was linear in both cases.     

Inhibition of growth and adhesion ofHelicobacter pylori using egg yolk antibodies

Helicobacter pylori is known as a key pathogen for chronic gastric and duodenal ulcers. Egg yolk antibody, IgY produced from chicken immunized withH. pylori antigen was tested for the inhibition of growth and adhesion ofH. pylori to gastric epithelial cell, AGS. The colony forming ofH. pylori was repressed by 30% using 1 mg/mL of IgY while that ofE. coli was only 7% with the same amount of IgY, which showed the growth inhibition ofH. pylori was mainly due to the specific interaction between IgY andH. pylori. The inhibition ofH. pylori adhesion to AGS was as high as 90% using 0.5 mg/mL of antibody only. More than 80% ofH. pylori attached to AGS could be detached treating with the same amount of IgY for one and a half hr. However, this effect was severely dependant on theH. pylori strains tested. The strain used for immunization of chicken was very sensitive to the antibody treatment but changing the test strain generally showed a variation in adhesion inhibition between 15 and 80%. Further studies are necessary to employ the egg yolk antibodies for the treatment ofH. pylori in vivo.     

The effect of age, season and growth conditions on anti-inflammatory activity in Eucomis autumnalis (Mill.) Chitt. Plant extracts

Eucomis (Family Hyacinthaceae) bulbs are greatly valued in traditionalmedicine for the treatment of a variety of ailments, predominantly thoseinvolving pain, fever and inflammation. The COX-1 assay was used to screenethanolic extracts prepared from the dried leaves, bulbs and roots of E. autumnalis (subspecies autumnalis) to determine the variation ofanti-inflammatory activity with age and season of harvest. Young plantswere found to have large amounts of COX-1 inhibitory activity, particularlyin the leaves. As the plant matured, greater activity was associated with thebulb and root extracts. The anti-inflammatory activity of the leaf, bulb androot extracts varied slightly throughout the year, with the greatest levelsdetected towards the end of the growing season, shortly before the onsetof dormancy. A seaweed application (Kelpak) decreased the anti-inflammatory activity of the leaf, bulb and root extracts, while increasedtemperature/increased light intensity had no significant effect on theCOX-1 inhibitory activity of the leaf extracts. The bulb extracts fromtreated plants harvested towards the end of the growing season showed asignificant decrease in anti-inflammatory activity, while the anti-inflammatory activity of the corresponding not root extracts increased.     

Effects of germanium dioxide,an inhibitor of diatom growth,on the microscopic laboratory cultivation stage of the kelp, <Emphasis Type="Italic">Laminaria saccharina</Emphasis>

The kelp, Laminaria saccharina, is an economically and biologically valuable seaweed used in integrated multi-trophic aquaculture. The development of the initial microscopic phase of the life cycle of this species is carried out in the laboratory. A treatment of germanium dioxide (GeO₂) can be applied to algal laboratory cultures to inhibit the growth of contaminating diatoms. Previous studies reported inhibitory effects also affecting the macroscopic stage of the life cycle of L. saccharina, the sporophyte, but the effects on the microscopic gametophytic life stage are unknown. To determine the effects, cultures of L. saccharina were treated with varying concentrations of GeO₂ and the resulting lengths and surface area of the juvenile sporophytes produced were measured. It was determined that GeO₂ follows a typical dose-effect pattern, increasing the growth rate of juvenile sporophytes until a critical point at which an inhibitory effect was observed. To obtain juvenile sporophytes ready for deployment to aquaculture sites in the shortest time and to successfully reduce diatom contamination, it was determined that a concentration between 0.10 and 0.50mL of GeO₂ per litre of seawater, applied at day 8, was the most efficient.