Affinity thermoprecipitatin: Contribution of the efficiency of ligand-protein interaction and access of the ligand

Conjugates to two thermoprecipitating polymers, poly(N-vinyl caprolactam) and poly(N-isopropylacrylmide), with soybean trypsin inhibitor, Cibacron Blue 3GA, Cu-iminodiacetic acid, and p-aminobenzamidine were synthesized. The interaction of these conjugates with trypsin and lactate dehydrogenase was studied. Coupling of the ligand to a polymer resulted in a 100-1000-fold decrease in enzyme-affinity. Rough theoretical estimates revealed that a successful affinity precipitation required that the binding of a target protein and a ligand coupled to a polymer have binding constants on the order of 10(-7)-10(-8) M. Such strong affinity of low molecular weight ligands that can provide binding constants of 10(-9)-10(-11) M or alternatively multipoint attachment of the target protein molecule. The ligand in the ligand-polymer conjugate is still accessible to the protein after thermoprecipitation, and the latter can bind with the particle of the dispersion of thermoprecipitated ligand-polymer precipitate may result in stripping of enzyme molecules from the surface of the particles. (c) 1993 Wiley & Sons, Inc.     

应用鼠单抗显示鼠组织中特异性抗原的免疫细胞化学方法

作者在应用小鼠抗肾综合征出血热病毒(HFRSV)单克隆抗体(MAbs)免疫细胞化学法定位HFRSV自然感染大鼠组织中的病毒抗原时发现,部分大鼠组织的特异性HFRSV MAbs的染色切片及无关MAb对照和空白对照染色的切片中均出现血液。血细胞,血管壁,单核吞噬细胞系统,肾小球毛细血管及散在的心肌和肝细胞阳性,说明组织中有交叉反应。免疫双扩试验,羊抗鼠IgG桥抗可与大鼠Ig产生免疫沉淀反应,提示,组织中的交叉反应是由桥抗与大鼠细胞中本身存在的Ig(EIg)反应所致。为了避免EIg对特异性病毒抗原染色的干扰,我们以HFRSV MAbs预先分别与二抗羊抗鼠IgG结合,再用正常鼠Ig结合可能存在的二抗中尚未结合MAbs的Fab结合位点,制成一抗分子复合物,并以此作为一个新的相当于羊源性单克隆抗体孵育组织,以抗羊抗体连接羊PAP的多重PAP法显示一抗分子复合物的特异性抗原结合位点,结果显示该方法具有一抗的特异性,以有多重PAP法的敏感性,可有效的避免因EIg产生的交叉反应。用小鼠抗大鼠Kappa轻链MAb按同样方法制备一抗分子复合物用于染色证实,产生交叉阳性的部位确为大鼠组织中的Ig。病毒抗原及Ig的定位结果说明,HFRSV自然感染大鼠组织中的病毒抗原有广泛分布,也存在免疫复合物,但后者有别于HFRS人体组织的分布,另外,心肌及肝细胞Ig阳性说明该组织存在因HFRSV感染所造成的组织损伤,该损伤可能与机体的免疫反应有关。     

应用病毒感染细胞酶联免疫吸附试验检测肾综合征出血热病毒抗原和抗体

应用病毒感染细胞酶联免疫吸附试验(VIC-ELISA)检测肾综合征出血热病毒(HFRSV)感染性滴度比双抗体间接ELISA和间接免疫荧光法(IFA)分别敏感10倍和100倍。VIC-ELISA检测兔抗HFRSV抗体的滴度比双抗体间接夹心ELISA和IFA分别敏感1.6倍和8倍。VIC-ELISA能敏感、快速、有效地检测HFRSV抗原和抗体。     

应用单克隆抗体免疫细胞化学法分析尸检组织中肾综合征出血热病毒G2，NP及HA结构蛋白抗原

本文应用15株分别抗肾综合征出血热(HFRS)病毒糖蛋白(Glycoprotein Ⅱ G2),核蛋白(Nuclcocapsid,NP)及血凝素(Hemagglutinin,HA)抗原的单克隆抗体免疫细胞化学方法对19例HFRS尸检病例的16种组织中的病毒抗原进行了定位和分布的研究及抗原分析。结果表明,死于早期HFRS人体组织内病毒抗原量大,分布广泛,主要以可溶性和颗粒性两种形式存在,前者存在于细胞内和细胞外,呈G2,NP及HA抗原阳性,是参与形成免疫复合物的主要抗原形式;后者是以单纯病毒NP或HA抗原阳性的病毒包涵体(IB)形式存在于细胞内,是病毒直接作用所致细胞病变的标志,IB的广泛分布但只有极个别阳性细胞发生坏死,说明该病毒具有泛嗜性感染和弱致细胞病变能力的特性。抗原分析结果显示,组织细胞中病毒抗原的表达及其抗原量受宿主细胞的种属,组织结构特点及病期的影响,也存在个体差异以及因感染病毒株或血清型不同产生差异的可能性。病毒抗原染色形态学证实,NP上具有HA抗原位点,其抗原决定簇有三类,其中的某些抗原决定簇可因病毒宿主动物或细胞的种属不同,表达也不同。HA抗原在人体组织细胞中的高表达和广泛分布也说明HFRSV对人体有很强的侵袭力。     

小鼠早期胚胎性别鉴定的研究

用改进的细胞遗传学方法制备染色体标本,鉴定了小鼠晚期桑椹胚(晚桑)、囊胚和扩展囊胚(扩囊)的性别。在实验中,利用小鼠早期胚胎染色体标本制备的理想实验参数,鉴定了242枚小鼠晚桑、囊胚和扩囊的性别,性别鉴定成功率分别为80.4％、99.0％和94.5％。以细胞遗传学方法鉴定小鼠早期胚胎性别的结果为标准,得出用间接免疫荧光法和PCR(聚合酶链反应)扩增SRY(y染色体的性别决定期)部分序列法分别鉴定100枚和26枚小鼠胚胎性别的准确率相应地为74.0％和92.3％。     

The effect of salinity on the dominance-diversity relations of experimental coastal macrophyte communities

Salinity and water regime have previously been recognised as the main environmental factors controlling the abundance of coastal submerged macrophytes in temporarily-flooded marshes in the Camargue. The effects of these environmental variables, which are considered interrelated, are tested experimentally by subjecting experimental macrophyte communities from six temporarily flooded marshes to different levels of salinity (from 0 to 6 g/1 Cl^?). Communities subjected to high salinity levels (4 and 6 g/1 Cl~) showed a decrease in species richness and in biomass of all species involved. The species that most frequently dominate these communities, Chara áspera and Zannichellia pedunculata, are tolerant of salt and dominate over the entire salinity range. Three species groups can be distinguished based on the distribution of their biomass and centre of gravity of distribution over the salinity range: (1) non-salt-tolerant species, ‘glyco-phytes’, (2) moderately salt-tolerant species and (3) very tolerant species (‘halophytes’). A species ordination based on the experiments appeared to give results close to those previously obtained from field data.     

Purification and characterization of NADPH-cytochrome P-450 reductase from rat epidermis

NADPH-cytochrome P-450 oxidoreductase (P-450 red) transfers reducing equivalents from NADPH to cytochrome P-450 (P-450) in the monooxygenase system. Detergent solubilized proteins from the membrane fraction of neonatal rat epidermis were purified by 2′,5′-ADP-agarose affinity column chromatography. The purified protein showed an apparent homogeneity on sodium dodecylsulfate-polyacrylamide gel electrophoresis and molecular weight was estimated to be 78 kDa. NADPH-cytochrome c reductase activity increased by 95-fold in the purified enzyme. Epidermal P-450 red in vitro reconstituted benzo(a)pyrene hydroxylase activity in a dose dependent manner with P-450 purified from either rat liver or epidermis. Western blot analysis demonstrated that epidermal P-450 red immunologically cross reacts to liver P-450 red. Immunohistochemical staining showed that the enzyme was predominantly localized in the epidermis. The intensity of immunohistochemical staining of rat skin sections and tissue distribution did not change in the skin treated with β-naphtoflavone, which results in a substantial increase in P-450 1A1 activity. Quantitative assessment of P-450 red in treated and untreated epidermis also showed no change. These findings indicate that constitutive P-450 red, fully capable of supporting P-450, exists in rat epidermis, and can function in metabolism of endogenous and exogenous compounds.     

Keratinolysis and its morphological expression in hair digestion by airborne fungi

The morphological expression of keratinolysis in fungi isolated from the air of Torino (98 isolates belonging to 36 species) was studied. Light microscopy on whole material and on semithin sections, as well as scanning electron microscopy was used. There were 19 keratinolytically active species, with seven in the genusChrysosporium (C. indicum, C. keratinophilum, C. pannicola, C. tropicum, C. an.Arthroderma cuniculi, C. an.Pectinotrichum llanense, C. an.Renispora flavissima), four in the genusMalbranchea (M. arcuata, M. fulva, M. sulphurea, M. st.Uncinocarpus reesii), and three in the genusTrichophyton (T. mentagrophytes, T. rubrum, T. terrestre). In addition there wereAphanoascus fulvescens, Beauveria bassiana, Geomyces pannorum v.pannorum, Gymnoascus umbrinus andMyceliophthora vellerea. Most of these species were capable of developing structures related to surface erosion and radial penetration contemporaneously. HoweverGymnoascus umbrinus, Myceliophthora vellerea, an isolate ofC. indicum, C. tropicum andTrichophyton mentagrophytes demonstrated only surface erosion. Different isolates of one species can vary in their production of invasive structures and in degree of keratinolytic activity. Thus such activity, like many biochemical activities of fungi, does not appear to be a constant or rigorously species-specific character.     

33个黄花菜品种及3种野生黄花菜的过氧化物酶同工酶分析

１９８４－１９８７年先后全国１６个省区收集了３３个黄花菜（ＨｅｍｅｒｏｃａｌｌｉｓｃｉｔｒｉｎａＢａｒａｎｉ）品种和３个野生黄花菜品种。８７－９４年连续７年观察了各品种的主要形态特征,农艺性状。利用聚丙烯酰胺凝胶电泳技术分析比较了各品种的过氧化物酶同工酶谱。结果表明,凡形态上差异较大者,过氧化物酶谱同工酶谱差异也大;形态上无明显差异者,过氧化物酶同工酶谱也基本相同,表现了很强的对应性。各地栽培的黄花菜中普遍存在着同物异名或同名异物现象。收集的３３个黄花菜品种整理归并为２５个不同的品种;１７号早黄花（野生）、１９号四月花（栽培）均为北萱草（Ｈｅｍｅｒｏｃａｌｌｉｓｅｓｃｕｌｅｎｔｓ）,１８号野黄花为折叶萱草（Ｈｅｍｅｒｏｃａｌｌｉｓｐｌｉｃａｔａ）。为研究黄花菜品种的亲缘起源关系及进行杂交育种工作提供了一些理论依据。