补肾明目冲剂抗氧化作用实验研究（Ⅱ）

探讨补肾明目冲剂处缓和预防机体组织细胞生理衰老的机理。方法：选用自然衰老的１６月龄Ｗｉｓｔａｒ大鼠，用补肾明目冲剂灌胃３０天，维生素Ｅ为阳性对照组，灌胃结束后，取大鼠心，脑，晶状体蛋白和视网膜脉络膜组织。用丙二醛法测定脂质过氧化物含量；用亚硝酸盐试剂盒检测超化物歧化酶活性；用ＤＴＮＢ法测定谷光甘肽过氧化物酶活性；用钼酸铵显色法测定过氧化氢酶活性；用Ｓｃｈｉｆｆ碱荧光法测定脂褐素含量。     

Radioimmunoassay of sheep alpha- and gamma-crystallins II. Organ specificity of lens crystallins

The α- and γ-crystallin content in sheep ocular tissues was assayed by radioimmunoassay.In the normal sheep eye, the amounts assayed in extralenticular tissue extracts were found to be close to the lower limit of sensitivity of the assays, 2 ng/ml. making the nature of the measured effects uncertain. The concentrations found, indicated a crystallin content of less than 0·05 parts per million wet weight tissue. This concentration is so low that it is considered unlikely that the crystallins are produced outside the lens.Freezing and thawing of the intact eye caused leakage of crystallins from the lens to the surrounding tissues. Because of the enormous concentration gradient of crystallins across the lens capsule, even minute damage to the capsule was shown to cause crystallin contamination of the surrounding tissues. One sheep with Peter's anomaly in one eye had increased amount of crystallins outside the lens in both eyes.     

The stability of human acidic beta-crystallin oligomers and hetero-oligomers

Crystallins are bulk structural proteins of the eye lens that have to last a life time. They gradually become modified with age, denature and form light scattering centres. High thermodynamic and kinetic stability of the crystallins enables them to resist unfolding and delay cataract. Here we have made recombinant human betaA1-, betaA3-, and betaA4-crystallins. The betaA3-crystallin formed higher oligomers that lead to precipitation at ambient temperature. Heat-induced precipitation of betaA3-crystallin was compared with human and calf betaB2-crystallins, showing that the human proteins start to precipitate above 50 degrees C while the calf betaB2-crystallin stays in solution even when unfolded. The stabilities of these human acidic beta-crystallin homo-oligomers have been estimated by measuring their unfolding in urea at neutral pH. BetaA3/1/betaB1 and betaA4/betaB1-crystallin hetero-oligomers have been prepared from homo-oligomers by subunit exchange. The resolution of the methodology used was insufficient to detect a stabilization of the betaA4-crystallin subunit in the hetero-oligomer, the betaA1-crystallin subunit was clearly stabilized by its interaction with betaB1-crystallin. Circular dichroism and fluorescence spectroscopies show that homo-dimer surface tryptophans become buried in the betaA3/1/betaB1-crystallin hetero-dimer concomitant with changes in polypeptide chain conformation.     

Distribution of Human Lens Crystallins and Their Sulphydryl Contents of Different Age in Two—dimension Electrophoresis

Purpose: To analyze water-soluble(WS) human lens proteins of fetus, adult and age-related cataract by two-dimensional IEF/SDSPAGE electrophoresis. Methods: DACM [ N- (7-Dimethylamino-4-methyl-3-coumarinyl) maleimide ] was used to determine the lens proteins sulphydryl (SH) content.Result: Protein SH contents in WS lens proteins have no significant difference among fetus, adult and age-related cataract lens. This is different from the relative published results obtained in lens proteins of animal cataract model using similar SH detecting methods.Conclusions: IEF/SDS-PAGE electrophoresis demonstrated that there were much more fragmentation of crystallins during lens development and cataractogenic process. It is suggested that this phenomenon is likely to be due to further conformational changes in the fragmented cyrstallins during aging and cataractogenic process. Eye Science 1999; 15:32-35.     

Steroid adduct formation with lens crystallins

Background : Development of steroid cataract is a likely outcome following prolonged exposure to glucocorticoids. It has been suggested that formation of steroid-protein adducts is a key event in this lens opacification. In order to explore this possibility, we have monitored the reaction of bovine lens proteins with glucocorticoids and examined the effects of adduct formation on their structures. Methods : Bovine lens proteins were incubated with high (10^?4 M) and low (10^?8 M) concentrations of dexamethasone or prednisolone for up to 56 days at 37 degrees Celsius. Changes in molecular size and solubility of the crystallins and their polypeptide subunits were examined using gel permeation chromatography and SDS gel electrophoresis. Conformational changes were assessed with the aid of tryptophan fluorescence spectroscopy and oxidation was monitored by measuring protein sulphydryl content. Results : Covalent incorporation of glucocorticoids was observed for all crystallins with relative reactivities for α-: β-: γ-crystallin of 20: 5: 1. The maximum incorporated was one steroid molecule per 40 to 50 subunits of α-crystallin. The proportions and sizes of the soluble crystallins and their subunits were unchanged. Protein sulphydryl contents decreased by eight to 10 per cent more than controls but no intermolecular disulphide bonds were detected. There were no alterations in tryptophan microenvironments. Conclusions : Steroids form adducts with lens proteins, in particular α-crystallin, but it appears unlikely that this reaction is responsible for steroid cataract formation.     

药物抑制兔眼晶体囊膜上皮细胞增生效果评价

探索细胞生长抑制制药物预报后囊混浊的可能性。方法（１）晶体后囊膜皮细胞体外培养，传代在２４孔内用不同浓度的氟脲嘧啶、阿霉素培养２４ｈ、７２ｈ做活细胞计数，求出半效抑制量；（２）兔眼晶体囊外摘除加工人晶体植术后，连续应用５－Ｆｕ、ＡＤＭ５次，