人工流产刮宫物中绒毛膜促性腺激素的提取和纯化

以健康妇女人工流产的刮宫物为原料,经50%乙醇溶液抽提、醋酸钠50%乙醇缓冲溶液分级沉淀、硫酸铵分段盐析,最后用DEAE—纤维素柱层析纯化.终产品放免活性为3500IU/mg,产品总回收率为40～50mg‰.终产品经PAG圆盘电泳分析,只出现1条电泳区带,并与纯度相近的尿HCG产品的电泳特性一致.     

家蚕丝素-RGD融合蛋白的盐析纯化工艺探讨

利用基因重组技术合成了具有良好细胞粘附活性的重组家蚕丝素-RGD融合蛋白质(Silk-RGD),探讨了其盐析的工艺条件。研究发现,在融合蛋白等电点pH8附近,当硫酸铵在溶液中的饱和度较低(10%)时,其蛋白质沉淀量随着温度的升高而增加;当硫酸铵的饱和度升至20%时,蛋白质沉淀量不再随着温度的变化而变化,但与低饱和度硫酸铵时相比,沉淀量明显增加;当硫酸铵的饱和度升至30%或以上时,沉淀中杂蛋白含量明显增多,不利于目的蛋白的纯化。Silk-RGD融合蛋白相对合理而且易操作的盐析纯化条件为:pH8,硫酸铵在溶液中的饱和度为20%,室温(约20℃)。利用该工艺条件,可以低成本、高效率地生产Silk-RGD融合蛋白,从而满足新型材料规模化开发的需要。     

An attempt to determine the tissue origin of equine serum alkaline phosphatase by isoelectric focusing.

The main purpose of this study was to ascertain whether isoelectric point determination of alkaline phosphatase (AP) using an isoelectric focusing technique on agarose gels could define the isoenzymes present in healthy equine serum. The isoelectric points of AP extracted from nine tissues ranged from pH 3.5 to 7.5 with all tissues having multiple bands. There was considerable similarity in band pattern among tissues, with only pancreatic and colostral AP having substantially different isoelectric points from the others. Sera contained thirteen bands with isoelectric points ranging from pH 3.5 to 6.2 and as each band was common to more than one tissue it was not possible to define the tissue origin of these by direct comparison with tissue patterns. The intensity of all serum bands declined as foals aged, with the greatest decrease in bands 4 and 5 (numbered from the anode). There was no relative change in the banding pattern between early and late pregnant mares or in the sera of two foals before and after ingestion of colostrum. The mean (+/- SD) total serum AP activities of young foals (1676 +/- 1100 IU/L), three month foals (402 +/- 64 IU/L) early pregnant (190 +/- 54 IU/L) and late pregnant mares (109 +/- 26 IU/L) were significantly different from each other whereas colostral ingestion in two neonatal foals had no effect. We concluded that equine AP is a very heterogeneous protein and that normal horse sera do not contain significant renal or small intestinal derived AP. However isoelectric focusing alone could not differentiate bone from liver derived AP in sera.     

牛初乳中乳铁蛋白粗提工艺的优化研究

利用盐析和有机溶剂沉淀相结合的方法对牛初乳中的乳铁蛋白进行初步的分离,采用单因素试验和正交试验的方法对影响分离效果的因素进行了试验研究,研究结果表明:用pH值8.5、80%的硫酸铵溶液进行盐析,然后用60%的乙醇浓度进行沉淀,分离的效果较好。SDS-PAGE凝胶电泳结果表明,乳铁蛋白的纯度大约为48%,分子量大约为78000Da。     

Purification of the phospholipase D of Corynebacterium pseudotuberculosis by recycling isoelectric focusing

Recycling isoelectric focusing was investigated as a method for purification of phospholipase D (PLD) from cultures of Corynebacterium pseudotuberculosis. Supernatant fluids from cultures of equine isolate 155 in brain-heart infusion broth were dialyzed against distilled water, concentrated by lyophilization, and fractionated by preparative isoelectric focusing in free solution in a pH 3 to 13 gradient with 6M urea. Protein concentration, pH, and PLD activity of the 10 resulting fractions were determined. Two PLD activity assays were used: release of 14C choline from labeled sphingomyelin and synergistic hemolytic activity with Rhodococcus equi factors. Enzyme activity focused in 2 fractions at pH 8.5 to 9.8. The synergistic hemolytic assay was simple and rapid for detecting PLD in partially purified fractions. Electrophoretic examination of the fraction containing the highest concentration of PLD activity revealed protein bands at 14, 21, and 31.7 kD Mr, suggesting purification to near-homogeneity. Proteins from the 31.7-kD band were labeled by antibodies in serum from a goat with chronic C pseudotuberculosis infection.     

Biological potency and radioimmunoassay of canine calcitonin

Calcitonin (CT) is a major calcitropic hormone. Because of low cross reactivity of canine CT (cCT) in radioimmunoassays (RIA) developed for other species, a homologous RIA is needed. Synthesis of cCT allowed study of its biologic potency using a rat bioassay and its plasma half-life in dogs. The availability of cCT also made possible the development of a homologous RIA for measurement of basal and stimulated plasma CT concentrations in dogs. The biologic potency of the synthesized cCT in rats is 24 IU/mg of peptide, which is low in comparison with the 4,000 IU/mg of the salmon CT standard. In the dog, an even lower potency of 4.4 IU/mg of cCT was found. Measurement of the disappearance of iv-injected radioiodinated or nonradioiodinated cCT revealed a short biologic half-life of less than 3 min, followed by a long half-life of 20 min. A polyclonal antiserum against synthetic cCT was raised in a goat. Using a final antiserum dilution of 1:12,000 and 125I-labeled synthetic cCT, the RIA had a detection limit of 6.5 ng/l. The antibody did not crossreact with standard human CT and had <0.1% cross reactivity with porcine CT. For measurement of plasma cCT concentrations, an extraction procedure was developed using ethanol. Dilutions of synthetic cCT and canine plasma extracts revealed parallelism over a wide range of concentrations. Size exclusion chromatography of canine plasma extracts on Biogel P-10 revealed a single cCT peak at the same position as [125I]-cCT, showing that there was little interference by other proteins or cCT prohormone. Basal plasma CT concentrations were 12-80 ng/l, and there was an 8- and 20-fold increase after calcium (1 and 2.5 mg/kg body weight) bolus infusion.     

Biological potency and radioimmunoassay of canine calcitonin

Calcitonin (CT) is a major calcitropic hormone. Because of low cross reactivity of canine CT (cCT) in radioimmunoassays (RIA) developed for other species, a homologous RIA is needed. Synthesis of cCT allowed study of its biologic potency using a rat bioassay and its plasma half-life in dogs. The availability of cCT also made possible the development of a homologous RIA for measurement of basal and stimulated plasma CT concentrations in dogs. The biologic potency of the synthesized cCT in rats is 24 IU/mg of peptide, which is low in comparison with the 4,000 IU/mg of the salmon CT standard. In the dog, an even lower potency of 4.4 IU/mg of cCT was found. Measurement of the disappearance of iv-injected radioiodinated or nonradioiodinated cCT revealed a short biologic half-life of less than 3 min, followed by a long half-life of 20 min. A polyclonal antiserum against synthetic cCT was raised in a goat. Using a final antiserum dilution of 1:12,000 and 125I-labeled synthetic cCT, the RIA had a detection limit of 6.5 ng/l. The antibody did not crossreact with standard human CT and had <0.1% cross reactivity with porcine CT. For measurement of plasma cCT concentrations, an extraction procedure was developed using ethanol. Dilutions of synthetic cCT and canine plasma extracts revealed parallelism over a wide range of concentrations. Size exclusion chromatography of canine plasma extracts on Biogel P-10 revealed a single cCT peak at the same position as [125I]-cCT, showing that there was little interference by other proteins or cCT prohormone. Basal plasma CT concentrations were 12-80 ng/l, and there was an 8- and 20-fold increase after calcium (1 and 2.5 mg/kg body weight) bolus infusion.     

碳源和氮源对黑曲霉产木聚糖酶的影响

研究了碳源、氮源以及其它因子对黑曲霉(Aspergillusniger)A3菌株产木聚糖酶的影响。结果表明,当迟效碳源为玉米芯70g/l、速效碳源为葡萄糖10g/l,氮源为NaNO33g/l和(NH4)2SO43g/l,麸皮5g/l,用无氮的Mandels无机盐溶液配制,起始pH值5.5～6.0,并添加中和剂CaCO3调控发酵pH值,250ml三角瓶装液量为30ml时,黑曲霉A3液体发酵酶活力达405.6IU/ml。采用玉米芯代替半纤维素作为主要碳源,可以大大降低生产成本,减少环境污染,有利于工业化生产。     

不同处理凤仙花对瘤胃微生物体外产甲烷及发酵特性的影响

本研究旨在分析不同处理凤仙花(Impatiens balsamina)对瘤胃微生物体外产甲烷( CH4)及发酵特性的影响.试验采集装有永久性瘤胃瘘管牛瘤胃液,以稻草粉、玉米粉和黄豆粉为人工饲料,通过体外培养法研究了凤仙花水浸提液、乙醇浸提液和固体粉剂3种处理方式不同添加量(0.5％、1.0％和2.5％)对气体产生以及瘤胃发酵参数pH、氨态氮(NH3-N)和微生物蛋白质(MCP)的影响.结果表明,凤仙花固体粉剂累积产气量最高,平均比对照提高了56.8％,乙醇浸提液和水浸提液分别平均比对照提高了24.7％和14.1％;凤仙花明显提高CO2含量,降低CH4和pH,且添加量越大,效果越明显,2.5％固体粉剂完全抑制CH4生成.凤仙花明显提高MCP含量,降低NH3-N含量,其中固体粉剂效果最显著,水浸提液效果最差.固体粉剂3个添加量使MCP含量分别比对照提高了84.9％、139.7％和199.7％ (P ＜0.05),NH3-N含量分别比对照降低了41.1％、52.2％和55.4％ (P＜0.05).结果提示,体外培养条件下,凤仙花明显降低CH4生成、提高CO2生成,并促进NH3-N向MCP转化,且固体粉剂处理效果最好.     

Characteristics of the labile neurotoxin associated with nervous coccidiosis.

Reported are the results of preliminary attempts to characterize the molecular weight, heat sensitivity and other features of a labile neurotoxin identified in the serum of calves exhibiting neurological signs in association with coccidial enteritis. The labile neurotoxin activity is heat labile (60 degrees C for 30 min) and is lost upon exposure to acidic pH (5.5) and cysteine (1.75 g/100 mL serum). Activity can be recovered from the precipitate of a 30% wt/vol solution of (NH4)2SO4 in serum. Ultrafiltration trials suggest that labile neurotoxin activity may be linked to a molecule of over 300,000 MW.     

多级沉淀与生物净化结合模式的水质净化特征分析

对北京市一企业园区一处4个串联水池(A-D池)的多级沉淀与生物净化相结合的净化系统的水样进行了实地采样和水质分析,测定了pH、COD、TN、NH4+-N、NO2--N等5项水质指标。结果表明,各项主要污染指标去除率为68.9%~87.6%,净化后水质清洁,达到地表环境水质量标准Ⅱ类水质标准,净化后的水体可用于一般景观水体的补水。物理沉淀与生物净化具备协同作用,其中A、B池沉淀作用更明显,并主要针对TP净化,C、D池生物净化作用更明显,并主要针对COD和N类污染。但两类净化作用表现并不独立,沉淀具初期快速净化作用,生物净化具彻底净化作用。     

In Vitro Biomechanical Comparison of a Modified 5.5 mm Locking Compression Plate Fixation with a 5.5 mm Locking Compression Plate Fixation of Osteotomized Equine Third Metacarpal Bones

Objectives: To compare number of cycles to failure for palmarodorsal 4‐point bending of a modified 5.5 mm broad locking compression plate (M5.5‐LCP) fixation with a 5.5 mm broad LCP (5.5‐LCP) fixation used to repair osteotomized equine third metacarpal (MC3) bones. Study Design: In vitro biomechanical testing. Animal Population: Adult equine cadaveric MC3 bones (n=6 pairs). Methods: An 8‐hole, M5.5‐LCP, obtained by having a 1.0 mm thickness removed from the bone contact portion of the 5.5‐LCP, was applied to the dorsal surface of 1 randomly selected MC3 from each pair, and an 8‐hole, 5.5‐LCP was applied dorsally to the contralateral bone from each pair using a combination of cortical and locking screws. Plates and screws were applied using standard ASIF techniques to MC3 bones with a mid‐diaphyseal osteotomy. MC3 constructs had palmarodorsal 4‐point bending cyclic fatigue testing. Mean cycles to failure for each method were compared using a paired t‐test within each group. Significance was set at P<.05. Results: Mean±SD cycles to failure of the M5.5‐LCP fixation (188,641±17,971) was significantly greater than that of the 5.5‐LCP fixation (166,497±15,539). Conclusion: M5.5‐LCP fixation was superior to 5.5‐LCP fixation of osteotomized equine MC3 bones in resisting cyclic fatigue under palmarodorsal 4‐point bending. Clinical Relevance: This suggests that biological plate fixation is not the ideal choice for osteotomized equine MC3 bones.     

野桑蚕多酚氧化酶的部分生化特性分析

基于开发专一的酶抑制剂控制野桑蚕危害桑园的目的,采用硫酸铵分级沉淀及Sephadex G-100凝胶过滤等方法,纯化了野桑蚕(Bom byx mandarina)多酚氧化酶,纯化倍数为57.14倍。该酶对焦性没食子酸、邻苯二酚和L-多巴的米氏常数(Km)值分别为3.39、2.06和3.17 mmol/L,在pH 7.0、37℃时活性最高。利用硫脲、抗坏血酸等多种氧化酶抑制剂对该酶活性的抑制结果表明,所用抑制剂对其均有不同程度的抑制作用。此外,该酶对乙二胺四乙酸(EDTA)和金属离子比较敏感。     

Evaluation of hyaluronidase activity in equine and bovine sera and equine synovial fluid samples by use of enzyme zymography

OBJECTIVE: To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes. SAMPLE POPULATION: Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses. PROCEDURE: Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis. RRESULTS: Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7 Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints.     

Acute response of urine pH following ammonium chloride administration to dogs

To test the acidifying ability of the distal portion of the nephrons in healthy dogs, 0.2 g of NH4Cl/kg of body weight was given PO. Samples for venous blood gas analysis and urine pH were taken hourly for 6 hours. Systemic acidemia developed, as evidenced by a statistically significant (P less than 0.05) decrease in blood pH 1 hour after NH4Cl administration. Four hours after administration, mean urine pH decreased to a low of 5.16 +/- 0.1 and was less than 5.5 3 hours after administration. Changes in urine pH 2 hours after administration were statistically significant (P less than 0.05). In human beings, NH4Cl loading is used to detect patients with distal renal tubular acidosis (defective hydrogen ion secretion by the distal nephrons) and normal acid/base values. Distal renal tubular acidosis is diagnosed if urine pH fails to decrease to less than 5.5 after NH4Cl administration. On the basis of the findings of this study, a similar value would be valid for dogs.     

Phosphodiesterase activity in neutrophils from horses with chronic obstructive pulmonary disease

Neutrophils are recruited to the lungs of horses with chronic obstructive pulmonary disease (COPD) and exhibit increased activity after antigen challenge. Phosphodiesterase type4 (PDE4) inhibitors have been shown to attenuate human neutrophil activation. The aim of this study was to establish the PDE isoenzyme profile of equine neutrophils using isoenzyme selective inhibitors to determine if these compounds should be evaluated in horses with COPD. Total cAMP and cGMP dependent PDE activity was no different in neutrophils from normal (156.2+/-7.1 and 6.8+/-0.6 pmol/min/mg for cAMP and cGMP, respectively) and COPD susceptible horses (146.0+/-10.2 and 5.5+/-0.6 pmol/min/mg for cAMP and cGMP, respectively). The PDE4 inhibitors, CDP840 and rolipram, caused significant, concentration related and almost complete inhibition of PDE activity (IC(50) values=8.8+/-0.1 x 10(-9) and 7.3+/-0.2 x 10(-9)M for CDP840; 1.2+/-0.1 x 10(-6) and 1.1+/-0.1 x 10(-6)M for rolipram in normal and COPD susceptible horses, respectively). The inhibitory effects of the mixed PDE3/ PDE4 inhibitor, zardaverine were of similar magnitude and potency to rolipram. However, the limited inhibitory effects of the PDE3 inhibitor, siguazodan, suggest that zardaverine is acting primarily via PDE4 inhibition. These results indicate that PDE4 is the predominant isoenzyme present in the equine neutrophil and inhibition of PDE activity using selective PDE4 inhibitors may, therefore, modulate equine neutrophil activation in horses with COPD.     

Effect of glucose addition and N sources in defined media on fibrolytic activity profiles of Neocallimastix sp. YQ1 grown on corn stover

Cleavage of plant cell wall arabinoxylans occurs by the action of ferulic acid esterase (FAE) and acetyl esterase (AE), which cleave feruloyl groups substituted at the 5'-OH group of arabinosyl residues and acetyl groups substituted at O-2/O-3 of the xylan backbone, respectively. In this study, we examined the enzyme profiles of the anaerobic rumen fungus Neocallimastix sp. YQ1 for FAE, AE and polysaccharide hydrolases when grown on corn stover, a lignin-rich waste biomaterial. A 2 × 4 factorial experiment in 10-days pure cultures was used to test glucose addition (G(+) : glucose at 1.0 g/l, G(-) : no glucose) and four N sources (N1: 1.0 g/l yeast extract, 1.0 g/l tryptone and 0.5 g/l (NH(4))(2) SO(4); N2: 2.8 g/l yeast extract and 0.5 g/l (NH(4))(2) SO(4) ; N3: 1.6 g/l tryptone and 0.5 g/l (NH(4))(2) SO(4); N4: 1.4 g/l tryptone and 1.7 g/l yeast extract) in defined media. The optimal combinations of glucose and N sources to promote FAE and AE activity were G(+) N2 and G(+) N4, respectively. The peak activities of FAE and AE occurred on days 9 and 10, respectively. Addition of glucose and an increase in yeast extract and/or tryptone to a Hungate's medium favoured fungal production of volatile fatty acids, which could be just a consequence of more organic matter available to digest. This suggests that enzymatic release of ferulic acid by a synergistic action of lignin hydrolytic esterase and polysaccharide hydrolases may be essential for plant cell wall biodegradation in the rumen.     

不同生理时期梅花鹿血液GSH-Px含量测定及其纯化

研究不同生理时期梅花鹿全血中GSH-Px含量变化及其纯化技术,为开发鹿血抗衰老资源奠定理论基础.用DINB法测定梅花鹿的溶血液、血细胞内容物、血浆和血细胞细胞膜中GSH-Px含量并计算全血GSH-Px含量；利用10％～100％饱和度的(NH4)2SO4盐析和柱层析技术纯化GSH-Px;用SDS-聚丙烯酰胺凝胶电泳测定其亚基相对分子质量.试验结果表明:全血GSH-Px含量以生茸期最高为(1 973.07士25.43)U·mL-1,与配种期的(1 727.74士12.46)U·mL-1差异极显著(P＜0.01),与生茸前期的(1 961.83士16.54)U·mL-1差异不显著(P＞0.05)；GSH-Px的初始比活力为24.9 U·mg-1,经纯化后得到GSH-Px 0.37 mg· mL-1,最终比活力1 347.7U·mg-1,纯化倍数为54.1倍,回收率25.00％；鹿血GSH-Px亚基的相对分子质量为17.2 ku.结果提示生茸期鹿血的GSH-Px含量最高,开发利用价值最大,GSH-Px含量与生理特性相关；鹿血GSH-Px盐析条件为40％～80％饱和度的(NH4)2SO4,此时所得GSH-Px纯化倍数较高.     

骆驼刺青贮中胶红酵母菌分离鉴定和菌株特性研究

用青贮骆驼刺进行倍比稀释、涂布获得单菌落,单菌落在PDA培养基上划线纯化,获得3株革兰氏阳性菌,经菌落形态特征鉴定、生理生化特征鉴定及ITS（ITS1＋5.8S＋ITS2）序列同源性分析,结果表明：其中1株菌红色单菌落的细胞形态呈圆形或椭圆形且中央隆起,能够利用蔗糖、D-半乳糖等多种碳源和（NH4）2SO4、L-谷氨酸2种氮源,不利用KNO3,5.8S rDNA测序的序列全长616 bp,采用DNA Star软件分析序列与胶红酵母标准菌株ATCC 4054（NCBI登录号：KC881069）的5.8S rDNA基因序列同源性为99.8%,确定其为胶红酵母（Rhodotorula mucilaginosa）。     

绿豆芽中SOD的分离纯化研究

采用硫酸铵盐析,透析,抽真空,DEAE-纤维素A-52柱层析,从绿豆芽中提纯了超氧化物歧化酶,酶的纯 化程度达到每毫克蛋白2 855U,总酶活回收率为38.4％,纯化倍数为27.5。