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The Structural Proteins of the Rabbit Eye Lens After X-Irradiation

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The Structural Proteins of the Rabbit Eye Lens After X-Irradiation THE STRUCTURAL PROTEINS OF THE RABBIT EYE LENS AFTER X-IRRADIATION K.N.LIEM-THE THE STRUCTURAL PROTEINS OF THE RABBIT EYE LENS AFTER X-IRRADIATION PROMOTOR: DR. H.J.HOENDERS CO-PROMOTOR: PROF.DR. H. BLOEMENDAL. THE STRUCTURAL PROTEINS OF THE RABBIT EYE LENS AFTER X-IRRADIAT1ON PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE WISKUNDE EN NATUURWETENSCHAPPEN AAN DE KATHOLIEKE UNIVERSITEIT TE NIJMEGEN, OP GEZAG VAN DE RECTOR MAGNIFICUS, PROF.MR. F.J.F.M. DUYNSTEE, VOLGE; S BESLUIT VAN HET COLLEGE VAN DECANEN IN HET OPENBAAR TE VERDEDIGEN OP DONDERDAG 17 APRIL 1975 DES NAMIDDAGS TE 2 UUR PRECIES door KI AM NIO LIEM-THE geboren te Soerabaya Druk: Offsetdrukkerij Faculteit der Wiskunde en Natuurwetenschappen Nijmegen S I I- 1.1 IN (J 1 N 1 Bij de isoleung v<ni crysidllines is het aan te bevelen urn ais eerste methode gebruik te maken van moleculaire /.even m plaats van ioneiiwisselaars. II Het woord "Inalin bodies" in leverbiOpten is een betere, meer algemene naamgeving dan de term Mallory bodies'. UI De door Honnell en Selander getrolcken conclusie dat bij zeeolifanten een uniforme homozygotie van de door hen genoemde loei aanwezig zou zijn, is onjuist. Mi. D..nnell en i? K Selander, Scicnce [84.90H-909 (1974) IV Het is onwaarschijnlijk dat de "fractie f' in konmginnegelei van de honingbij ( Royal jelK I een "crjongende werking heeft op de menselijke huid. De door Satoh verstrekte gegevens over de verdeling van lenseiwitten in de humane lens. kunnen slechts zeer globaal worden beschouwd. K Satoh. I \p. Cye RL-S. 14. 53-57(1972) VI De geleidelijke atrofie van de hepatocyten gepaard gaande met ductulaire proliferaties in de lever van de gastheer is het best te bestuderen in het model van de heterotype, auxiliare levertransplantatie volgens Hess. F. Hess. C. Jcrusalem en M.N. va . der Heyde. Arch. Surg. 104. 76-80(1972) Vil De verschillen in heterogeniteit tussen de 40 S ribosornale subunits van tumur ascites celltn groeiend in vivo respectievelijk in vitro kunnen herleid worden tot de verschillen in fysiologische condities van deze cellen. M. Sameshima i?n M. Izawa, Ri' chini. Biophys Acta 378. 405^*14 (1975) VIII De tabel van Darnall en Kiotz over de subunits van eiwitten verliest aan waarde door de soms zeer oude referenties. D.W. Damall en I.M. Klot?., Arch. Biochim. Biophys. 166, 651-682 (1975) IX Het moet worden betwijfeld, of de intracellulaire pH of electrolyt-concentra- tie in de pancreas zoals bepaald door Swanson en Solomon met behulp van analyses op het gehele weefsel, inderdaad van toepassing zijn op de ductulaire cellen, omdat die slechts een klein deel van het totale weefselvolume innemen. C.H. Swanson en A.K. Solornon, J. Gen. Physioi. 62, 407-429 (1973) C.H. Swanson en A.K.. Solomon, J. Gen. Physioi. 65, 22-45 (1975) X Lasser en Balasz wekken de indruk dat alle membranen in de lens van het tight junction type zijn. Dit wordt weersproken door verschillende resultaten, zowel van henzelf als van anderen. A. Lasser en-E,A. Balasz, lixp. Eye Res. 13, 292-308 (1972) I. Dunia, C.S. Ghosh, F..L. Benedetti, A. Zwecrs en H. Bloemendal, FEBS Letters, 45, 139-144(1974) Nijmegen, 17 april 1975 K.N.Liem-The Aan mijn ouders Aan mijn man CONTENTS Chapter 1 THE EYE LENS Normal lens 9 Dimensions 9 Chemistry 9 Morphology 10 Function 10 Cataractous lens 10 General considerations 10 Cure 11 Problems in research 11 Experimental X-ray cataract in rabbit lens 11 Human senile cataract 1J Aim of' study 14 2 CHARACTERIZATION OF THE SOLUBLE PROTEINS FROM RABBIT EYE LENS Exp. Eye Res. 18:143-152(1974) Introduction 19(143) Materials and Methods 19(143) Results 20(144) Discussion 25 (149) 3 HM-CRYSTALLIN AS AN INTERMEDIATE IN THE CONVERSION OF WATER-SOLUBLE INTO WATER-INSOLUBLE RABBIT LENS PROTEINS Exp. Eye Res. 19:549-557(1974) introduction 29 (549) Materials and Methods 30(550) Results 31(551) Discussion 35 (555) 4 FURTHER CHARACTERIZATION OF HM-CRYSTALLIN IN RABBIT LENS Exp. Eye Res. In press (1975) Introduction 39 Materials and Methods 40 Results , 41 Discussion 48 Chapter 5 X-RAY INDUCED CATARACT IN RABBIT LENS Hxp. Eye Res. In press (1975) Introduction 51 Materials and Methods 52 Results 54 Discussion <•> 2 6 CHANGES IN THE PROPORTION AND COMPOSITION OF STRUCTURAL PROTEINS DURING THE DEVELOPMENT DF X-RAY CATARACT IN RABBIT LENS To be submitted to Exp. Eye Res. Introduction ft? Materials and Methods <^ Results 69 Discussion 7S 7 CHARACTERIZATION OF CRYSTALLINS IN NORMAL AND CATARACTOUS HUMAN EYE LENS To be submitted to Exp. Eye Res. Introduction S3 Materials and Methods 85 Results 87 Discussion 96 SUMMARY AND CONCLUDING REMARKS 101 SAMENVATTING EN CONCLUSIES 105 NAWOORD 109 Chapter 1 The Eye Lens Normal lens Dimension* The vertebrate lens is a transparent, avascular, semi-solid body having a biconvex shape. In the three-month-old rabbit lens, the central axis from anterior to posterior pole is about 7 mm in diameter, while the equatorial diameter is about 10 mm. The weitdu of a three-month-old rabbit lens is approximately 300 mg. The average dimen- sions of a mature human lens are 4 and 10 mm for its thickness and equatorial diameter, respectively. The lens wet weight is approximately 200 mg. C/wmisriy Chemically, the lens essentially consists of protein and water. The protein is ver, abundant, forming approximately 55% of the total wet weight. M6rner( 1894) divided the proteins from hnvine lenses into 2 fractions: soluble and insoluble protein (albu- moid). The soluble protein was further divided into three fractions, now called a-, /3- and 7-crybtallin. Krause (1932 and 1934) estimated that albumoid comprises about 12.5%, a-crystallin 31.7%, 0-ciystallin 53.4* and 7-crystallin 1.5% of the total protein. In addition he found minor amounts of glyco- and nucleoprotein. Some of the proper- ties (amongst others molecular weight, electrophoretic mobility, thiol content, N- terminal amino groups) of the crystallins have beer, determined (for a detailed report see Waley, 1969); especially a-crystallin was investigated throughly (reviewed by Bloemendal, 1972). Earlier papers reported that a-crystallin consists of four subunits, namely two acidic and two basic subur.its (Schoenmakers and Bloemendal, 1968; Bjork, 1968). Recently it has been shown that only cortical a-crystallin has four subunits, whereas nuclear a-crystallin reveals a more complex subunit structure (van Kleef, Nijzink-Maas and Hoenders, 1974). The amino-acid sequence of the polypeptide chains ceA2 and oB2 have been elucidated (van der Ouderaa, de Jong and Bloemendal, 1973 and van der Ouderaa, Hilderink, de Jong and Bloemendal, 1974). Since some years the existence of Mgh molecular weight proteins is known (Spector, U, Augusteyn, Schneider and Freund, 1971). Their polypeptide chain composition is similar to that of a-crystaliin. Morner (1894) was the first who observed that the water-insoluble albumoid is mainly found in the nucleus (older part) of the lens. The decreasing amount of a-crystallin and the increasing amount of albumoid found, going from the outer to the inner part of the lens, led to the suggestion that a-crystallin was being converted into albumoid in vivo (Krause, 1934). The close relationship between a-crystallin and aibu- moid was evidenced by applying electrophoretical methods, immunochemical and amino-acid analysis (see Waley, 1969). The decrease in soluble and increase of insoluble proteins seems to be a function of age. Morphology The lens is built up of three parts: the capsule, the epithelium and the fibre cells. The capsule is a tough, clear membrane, entirely surrounding the rest of the lens. The epithelium extends as a monola\er imderne.ith tli. antenor cansule There jie nr epithelial cells on the posterior suhcapsular surface of the lens The lens fibre '.ells originate from the tens epithelium. In a /one just antenoi to the equator, wnich ii Schematic drawing of part of the •epithelial;--ceils lens. (From Papaconstatinou, J.: germinaiive Molecular aspects of lens ceil region differentiation. Science 156, 338, 1967! region of cellular elonaation ;ortex fibreceHs copsuie called the germinative region, the epithelial cells divide at a high rate. At the equator these cell? are elongating to form fibre cells. This process continues until the end of life, although the rate decreases. The lens thus contains essentially one type of cells, all formed in the gemrinative region. The outer layers (cortex) represent the newer part of the lens and the central region (nucleus) the older part. There is no mechanism known whereby the lens can rid itself, on a cytological scale, of abnormal, damaged or non- viable cells. So it is a unique tissue, as the entire history of its continuous growth is preserved in it ^ _ :.: ,,,/,ra:,.ra,,,,,i;_.„,„.„„ _m. __ ,. Function The-sole function of the lens seems to be the refraction of image-bearing light beam in a controllable fashion. It is essential, therefore, that the lens maintains its transparency. Proper function may fail, for instance, if it becomes opaque. Cataractous lens General considerations The opacification of the lens resulting in reduction or loss of vision is called cataract. The etiology of cataract is complex. Degenerative changes accompanying aging processes, intraocular disease, genetic, metabolic, endocrine, chemical, radiation 10 and traumatic disorders n iy be causes of cataract formation. According to Sorsbv (1962) each year some 1.2 million people throughout the world develop visual impair- ment due to cataract What we know now is that the clinical opaque appearance of the cataractouslens is the result of three kinds of disturbances, namely: the presence of very high molecular weight crystalling accumulation ofirreversibly precipitated lens protein and of the numerous Lregular zones with different refractive indices caused by water clefts All three rypes of disturbances scatter light.
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