Purification and characterization of a 630 kDa bacterial killing metalloprotease (KilC) isolated from plaice Pleuronectes platessa (L.), epidermal mucus

Antibacterial chemicals in the mucus of fish such as lysozyme, lectins, peptides and proteases provide an efficient first line of defence against pathogens. This study shows that there are at least three antibacterial proteins in plaice skin mucus in addition to lysozyme. One of these proteins is responsible for approximately 74% of the antibacterial activity and is a 630 kDa protease complex designated KilC (bacterial killing metalloprotease C). Purified KilC kills the bacteria Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa efficiently. The protease activity of KilC is dependent upon the divalent cation Mg(2+) and shows pH dual optima of 5.0 and 8.0. The enzyme has a temperature optimum of 25 degrees C and is made up of at least five different sized peptides. Studies with protease inhibitors show that the catalytic site of KilC may be cysteine- or serine protease-like. KilC may kill bacterial cells by acting directly upon the bacteria or by producing low molecular weight bioactive compounds such as peptides.     

A comparison of changes in stable isotope ratios in the epidermal mucus and muscle tissue of slow‐growing adult catfish

Although stable isotope analysis is a powerful tool for determining diet, migration patterns and the structure of food webs in aquatic systems, the slow response of isotopic ratios in the widely used muscle tissue often hampers this approach, particularly in slow‐growing or adult fishes. We conducted a diet‐switch experiment to compare the changes in the stable carbon isotope ratio (δ¹³C values) in the epidermal mucus and muscle tissue of five‐year‐old catfish (Silurus asotus). The isotope ratios in noninvasively sampled mucus changed more rapidly than those in the muscle tissue. As isotopic change in mucus was relatively rapid, this technique can be used over a finer timescale than traditional isotopic analyses using the muscle tissue. The isotopic change half‐life in our experimental condition was 200 days, which would not be short enough for some research purposes. Examining mucus along with the muscle would enable food habits of slow‐growing fishes to be determined over different timescales. However, the rate of isotopic change in the mucus was negatively affected by the size of fish and was slower than the previously reported rates in juvenile steelhead. These findings suggest that mucus turnover rates need to be determined prior to the field data interpretation.     

黄鳝体表粘液中基因组DNA的提取与分析

黄鳝基因组DNA一直采用抽血或剪取肌肉来提取,但是采血或剪取肌肉后黄鳝成活率很低。为了保留亲本,尝试从黄鳝体表粘液中提取DNA,并采用紫外分光光度计对提取到的DNA的纯度和浓度进行了检测。结果表明,用蛋白酶K消化,酚仿抽提后从粘液中得到的DNA的纯度与从肌肉中提取到的相差不大,但是提取到的量较少。为了验证提取的DNA是不是黄鳝基因组DNA,用黄鳝actin引物和18S r RNA基因引物扩增之后检测到了粘液中的较亮的DNA片段。为了进一步确认检测到的是黄鳝的DNA,我们又对得到的DNA进行了扩增,并得到的产物进行了克隆和测序。结果表明,粘液中提取到的DNA是黄鳝的基因组DNA。随后又选择了60尾黄鳝,提取其粘液之后对其成活率进行统计,养殖1个月后黄鳝的成活率都在95%以上。此黄鳝体表粘液提取DNA方法能提取高质量的DNA,可用于用于PCR鉴定。     

鸭跖草营养器官的解剖学研究

本文报道了药用植物鸭跖草的根、茎和叶的解剖结构。鸭跖草的木质部为六原型，韧皮部与木质部排列，根皮层中分布有粘液细胞和晶体细胞。其内皮层具凯氏带。茎中维管束散生，许多近表皮薄壁组织细胞中含有分散的或成束的多个晶体，茎中央薄壁组织中含有粘液细胞和单宁细胞。叶为两面叶，叶肉组织分栅栏组织和海绵组织，栅栏组织细胞一层，叶中含有粘液细胞和晶体细胞。     

母牛发情周期和妊娠早期宫颈-阴道粘液电导率变化规律的研究

 本文应用自行设计安装的MFR-1型母牛发情鉴定及早期妊娠诊断仪，将特制探测电极插入母牛阴道穹窿部，测定了94头母牛宫颈-阴道粘液的电导率（CVMEC）。结果表明，母牛在发情周期和妊娠早期CVMEC呈规律性变化。发情周期正常的母牛，CVMEC在间情期处于较低水平（5.64mS/cm），前情期开始上升（8.93mS/cm），发情期达峰值（13.40mS/cm），后情期开始下降（9.84mS/cm），最后降至间情期水平。输精后，未妊与妊娠母牛CVMEC的变化规律不同。在发情周期的各时期，未妊母牛高于妊娠母牛；未妊母牛在输精后21天左右返情，CVMEC再度出现峰值；妊娠早期母牛则保持与间情期相近的较低水平（5.08mS/cm）。笔者认为，母牛CVMEC的规律性变化的生理学基础是体内激素水平和宫颈-阴道粘液的周期性变化，据此进行发情鉴定和早期妊娠诊断是可行的。与目前使用的方法相比，CVMEC法更具有实用价值。     

Evaluation of three hormonal protocols for anovulatory lactating cows under regulations restricting the use of estrogenic compounds

When European Union regulations restricted the use of estrogenic compounds in food‐producing animals, refined hormonal protocols were no longer applicable for anovulatory cows. However, Ovsynch and its adaptations are routinely and uniformly applied to all cows regardless of ovarian function. To evaluate their efficacy on anovulatory cows, 143, 147 and 144 anovulatory cows received Ovsynch, Presynch and G6G protocols, respectively. In comparison, 150 cyclic cows were bred without using a synchronized protocol. Results showed that cows in the Presynch group had luteolysis responding to the last prostaglandin F_2α (PGF_2α) injection greater than the Ovsynch group. The serous progesterone levels at the first gonadotropin‐releasing hormone of Ovsych and the last PGF_2α injection was greater in the G6G group than the other two hormonal treatment groups. Concentrations of Ca²⁺ and total protein in cervical mucus in all three hormone‐treated groups before artificial insemination (AI) were significantly different from the controls. The G6G group obtained a greater pregnancy rate compared with Ovsynch and Presynch, but significantly less than the controls. For open cows in the Ovsynch group, estrus rate within 24 days after the first AI was significantly less than the controls. In conclusion, the G6G treatment resulted to better reproductive performance in anovulatory cows.     

Epidermal proteases of the Japanese eel

A fluorescent-sensitive assay was used to demonstrate the protease activity in the dorsal skin of Japanese eel (Anguilla japonica). Two distinct extracts were separately prepared from skin mucus and epidermal cell layers, with no mutual contamination. The epidermal extract was sensitive to various substrates, whereas there was no, or only marginal, susceptibility to the same substrates for the mucous extract. Optimum hydrolysis pHs of the epidermal extract was variable and below pH 7.0, and the optimum hydrolysis temperatures were between 40 and 50 °C. In addition, Tos-Phe-Ch₂Cl, chymostatin, CdCl₂, CuCl₂, HgCl₂ and ZnCl₂ inhibited protease activities to different extents. Several other reagents specifically affected the protease activities, and their induced effects were useful for the identification of epidermal proteases. The findings indicate that a proteolytic factor, exhibiting various enzymological specificities, is retained within epidermal cell layers of Japanese eel. This factor is composed of 4 distinct proteases, such as cathepsins L and B-like proteases, a serine protease and an aminopeptidase.     

Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel (Anguilla japonica) for its use in aquaculture

In the present study, a potential Lactobacilli probiotics were isolated from Japanese eels (Anguilla japonica) and characterized and evaluated for their possible use in eel farming. Sixteen Lactobacilli were isolated from intestines of Japanese eels, using selective media. The lactobacilli strains (represented as PL1 to PL16) were screened by their ability to produce digestive enzyme. Among these, three strains (PL11, PL13 and PL16) producing four digestive enzymes (amylase, cellulase, protease and phytase) simultaneously were characterized further using API ZYM kit. From these, PL11 (Lactobacillu (L.) pentosus) was identified as potential probiotics candidate producing 15 enzymes among 20 tested. Further examination of biological activities of PL11 revealed tolerance against pH, artificial bile juice and antibacterial activity against several fish pathogenic bacteria. The in vitro competitive exclusion assay also revealed 88.4% reduction in adhesion of fish pathogen (Edwardsiella tarda) by PL11 to host intestinal mucus. In vitro incubation of Japanese eel foregut with Baclight‐labelled PL11 showed colonization of the enterocyte surface by confocal and scanning electron microscopy. In summary, PL11 isolated from eels could serve as a potential probiotics with acid and bile tolerance, production of digestive enzymes, antibacterial activity and inhibition of fish pathogen adhesion to intestinal mucus.     

滴鼻免疫疫苗对SPF鸡气管功能影响的研究

为研究疫苗滴鼻免疫对鸡气管功能的影响,本研究对SPF鸡滴鼻免疫新城疫疫苗后气管纤毛摆动速率、气管形态结构和黏液分泌进行了研究。80只7日龄SPF鸡随机分为对照组和试验组,每组40只,试验组SPF鸡滴鼻免疫新城疫弱毒疫苗,对照组滴鼻生理盐水;于免疫后的第0、2、4和6天,每组每次随机剖杀10只鸡,应用红细胞示踪法测定气管纤毛摆动速率,采集气管制备石蜡组织切片进行HE和奥新蓝pH 2.5和pH 1.0染色。结果发现,疫苗免疫后的第0和2天试验组SPF鸡纤毛摆动速率较对照组升高,但差异不显著（P>0.05）;滴鼻免疫后SPF鸡气管结构完整,无明显损伤;滴鼻免疫后试验组SPF鸡气管酸性黏液分布容积在免疫后的第0和2天显著高于对照组（P<0.05）,而后明显降低,于免疫后的第6天又有所升高,但差异不显著（P>0.05）;硫酸化黏液分布容积在免疫后的第0和2天显著升高（P<0.05）,而后降低并与对照组相一致。结果表明,新城疫疫苗滴鼻免疫后前两天SPF鸡气管黏液分泌增多,而后恢复到正常水平,疫苗的免疫对气管形态结构和纤毛的摆动速率无明显影响。