Use of the international prognostic index and the tumor score to detect poor-risk patients with primary mediastinal large B-cell lymphoma: a study of 37 previously untreated patients

How can enzymes function in the centre of a crowded lens over the many decades of an individual's life when the same proteins are usually turned over in a period of days or h in most other tissues? The discovery that alpha-crystallin could function as a molecular chaperone in-vitro has led to the hypothesis that alpha-crystallin could protect enzyme activities against various stresses. In the laboratory the authors have focused on the effect of alpha-crystallin on the activity of enzymes upon exposure to a chemical or thermal stress. The authors have demonstrated that enzymes are rapidly inactivated by sugars, sugar phosphates, steroids and cyanate. These compounds are elevated in diseases such as diabetes, diarrhoea and renal failure, all of which are risk factors for cataract. alpha-Crystallin has been shown to protect specifically against both chemically- and thermally-induced inactivation. Some enzymes are protected with a stoichiometry of one or two enzyme molecules protected per alpha-crystallin aggregate, consistent with a chaperone-like structure. However with other enzymes a more efficient protection occurs consistent with a micellar structure or binding on the outside of alpha-crystallin molecules. Investigation of complex formation indicates that although stable complex formation between enzymes and alpha-crystallin may be involved in protection of enzymes against thermal inactivation, protection against chemically-induced inactivation may be more dynamic in nature.     

Bovine and human alpha-crystallins as molecular chaperones: prevention of the inactivation of glutathione reductase by fructation

With no measurable protein synthesis occurring in the centre of the lens, structural proteins and enzymes there will need to be stable for many years, if not decades, in order to maintain lens integrity and function. Recent work has indicated that alpha-crystallin, which is sequentially related to heat shock proteins, has chaperone-like properties in that it is capable of preventing heat-induced aggregation of various proteins, including other crystallins. Thus this universal vertebrate lens protein may contribute to maintenance of lens integrity by protecting other lens proteins from non-enzymic insults or the consequences thereof. We previously showed that the enzyme glutathione reductase was inactivated in a time-dependent manner when incubated with various sugars, suggesting glycation was responsible for this effect. In this paper we confirmed that this was the case. Using this enzyme model system, the inclusion of either bovine or human alpha-crystallin protected against the inactivation of glutathione reductase by fructation. This action was specific, with control proteins displaying no such protection. Use of high performance liquid chromatography supported the fact that alpha-crystallin did not act simply by mopping up free sugar but rather maintained the activity of the modified enzyme. Dose-dependent experiments indicated that human alpha-crystallin was more effective than its bovine counterpart, which might be expected considering the much longer lifespan of humans. The stoichiometry of the protection by both alpha-crystallins indicated that alpha-crystallin with glutathione reductase was not acting like GroEL as a large complex with a hydrophobic pore, but rather that individual subunits may be capable of acting as chaperones.     

Inactivation of Cu,Zn-superoxide dismutase by intermediates of Maillard reaction and glycolytic pathway and some sugars

Human Cu,Zn-superoxide dismutase (SOD) was incubated with various intermediates of the Maillard reaction and glycolytic pathway (arabinose, glyoxal, glycolaldehyde, glyceraldehyde, glyceraldehyde 3-phosphate, and dihydroxyacetone) and some reducing sugars (sorbose, xylose, and ribose). The change of the activity and the molecular weight were measured and compared with that of SOD incubated with glucose or fructose. Sorbose, xylose, and ribose decreased the activity with a rate comparable to fructose. Site-specific and random fragmentation were observed upon the incubation with them. Arabinose showed a similar inactivation rate as glucose. The intermediates other than arabinose had a high inactivation rate. Especially, glyceraldehyde, glycolaldehyde, and glyoxal most strongly lowered the activity in a concentration-dependent manner and a significant inactivation was recognized even at 1 mM level. SDS-PAGE band patterns indicated that the inactivation by those carbonyl compounds occurred by both crosslinking and site-specific fragmentation of SOD.     

An impediment to glutathione diffusion in older normal human lenses: a possible precondition for nuclear cataract

Human age-related nuclear cataract is associated with progressive and widespread oxidation of proteins, particularly in the centre of the lens. The reasons for the onset of cataract and why this disease should take place only in the lenses of older individuals remain unclear. However, a common feature of nuclear cataract is the low concentration of reduced glutathione (GSH) in the centre of the lens. GSH is the principal lenticular antioxidant of the lens and it is synthesized and regenerated in the lens cortex. In this study we investigated the diffusion of glutathione within the human lens as a function of age. Normal human lenses were incubated in artificial aqueous humor containing [35S]cysteine and the label was metabolically incorporated into GSH. After 48-h incubation, lenses were sectioned and phosphorimaging was used to determine the distribution of 35S label. In young lenses, label appeared to diffuse uniformly throughout the whole lens. By contrast, in lenses over the age of 30, very little 35S had penetrated to the centre of the lens. A distinct zonal pattern of label distribution was noted in the older lenses after 48 h incubation, which had dimensions of approximately 7.2 mm (diameter) by 2.8 mm (axial). In some older lenses this pattern was noticeable even after 96-h incubation. Thus a barrier to the diffusion of GSH was observed in older normal lenses which was not present in younger lenses. Furthermore, the internal zone thus delineated has dimensions that coincide with those of the coloured and sclerotic zone present in nuclear cataract lenses. Since nuclear cataract is a disease of the elderly, and maintenance of GSH is known to be vital for lens clarity, we propose that the development of a barrier to the movement of GSH from its site of synthesis and regeneration in the cortex, into the nucleus in older normal lenses, may over time allow oxidative modification of protein to take place in the nucleus, resulting ultimately in nuclear cataract.     

Effects of semotiadil, a novel Ca2+ channel antagonist, on the electrical activity of Langendorff-perfused guinea pig hearts in comparison with diltiazem, amlodipine and nifedipine

In view of renewed interest in the lens epithelium as the initiation site for cataract development, it seemed timely to review recent studies which appear to establish UV damage in the lens epithelium as the cause of UV cataract. While UV photons can and do interact with lens proteins in the cortex and nucleus, experimental results from cultured lenses and tissue cultured epithelial cells also demonstrate both mutagenic and cytotoxic effects in the epithelium. This minireview examines UV-induced changes in lens physiology that appear to follow epithelial cell damage, including inactivation of critical enzymes of transport and metabolic processes. Changes in membrane function include altered cation transport, increased permeability, and altered biosynthesis. One potential scenario for the propagation of damage from the epithelium to the underlying fiber cells includes calcium elevation, an early event in cataract development and critical to many physiological processes.     

From lens regeneration in the newt to in-vitro transdifferentiation of vertebrate pigmented epithelial cells

Through studies to clarify the cellular origin of lens regeneration in the newt, the pigmented epithelial cells of the iris and the retina of many vertebrate species have been shown to possess a dormant potency to transdifferentiate into the lens. The method of in-vitro culture of pigmented epithelial cells has been optimized to enable detailed studies of the transdifferentiation process by molecular techniques. Growth factors and extracellular matrix components are found to be important in the control of the transdifferentiation process. New systems for in-vitro culture are introduced, while prospects for renewed in-vivo studies using newts are given.     

Structural perturbation of alpha-crystallin and its chaperone-like activity

alpha-Crystallin is a multimeric lenticular protein that has recently been shown to be expressed in several non-lenticular tissues as well. It is shown to prevent aggregation of non-native proteins as a molecular chaperone. By using a non-thermal aggregation model, we could show that this process is temperature-dependent. We investigated the chaperone-like activity of alpha-crystallin towards photo-induced aggregation of gamma-crystallin, aggregation of insulin and on the refolding induced aggregation of beta- and gamma-crystallins. We observed that alpha-crystallin could prevent photo-aggregation of gamma-crystallin and this chaperone-like activity of alpha-crystallin is enhanced several fold at temperatures above 30 degrees C. This enhancement parallels the exposure of its hydrophobic surfaces as a function of temperature, probed using hydrophobic fluorescent probes such as pyrene and 8-anilinonaphthalene-1-sulfonate. We, therefore, concluded that alpha-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30 degrees C involving enhanced or re-organized hydrophobic surfaces of alpha-crystallin is important for its chaperone-like activity. We also addressed the issue of conformational aspects of target proteins and found that their aggregation prone molten globule states bind to alpha-crystallin. We trace these developments and discuss some new lines that suggest the role of tertiary structural aspects in the chaperone process.     

Alpha-crystallin, a molecular chaperone, forms a stable complex with carbonic anhydrase upon heat denaturation

Alpha-crystallin, a major eye lens protein of vertebrates has been characterized as a molecular chaperone based on its ability to inhibit the aggregation of proteins undergoing thermal denaturation (Horwitz, J., Proc. Natl. Acad. Sci. USA 1992, 89, 10449-10453). To understand the mechanisms underlying this chaperone-like activity, the present study addressed molecular interactions between alpha-crystallin and its target proteins. Using carbonic anhydrase as a model target protein, we demonstrate complex formation between the 2 proteins upon heating, as assessed by the criteria of agarose gel electrophoresis, immunoprecipitation, ultrafiltration and gel filtration chromatography. The complex of alpha-crystallin and carbonic anhydrase is stable, at room temperature and at 4 degrees C, for over 18 hours, and is non-covalent in nature. The results also indicate that alpha-crystallin binds the early non-native form of the target protein.     

Protein associated with human lens 'native' membrane during aging and cataract formation

Plasma membrane contains extrinsic as well as intrinsic proteins. Changes in the extrinsic proteins of lens membrane during human aging and cataract formation have not been investigated in detail. Unlike previous studies which examined lens membrane after being stripped of extrinsic proteins by treatment with chaotropic agents, we have isolated whole or 'native' lens membrane on a sucrose gradient by ultracentrifugation of the total water-insoluble protein. Essentially all of the water-insoluble protein from young to aged to cataractous human lens appeared membrane associated. In young lens (20-37 years old), most of the membrane banded at the 25/45% sucrose interface fraction. This fraction contained relatively little urea-soluble protein and likely represents fiber-cell plasma membrane with its physiologically associated extrinsic and intrinsic proteins. With aging (62-80 years old), about one-third of the membrane, as judged by the distribution of cholesterol, banded at a much higher density (50/58% sucrose fraction). The higher density was due to a great increase in the membrane's relative protein content (protein/cholesterol). Although this extra protein was composed of both urea-insoluble and -soluble fractions, the urea-soluble protein predominated in all lenses. Cataractous lens differed from aged-clear lens in that much more of the total membrane (70-75%) had shifted to the high density and participated in this massive binding of cytosolic proteins. Although alpha-crystallin was the principal extrinsic-membrane protein in young lens, high molecular weight aggregate of modified (acidic) crystallins accounted for the increased extrinsic protein in aging. The extrinsic proteins bound to both clear-aged and cataractous lens membrane were aggregated. In conclusion, examination of human lens native membrane fractions revealed that the association of crystallins with membrane in both aging and cataracts was much greater than previously recognized and most of this increased protein was non-covalently bound to the membrane. Much more of the lens total membrane from cataractous than clear-aged lens was involved in this massive protein association and the protein bound to cataract membrane appeared more highly aggregated.     

alpha-crystallin stabilizes actin filaments and prevents cytochalasin-induced depolymerization in a phosphorylation-dependent manner

alpha-crystallin, a major lens protein of approximately 800 kDa with subunits of about 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress-induced damage and to share sequence similarity with small heat-shock proteins, sHsp. It is now demonstrated that this chaperone effect extends to protection of the intracellular matrix component actin. It was found that the powerful depolymerization effect of cytochalasin D could be almost completely blocked by alpha-crystallin, alpha A-crystallin or alpha B-crystallin. However, phosphorylation of alpha-crystallin markedly decreased its protective effect. It is suggested that phosphorylation of alpha-crystallin may contribute to changes in actin structure observed during cellular remodeling that occurs with the terminal differentiation of a lens epithelial cell to a fiber cell and contributes to cellular remodeling in other cell types that contain alpha-crystallin species. This communication presents biochemical evidence clearly demonstrating that alpha-crystallin is involved in actin polymerization-depolymerization dynamics. It is also shown that alpha-crystallin prevented heat-induced aggregation of actin filaments. alpha-crystallin was found to stabilize actin polymers decreasing dilution-induced depolymerization rates up to twofold while slightly decreasing the critical concentration from 0.23 microM to 0.18 microM. Similar results were found with either alpha-crystallin or its purified subunits alpha A-crystallin and alpha B-crystallin. In contrast to the experiments with cytochalasin D, phosphorylation had no effect. There does not appear to be an interaction between alpha-crystallin and actin monomers since the effect of alpha-crystallin in enhancing actin polymerization does not become apparent until some polymerization has occurred. Examination of the stoichiometry of the alpha-crystallin effect indicates that 2-3 alpha-crystallin monomers/actin monomer give maximum actin polymer stabilization.     

Perspective on allogeneic melanoma lysates in active specific immunotherapy

PURPOSE: To compare calcium ionophore-induced cataract formation and in vitro light scattering in cultured lenses from guinea pig and rabbit. METHODS: Lenses from guinea pig and rabbit were cultured for 5 or 6 days with calcium ionophore A23187. To assess the involvement of calpain in cataract formation; SDS-PAGE, immunoblotting and calcium determinations were performed. For in vitro light scattering, lens soluble proteins from rabbit were hydrolyzed for 24 h by either endogenous lens calpain, or by addition of purified m-calpain and then further incubated for up to 10 days. Light scattering was measured daily at 405 nm. RESULTS: Lenses from younger guinea pigs cultured in A23187 first developed outer cortical opacities followed by nuclear cataract. Total calcium was markedly increased by A23187 in lenses of all ages. Proteolysis of crystallins and alpha-spectrin were observed in nuclear cataract in younger guinea pigs. This was attenuated with age, in association with the attenuation of cataract formation with age. Calpain 80 kDa subunit in the lenses cultured with A23187 was also decreased. Co-culture with SJA6017 or E64d (reversible and irreversible inhibitors of calpain, respectively) reduced A23187-induced nuclear opacities, proteolysis of crystallins and alpha-spectrin, and loss of calpain without affecting increased total calcium. In contrast, rabbit lenses cultured in A23187 did not develop nuclear cataract, although biochemical changes in cultured rabbit lenses were similar to those in cultured guinea pig lenses. Furthermore, no appreciable in vitro light scattering occurred in soluble proteins from rabbit lenses after activation of endogenous m-calpain, or after addition of exogenous purified m-calpain, although crystallins were partially hydrolyzed by calpain. CONCLUSIONS: Both rabbit and guinea pig lenses undergo calpain-induced proteolysis upon elevation of lenticular calcium. However, factors in intact guinea pig lenses may promote light scattering and insolubilization after proteolysis by calpain, but these factors were not functional in rabbit lenses. Discovery of the factors promoting light scatter and insolubilization after proteolysis will help to explain the role of certain crystallin polypeptides in cataract formation.     

Phylogeny of wapiti, red deer, sika deer, and other North American cervids as determined from mitochondrial DNA

alpha-crystallin acts as a molecular chaperone by preventing the aggregation of proteins. Although the mechanism for this activity is not understood there is a proposition that temperature activation at or above 30 degrees C of alpha-crystallin is an absolute requirement, thereby suggesting a conformational transition as a trigger for the activity. In an attempt to unravel the putative temperature-activity relationship, the chaperone-like activity of alpha-crystallin was studied at a number of temperatures above and below 30 degrees C. Chaperone activity was monitored against aggregation of the insulin-B chain induced by cleavage of disulfide bond of insulin and also against photo-aggregation of gamma-crystallin. Contrary to the above notion, the results indicate that alpha-crystallin does not require thermal activation for its chaperone function and that it can efficiently function as a molecular chaperone even at temperatures below the previously reported transition temperature.     

Elemental studies in rat lens during galactose cataract reversal

Alterations in elemental composition of the normal lens have been reported to accompany galactose cataract development in rats. In this report we present the changes in regional distribution of Na, K, Cl, P, S and Ca during the reversal of galactose-induced cataracts. Elemental X-ray maps of lenses from young female Sprague Dawley rats fed 50% galactose for 20 days were examined at 0, 20, 40 and 90 days following the transfer of galactose fed rats to Purina Rat Chow diet. Reinstatement of normal elemental distribution accompanied the progression of lens transparency. By 90 days on the rat chow diet, K had increased and Na, Cl and Ca had decreased so that a near normal lenticular distribution of these elements was established. The reinstatement of elemental distribution during cataract reversal followed a pattern similar to that observed for alterations during cataract development, initiating near the equatorial surface and expanding centrally. The correlation between the alterations in the distribution of the elements studied and our previously reported morphological investigation of lenses during galactose cataract reversal is discussed in this report.     

The hydroxyl radical in lens nuclear cataractogenesis

Cataract is the major cause of blindness; the most common form is age-related, or senile, cataract. The reasons for the development of cataract are unknown. Here we demonstrate that nuclear cataract is associated with the extensive hydroxylation of protein-bound amino acid residues, which increases with the development of cataract by up to 15-fold in the case of DOPA. The relative abundance of the oxidized amino acids in lens protein (assessed per parent amino acid) is DOPA > o- and m-tyrosine > 3-hydroxyvaline, 5-hydroxyleucine > dityrosine. Nigrescent cataracts, in which the normally transparent lens becomes black and opaque, contain the highest level of hydroxylated amino acids yet observed in a biological tissue: for example, per 1000 parent amino acid residues, DOPA, 15; 3-hydroxyvaline, 0.3; compared with dityrosine, 0.05. The products include representatives of the hydroperoxide and DOPA pathways of protein oxidation, which can give rise to secondary reactive species, radical and otherwise. The observed relative abundance corresponds closely with that of products of hydroxyl radical or metal-dependent oxidation of isolated proteins, and not with the patterns resulting from hypochlorite or tyrosyl-radical oxidation. Although very little light in the 300-400-nm range passes the cornea and the filter compounds of the eye, we nevertheless also demonstrate that photoxidation of lens proteins with light of 310 nm, the part of the spectrum in which protein aromatic residues have residual absorbance, does not give rise to the hydroxylated aliphatic amino acids. Thus the post-translational modification of crystallins by hydroxyl radicals/Fenton systems seems to dominate their in vivo oxidation, and it could explain the known features of such nuclear cataractogenesis.     

Advances in computer technology: impact on the practice of medicine

Advances in computer technology provide a wide range of applications which are revolutionizing the practice of medicine. The development of new software for the office creates a web of communication among physicians, staff members, health care facilities and associated agencies. This provides the physician with the prospect of a paperless office. At the other end of the spectrum, the development of 3D work stations and software based on computational chemistry permits visualization of protein molecules involved in disease. Computer assisted molecular modeling has been used to construct working 3D models of lens alpha-crystallin. The 3D structure of alpha-crystallin is basic to our understanding of the molecular mechanisms involved in lens fiber cell maturation, stabilization of the inner nuclear region, the maintenance of lens transparency and cataractogenesis. The major component of the high molecular weight aggregates that occur during cataractogenesis is alpha-crystallin subunits. Subunits of alpha-crystallin occur in other tissues of the body. In the central nervous system accumulation of these subunits in the form of dense inclusion bodies occurs in pathological conditions such as Alzheimer's disease, Huntington's disease, multiple sclerosis and toxoplasmosis (Iwaki, Wisniewski et al., 1992), as well as neoplasms of astrocyte origin (Iwaki, Iwaki, et al., 1991). Also cardiac ischemia is associated with an increased alpha B synthesis (Chiesi, Longoni et al., 1990). On a more global level, the molecular structure of alpha-crystallin may provide information pertaining to the function of small heat shock proteins, hsp, in maintaining cell stability under the stress of disease.     

Calcium-induced changes on crystallins in organ-cultured porcine lens

In this study, intact porcine lenses were cultured in vitro for 7 days supplemented with commercial balanced salt solution (BSS) which is usually used as an irrigation solution during intraocular surgery, and the lenses were maintained under various culture conditions, e.g. temperature and CO2 concentration. The intact porcine lenses after 7 days culture were analyzed with optical density scanner, gel permeation chromatography on TSK HM-55 column and SDS-PAGE (polyacrylamide gel electrophoresis). It was found that lenses exhibited the least opacity when lenses were cultured with Ca(+2)-free BSS buffer, CO2-free incubator and maintained at a temperature of 25 degrees C. After the lenses were cultured with Ca(+2)-free BSS or BSS medium, the composition of crystallins in lenses was separated with TSK HM-55 column. It was indicated that the percentage of high molecular weight (HMW) protein and (alpha-crystallin increased, and gamma-crystallin decreased in lenses incubated with BSS medium compared with lenses incubated with Ca(+2)-free BSS medium. Following an increase in the concentration of calcium in the medium from 4.3 mM, 20 mM, 50 mM, 100 mM to 200 mM, the opacity of the lens was measured with a densitometer. The changed percentage of various crystallins was similar to lenses with BSS media that increased in HMW protein and alpha-crystallin, decreasing in gamma-crystallin. In the case of lens protein pattern, the crystallin washed from TSK HM-55 gel was separated with SDS-PAGE (polyacrylamide gel electrophoresis). It was indicated that some of proteins disappeared when lenses were incubated with various concentrations of calcium. The vanished pH proteins were 20.5 kDa at 50 mM calcium, 20.5 kDa and 21 kDa at 100 mM, 20.5 kDa, 21 kDa, 22 kDa and 23 kDa at 200 mM which were compared with the protein bands in the presence of 20 mM calcium in BSS medium. This study indicates that the commercial balanced salt solution (BSS) which is usually used as an irrigating solution during intraocular operations may increase the risk for lens opacity because of the calcium contained in the solution.     

Increased levels of advanced glycation endproducts in the lenses and blood vessels of cigarette smokers

BACKGROUND: Advanced glycation endproducts (AGEs) arise from the spontaneous reaction of reducing sugars with the amino groups of macromolecules. AGEs accumulate in tissue as a consequence of diabetes and aging and have been causally implicated in the pathogenesis of several of the end-organ complications of diabetes and aging, including cataract, atherosclerosis, and renal insufficiency. It has been recently proposed that components in mainstream cigarette smoke can react with plasma and extracellular matrix proteins to form covalent adducts with many of the properties of AGEs. We wished to ascertain whether AGEs or immunochemically related molecules are present at higher levels in the tissues of smokers. MATERIALS AND METHODS: Lens and coronary artery specimens from nondiabetic smokers and nondiabetic nonsmokers were examined by immunohistochemistry, immunoelectron microscopy, and ELISA employing several distinct anti-AGE antibodies. In addition, lenticular extracts were tested for AGE-associated fluorescence by fluorescence spectroscopy. RESULTS: Immunoreactive AGEs were present at significantly higher levels in the lenses and lenticular extracts of nondiabetic smokers (p < 0.003). Anti-AGE immunogold staining was diffusely distributed throughout lens fiber cells. AGE-associated fluorescence was significantly increased in the lenticular extracts of nondiabetic smokers (p = 0.005). AGE-immunoreactivity was significantly elevated in coronary arteries from nondiabetic smokers compared with nondiabetic nonsmokers (p = 0.015). CONCLUSIONS: AGEs or immunochemically related molecules are present at higher levels in the tissues of smokers than in nonsmokers, irrespective of diabetes. In view of previous reports implicating AGEs in a causal association with numerous pathologies, these findings have significant ramifications for understanding the etiopathology of diseases associated with smoking, the single greatest preventable cause of morbidity and mortality in the United States.     

Selective inactivation of glyceraldehyde-3-phosphate dehydrogenase by vinyl sulfones

Incubation of glyceraldehyde-3-phosphate dehydrogenase with vinyl sulfones resulted in a pseudo first-order loss of enzyme activity. The selective inactivation of the enzyme by vinyl sulfones is suggested from the structural requirement analysis and the enzyme susceptibility test. The enzyme inactivation was strongly reduced in the presence of NAD or glyceraldehyde-3-phosphate, and the prior treatment of the enzyme with 5,5'-dithio-bis-(2-nitrobenzoic acid) prevented the enzyme from the inactivation by vinyl sulfones (> or = 90%). Moreover, the early rapid phase of inactivation was much more responsive to L-cysteine reactivation, compared with the slower phase. Based on these results, it is proposed that vinyl sulfones inactivate the enzyme by inducing the oxidation of cysteine residue and/or covalent binding to cysteine residue in active site.     

Lens glutathione, lens protein glycation and electrophoretic patterns of lens proteins in STZ induced diabetic rats

As diabetes is a very complex disease, with the pathological symptoms varying with age, diabetic type and means of control, it still warrants many in vivo and in vitro studies. During hyperglycaemia, increases in the sorbitol pathway, nonenzymatic glycosylation of lens proteins and damage to antioxidant systems have been reported to cause opacification of the lens leading to cataract formation. In this study, intracapsular extracts of lenses from STZ induced diabetic female rats were examined. Total protein, glutathione and nonenzymatic glycosylation were determined by the Lowry, Ellman reagent and thiobarbituric acid methods respectively. Laemmli protein electrophoresis was also carried out on the lens homogenates. After a period of as short as 5 weeks, a decrease in lens glutathione, and an increase in nonenzymatic glycosylation of lens proteins were found. The electrophoresis showed an increase in proteins of high molecular weight.     

p53 oncogene in the laryngeal cancer

The effect of a novel flavonoid, venoruton (a mixture of mono-, di-, tri- and tetrahydroxyethylrutosides) has been investigated in healthy rat lenses and compared with diabetic cataract modelled in vitro. One mM venoruton was added to medium simulating healthy and diabetic conditions for the incubated lenses; damage was followed by either stereoscopic photography of the lenses under a Cooperative Cataract Research Group operating microscope or with our recently developed method: the leakage of lactate dehydrogenase (LDH) into the lens culture media. The increased LDH activity in the medium and observable development of the opacity were correlated with cell damage, which has been found to be associated with globular degeneration and cataract formation. The extent of opacification and LDH release is reduced if 1 mM venoruton is included in the medium. The protective effect may be related to antioxidant activity against reactive oxygen species: decreased luminol luminescence was shown after venoruton addition to either superoxide-generating hypoxanthine plus xanthine oxidase, or hydrogen peroxide.