Purification and characterization of high antioxidant peptides from duck egg white protein hydrolysates

The hydrolysate from duck egg white protein (DEWP) prepared by “SEEP–Alcalase” at degree of hydrolysis (DH) value of 21% (namely HSA₂₁) exhibited high antioxidant capacity in different oxidation systems. A consecutive chromatographic method was then developed for separation and purification of HSA₂₁, including ion-exchange chromatography, macroporous adsorption resin (MAR) and gel filter chromatography. The final peptides “P_21-3–75-B” were obtained with significantly enhanced antioxidant activity (p < 0.05). It was further confirmed that the product mainly consisted of five oligopeptides (Mr: 202.1, 294.1, 382.1, 426.3, and 514.4 Da). Furthermore, the antioxidant activity of P_21-3–75-B kept stable after in vitro digestive simulation. Antioxidant capacity of the purified peptides was closely related to the molecular mass, hydrophobic amino acid residues, acidic amino acid and some antioxidant amino acids. This research provided a valuable route for producing new natural-source peptides with strong antioxidant capacity and high nutritious value for our daily intake.     

A Duplex Real-Time PCR Assay for the Simultaneous Detection of Porcine Circovirus 2 and Circovirus 3

Porcine circoviruses (PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry.     

Ultrastructure des cellules de Leydig et des cellules de Sertoli au cours du cycle testiculaire du Canard Pékin

Résumé L'ultrastructure des cellules de Leydig et des cellules de Sertoli du testicule du Canard Pékin a été étudiée au cours de la phase printanière du cycle sexuel, soit de janvier à juillet. Parallèlement on a effectué chez les mêemes animaux la recherche histochimique de la ⁵-3 -hydroxystéroïdedeshydrogénase (⁵-3 -HSDH) ainsi que le dosage, par chromatographie en phase gazeuse des stéroïdes androgènes dans le plasma veineux périphérique et dans le testicule.Les cellules de Leydig du Canard possèdent les organites cytoplasmiques spécifiques des cellules stéroïdogènes (reticulum lisse, mitochondries à crêtes tubulaires) ainsi que d'autres structures souvent rencontrées dans ce type cellulaire (microfilaments, vacuoles, granules denses). Les cellules de Sertoli contiennent un reticulum agranulaire moins développé que celui des cellules de Leydig et, très rarement, des mitochondries à crêtes tubulaires. Ces divers organites cytoplasmiques subissent un cycle saisonnier. La différenciation du reticulum lisse et des crêtes mitochondriales tubulaires commence en janvier et atteint son optimum en mars. Leur régression s'amorce en avril; d'abord accompagnée de structures dégénératives transitoires; elle conduit à la dispartion totale de ces organites en mait. Aucun indice de nécrose n'est observé dans ces cellules. Histochimiquement, une activité ⁵-3 -HSDH est présente dans les cellules de Leydig et, à un degré moindre, dans les tubes séminifères. Son intensité varie au cours du cycle.La confrontation de l'étude morphologique avec les résultats des dosages hormonaux montre qu'il existe une bonne corrélation entre le développement puis la régression du reticulum lisse et des crêtes tubulaires des mitochondries ainsi que des critères histochimiques de la ⁵-3 -HSDH d'une part et l'évolution de la testostérone plasmatique et testiculaire d'autre part. De plus on observe une augmentation du rapport testostérone/⁴-androstènedione testiculaire parallèlement au développement des organites cytoplasmiques. Ces organites semblent donc bien impliqués dans la synthèse et la sécrétion de la testostérone chez le Canard.

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Ultrastructure of Leydig and Sertoli cells in the testicular cycle of the Pekin duckBiochemical and cytoenzymological correlations

Summary Leydig and Sertoli cells of the testis of the Pekin duck were studied ultrastructurally during the spring phase of the sexual cycle, from January to July. Simultaneously, in the same animals, ⁵-3 -hydroxysteroiddehydrogenase (⁵-3 -HSDH) activity was ascertained histochemically and androgenic steroids of the plasma and testes were assayed by gas-liquid chromatography.The Leydig cells of the duck possess cytoplasmic organelles specific to steroidogenic cells (smooth reticulum, tubular mitochondria) as well as other structures often found in this cell type (microfilaments, vacuoles, denses bodies). The Sertoli cells contain an agranular reticulum that is less developed than that of the Leydig cells, and rarely show mitochondria with tubular cristae. These various cytoplasmic organelles undergo a seasonal cycle. The differentiation of the smooth reticulum and the mitochondrial tubular cristae begins in January and reaches a maximum in March. They begin to regress in April, at first with transitory degenerative structures, and then by total disappearance of these organelles by May. No indication of necrosis is observed in the cells. Histochemically ⁵-3 -HSDH activity is present in the Leydig cells, and to a slightly lesser degree in the seminiferous tubules. The intensity varies during the cycle.The comparison of the results of the morphological study with the hormone assays shows that a good correlation exists with the development and regression of the smooth endoplasmic reticulum and tubular cristae in the mitochondria, as well as the histochemical criteria of the ⁵-3 -HSDH on one hand, and the levels of plasma and testicular testosterone on the other hand. In addition there is an increase in the ratio of testicular testosterone to ⁴-androstenedione which parallels the development of the cytoplasmic organelles. These organelles thus seem to be implicated in the synthesis and secretion of testosterone in the duck.

Nous tenons à remercier très vivement Mme G. Collenot qui nous a initiées aux techniques d'histoenzymologie et nous a très gentiment permis de faire cette partie de nos recherches dans son laboratoire. Nous remerçions également M. Claude Pennarun, photographe, pour son excellente collaboration.     

贵州省石阡鸳鸯湖国家湿地公园越冬鸳鸯数量、分布及日行为初探

为了解贵州省石阡鸳鸯湖国家湿地公园越冬鸳鸯Aix galericulata的种群密度、日行为特征及人为干扰对其的影响,于2017年10月—2018年3月,采用直接计数法对鸳鸯数量、性比及人为干扰下的种群密度进行了22次调查。越冬中期,种群数量稳定在755只±17只,性比为1.14∶1,各条河沟的数量之间的差异具有高度统计学意义(X^2=84.041,df=5,P<0.01),即大河、小河与坝坎为鸳鸯分布及活动的主要区域;种群密度与人为干扰次数呈极显著负相关(R=-0.799,n=90,P<0.01),在人为干扰次数为1次±1次的区域,种群密度为42只/hm^2±3只/hm^2～53只/hm^2±14只/hm^2,当人为干扰次数达到7次±3次时,几乎没有鸳鸯活动。同时采用瞬时扫描法对鸳鸯的8种越冬行为进行了34 d(442 h)的观察,共记录各行为136 933次。取食、休息和修整为鸳鸯的主要行为;除了修整,鸳鸯在保育区与非保育区的各行为时间分配差异有高度统计学意义(P<0.01);越冬日行为节律极具规律性,取食高峰在07∶00—08∶00、14∶00—15∶00和17∶00—18∶00;休息高峰在13∶00—14∶00和15∶00—16∶00;飞行高峰在06∶00—07∶00(飞回湖区)和18∶00—19∶00(飞往夜栖地);修整在各时间段发生均较为频繁。     

GST pull-down结合质谱筛选猪圆环病毒2型ORF4潜在互作蛋白

猪圆环病毒2型编码的ORF4蛋白是近年来发现的新蛋白。迄今为止,人们对ORF4所参与的细胞生物学过程知之甚少。本研究首先构建了带双标签的真核表达载体pCMV-N-Flag-GST,再将ORF4基因插入该载体中,形成pCMV-N-Flag-GST-ORF4。将质粒转染293T细胞表达ORF4后,通过GSTPull-down试验捕获细胞内潜在与ORF4互作的蛋白库。经SDS-PAGE分离及银染后,对所得的特异性条带进行质谱鉴定,筛选出5个与ORF4潜在互作的蛋白,包括丝氨酸/苏氨酸蛋白磷酸酶6催化亚基、α心肌蛋白、β肌动蛋白、SEC-14样蛋白5和肌球蛋白myosin 9。上述研究结果为深入揭示ORF4在病毒感染细胞过程中发挥的作用提供新的思路与方向。     

Evidence for activation of Amh gene expression by steroidogenic factor 1

The anti-Müllerian hormone gene (Amh) is responsible for regression in males of the Müllerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species.     

猪圆环病毒2型双拷贝感染性DNA的构建及体外拯救

感染性分子克隆是研究病毒复制和致病机制的有力工具。本研究应用PCR诱变技术解决了外源片段易于自连的难题,成功将2个PCV2SD1株全基因组(DQ346683)头尾相接插入到真核生物表达载体pSK的多克隆位点中,构建重组质粒pSK-2PCV2;另外课题组成功构建含单个PCV2全基因组的pSK-PCV2和自身环化质粒ds-PCV2。将所得3种质粒分别转染无PCV污染的PK-15细胞系,经10次连续传代后,间接免疫荧光试验检测显示三者均在细胞核中聚集大量的病毒抗原;经RT-PCR检测都有PCV2特异性基因转录;透射电镜下可观察到直径约为17～20nm的典型PCV2病毒粒子;经测序鉴定所拯救出的病毒与亲本病毒核苷酸同源性为100%。拯救出的PCV2与亲本病毒具有相同的病毒学及分子生物学特性。本研究应用PCR诱变技术成功构建PCV2双拷贝感染性克隆,并经体外拯救证实其具有感染性,为进行PCV2分子特性及致病机理研究打下了基础。     

北京鸭线粒体基因组全序列测定和分析

线粒体DNA作为遗传标记,已在家鸡(Gallus gallus)和家鹅(Anser anser)的研究中取得了重大进展,而对家鸭(Anas platyrhychos domesticus)的研究却很少.本研究参照近源物种线粒体基因组序列设计15对引物,通过PCR扩增、测序、拼接,获得北京鸭(A.platyrhychos)线粒体基因组全序列,初步分析其特点和各基因的定位.结果显示,北京鸭线粒体基因组全长16 604 bp,碱基组成为29.19%A、22.20%T、15.80%G、32.81%C,包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码控制区(D-loop),基因组成及排列顺序与其他鸟类相似.基于线粒体D-loop区全序列,用N-J法构建了7种雁形目鸟类系统进化树,结果表明,北京鸭与绿头鸭(A.platyrhychos)系统进化关系较近.     

Mitochondrial DNA divergence in the critically imperilled pygmy madtom,Noturus stanauli (Siluriformes: Ictaluridae)

The mitochondrial cytochrome b gene was sequenced for six individuals of the pygmy madtom, Noturus stanauli, a globally imperilled catfish, from both known localities in Tennessee (U.S.A.) separated by over 1055 river km. Phylogenetic and population genetic analyses revealed little divergence between these populations for this locus.     

Identification of a virulence-associated protein homolog gene and ISRa1 in a plasmid of Riemerella anatipestifer

Riemerella anatipestifer is the causative agent of polyserositis of ducks and geese. We have previously reported that a 3.9-kb plasmid, pCFC1, carries protein genes (vapD1 and vapD2) that are similar to virulence-associated genes of other bacteria. In the present study, we report the complete sequence of a second plasmid of 5.6 kb, pCFC2. pCFC2 has a 28% G-C content and three large open reading frames (ORFs). One of the ORFs (designated asVapD1) encodes a polypeptide that shares 53.9, 53.9, 48.3, 48.3 and 46.1% identity with virulence-associated proteins of Dichelobacter nodosus, Actinobacillus actinomycetemcomitans, Neisseria gonorrhoeae, Helicobacter pylori and Haemophilus influenzae, respectively. The second ORF encodes a putative DNA replication protein (RepA3) with 309 amino acids and a molecular mass of approximately 36 kDa. A novel insertion sequence (IS) element, designated ISRa1, was found on the plasmid pCFC2. ISRa1 was flanked by 15-bp imperfect inverted repeats (only one mismatched nucleotide). ISRa1 contained an ORF encoding a putative transposase of 292 amino acids. Southern blot analysis indicated that in R. anatipestifer strains examined, ISRa1 was present with 2-20 copies (at least). ISRa1 displayed a sequence approximately 35% homologous to the putative IS982 and RSBst-alpha from Lactococcus lactis ssp. cremoris SK11 and Bacillus stearothermophilus CU21. Three hybridization patterns of genomic DNA of eight R. anatipestifer strains with an ISRa1 probe indicated that ISRa1 might be a useful tool for epidemiological studies.