肝素钠对颈总动脉搭桥术犬纤溶系统的影响

选取10条雄性健康犬，随机分成对照组和治疗组，每组5条。实验动物模型采用同侧自体颈静脉行颈动脉45°搭桥。术后均采取常规治疗，治疗组给予肝素钠。分别于术前及术后1、2、3天在颈静脉采血，检测D－二聚体、血清纤维蛋白（原）降解产物（FDP）、血浆血小板α颗粒膜蛋白（GMP－140）、纤溶酶原激活剂的抑制物－1（PAI－1）和组织纤溶酶原激活剂（t—PA）等纤维蛋白溶解系统（纤溶）指标的变化。结果：治疗组与对照组相比，D－二聚体、GMP－140、PAI－1显著降低（P〈0．05），t—PA显著升高（P〈0．05），FDP变化不显著（P〉0．05）。结论：肝素钠可改善血管搭桥术后纤溶系统，纠正血液高凝状态，恢复抗凝系统活性。     

大黄注射液对犬血管搭桥术后纤溶系统的影响

为研究大黄注射液对犬血管搭桥术后纤溶系统的影响,将20只犬随机分为高(0.8 g.kg-1)、中(0.4g.kg-1)、低剂量组(0.08 g.kg-1)及对照组,同侧自体颈外静脉重建颈总动脉,头静脉滴注大黄注射液,ELISA定量分析测定术前及术后1、2、3、5、7、14、21 d纤溶系统各指标的变化。结果显示,用药后血小板α颗粒膜蛋白、D-二聚体、纤溶酶原激活剂的抑制物-1显著降低,组织纤溶酶原激活剂显著升高,且成剂量依赖关系;血清纤维蛋白(原)降解产物无显著变化。结果表明,大黄注射液能纠正搭桥术后血液高凝状态,恢复抗凝系统活性,预防血栓形成。     

应用一步亲和层析法纯化转基因山羊乳腺定位表达的组织型纤维蛋白溶酶原激活剂

对转基因山羊乳腺定位表达的羊奶中组织型纤维蛋白溶酶原激活剂 ( t PA)的分离纯化方法作了研究 ,摸索出一种简单可行的纯化方法。羊奶中 t PA先经冰乙酸和丙酮沉淀后 ,再通过 Benzamidin Sepharose 6B柱一步亲和层析 ,纯化倍数达 671 4.3倍 ,比活性 1 880 0 0 U/mg,活性回收率 2 6%。经 SDS-PAGE还原电泳分析 ,t PA纯度大于 95%,其中低分子双链蛋白占 90 %以上 ;Western-blotting检测证实 ,它和天然 t PA具有相同的抗原性 ;用纤维蛋白平板法检测 ,其比活性与天然 t PA相近 ,并且都可被 t PA单克隆抗体所抑制     

里氏木霉重组t-PA纯度、分子量及等电点鉴定

基因工程菌株里氏木霉(Trichodermareesei)生物合成的组织型纤溶酶原激活剂(tissue-typeplasminogenactivator,t-PA)经分离纯化后,采用还原SDS-PAGE和非还原PAGE分析达到了电泳纯,采用Superdex75PrepGrade凝胶过滤色谱分析达到了色谱纯;采用还原SDS-PAGE分析其相对分子质量为6.6×104,SDS-聚丙烯酰胺凝胶电泳纤维蛋白自显影法检测其相对分子质量为6.6×104,与相关文献报道的一致。等电聚焦电泳测定其等电点为4.24。     

蚁狮纤溶活性蛋白的发现及理化研究

本实验采用硫酸铵分段盐析法分离和提取得到了具有纤溶活性的蚁狮粗蛋白,使用纤维平板法检测目标蛋白纤溶活性,结果显示三个不同浓度梯度的粗蛋白均具有明显的纤溶活性;测得其最适温度为37℃,最适pH值约为6.0;金属离子Ba~(2+)、Cu~(2+)、Mg~(2+)、Mn~(2+)对蚁狮纤溶活性蛋白具有较强的抑制作用,Na~+对其抑制作用较小;抑制剂EGTA对粗蛋白抑制率较高,βME对其影响较小;使用BCA试剂盒测定蚁狮纤溶活性粗蛋白浓度为4mg/mL左右。     

新鲜和冷冻猪卵子体外成熟培养对纤溶酶原激活物存在的影响

对体外成熟培养不同时间段猪的新鲜和冷冻的卵子中是否能够释放纤溶酶原激活物(PAs)的情况进行了研究。被卵丘细胞包裹的卵子取自于囊状卵泡,用NUSU-23培养液进行体外培养。OPS冷冻法冷冻卵子。在成熟培养液的卵丘细胞通过用细管不断地吸吐的方法去除。用SDS和酶谱法对猪的卵丘卵母细胞和裸卵里的纤溶酶原激活物进行密度测定。结果显示:在猪新鲜的卵丘卵母细胞中能够检测出Upa、tPA和tPA-PAI。体外培养24h后,在裸卵中也可以检测出uPA的活性;体外成熟培养48h后,在卵丘卵母细胞中能够检测出tPA和tPA-PAI,但在裸卵中没有检测出PAs。在所有冷冻组的试验中,没有检测出PAs活性。     

蚯蚓纤溶酶活性研究

蚯蚓是优质的蛋白质饲料,但蚯蚓体内含有纤溶酶等生物活性物质,大量使用会造成动物机体的损害。为了保证蚯蚓作为蛋白饲料在畜牧业生产中的应用安全,本研究采用琼脂糖-牛血纤维蛋白原平板测定了蚯蚓水解液在不同时间、不同pH值和不同温度下的纤溶酶活性。结果表明,随着pH值降低和温度的升高蚯蚓纤溶酶的活性逐渐降低,但直至pH值为1时或温度达到100℃时纤溶酶仍具有较强的活性。因此认为,蚯蚓纤溶酶在胃液的酸性条件下或通常的环境温度下难以完全灭活,把蚯蚓作为蛋白饲料或饲料添加剂使用时应严格控制动物的摄入量,以保证养殖业的安全生产。     

中华圆田螺纤溶活性蛋白的 发现及活性研究

纤溶活性蛋白研究是当前研究之一,该蛋白具有良好的溶栓效果。中华圆田螺富含活性蛋白。本实验以中华圆田螺为研究对象,采用硫酸铵分段盐析分离和提取得到了活性粗蛋白。纤溶平板法证明其具有纤溶活性,并研究了温度、pH值、金属离子对其纤溶活性的影响。实验结果表明中华圆田螺活性蛋白最适作用温度为58℃;pH值为7～10范围内影响较小,活性较强;Na~+、K~+对其纤溶活性无影响,Ca~(2+)、Fe~(3+)对其有抑制作用。     

重组减毒沙门菌ΔcrpSL1344(pET28-tPA)在BHK细胞中的表达及活性检测

为了探讨原核表达质粒中的真核基因在减毒沙门菌中表达的多肽是否能在真核细胞中加工修饰成具有生物活性的蛋白,试验根据人t-PA的编码序列设计1对引物,扩增t-PA目的基因片段,构建pET28-tPA原核表达质粒,将表达质粒电转化导入减毒鼠伤寒沙门菌ΔcrpSL1344中并转染BHK细胞,应用SDS-PAGE技术检测t-PA表达情况,ELISA法检测其表达水平,琼脂糖平板溶圈法检测纤溶活性。结果表明:重组菌ΔcrpSL1344(pET28-tPA)在BHK细胞中有66.0 ku的蛋白表达,转染96 h的表达量为108μg/L,细胞裂解液和转染表达质粒的细胞培养上清液均有促纤溶活性。说明携带t-PA原核表达质粒的减毒沙门菌具有较强的促纤溶活性。     

隐性乳房炎对绵羊乳成分变化的影响

笔者研究了隐性乳房炎对同腺体水平绵羊产奶量及其成分的影响机制。采用36头单侧乳房被经过标记的凝固酶阴性葡萄球菌感染的以色列-阿萨福奶绵羊．选择另一侧未被感染的乳房为对照。研究结果表明：被感染一侧乳房的产奶量显著低于另一侧的产奶量(分别为0．36和0．76kg／次挤奶)，奶中体细胞记数和N-乙酰-β-氨基葡萄糖苷酶活性显著高于另一侧。被感染的乳腺中的纤溶酶原激活物和纤维蛋白溶解酶的活性显著高于另一侧的乳腺，而纤溶酶原活性及其与纤维蛋白溶解酶的比例却显著降低。Ca^2 浓度差异不显著，但被感染乳腺中Ca^2 活性却显著降低，（月示）蛋白胨浓度是未被感染乳腺中的2．4倍。被感染乳腺凝乳的产量显著低于未被感染的乳腺凝乳的产量。     

Efficiency and precision of electroimmunoassay for canine factor VIII-related antigen

Factors that improved the efficiency, precision, and sensitivity of the electroimmunoassay for canine factor VIII-related antigen were investigated. These included rapid electrophoresis, increased rocket heights, deletion of the gel-washing step, and doubling of the sample capacity per plate. The modified assay was reliable and accurate, could be completed within 3 hours, and permitted twice as many determinations. Other experimental variables that affected the assay were the type of agarose and sample diluent, storage conditions of samples before assay, source of antibody, and use of 2 mM calcium lactate.     

Use of newly developed assays for protein C and plasminogen in horses with signs of colic

Protein C content and plasminogen activity were measured in plasma from 100 horses with signs of colic. Data were analyzed by grouping horses 4 ways. Each horse was allotted to 1 of 2 outcome groups (survivors and nonsurvivors), 1 of 3 broad-category diagnosis groups (inflammatory disorders, strangulating obstructions, and all other gastrointestinal disorders), and 1 of 2 clinical management groups (medical and surgical). In a fourth grouping, all horses (although numbers of horses included in each subgroup were small) were assigned either to specific diagnostic groups that had high expectation for activated hemostasis (intestinal ischemia, endotoxemia, jugular thrombosis, peritoneal adhesions, and laminitis) or to a control group, in which active hemostasis was unlikely. Within 2 to 24 hours after admission, nonsurvivors developed lower protein C content than did survivors. Protein C content and plasminogen activity became low during hospitalization in horses with strangulating obstructions and in horses having surgery. The results from the grouping by specific diagnosis must be considered pilot data because the numbers of horses in each subgroup were small. Although not statistically significant, trends were noticed in protein C and plasminogen: (1) horses with intestinal ischemia and endotoxemia developed low protein C content and plasminogen activity, (2) protein C content became low in horses that developed peritoneal adhesions or laminitis, and (3) plasminogen activity became low in horses that developed jugular thrombosis. Low protein C content or low plasminogen activity, or both, may be useful as predictors for outcome and for these specific complications of equine colic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved agar plate assays of bovine lysozyme and haemolytic complement activity

Bovine serum and colostral whey samples were examined for lysozyme and haemolytic complement activity, employing agar plate techniques. The tests were carried out in agarose gel containing Micrococcus lysodeikticus (for lysozyme), and antibody sensitized rabbit erythrocytes (for complement), respectively. The confirmation of lysozyme (E.C.3.2.1.17) — dependent lysis has been presented elsewhere (Lie & Syed 1986), while heat-inaotivation and antibody to C3 were used in the present study to confirm that the haemolytic activity was attributable to the complement cascade. Repeatability and sensitivity of the described tests were found to be superior to those of photometric procedures. Staining and preservation techniques were developed which extended the applicability of the assay, as they made reading of results independent of time and resulted in the plates being very suitable for photography and storage.     

Interactions of phytate and myo-inositol phosphate esters (IP1-5) including IP5 isomers with dietary protein and iron and inhibition of pepsin

Phytic acid (IP(6)) and myo-inositol phosphate esters (IP(1-5)), including IP(5) isomers prepared chemically and enzymatically with bacterial and fungal phytases, were examined for their effects on protein aggregation of soy protein and β-casein, interaction with Fe(3+), and pepsin activity. The results indicated that the aggregating capabilities of IP esters (IP(1-6)) on the 2 proteins decreased dramatically from IP(6) to IP(5) and became negligible with IP(1-4). Among the IP(5) isomers tested, InsP(5)(1,2,3,4,5) produced by 6-phytase was slightly less powerful in aggregating protein than InsP(5)(1,2,4,5,6) produced by 3-phytase (P = 0.001). For protein hydrolysis, IP esters of IP(3-4) still showed inhibition of pepsin though to a lesser extent than IP(5-6). The in vitro data with IP(1-5) generated with microbial 3- and 6-phytases indicate that, for complete alleviation of pepsin inhibition, IP(6) needs to be broken down to IP(1-2.) In contrast to the aggregation with protein, the reactivity of IP(1-6) toward Fe(3+) decreased proportionally from IP(6) to IP(3.) Based on the radical decrease in turbidity of IP(6) -protein complex observed, as a result of IP(6) dephosphorylation to IP(5), a novel qualitative and semi-quantitative phytase plate assay was established using IP(6)-protein complex incorporated into an agarose petri-dish as substrate. Phytase activity was shown as the development of clear halos on the agarose plate with time. This simple phytase plate assay method can be used at animal farms, control laboratories, and even for the screening of engineered phytase variants. The current study, thus, stresses the importance of the efficient hydrolysis of IP(6) at lower pH range to alleviate the negative effect of phytic acid and its degradation products on protein and Fe(3+) digestion.     

Sensitivity of commercial prothrombin time reagents to detect coagulation factor deficiencies in equine plasma

The sensitivity of commercial prothrombin time (PT) tests was assessed based on a dilution series of equine pooled plasma (EPP) (experiment 1) and on 40 equine plasma samples with reduced activity of coagulation factors II, V, VII and X (experiment 2). Two different PT reagents (reagent 1, human placental thromboplastin; reagent 2, recombinant human tissue factor) were used according to the manufacturers' instructions (standard test, PT([ST])) and compared to a modified test procedure (modified test, PT([MT])) using sample dilution and fibrinogen addition. In all samples, sensitivity was lower (P<0.01) when using PT([ST]) with reagent 2 (0.20) than when using either PT([ST]) with reagent 1 (0.65) or PT([MT]) with both reagents (reagent 1, 0.60-0.75, reagent 2, 0.58-0.70, depending on sample dilution). The highest sensitivity was found for PT([MT]) when using a 1:20 sample dilution. In those samples in which at least one coagulation factor activity was decreased (by 20%; n=18), the sensitivity of PT([ST]) with reagent 2 (0.33) was found to be inadequate, in contrast to all other test procedures (0.83-0.94). This low sensitivity corresponded to shorter time intervals between different coagulation activity levels prepared by EPP dilution. The results indicate that adequate sensitivity of PT measurements in equine plasma can be achieved using a standard test procedure as long as a suitable reagent is used.     

Peroxidative protection of parenteral admixture by D-alpha-tocopherol

High lipid:low dextrose (HL:LD) parenteral admixtures (PAs) are becoming commonplace in the nutritional support of veterinary patients. Lipid peroxidation before administration appears to be an unwanted sequela of high lipid content in PAs that can lead to oxidative injury of biologic membranes in vivo. The purpose of this in vitro study was to measure hydroperoxides in HL:LD PAs and to determine the optimal dose of d-alpha-tocopherol to minimize peroxidation in these PAs during a 24-hr period. Detectable concentrations of hydroperoxides were present in all PAs. D-alpha-tocopherol appeared to significantly minimize peroxidation of HL:LD PAs in vitro. These results have clinical implications for parenteral feeding in critically ill patients.     

牛血红蛋白β亚基片段衍生抗菌肽的分子设计及其抗菌活性分析

【目的】 试验旨在获得分子质量小、溶血性低、稳定性好的抗菌肽。【方法】 以牛血红蛋白β亚基的氨基酸序列为基础,以抗菌肽的构效理论为指导,筛选出1条多肽（YKK-18）,并以其为模板肽设计了3条长度为18个氨基酸残基的改造多肽（LJ-1、LJ-2和DLK-3）,通过最小抑菌浓度的测定反映4条多肽的抗菌活性,采用琼脂糖扩散法分析有抗菌活性多肽的热稳定性、酸碱稳定性、盐离子稳定性和反复冻融稳定性,用分光光度法测定有抗菌活性多肽的溶血性。【结果】 设计的4条多肽均为带正电荷的亲水性多肽,与APD3数据库中收录的抗菌肽氨基酸序列相似性均低于50%,二级结构中均有较高含量的α-螺旋,改造多肽的两亲性程度均高于模板肽,模板肽对测试菌株没有抗菌活性,而改造多肽对测试的大肠杆菌、沙门菌、绿脓假单胞菌、葡萄球菌、白色念珠菌等均有不同程度的抗菌活性,其中DLK-3和LJ-1的抗菌活性优于LJ-2,但对奇异变形杆菌均没有抗菌活性,在100 ℃高温、pH 4.0~10.0、生理浓度的盐离子（Na⁺、K⁺、Ca²⁺、Mg²⁺、Fe³⁺、Cu²⁺）和14次反复冻融等条件下仍有较好的抗菌活性,DLK-3的溶血作用呈剂量依赖性,在200 μg/mL时的溶血率已超过50%,而LJ-1和LJ-2的溶血率在高浓度（200 μg/mL）时仍低于5%。【结论】 本研究结果为抗菌肽的分子设计和改造提供了参考,获得的抗菌肽LJ-1和LJ-2具有成为抗生素替代物的潜力。     

Alternative pathway of bovine complement: concentration of factor B, hemolytic activity and heritability

Concentrations of bovine factor B (Bbov) were determined by radial immunodiffusion in sera of 46 Holstein cows and heifers aged one to nine years. Mean values were 34.2 +/- 5.3 mg/100 ml. A hemolytic diffusion plate assay in agarose gel in presence of 10 mM EGTA and 5 mM Mg accurately measured concentrations of purified Bbov but gave higher mean values, i.e. 47.8 +/- 10.2 mg/100 ml, for concentrations of Bbov in whole sera. Hemolytic values obtained by the hemolytic diffusion plate assay, however, weakly correlated (r = .4539, p less than 0.01) with the serum concentration of Bbov measured by radial immunodiffusion. It was concluded that the hemolytic diffusion plate assay was not an accurate technique for the quantitative measurement of Bbov but a good assay for quantitation of the total hemolytic activity mediated via activation of the alternative complement pathway. It is suggested that the difference between the values obtained by the two tests for one particular serum is, to some degree, an expression of the ratio of amplification and restriction of the alternative pathway activity. No significant heritability (offspring and one parent) was detected for the hemolytic activity of serum. A heritability of 0.93 at a significance level of p less than 0.1 was determined for the serum concentration of Bbov.     

采用改良CTAB法提取柞树(Quercus L.)基因组DNA

为了获得质量较高的柞树基因组DNA,采用经过改良的十六烷基三乙基溴化铵(CTAB)提取法对几种常见柞树植物进行基因组DNA的提取,所得到的DNA样品经紫外分光光度计和琼脂糖凝胶电泳检测,纯度较高,D260 nm/D280 nm为1.75~1.80,应用于SSR和RAPD标记分析时,可以获得较好的扩增片段。     

干旱胁迫对紫花苜蓿叶片和根系多胺代谢的影响

采用盆栽试验方法研究了紫花苜蓿(品种:陇东) 叶片和根系中游离态腐胺(Put)、亚精胺(Spd)、精胺(Spm)及精氨酸脱羧酶(ADC)、鸟氨酸脱羧酶(ODC)、多胺氧化酶(PAO)活性的动态变化。结果表明,干旱胁迫下紫花苜蓿叶片和根系中的多胺含量增加,随着胁迫时间的延长, Put、Spd、Spm及ADC、ODC、PAO活性均呈先上升后下降的趋势,且随胁迫程度加重而增大。ADC、ODC活性与Put含量呈极显著正相关,参与了紫花苜蓿Put的合成代谢。PAO活性与Spd、Spm含量变化趋势相似且呈极显著相关关系,叶和根中多胺代谢显著相关,表明多胺代谢与紫花苜蓿抗旱性关系密切。