| Cataract is a frequently encountered disease and it is the most common cause of blind. Although lots of study of etiology of cataract has been done, it still can not meet the clinical need. Surgery is the only useful treatment to cataract. Secondary cataract, secondary glaucoma and cornea lesion are common complications of surgery. Because the exact etiology of cataract is still unclear, pharmaceutical treatment is difficult. The drugs used at present have some shortcomings, including low efficiency and low specificity and severe side effect. The pathological mechanism of cataract need further study. The proteins in determining the transparency of lens are critical to study and can be focused as the targets of treatment and prevention.A continuing growth of population and aged people in our country will lead to the increased morbility of cataract. The pathological mechanism of cataract need further study to prevent and treat the disease.Clinical study showed that senile cataract is the most common cataract. Aging related cataract is the major cause of the decrease of visual acuity in old people. A cloudy area is formed during aging process and opacity develops. This is called degenerative cataract. About 95% of Americans age over 85 years old have cataract.The function of lens is to focus light on retina to produce visual sense. The major components of lens are water and proteins. The transparency of the lens is maintained through constant water content, high concentration of water soluble protein, the correct structure of crystallins and complicated metabolism. Proteins make up eighty to ninety percent of the dry weight of the lens. The correct structure of the proteins and their interaction are the molecular basis of the transparency of the lens. Alpha crystallins, with molecular chaperone function, have fundamental role in the normal structure of lens. Beta B2 crystallin with a high content in the lens is another important protein in maintaining the normal structure and transparency of the lens.Molecular chaperones are a family of cellular proteins that mediate the correct assembly or disassembly of other polypeptides, and in some cases their assembly into oligomeric structures, but which are not components of those final structures. Alpha crystallins are special because they both have chaperone activity and are important structural proteins of the lens.The changes of alpha crystallins during aging process can directly influence the activity of the molecular chaperone function. The molecular chaperone function usually decreases as the aging process and water-insoluble high molecular weight aggregates are formed by crosslinking.During the development of the lens, the quantity and quality of the crystallins will change as the results of the aging process of the internal and environmental factors. Once the correct structure of the crystallins is damaged, the lens will change from transparency to cloudy and a senile cataract is finally formed.Having a senile cataract is a normal part of the aging process of human body. The mechanism involved in the senile cataract need to be elucidated and some methods can be taken to treat and prevent the senile cataract.Previous studies have showed the changes of quantity and quality of crystallins, but it was unspecific. For example, the changes of beta crystallins have been reported, but the exact crystallin, PB2 or PB3 was not specified. The changes of different crystallins during the aging process are different, therefore it needs to determine the exact changes of each crystallins.We studied the changes of water soluble proteins and water insoluble proteins during the aging process. The changing pattern of the major crystallins was described. Some important crystallins in the aging process were chose to further study their physiological and biochemical properties. The role of these proteins in cataract formation was studied, and a series of compounds were chosen to act as anti-degeneration agents. The efficient way to stabilize the protein structure and the transparency of the lens are discussed.Part onePhasic change of water soluble crystallins with aging in ratsThe function of lens is to focus light on retina to produce visual sense. The composition and correct structure of the crystallins and their interaction are the molecular basis of the transparency of the lens. The three dimensional structure of lens depends on the correct alignment of crystallins. If the composition of the crystallins is changed, the normal structure of lens will be damaged and the transparency of lens will be lost, therefore cataract forms.The transparency of lens not only depends on the composition of the crystallins but also depends on the metabolism of the proteins. Studies have showed that the synthesis or the metabolism of the crystallins changed obviously during the aging process. If these changes are balanced in some extent, the structure of the crystallins will not be changed and the transparency of the lens will be maintained. Water soluble proteins decrease and the water-insoluble proteins increase as the aging process, and the adequate proportion of the proteins in the lens and fibrous membrane of the cortex is damaged. As a result, some scattering centers are formed and the scattered light is enhanced with a decrease of transmitted light.The study of changing pattern of the water soluble protein during the aging process will be helpful to determine the changing law of the major crystallins; will be helpful to determine the changing law of alpha chaperone crystallins and its relationship with other proteins; and will be helpful to determining the most important changing age of the lens by observation of the degeneration products of the crystallins. The mechanism based on the changes of water soluble proteins may be helpful to the prevention of senile cataract and anti aging process.Six groups of SD rats (age 1 d, 8 d, 2 weeks, 8 weeks, 8 months and 1. 5 years) were raised routinely. Water soluble crystallins of each group were extracted and separated by two-dimensional polyacrylamide gel electrophoresis. After Coomassie blue staining, the changing patterns of crystallins were scanned and analyzed. Results:1. Phasic changesOut of the eighteen water soluble major rat crystallins tested in each group, seven showed gradual phasic changes in relative quantity of crystallins:(1) Increasing: PE^ aB2^ C1A2(2) Decreasing: p7(pi). pg. yi. 3> Ys. 6(yC,yD)(3) relatively stable : pAi, pA3, pB5(4) irregular: other proteins2. The changes of relative quantity of the major crystallins:(1) PB7 increased as the aging process (r=0.938, p<0.01) .(2) The relative quantity of chaperones of 01A2 and (XB2 increased as the aging process. (r=0.890, p<0.05; r=O.93O, jxO.Ol).(3) The changes of pB2 are positively associated with (XA2 and aB2(r=0.949, p<0.01; r=0.986,p<0.01) .(4) The ration of PB2AXA2 (n= 10) increased as the aging process.3. Description of other the major crystallins:(1) The total relative quantity of water soluble protein of each group is stable.(2) Posttranslational modification of crystallins is obvious after 8 weeks old.(3) The gamma crystallins lost dramatically after middle age.Other previous study showed that beta crystallins decreased as the aging process, but our results showed that the quantity of PB2 increased as the aging process. What is the pathophysiological role of PB2 in aging and cataract formation? This is what we will further explore in the next part.Part two Phasic change of major urea soluble crystallins with aging in ratsPhasic changes of water soluble crystallins were showed in the first part. So what happened to the water insoluble proteins? How about the three important crystallins including PB2, showed a special changing pattern in water soluble protein, aA2 and (1B2? hi this part, we studied the changing pattern of water insoluble crystallins andcompared with water soluble crystallins. The role of phasic change of crystallins with ageing in the formation of aging cataract will be further studied.The lens was prepared as the first part described. After the water soluble extraction, 8M urea was added to the water insoluble precipitation to extract the protein again. Two-dimensional polyacrylamide gel electrophoresis of the urea soluble proteins was done to observe the phasic change pattern of urea soluble proteins. Changing pattern of most of the crystallins was described and quantitative changes of (1A2 and PB2 were analyzed.Results:(1) The relative quantity of PB2 in urea soluble crystallins increased as the aging process, while chaperone crystallin 0A2 keep relatively stable.(2) The quantitative changes of ctA2> PB2 were not so obvious as compared with their water soluble counterparts.(3) aAi, the posttranslationally modified product of (XA2 increased significantly after 8 weeks.(4) aBi, the posttranslationally modified product of (XB2 also increased significantly after 8 weeks.(5) Several degeneration products of PB2 were detected as showed in the 2-D gel analysis that several new protein spots appeared under the position of PB2.(6) The gamma crystallins slowly decreased after middle age.To our surprise, urea soluble PB2 increased with age and a similar result was showed in the water soluble PB2. As we known, these two changes are contradictory to the development of cataract. The increased water soluble PB2 was helpful to maintain the normal structure and the transparency of lens. The increase of water insoluble PB2 was the result of various damages to the crystallins and the increase of water insoluble protein will decrease the transparency of the lens, leading to senile cataract.The increase of the posttranslationally modified protein of the two chaperone proteins aA2 and aB2 showed that the water insoluble proteins changed significantly both in quantity and in quality after the middle age. These results showed that thedegeneration of crystallins subjected to the internal and external influence factors was increased after maturation period. These changes may be further accelerated in the old stage. It is suggested that some treatment methods should be taken to decrease the degeneration of crystallins.Part threeCloning and expression of rat pB2 crystallin and preliminary study ofits biochemical property.Crystallins are composed by three major groups: a, P and y and their roles in the aging process are different. Lots of work has been done to study the property and physiological role of a crystallins, especially about their chaperone function. The chaperone activity of a crystallins decreased as aging. Our results showed that both the water soluble and the water insoluble a crystallins increased as aging and the crystallin can be posttranslationally modified to become water insoluble. The total amount of water soluble a crystallins decreased as the aging process and this will decrease their chaperone activity.Only a little work has been done to study the function and property of beta crystallins at present. We chose beta B2 crystallin as the major target to study.In order to study the biochemical and physical property of PB2, we cloned the PB2 gene and expressed in a prokaryotic system and purified the protein. The cDNA of PB2 was cloned into pMD18-T vector. The PB2 gene was then cloned into the prokaryotic system pGEX-4T-l. IPTG was used to induce expression. The E. coli were collected and PB2 protein was purified by Glutathione Sepharose 4B affinity chromatography. The purified protein was identified by Westem-blot. Thermal stability of PB2 was done. Our results showed that PB2 was thermal instable.Beta B2 was expressed and purified in this part study and the purified protein can be used to study its function in vitro. Our preliminary results showed that the thermal stability of PB2 was rather low, which further verified that PB2 crystallin played an important role in the formation of senile cataract.Part fourA group of compounds as anti-degenerative agents of crystallins andtheir clinical prospectThe transparency of lens depends on the quantity and quality of water soluble proteins, especially the structural proteins such as p$B2^ Ys,6(yC, yD^ yii4> 723 and chaperones such as 01A2 and 01B2. The changes of quantity and quality of water soluble proteins can influence the transparency of lens and lead to cataract. Therefore, agents that can protect crystallins from degeneration can be used to delay the occurrence of cataract or to treat cataract.Lots of drugs have been developed to treat or prevent cataract. There are still some shortcomings of current drugs. Pharmaceutical mechanism of some drugs is not clear. Single drugs are not effective enough to treatment. Most of the drugs used clinically are oral drugs. Low dosage is difficult to reach the lens and if high dosage is used, the side effect will be prominent.The effective way to prevent and treat cataract is still the first need of clinician and this must be based on the pathological mechanism of cataract. At present, it is difficult to reach satisfying effect just using single drug. We hypothesize that combined usage of compounds to treat cataract will be more effective.Based on our previous study and the characteristic composition of amino acid of crystallins, the major crystallin PB2 is chosen as pharmaceutical target because it is fragile to modification of internal and external factors and easily become water insoluble from water soluble. We designed several groups of compounds based on the character of the target protein to protect PB2 crystallin from the modification effect. Experimental animal model will be done to verify our hypothesis and a new method to prevent and treat cataract will be found.Our drugs are used to protect the crystallins from degeneration effect of glycation, oxidation, cross linkage and thermal aggregation. Several clinically used drugs (not used for the treatment of cataract) and 1-2 unused agents will be used together as topica. Liposome will be used as the drug carrier to enhance the lens...
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