Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa)

The classification of some of the extractable birch pollen antigens as allergens was established by crossed radioimmunoelectrophoresis (CRIE). In CRIE the major allergen (antigen 23) exhibited the strongest “radiostaining” and only a few other components of birch pollen extract were visibly radiostained. The major allergen and a preparation containing mainly the minor allergens, antigens 25 and 19, were isolated from a crude aqueous birch pollen extract by a combination of anion-exchange, size-exclusion, and chelate chromatography. Antigen 23 was purified to near homogeneity. The molecular weights and the pIs of antigens 23, 25, and 19 were determined to be 17,000 daltons, pI 5.25 (5.5, 5.0); 25,000 daltons, pI 5.0 (4.9, 5.4); and 29,000 daltons, pI 6.2 (5.4), respectively. The classification of antigen 23 as the major allergen in birch pollen was supported by results of RAST inhibition experiments, RAST screening, and skin prick testing.     

一种检测血清β脂蛋白谱的新方法

目的建立一种分离血清β脂蛋白(βLp)亚组分的新方法———二阶双梯度聚丙烯酰胺凝胶电泳(s-DGPAGE)法。方法在不同浓度梯度凝胶中匹配一定的pH梯度,借助浓度梯度的分子筛作用和pH梯度的聚焦作用,促进βLp亚组分分离,通过凝胶成像系统和光密度扫描仪完成βLp谱的记录和量化分析。血清βLp的分离采用一阶DGPAGE。结果s-DGPAGE法能将血清βLp分离出4种亚组分,分别命名为βLp1、βLp2、βLp3和βLp4,并可量化出各亚组分在βLp和血清脂蛋白整体中的相对含量。结论s-DGPAGE法能检出血清βLp的不均一组成,反映机体血清βLp的动态平衡状态,是一种简便实用的新方法。     

2种菌株来源的左旋门冬酰胺酶的鉴别和效价测定研究

目的:探讨由大肠埃希菌(E.coliASI.357)和欧文菌(Erwinia carotovora)制备的左旋门冬酰胺酶的鉴别方法,以及效价测定的最适酶促反应条件。方法:采用高效液相色谱法及等电聚焦电泳法对2种菌株来源的左旋门冬酰胺酶进行鉴别;考察缓冲液的种类及pH值对左旋门冬酰胺酶效价测定的酶促反应的影响。结果:大肠埃希菌和欧文菌2种来源的左旋门冬酰胺酶的色谱图有差别,主峰保留时间分别为11.0、11.8min,等电点色带分别为4.65～5.1、7.1～8.20;效价测定的最适酶促反应条件分别是三羟甲基氨基甲烷盐酸缓冲液(pH=9.0)、0.2mol·L-1磷酸盐缓冲液(pH=8.0)。结论:2种菌株来源的左旋门冬酰胺酶的等电点有显著差别,效价测定的最适酶促反应条件亦不相同,应作为2个品种制定质量标准。     

Lowering of pI by acylation improves the renal uptake of Tc-labeled anti-Tac dsFv: effect of different acylating reagents

Anti-Tac disulfide-stabilized variable region fragment (dsFv) was labeled with ^99mTc by a preformed chelate approach using ^99mTc-MAG3-trifluorophenyl (TFP) ester. Simultaneously it was acylated with TFP-lactate or succinic anhydride to decrease the isoelectric point of dsFv (pI 10). Acylation of dsFv (0.04 mM) with the lactate at a 73 times molar excess reduced the pI to 5.0–6.7, whereas acylation with succinic anhydride at a 30 times molar excess reduced the pI to 4.9–8.7. Comparative biodistribution studies performed in mice (n = 5) showed the reduced renal accumulation of the ^99mTc proportional to the pI reduction. The effect of the pI on the reduced renal uptake was especially pronounced at 15 min postinjection. The reduced renal uptake was also reflected in the reduced whole-body retention, indicating that lowering the pI inhibited the tubular reabsorption of the labeled dsFv.     

大鼠脑阿片受体的溶脱与提纯

大鼠脑(去小脑)突触膜在含二硫苏糖醇和胰酶抑制剂的Tris中用CHAPS溶脱。将溶脱液先后通过自制的阿片类配体10b和10cd 2个亲和层析柱,均用纳洛酮溶液洗脱。洗脱液再经过麦胚凝集素亲和层析和N-乙酰葡萄糖胺洗脱。进一步用制备型粒状凝胶等电聚焦电泳纯化和浓缩,所用凝胶为SG200,在pH5和pH7.8各有一蛋白质峰。淋冼所得样品用银染法测蛋白质含量,并用RRA测与~3H-伊托啡结合的活性,结果表明二者都是OPR,纯化程度达80 000倍以上。分子量用凝胶过滤法测定,pH5和pH7.8样品中的OPR分子量分别为52kD和42kD。pH5中OPR经用与~3H-羟基芬太尼结合的活性鉴定为μ-型受体。     

THE IMMUNOCHEMICAL CHARACTERISATION OF CIRCULATING IMMUNE COMPLEX CONSTITUENTS IN CANDIDA ALBICANS OSTEOMYELITIS BY ISOELECTRIC FOCUSING, IMMUNOBLOT, AND IMMUNOPRINT

Abstract Circulating immune complexes present in the serum of a patient with systemic lupus erythematosus, end-stage renal failure, and thoracic vertebral Candida albicans osteomyelitis were sequentially analysed by isoelectric focusing, immunoblotting, and immunoprinting. Candida antigens (including mannoproteins), Clq and C₃ complement components, and specific anti-Candida antibody were detected within polyethylene glycol precipitated complexes. An antigen of 47K molecular weight was amongst those demonstrated by polyacrylamide gel electrophoresis to be present within the complexes. It has been proposed elsewhere that a serologic response to a 47K protein is predictive of recovery from Candida albicans infection. Free antibody had specificity for Candida antigens ranging in molecular weight from 18K to more, than 100K including a 47K component. The analytical techniques employed allowed rapid and precise identification of the components of one particular immune complex system and will be widely applicable to the dissection of other diverse systems.     

The identification of potential alternative biomarkers of nitrofurazone abuse in animal derived food products

Semicarbazide (SEM) was considered to be a characteristic protein-bound side-chain metabolite of the banned veterinary drug nitrofurazone and used as a marker of nitrofurazone abuse. It was recently discovered that SEM can arise in food from sources other than nitrofurazone. This uncertainty over the source of SEM may be overcome if alternative markers specific to tissue-bound nitrofurazone residues can be determined. The structure of nitrofurazone metabolites in vivo and particular proteins to which they are bound are not known. These proteins with altered structure due to the presence of the drug metabolites can be considered as potential alternative biomarkers of nitrofurazone abuse. The proteins implicated in the in vivo binding of nitrofurazone were separated and identified. A crude mixture of proteins extracted from the liver of a rat treated with the drug was separated using a series of different techniques such as preparative isoelectric focusing and size exclusion HPLC. Multiple fractions were assayed by LC-MS/MS to detect the presence of SEM. The proteins containing SEM residues were identified by peptide mass mapping using trypsin digestion and MALDI-TOF. The first protein identified as containing high concentration of SEM was albumin. It was also shown that low molecular weight species within a protein mixture whose main constituent was glutathione S-transferase contained a high concentration of SEM. The chemical composition of these components is under investigation. Preliminary data suggest the SEM forms part of a nitrofurazone metabolite conjugated to glutathione.     

温带臭虫和热带臭虫生活史各期蛋白质和酯酶的初步分析

本文利用等电点聚焦电泳技术报道了温带臭虫和热带臭虫生活史各期蛋白质和酯酶的初步分析。实验结果表明:(1)蛋白质带谱:温带臭虫雌成虫、若虫和卵各呈现28、30和29条;热带臭虫雌成虫、若虫和卵各呈现32、33和29条;(2)酯酶同工酶谱:温带臭虫雌成虫、若虫和卵各显示12、12和11条;热带臭虫雌成虫、若虫和卵各显示18、12和11条;(3)热带臭虫生活史各期的蛋白质及酯酶带数目皆比温带臭虫生活史各期为多;(4)两种臭虫若虫期的蛋白质带数目皆比各自其它生活史各期为多。     

Urinary clearance of albumin is critically determined by its tertiary structure

The excretion of serum albumin in the urine is considered the net result of renal glomerular filtration and tubular uptake. During routine experiments, we observed that a batch of tritium-labeled albumin yielded anomalous results, being excreted in the urine of isolated perfused kidneys at 10 times the rate of normal tritiated albumin. This anomalous albumin, when simultaneously studied with normal carbon 14-labeled albumin, exhibited 10 times greater excretion than normal [(14)C]albumin. Anomalous albumin could not be reversed to normal albumin by means of conditioning with blood. In vivo clearances of anomalous albumin could not be quantitated because anomalous albumin is degraded during circulation. Anomalous albumin appeared to have the same molecular size (as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, capillary electrophoresis, and gel chromatography) and isoelectric-point profile (2-dimensional electrophresis) as normal albumin. Normal albumin could be transformed to anomalous albumin with alkali/heat treatment. Reverse-phase high-pressure liquid chromatography analysis of fragments from tryptic digests of anomalous albumin, alkali/heat-treated albumin, and normal albumin suggest that anomalous albumin and alkali/heat-treated albumin have altered tertiary structure, possibly as a result of denaturation and disulfide exchange. These studies show that the tertiary structure of albumin, beyond simple size and charge, is a critical determinant for albumin processing by the kidney and suggest that a specific albumin-recognition event by the kidneys is critical to normal renal handling of albumin.