Apoptosis in Cooled Porcine Oocytes: Role of Calcium (Ca2+) and Ca2+-dependent Enzymes

Veterinary Research Communications -     

Isolation and purification of a diagnostic antigen for bovine cysticercosis by hydrophobic chromatography

An ammonium sulfate fraction of Taenia hydatigena cyst fluid (ThFAS) was further fractionated by hydrophobic interaction chromatography, using alkylagarose and omega-amino alkylagarose columns, in an effort to isolate and purify a specific diagnostic antigen in the ThFAS preparation. The less than 12 kDa antigen was found to have an affinity for immobilized alkanes with chain length of six carbons or greater. The antigen was recovered in an ethylene glycol eluate from a hexylagarose column then analyzed by Western blot; it reacted with bovine and human cysticercosis infection sera and with specific monoclonal antibodies but not with control sera or fascioliasis infection sera. When the eluate was used as coating antigen in a plate ELISA assay no false positive reactions were seen in sera from cattle infected with Fasciola hepatica; false positive reactions were observed for the unfractionated ThFAS antigen preparation.     

苜蓿草粉对禾花鲤蛋白酶和淀粉酶活性影响的研究

选取来源一致、规格整齐的2龄禾花鲤1 200尾,采用单因子完全随机设计,分为5个处理,每处理3个重复,每重复80尾鱼,分置于15个试验池中。各处理苜蓿Medicago sativa草粉的添加量分别为0（对照组）、40（试验Ⅰ组）、80（试验Ⅱ组）、120（试验Ⅲ组）和160 g/kg（试验Ⅳ组）,正试期50 d,观测苜蓿草粉对禾花鲤前、中、后肠及肝胰脏蛋白酶和淀粉酶活性的影响。结果表明：1）添加苜蓿草粉对前肠蛋白酶活性有显著影响 (P<0.05),其中试验II组显著高于对照组和试验Ⅳ组,但和试验Ⅰ、Ⅲ组差异不显著 (P>0.05)。添加苜蓿草粉对中、后肠及肝胰脏蛋白酶活性均无显著影响 (P>0.05),但它们有相同的变化趋势,即试验Ⅰ、Ⅱ、Ⅲ组鱼的蛋白酶活性均高于对照组。2）鲤鱼的前肠、中肠、后肠蛋白酶活性依次减弱,而肝胰脏的蛋白酶活性远低于肠道。3）添加苜蓿草粉后前肠及中肠淀粉酶活性比较,试验II组显著高于对照组 (P<0.05),其余各组与对照组差异不显著 (P>0.05),后肠淀粉酶活性与对照组无显著差异 (P>0.05)。添加苜蓿草粉能提高鲤鱼肝胰脏淀粉酶活性,其中试验Ⅱ组显著高于对照组 (P<0.05),其余试验组与对照组无显著差异 (P>0.05)。4）禾花鲤前、中、后肠及肝胰脏4部分淀粉酶的活性有较大的不同,肝胰脏>后肠>中肠>前肠。     

Na(+)-dependent transport of D-xylose by bovine intestinal brush border membrane vesicles (BBMV) is inhibited by various pentoses and hexoses

To detect whether pentoses and hexoses occurring in rumen bacteria or in hemicellulose ingested with feed and partly released in the small intestine have an affinity for the Na(+)-dependent glucose transporter of the bovine intestinal brush border membrane (BBM), we investigated whether these monosaccharides inhibit Na(+)-dependent transport of 14C-labelled D-xylose across the BBM using brush border membrane vesicles (BBMV) isolated from the mid-jejunum of cows. We used D-xylose as the transport substrate, because it has a low affinity for the Na(+)-dependent glucose transporter and thus its uptake into BBMV is more efficiently competitively inhibited by other sugars than that of D-glucose. D-Ribose, D-mannose and L-rhamnose occurring in rumen bacteria significantly inhibited Na(+)-dependent uptake of D-xylose into BBMV, but their inhibitory effect was less than that of D-glucose, D-xylose and phlorizin. This also applied to L-arabinose (and D-arabinose), which is, like D-xylose and D-galactose, a constituent of hemicellulose, and to 2-deoxy-D-glucose. Of all monosaccharides tested, only D-fructose did not affect Na(+)-dependent D-xylose transport. It is concluded that some pentoses and hexoses occurring in rumen bacteria (D-ribose, D-mannose and L-rhamnose) or hemicellulose (L-arabinose and D-xylose) have a low affinity for the Na(+)-dependent glucose transporter of the bovine BBM and may therefore be absorbed from the jejunum when released in the small intestine.     

Specific in-vitro lymphocyte immunostimulation activities of Brucella abortus fractions obtained by column chromatography

A Brucella abortus soluble antigen (BASA) preparation and fractions obtained thereof, by column chromatography, were compared in terms of their ability to induce specific lymphocyte stimulation responses (LSR) in lymphocytes from cattle infected with B. abortus. Endotoxin and protein contents and the fractions were determined. The LSR induced by BASA and the fractions were compared in terms of correctly identifying samples from infected and non-infected cattle. The sensitivity and specificity for each preparation were determined and these two attributes were then correlated with endotoxin and protein content. The results suggest that lymphocyte stimulation had greater association with relative protein content than endotoxin content of the antigen preparations.     

喀斯特山区不同草地土壤养分与酶活性特征

为探究喀斯特山区植被恢复过程中不同草地的土壤性质,研究了贵州喀斯特山区不同草地土壤酶活性和土壤养分含量的变化规律。结果表明:土壤酶活性和土壤养分含量随着土层厚度增加而减少;过氧化氢酶和磷酸酶活性在3种草地中的变化趋势为林草间作地>荒草地>退耕还草地,脲酶活性在0~10 cm土层的变化趋势为林草间作地>荒草地>退耕还草地,10 cm以下土层的变化趋势为林草间作地>退耕还草地>荒草地,蔗糖酶活性为荒草地>林草间作地>退耕还草地,蛋白酶活性为林草间作地>退耕还草地>荒草地,多酚氧化酶活性在3种草地中的变化各异;土壤养分含量在3种草地土壤中表现为林草间作的高于荒草地和退耕还草地。相关性分析得出:不同草地土壤养分含量与土壤酶活性的关系不同,正负兼有。研究结果表明:随着地表植被逐渐恢复、生物多样性逐渐增加的林草间作地土壤酶活性与土壤养分含量都较高,喀斯特山区生态环境逐渐得到了改善。     

High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.     

小肽制品对南美白对虾蛋白酶和淀粉酶活力的影响

有关对虾消化酶的研究已有大量的报道,但多集中在中国对虾和日本对虾上(潘鲁青和王克行,1997;潘鲁青和王伟,1997;孙建明等,1995;刘玉梅等,1991),对南美白对虾消化酶的研究未见报道。饵料成分的变化对对虾消化酶活力的影响亦仅见于中国对虾(罗日祥,1997)。本试验研究南美白对虾不同消化器官的消化酶酶活力以及小肽制品对南美白对虾蛋白酶和淀粉酶活力的影响,以期为南美白对虾饲料的配制及小肽制品的促生长机理提供依据。1.1试验设计采用单因子浓度梯度法,在基础饲料中分别添加0.1%、0.5%、1.0%、1.5%、2.0%、2.5%的小肽制品(美国华达公司提供…     

The use of metal chelate affinity chromatography on the isolation of Leishmania chagasi promastigote hydrophobic proteinases

In this work, we have assessed the possibility of isolating metalloproteinase fractions from infective Leishmania chagasi promastigotes. Our strategy was the association of the Triton X-114 method with iminodiacetic chromatography enriched with Zn2+. Thus, by using acid conditions, it was possible to isolate two fractions containing two polypeptides, 59 and 63 kDa. The enzymatic activity assay indicated that the two fractions and the two polypeptides had proteinase activities. In addition, it was proposed that those proteinase activities were affected by the presence of 1,10-phenanthroline, a metalloproteinase inhibitor. With this gentle chromatography strategy proposed it is possible to obtain metalloproteinases from L. chagasi in folding that preserve the enzyme activity. This is important for further studies on pathological complications observed in visceral leishmaniasis.     

Calmodulin is involved in the occurrence of extracellular Ca2+-dependent full-type hyperactivation in boar ejaculated spermatozoa incubated with cyclic AMP analogs

In mammals, hyperactivation is essential for sperm fertilization with oocytes in vivo. Two types of hyperactivation “full-type and nonfull-type patterns” can be observed in the spermatozoa from boars, bulls, and mice. We have a hypothesis that the full-type hyperactivation is a physiological (in vivo) pattern and are elucidating its molecular bases. The aims of this study were to detect calmodulin in boar sperm flagella by Western blotting and indirect immunofluorescence and to investigate effects of extracellular Ca²⁺ and calmodulin antagonists “W-7 and W-5 (W-5; a less potent antagonist)” on the occurrence of full-type hyperactivation in boar spermatozoa. Calmodulin was specifically detected as the 17-kDa antigen in the flagella and postacrosomal region of the heads. Full-type hyperactivation could be induced effectively in the samples incubated with 3.42 mM CaCl₂ for 120–180 min, and it was significantly reduced in the concentration-dependent manners of W-7 and W-5. Suppressing effects of W-7 on the full-type hyperactivation were stronger than those of W-5. These observations indicate that flagellar calmodulin is involved in the occurrence of extracellular Ca²⁺-dependent full-type hyperactivation in boar spermatozoa. This is the first indication of the intracellular Ca²⁺-sensing molecule which can function in the full-type hyperactivation.     

蛋白酶对中华草龟生长性能、形态性状及消化酶活性的影响

试验研究饲料中添加不同剂量的蛋白酶对中华草龟(Chinemys reevesii)生长性能、形态性状及消化酶活性的影响。选取810只初始均重为(5.7±0.1) g的中华草龟,随机分为3组,每组3个重复,每个重复90只。日粮中蛋白酶的添加剂量分别为0(G0组)、150 mg/kg(G15组)和200 mg/kg(G20组)。养殖周期为56 d。结果表明,与对照组相比,G15组中华草龟的背甲宽、肝脏和肠道胰蛋白酶的活性显著增加(P0.05),肝体比显著降低(P0.05);G20组腹甲长、壳高和肝脏中胰蛋白酶活性显著提高(P0.05),肠道中胰蛋白酶活性显著降低(P0.05)。G15组和G20组中华草龟的增重率分别提高了11.55%和2.88%,饲料系数降低了10.32%和3.14%,但与对照组无显著差异(P0.05)。综上所述,饲料中添加蛋白酶可提高中华草龟生长性能、形态学指标以及胃肠道消化酶活性,蛋白酶适宜添加量为150 mg/kg。     

Bovine polymorphonuclear neutrophil-mediated phagocytosis and an immunoglobulin G2 protease produced by Porphyromonas levii.

Acute interdigital phlegmon (AIP) is a commonly occurring anaerobic bacterial infection in cattle. This study examined in vitro the interaction of bovine polymorphonuclear granulocytic neutrophils (PMN) from blood with bacterial species involved in AIP. Polymorphonuclear neutrophils were purified from whole bovine blood, exposed to one of the three putative etiologic agents of AIP and comparatively assessed for phagocytosis using light microscopy. Fusobacterium necrophorum and Prevotella intermedia were effectively phagocytosed by PMN, but Porphyromonas levii was phagocytosed significantly less effectively by PMN. The effect of high titre anti-P. levii bovine serum on antibody-mediated phagocytosis by PMN was also evaluated. High titre serum increased the efficiency of phagocytosis of P. levii by bovine PMN. This was independent of heat labile complement factors. Antibodies specific for P. levii were assessed for protease activity capable of cleaving bovine immunoglobulins (IgG, IgG1, IgG2, and IgM). Partially purified supernatant from broth cultures of P. levii were incubated with biotinylated immunoglobulins (Igs). Samples were taken from times 0 to 72 h and examined using SDS-PAGE followed by Western blot analysis. Streptavidin-alkaline phosphatase and NBT-BCIP were used to visualize the Igs for heavy and light chains as well as lower molecular weight fragments of these glycoproteins. Porphyromonas levii produced an immunoglobulin protease which readily cleaved bovine IgG into fragments, but did not act against IgM. Specifically, the enzyme may be a significant virulence factor as it may act to neutralize the antibodies demonstrated necessary for effective PMN-mediated phagocytosis.     

血凝法测定饲料中植物凝集素含量

通过研究植物凝集素(lectin)的血细胞凝集活性与其浓度的关系，提出了用血凝法测定饲料中lectin的新方法，并探讨了试样粉碎粒度、脱脂方法、脱脂时间、抽提时间、抽提次数等测定条件对测定结果的影响。结果表明，lectin的活力与浓度之间存在高度线性相关(R^2=0．99)。血凝法测定饲料中lectin的含量时，以样品粉碎过40目、样品与30～60℃石油醚或乙醚比例(W／V)1：8脱脂8h、样品与生理盐水(W／V)1：8抽提8h为宜。在上述条件下测定，回收率可达88％～102％。经过对8种不同的饲料及原料样品lectin含量的测定，平均相对偏差为0．9％～2．4％，变异系数为1．3％～3．3％。结果表明，利用血凝法可快速测定饲料中的lectin含量。     

钌红对玻璃化冷冻牛卵母细胞线粒体Ca2+的调控研究

玻璃化冷冻会严重损伤哺乳动物卵母细胞的线粒体功能,进而极大地限制了其解冻后的发育能力。为此,本试验设置3个钌红（RR）处理组,即牛卵母细胞用含0.5、1、2 μmol／L RR的玻璃化冷冻液进行冷冻,解冻后放入含0.5、1、2 μmol／L RR的体外成熟液中继续培养0.5 h,同时,新鲜卵母细胞一部分不进行冷冻,一部分用不含RR的冷冻液进行玻璃化冷冻,分别作为新鲜对照组和玻璃化冷冻对照组,然后共检测5组牛卵母细胞线粒体Ca²⁺水平、ATP含量及孤雌激活后胚胎的发育能力,进而研究RR对玻璃化冷冻牛卵母细胞线粒体Ca²⁺水平的调控作用。结果显示：①玻璃化冷冻显著提高了牛卵母细胞中线粒体Ca²⁺水平（P＜0.05）,而2 μmol／L RR处理组线粒体Ca²⁺水平显著低于冷冻对照组（P＜0.05）,但与新鲜组相比无显著差异（P＞0.05）;②玻璃化冷冻显著降低了牛卵母细胞中ATP含量（P＜0.05）,2 μmol／L RR处理组卵母细胞中ATP含量显著高于冷冻对照组及0.5、1 μmol/L RR处理组（P＜0.05）;③玻璃化冷冻对照组卵裂率、囊胚率显著低于新鲜对照组（P＜0.05）,1 μmol／L处理组卵裂率、囊胚率与新鲜对照组相比无显著差异（P＞0.05）。综上所述,RR处理能显著抑制解冻后牛卵母细胞线粒体Ca²⁺流入,保护线粒体功能,提高其发育能力。本试验结果为正向调控玻璃化冷冻卵母细胞线粒体Ca²⁺水平,进而提高其发育能力,促进玻璃化冷冻卵母细胞的广泛应用提供了参考依据。     

Quantification of Mannheimia haemolytica leukotoxin by indirect ELISA

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of extracellular leukotoxin (LKT) produced in chemostat culture of Mannheimia haemolytica in a serum-free culture medium. Leukotoxin purified with preparative SDS-PAGE was used for the production of chicken polyclonal antibodies (PAb) that served as the primary detecting antibody. Excising the LKT protein from an analytical SDS-PAGE gel proved an efficient technique for the purification of the toxin. Consequently, the 102 kDa LKT polypeptide purified in this manner served as reference toxin and the resulting calibration curve was modelled using a four parameter logistic fit to relate absorbance to LKT protein concentration. The lower detection limit corresponded to an LKT concentration of 14.5 ng ml(-1). The presence of SDS, serum albumin and the coating pH had a distinct effect on the absorbance values of the indirect ELISA.     

肉鸡腹水综合征患鸡右心组织钙沉积和Ca2+-ATPase活性变化

右心肥大衰竭是腹水综合征患鸡发病的重要环节之一,而心肌细胞内Ca^2＋浓度在调节心脏收缩和舒张功能及其生长方面都起着重要作用。本试验应用右心导管法测定AS患鸡右心压力变化情况,采用焦锑酸钾沉淀法、电镜酶细胞化学法研究AS患鸡右心组织Ca^2＋和钙泵（Ca^2＋-ATPase）活性变化及其精确定位。结果显示AS组肉鸡右心室舒张压极显著高于对照组（P〈0.01）,同时,右心室内压最大变化速率也极显著降低（P〈0.01）;对照组肉鸡右心组织偶见少量散在的Ca^2＋沉淀颗粒,低温诱发AS患鸡右心组织发生了明显的钙沉积;对照组肉鸡的右心组织Ca^2＋-ATPase以高电子密度颗粒分布于肌浆网、线粒体膜等处,AS患鸡心脏组织的Ca^2＋-ATPase的电子密度颗粒显著减少或缺失。本研究揭示,在低温条件下AS患鸡具有明显的右心舒张功能障碍,Ca^2＋浓度增高和Ca^2＋-ATPase功能抑制可能在其中起着重要作用。     

Quantification of Ehrlichia ruminantium by real time PCR

Ehrlichia ruminantium (ER) is the causative agent of Heartwater, one of the most common tick-borne diseases affecting ruminants in African countries and West Indies. Although ER can be used as an inactivated vaccine for wild and domestic animals, there are currently no easy and reliable methods for the quantification of this obligate intracellular bacterium. This report describes the development of a SYBR Green I based real time PCR protocol for the quantification of ER for vaccine production purposes. The method was validated for four ER strains. The external-standard-based PCR protocol developed has a large dynamic quantitative range allowing accurate ER measurement in samples containing from 10(2) to 10(8) gene copies; the method is also reproducible and precise, with intra- and inter-assay coefficients below 5%. The detection limits were validated for samples collected from bovine aortic endothelial cell culture bulks, which are commonly used to produce the ER vaccine. In contrast to the methods based upon protein content, no interference from the host cells in ER quantification was observed. Furthermore, the extended applicability of the new technique was demonstrated by monitoring ER production in cell culture thus rendering it a valuable tool to ensure consistency between vaccine lots and to evaluate optimal vaccine dosage.