北极微生物的分离及抗菌抗肿瘤活性筛选

采用多种培养基对北极地区(海洋沉积物、湖泊沉积物及土壤)微生物进行分离,并采用琼脂平板打孔法和MTT法进行了抗菌、抗肿瘤活性菌株的筛选.共分离极地微生物151株,筛选结果显示,12.4%的菌株有抗菌活性,6.2%菌株有抗肿瘤活性,其中3株同时具有抗菌、抗肿瘤活性,表明极地有良好的微生物药用资源.     

两株海洋微生物抗肿瘤代谢产物的筛选及其菌种鉴定

对5株海洋微生物进行发酵培养,发酵液离心后,收集上清液,采用饱和正丁醇萃取制备海洋微生物的代谢产物,采用MTT活性跟踪法,以BEL-7402、RKO、A549、U251和MCF-7等细胞系为模型,对代谢产物进行抗肿瘤活性的筛选。结果显示,菌株S-1和N16代谢产物的活性组分具有较好的抗肿瘤活性:菌株S-1对MCF-7、U251和BEL-7402三种肿瘤细胞的IC50值分别为44、102和82μg/ml;菌株N16对MCF-7和BEL-7402两种肿瘤细胞的IC50值分别为84、133μg/ml。对菌株S-1和N16进行了16S r RNA序列分析、生理生化特征鉴定,结果显示,菌株S-1为短芽孢杆菌属(Brevibacillus sp.),N16为芽孢杆菌属(Bacillus sp.)。通过显微镜观察了菌株Brevibacillus sp.S-1和Bacillus sp.N16代谢产物的活性组分对BEL-7402细胞形态的影响,表明两株菌的活性组分可以改变肝癌细胞BEL-7402的细胞形态,抑制细胞增殖。该研究为新型抗肿瘤先导化合物的发现提供了新的海洋微生物种质资源。     

一株南极大磷虾共附生细菌的分离鉴定及一氧化氮靶向抑制活性筛选

从我国南极磷虾科学探捕项目所采集的南极大磷虾(Euphausia superba)样品中分离纯化到一株共附生细菌,对其进行了细菌多相分类学分析、发酵培养、代谢产物提取及HPLC分析,并对其发酵粗提物进行了针对多种重大疾病药物靶标一氧化氮(N0)抑制活性的靶向筛选.结果表明,该菌株与其进化关系最近的需盐杆菌(Salege...     

一株南海柳珊瑚共附生细菌的鉴定及其产活性物质的培养条件优化

对一株南海柳珊瑚共附生微生物菌株(中科院南海海洋研究所,菌种编号00856)进行了分类,并对其所产的生物活性物质特性进行了研究。依据形态及16SrRNA基因序列分析,海洋细菌00856归于芽孢杆菌属(SCSIO 00856)。利用所选择的细菌培养基,在一定的培养条件(28℃,pH 7.0)下,该菌株的次级代谢产物对酿酒酵母、金黄色葡萄球菌有一定程度的抑制活性。同时,在培养6d时,该菌产生的生物活性物质对酿酒酵母抑菌效果最好。利用滤纸片法测定产物活性,以抑菌物质高产为目标,利用正交试验的方法优化了培养基的配比。结果表明,用酿酒酵母作为指示菌,以海盐素26.25g/L、葡萄糖12g/L、酵母浸粉3g/L、磷酸二氢钾3g/L、麦芽浸粉5g/L为培养基发酵培养6d,海洋细菌00856产抑菌活性物质最多。     

南极钩虾Gammarus sp.肠道共附生菌的分离及Streptomyces xiamenensis YF008的化学成分研究

南极极端生境中蕴含着发掘特殊生物活性天然产物的巨大潜力。对采自南极海域水深6 000 m海区钩虾(Gammarus sp.)的肠道共附生微生物进行分离,并对一株有价值菌株的代谢产物进行研究。采用稀释涂布法进行菌株分离,利用形态学观察及16S rRNA测序进行菌株种属鉴定;综合运用薄层色谱、正相硅胶柱色谱、凝胶柱色谱、反相ODS柱色谱和半制备高效液相(HPLC)等色谱手段分离纯化得到单体化合物,采用波谱学和质谱技术鉴定其化学结构。从钩虾中分离得到17株菌,并将其从分类学角度归属于9个属,其中细菌11株,放线菌6株。从放线菌Streptomyces xiamenensis YF008的发酵产物中分离得到8个单体化合物,分别鉴定为：1(10)E,5E-germacradiene-2,11-diol (1),N-[2-(4-hydroxyphenyl)] ethylacetamide (2),环(L-苯丙氨酸-甘氨酸)(3),环(L-苯丙氨酸-L-丙氨酸)(4),环(D-苯丙氨酸-L-异亮氨酸)(5),环(L-羟脯氨酸-L-苯丙氨酸)(6),环(D-亮氨酸-L-脯氨酸)(7),环(L-苯丙氨酸...     

一株产吡咯喹啉醌南极海洋细菌的分离鉴定及生物活性

从采集自南极FAO 48.1海域海水样品中分离纯化到一株产吡咯喹啉醌(PQQ)海洋细菌,对其进行了细菌分类学鉴定、摇瓶培养发酵、发酵产物提取及HPLC定量分析,并对纯化的PQQ进行了针对一氧化氮(NO)抑制活性及羟自由基(.OH)清除抗氧化活性评价。结果表明,该菌株与副球菌属(Paracoccus sp.)内菌株的16S rRNA基因序列同源性较高。综合多个分类学指标的分析结果,将其鉴定为属于副球菌属,命名为Paracoccus sp.PQ-08。生物活性评价结果表明,PQQ可显著抑制人脐静脉内皮(ECV-304)模式细胞内诱导型一氧化氮合成酶(iNOS)在mRNA转录水平及蛋白水平的表达量,明显降低胞内的NO释放量。当其作用浓度为100μM时,对iNOS蛋白表达的最高抑制率为67.9%(P<0.05)。此外,实验还证实PQQ具有较强的清除.OH的抗氧化活性。     

具抑菌活性海洋微生物的筛选

从采集于上海郊区海域的海水和海泥样品中分离出 193株海洋细菌，从中筛选抑菌活性菌株。 分别利用琼脂块法和管碟法对分离出的菌株进行初筛和复筛，结果显示，有 29株海洋细菌具有抑菌活性， 占总分离菌株的 15%，其中抑菌能力较强的 G4，抑菌谱较广，能同时抑制革兰氏阳性和阴性指示菌。     

微小亚历山大藻amtk-4共附生菌群多样性及其产毒新种Z1-D的sxtA1基因进化研究

藻菌关系是揭示赤潮生消与防控、赤潮毒素产生机制的关键,而产毒甲藻共附生菌群多样性及可培养菌株的获得是解析藻菌关系的前提。微小亚历山大藻(Alexandrium minutum)是全球性典型赤潮甲藻,其产生的麻痹性贝类毒素(PSP)危害巨大,但目前对其共附生菌群尚缺乏系统性研究。通过高通量测序首次解析了产毒微小亚历山大藻amtk-4共附生菌群的物种种类及相对丰度信息;分离获得可培养菌株并对筛选获得的产毒细菌新种Z1-D的毒素合成基因sxtA1进行了基因进化分析,结果表明,amtk-4共附生菌群包括85个OTU,其中包括10门、20纲、40目、59科及87属。其6个优势属包括Phycisphaeraceae科未知属(11.8%)、Muricauda属(10.3%)、腐螺旋菌科未鉴定属(9.1%)、Hyphomonadaceae科未鉴定属(8.9%)、Haliea属(5.7%)及红细菌科未鉴定属(5.1%)。amtk-4共附生菌群中未鉴定属比例高达53.4%。藻生长稳定期所分离获得的可培养细菌数量及种类最多。5株细菌中菌株Z1-D及Z1-4经分子鉴定分别为亚硫酸杆菌属(Sulfitobacter)及Mesorhizobium属新种。其中Z1-D发酵代谢产物含微量石房蛤毒素(STX),其基因片段orf-01498与蓝藻sxtA1基因高度同源,与产毒蓝藻间可能存在着基因共同进化。     

一株海洋源细菌素产生菌筛选及细菌素性质研究

化学食品防腐剂潜在的副作用使其使用受到越来越严峻的挑战,微生物产天然食品防腐细菌素成为关注的热点。本研究以L.monocytogenes、E.coli、S.aureus、B.subtilis、V.parahemolyticus、A.niger为指示菌,以牛津杯法评价抗菌活性,以南海海洋动物肠道作为分离材料,通过抗菌作用的排酸实验、过氧化氢排除实验以及蛋白酶敏感实验进行细菌素产生菌的筛选,并进行了细菌素物质的热稳定性与酸稳定性的研究。筛选出1株具有抗菌活性强,抗菌谱广的细菌素产生菌A0705,其发酵液在-20~100℃处理30 min后,再在p H2.5~11.5的范围内37℃温浴2 h,仍保持较稳定的抗菌活性,经16S r DNA序列同源性比对及系统发育树分析,鉴定菌株A0705为芽孢杆菌(Bacillus sp),该菌是1株值得进一步研究与开发的海洋源细菌素产生菌。     

分离自腌干鱼的抗氧化发酵菌株的筛选及鉴定

以符合耐盐、耐高低温、产酸等要求和过氧化氢耐受能力为指标,对分离自传统腌干鱼制品的29株乳酸菌进行初筛,再以抗脂质过氧化率、羟自由基清除率和还原力为复筛指标评价了菌株不同组分的体外抗氧化能力,以期获得具有优良抗氧化活性的发酵菌株。结果表明,15株乳酸菌符合腌干鱼发酵要求,菌株间的抗氧化活性差异明显,其中以L4、L11和L21综合抗氧化活性最好。经VITEK-2微生物鉴定系统和16S r DNA分子鉴定,确定L4、L11和L21分别为戊糖片球菌(Pediococcus pentosaceus)、植物乳杆菌(Lactobacillus plantarum)和干酪乳杆菌(L.casei)。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.