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A series of 2,4-dioxo-thieno[2,3- d ], [3,2- d ] and [3,4- d ]pyrimidin-1-acetic acids ( 2 ) with a benzyl moiety at the N-3 position were prepared and tested in vitro for aldose reductase inhibitory activity against partially purified enzyme from rat lens. Some of these compounds were also evaluated for inhibition of sorbitol accumulation in the sciatic nerve or lens of streptozotocin-induced diabetic rats in vivo . Among the synthesized compounds, several showed potent aldose reductase inhibitory activity with IC 50 s in the 10 −8 M range. Particularly, the potencies of non-substituted thieno- ( 2a and 2aa ), 5-methylthieno- ( 2c ), 5,6-dimethylthieno-( 2g ), 6-isopropylthieno- ( 2j and 2k ), 6-chlorothienopyrimidine ( 2q ) and benzothienopyrimidine ( 2ac ) analogs were approximately equipotent to FK-366 ( 1A ) and Ponalrestat ( 1B ) as references. Although most compounds were inactive in vivo , 2 compounds, 2k and 2q , possessed moderate in vivo activity.

The formation of excess sugar alcohol mediated by aldose reductase (AR) and its intracellular accumulation in lens with resultant hydration is thought to be the initiating mechanism in the pathogenesis of diabetic and galactosemic cataracts. AR is also involved in other diabetic complications including retinopathy and neuropathy. Therefore, there is heightened interest in developing effective AR inhibitors (ARIs) for possible clinical use in human diabetes. However, the evaluation of these drugs for potential clinical use requires that the compounds be evaluated in appropriate target tissues since AR from different tissues is known to exhibit differential susceptibility to ARIs. The relative efficacy of ARIs in human lens epithelium (HLE) and human retinal pigment epithelium (HRPE) was studied by measuring the degree of inhibition of galactitol formation at various concentrations of ARI following incubation of cells in high galactose media for 72 hrs. Regardless of the structural characteristics of the ARIs investigated, higher doses were required to inhibit polyol synthesis in HRPE as compared to HLE cells. Based on ED50 values, dose required for 50% inhibition, the order of potencies against both HLE and HRPE enzymes was AL-4114 greater than AL-3152 greater than AL-1576 greater than tolrestat greater than statil greater than sorbinil. Since some ARIs are known to be bound to plasma proteins, it is conceivable that the observed differences in ED50 values could be due to differential binding to serum proteins in the culture medium. This possibility was examined by employing cultures of dog lens epithelium (DLE). These cells, which synthesize much higher levels of galactitol than HLE and HRPE, could be maintained in serum-free media for short periods (4 hrs) of time. The results, which demonstrate that the extent of polyol inhibition was the same in the presence or absence of serum, suggest that the differences in the potency of the inhibitors may reflect their inherent activity against AR in HLE and HRPE cells.

An increased prevalence of cataract is associated with diabetes. Biochemical studies of diabetic lenses have revealed a variety of metabolic abnormalities including changes in the levels of electrolytes, glutathione, nucleotides and sugars. Similar biochemical changes have also been observed in cataracts associated with galactosaemia, suggesting that these sugar cataracts have a common biochemical aetiology. The common biochemical factor found to initiate both types of sugar cataract is the formation of sugar alcohols (polyols) from either glucose or galactose by the enzyme aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21). Increased intracellular levels of these polar alcohols have a hyperosmotic effect which leads to lens fibre swelling, vacuole formation and subsequent opacification. The process of sugar cataract formation in animals can be prevented by inhibiting aldose reductase.

Previous evidence has shown clearly that sugar cataract formation results from unusually high intracellular levels of polyol. Documentation of polyol-related histological changes in the cortical fiber cells of the equatorial zone has been extensive. However, little attention has been given to the early changes in the lens epithelial cells, in spite of the fact that the highest level of aldose reductase is found in this layer of the lens. Also, cultured lens epithelial cells exposed to high sugar levels exhibit rapid accumulation of polyol and show ultrastructural alterations. Therefore, a study was designed to evaluate the role of the lens epithelium in sugar cataract formation. Specifically, an attempt was made to localize the earliest fine structural lesions in intact lenses of galactose-fed rats and to test their relation to aldose reductase. Rats were fed either a normal diet or a 50% galactose diet with or without sorbinil, an aldose reductase inhibitor. Rats were killed at varying periods of time ranging from 6 to 96 hr, and the eyes were processed for light and electron microscopy. The first detectable abnormalities occurred after 36 hr of galactose feeding, and were limited to the central lens epithelium. Cell edema, apparent dilution of cytoplasm, rounding of nuclei, aberrant intracellular vacuoles, and loss of normal tortuosity of cell boundaries were the salient lesions. No changes were detectable in the equatorial zone until 48 hr, and no deviation from the control structure was found in any of the rats treated with an aldose reductase inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent evidence obtained from both galactosemic dogs and rats indicated that aldose reductase inhibitors could prevent several capillary lesions which were similar to those typical of diabetic retinopathy in humans. The present study demonstrates that diabetes-like histopathological changes in the intact retina, which were not visible in the vessel whole mounts used previously, can also be prevented. Sprague-Dawley rats were fed diets containing 50% galactose with or without an aldose reductase inhibitor (tolrestat). After 28 months of galactose feeding, the findings from retinal transections examined by light and electron microscopy were consistent with reports on vessel whole mounts, but showed several additional changes. There was a marked increase in the thickness of the retinal inner limiting membrane, as well as in the capillary basement membranes. There was extensive gliosis and disruption of retinal layers as well as pericyte degeneration, endothelial cell proliferation, accellularity, capillary dilation, and microaneurysm formation. The contents of pericyte compartments in the capillary wall were often replaced with non-pericyte cytoplasm, which appeared glial cell-like. Many of the lumens of acellular capillaries were occluded with debris from degenerating endothelial cells or with cytoplasm possibly originating from glial cell processes. Structures suggestive of degenerated microaneurysms were present mainly in the inner nuclear and outer plexiform layers. The microaneurysms and other major changes were limited to the central and paracentral retina. All these retinal lesions were prevented with orally administered tolrestat. Thus, long-term galactosemia in rats induced histopathologically visible angiopathies, simulating those occurring in background diabetic retinopathy in humans, and these were prevented by treatment with an aldose reductase inhibitor.

Human lens epithelial (HLE) cells in tissue culture accumulated significant levels of galactitol when they were cultured for 72 hr in medium containing 30 m m d -galactose. Polyol accumulation was accompanied by the appearance of vacuoles as seen by transmission electron microscopy. The number and size of intracellular vacuoles increased when the culture period was extended to 7 days. In addition, polyol accumulation was accompanied by loss of myoinositol. None of these changes occurred in cells exposed to 30 m l -galactose which is not a substrate for aldose reductase. The accumulation of galactitol, intracellular vacuole formation and loss of myoinositol observed in d -galactose-exposed cells were prevented by the inclusion of the aldose reductase inhibitor, sorbinil, in the culture medium. Comparison of the relative efficacies of two aldose reductase inhibitors indicate that AL 1576 is nearly 20 times more potent than sorbinil in inhibiting the human lens enzyme. It is concluded that vacuole formation in HLE cells is due to the osmotic effect of polyol formation brought about by the action of aldose reductase and that the etiology of human diabetic cataract may also involve the polyol pathway.

• Vascular changes associated with early diabetic retinopathy that include the selective degeneration of pericytes, the formation of microaneurysms and acellular capillaries, and vessel dilation have been experimentally investigated in age-and sex-matched beagle dogs fed a 30% galactose diet and treated with or without the aldose reductase inhibitors sorbinil and/or M79175. Eyes from dogs in each group were periodically enucleated during a 36-month period and their retinal capillaries were examined as trypsin-digested flat preparations. These studies reveal that the destruction of retinal pericytes to form pericyte ghosts is the earliest observable retinal vessel change occurring after 19 to 21 months of galactose feeding. By 24 months, both an irregular distribution of endothelial cell nuclei near pericyte ghosts and the presence of acellular capillaries containing neither endothelial cells nor pericytes can be observed. This was followed by the histologic appearance of microaneurysms after 27 months and the funduscopic appearance of intraretinal hemorrhages after 33 months. Varicose enlargements of capillaries were also observed in the trypsin-digested preparations from dogs fed galactose for 33 to 36 months. All of these changes are linked to the initial aldose reductase-associated destruction of pericytes. The onset and progression of these retinal changes were retarded in a dose-dependent manner with aldose reductase inhibitors.

The development of the concept that aldose reductase (AR) is involved in diabetic complications is presented from its early beginning when Dr Van Heyningen first found polyols in sugar cataracts in 1959. The evolvement of the polyol theory of sugar cataract is described. The prevention of sugar cataract formation by aldose reductase inhibitors dramatically demonstrates the role of this enzyme in sugar-induced cataracts. The possibility of AR involvement of other diabetic complications was an obvious extension of the polyol theory. The use of AR inhibitors in preventing diabetic changes in nerve and retina in animals strongly suggest that AR may play a role in the development of other diabetic complications.

The in vitro effects of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, sphingomyelin, lysophosphatidyl choline, lysophosphatidyl ethanolamine, lysophosphatidyl serine, glycerophosphoryl choline, and phosphatidic acid on rubidium uptake and amino acid transport of rat lens are presented. None of these lipids affected the rubidium uptake mechanism; however, a large increase in the apparent leak-out of rubidium was observed with lysophosphatidyl choline. Lysophosphatidyl choline also inhibited all of the amino acid transport systems (A, ASC, L, Gly, Ly and β) as well as the uptake of inositol.

Changes in crystallin synthesis and crystallin mRNA content were followed in the epithelial and fiber cells of rat lenses during the development and reversal of galactose cataracts. Crystallin synthesis was not lowered in the lens epithelial cells throughout the 18 days of galactose feeding. By contrast, crystallin synthesis appeared partially reduced in the fiber cells of cultured lenses after the rats had been on a diet of 50% galactose for 6 days, and was essentially arrested in the lens fiber cells after the rats were fed galactose chow for 12 or 18 days. In vitro translation tests in a rabbit reticulocyte lysate demonstrated that control lenses contained α-, β- and γ-crystallin mRNAs in the cortical and nuclear fiber cells. The lens fiber cells of the 6-day galactosemic rats still possessed a full complement of crystallin mRNAs but those of the 15-day galactosemic rats had no detectable crystallin mRNAs. After 12 days the galactosemic lenses had lost approximately half of the crystallin mRNAs from the cortical fiber cells and all of the mRNAs from the nuclear fiber cells. Both crystallin synthesis and crystallin mRNA content recovered in the cortical fiber cells, but not in the nuclear fiber cells, during reversal of the galactose cataract. These results indicate that the reduction in crystallin synthesis is confined to the fiber cells and occurs initially by a decreased efficiency of utilization of crystallin mRNAs and subsequently by the degradation of the mRNAs. Previous experiments provide evidence that the impaired utilization of the crystallin mRNAs is due to the alterations in electrolytes in the cataractous lenses.

The proteins of the Rhesus monkey (Macaca mulatta) lens were studied and characterized. In general, there is a similarity of the monkey lens to the human lens particularly in the alpha- and beta-crystallins and the membrane proteins, although the amount of beta 1-crystallin may be greater in the monkey. There is immunological cross-reactivity in the crystallins between monkey and human lenses including the monkey low molecular weight proteins and the human low molecular weight proteins. The monkey lens low molecular weight material showed three differently sized proteins by gel exclusion chromatography. In this respect, the monkey lens proteins are similar to the human. However, the monkey lens low molecular weight proteins differ from the human low molecular weight proteins in charge as well as molecular weight determined by SDS-PAGE. One of the monkey low molecular weight proteins is more prevalent in the nucleus than in the cortex suggesting that this protein may be more important to the embryonic lens that to the adult lens.

The relevance of nonenzymatic glycosylation of lens proteins to cataract formation was studied in rats on a normal and high galactose diet, treated with and without sorbinil, an aldose reductase inhibitor. All galactosemic rats not receiving sorbinil had cataracts; none receiving sorbinil had cataracts. Lens homogenate was treated with a 200 fold molar excess of [3H]-borohydride and the extent of glycosylation was estimated from radioactivity incorporation and quantitation of hexitol-lysine adduct after extensive dialysis. We found no differences in the radioactivity uptake nor the amounts of hexitol-lysine in the lenses of galactosemic rats treated with and without sorbinil. Thus, nonenzymatic glycosylation was not responsible for the sugar-induced cataracts.

Weanling male Sprague-Dawley rats were given a 50% galactose diet with or without an aldose reductase inhibitor (0.04% tolrestat, Ayerst) for 88 weeks. Quantitative computer planimetry on electron micrographs of perpendicularly transected retinal capillaries showed mean basement membrane thicknesses to be 263 +/- 50 nm in galactose-fed rats, 153 +/- 10 nm in galactose-fed rats given tolrestat, and 114 +/- 16 nm in rats given control feed. The retinal capillary basement membrane thickening in galactose-fed rats was accompanied by other ultrastructural alterations mimicking changes observed in diabetic microangiopathy, such as multi-lamination and the formation of vacuoles and dense inclusions. The results extended previously reported (44-week) findings, showed greater galactose-induced changes in capillary basement membranes, and demonstrated the preventative effect of an aldose reductase inhibitor.

Sephadex gel filtration and SE-Sephadex chromatography of cattle and calf lens extracts revealed major differences in the protein patterns of the γ-crystallin fraction. With aging of the lens apparently a new form of γ-crystallin emerges. The older bovine lens contains two forms of γ-crystallin: one which is identical with calf lens γ is found in the nucleus; the second γ is found in the cortex and is of larger molecular weight than the calf lens γ. Amino acid analyses, N -group determinations and amide- N assays have further characterized differences and similarities between these two forms of γ-crystallins.

Two structurally unrelated aldose reductase inhibitors (sorbinil and tolrestat) were used for assessing the possible role of aldose reductase in the thickening of retinal capillary basement membranes of rats. Rats were fed either a normal diet, a 50% galactose diet, or a 50% galactose diet with the addition of an aldose reductase inhibitor. Micrographs of capillaries in the outer plexiform layer of the retina were analysed. This showed that the basement membrane thickening and the ultrastructural changes in the retinal capillaries that were induced by the galactose diet were inhibited by the addition of an aldose reductase inhibitor. Thickening of the basement membrane may be related to other tissue lesions in diabetic retinopathy. Therefore, its prevention might have a favourable influence on other aspects of this retinopathy.

Cataracts associated with diabetes and galactosemia are characterized by their rapid onset and bilateral appearance. These cataracts display similar morphology and histology and have common biochemical mechanisms initiating the cataractous processes. An understanding of these biochemical mechanisms has been aided both by the ability to reproduce these cataracts in various animal models and by the development of potent inhibitors of aldose reductase. This is a US government work. There are no restrictions on its use.

The final peak from the gel filtration fractionation of human lens proteins has been called either low-molecular-weight protein or ψ-crystallin. Since this fraction has been shown to be heterogeneous we have attempted to determine which of its components are really γ-crystallin. At least four distinct components have been found with molecular weights determined as 24000, 21000, 19000 and 10000. The 19000 dalton species was isolated and shown to be ψ-crystallin on the basis of immunochemical reactivity, conformational analysis by ultraviolet circular dichroism and molecular size and charge. The 10000 dalton species increases with age in the human lens and probably is the product of degradation of lens cyrstallins. It does not show cross-reactivity with antiserum specific for γ-crystallin. The 24000 and 21000 dalton components have not been separated from each other. The mixture of these two components clearly contains material related to β-crystallin immunochemically, but reacts only very weakly, if at all, with anti α-crystallin serum.

The non-protein amino acid ornithine is readily accumulated into rat lenses cultured in TC-199 bicarbonate medium. This accumulation, measured by the lens water/lens medium ratios of radiolabeled ornithine, appears to be the result of an apparent energy-dependent basic amino acid transport system. Moreover, competition studies suggest that increased levels of ornithine can depress the concomitant lenticular accumulation of arginine and lysine. Examination of the trichloroacetic acid protein precipitates of these cultured lenses indicates that the radiolabel from ornithine can also be incorporated into lens proteins as proline. This results from the conversion by ornithine aminotransferase (EC 2. 6. 1. 13) of radiolabeled ornithine to delta1-pyrroline-5-carboxylic acid (P5C), which is subsequently converted to proline by P5C reductase (EC 1. 5. 1. 2). This incorporation of radiolabel into cultured lens proteins is reduced upon addition of either 1 m m proline or P5C to the culture medium or inhibited by the addition of the protein synthesis inhibitor, puromycin.

A radioimmunoassay was developed for mouse α- and γ-crystallins. A standard inhibition curve against known quantities of the crystallins served as a basis for quantitating unknown samples. The sensitivity of the assay was 2 ng for α-crystallin and 4 ng for γ-crystallin. It was possible to quantitate the amount of γ-crystallin present in the cultures from normal mouse and Nakano mouse lenses. The results were consistent with the previous immunofluorescent observation of γ-crystallin localization in the lentoid bodies. In addition, changes in the lens crystallins in normal and Nakano mouse lenses were followed. A dramatic decrease of γ-crystallin occurred during cataract development, however the level of α-crystallin in the soluble lens protein remained unchanged during this period.

Ocular lenses in Nakano mice showed marked changes in synthesis, degradation and leakage of protein during cataractogenesis. The cataract-associated changes included the differential lowering of crystallin synthesis, the cleavage of crystallin polypeptides to lower molecular weight forms and the leakage of crystallins from cultured lenses. Ouabain treatment of normal lenses induced these alterations, suggesting that changes in the intracellular levels of Na+ and K+ affect the anabolism and catabolism of protein during cataract formation.

Certain biochemical and morphological changes involved in the formation of osmotic cataracts in the Nakano mouse and galactosemic rat lenses also occur in vitro. Incubation of young, normal, albino mouse lenses in glucose-free medium for 48 hr resulted in a gain in wet weight due to dramatic hydration. The glucose deprived lenses showed a marked decrease in the soluble protein content as is noted in cataract formation in vivo. Chromatography of the soluble protein demonstrated a substantial increase in heavy molecular weight material in the lenses incubated without glucose in comparison to the fresh lenses. A decrease in βH-crystallin was also striking and might be correlated with the disappearance of a 31 000 mol. wt polypeptide band seen by SDS-PAGE gel. Interestingly, the membrane polypeptides in the in vitro incubated lenses mimicked changes observed during cataract formation in vivo. A 23 000 mol. wt polypeptide band became prominent in the membrane fraction of these lenses and there was a concomitant decrease in the 26 000 mol. wt component. Messenger RNAs were present in the glucose deprived lenses as shown by cell-free translation in vitro, although no [35S]methionine incorporation into crystallin was noted. Both the hexokinase and glyceraldehyde phosphate dehydrogenase activities in the glucose deprived lenses fell rapidly and were virtually absent at the end of 48-hr incubation period. The data in this report suggest that the glucose deprived incubated mouse lens may serve as an in vitro model to the study of some biochemical and morphological changes occurring in the development of osmotic cataract in vivo.

Rat lenses in organ culture which are exposed to bovine rod outer segments (ROS) or to the major fatty acid of ROS, docosahexaenoic acid, are impaired in their ability to accumulate radiolabeled compounds which lenses normally accumulate by active processes. The extent of lens damage correlates well with the extent of lipid peroxidation in the culture medium as assessed by the thiobarbituric acid assay. Addition of vitamin E to the medium inhibits the effect on the lens while addition of Fe-ADP complexes potentiates the effect. Thus, the lens damage appears to be attributable to toxic species generated by peroxidation of the polyunsaturated lipid added to the culture medium. Toxic aldehyde products appear to be major mediators of the lens damage, since semi-carbazide, which avidly reacts with aldehydes, can protect lenses in this system. These findings may have relevance to the cataracts clinically associated with retinal degenerative diseases such as retinitis pigmentosa. The highly membranous photoreceptor cells are extremely rich in polyunsaturated lipid. Degeneration of these cells, which is the primary pathology in such diseases, would likely lead to peroxidation with generation of toxic products within the eye. Such products could potentially produce secondary damage to other ocular structures including the lens.

Lens epithelial cells from normal and congenital cataractous mice strains were cultured under similar conditions. Both normal and cataractous cells actively propagated and reached confluency on the eleventh day. These cells, thereafter, underwent morphological changes characterized by cell elongation, aggregation and formation of lentoid bodies at about 15 days. Electron microscopy revealed these lentoid bodies to consist of immature lens cells. These structures derived from cataractous cells had numerous vacuoles in the cytoplasm much more so than in the normal lens cells. In addition, some lentoid bodies closely resembled mature fibers of the intact lens. It was also demonstrated that these lentoid bodies showed positive immunofluorescence when reacted with fluorescent antiserum to γ-crystallin. There were certain differences observed between the cultured cells derived from normal lens and Nakano cataract. The disappearance of organelles and denucleation process were delayed in the lentoid bodies found in cultured Nakano cells when compared to normal cell culture. In addition a second type of lentoid body, although present as a minor population, was observed in the Nakano cell culture. Other subtle differences were observed during the course of culturing normal and cataractous lens cells.

Three classes of β-crystallin, which have previously been named β 1 , β 2 and β 3 -crystallins, have been isolated from normal human lenses by gel filtration on Sephadex G-200. They have been compared to each other in terms of physico-chemical, immunochemical and conformational parameters. All three β-crystallins have similar secondary and tertiary structures as seen by ultraviolet circular dichroism. They appear to be very closely related immunochemically. All three are quite heterogeneous but have similar isoelectric point ranges which are more acidic than that of bovine β-crystallin. β 1 , β 2 and β 3 appear to be composed in large part of identical subunits, but there are differences in the distributions of several minor polypeptides as visualized by electrophoresis in the presence of sodium dodecyl sulfate. The major distinction among the three β-crystallin classes is in molecular weight. All three classes contain a minor polypeptide or group of polypeptides of approximately 43 000 daltons which have not been seen in the β-crystallin from other species. The 43 000 dalton polypeptides were isolated from each β-crystallin class. Preliminary analysis of these preparations by isoelectric focusing and immunodiffusion was done.

Evidence linking the enzyme aldose reductase (alditol:NADP + oxidoreductase, EC 1.1.1.21) to the pathogenesis of several diabetic complications is rapidly mounting. The results of several animal studies combined with preliminary reports of ongoing clinical trials indicate that inhibition of aldose reductase produces a beneficial effect against such diabetic complications as neuropathy, cataract, corneal epitheliopathy, retinopathy, microangiopathy, and possibly nephropathy. 1,2 The observations that aldose reductase inhibitors appear to provide a new direct mode of treatment for the control of diabetic complications—a method independent of the insulin-related control of blood glucose levels—has spurred interest in the development of more potent and selective inhibitors. That goal can be more easily realized through an understanding of how these inhibitors interact with the aldose reductase protein. This requires insight into the steric and electronic requirements of both the inhibitors and the enzyme site where they bind (inhibitor site). Through the use of computer molecular modeling, molecular orbital calculations, known structure-activity relationships (SAR), protein modification reagents, and irreversible inhibitors, specific structural, and electronic similarities among the apparently structurally diverse aldose reductase inhibitors (ARIs) have been observed. 3,4 In turn, these studies have led us to postulate the pharmacophor requirements of the ARI site.

In diabetic cataracts aldose reductase initiates the cataractous process by converting glucose to sorbitol. The ensuing osmotic change, caused by sorbitol accumulation, adversely affects the lens permeability barrier so that the distribution within the lens of electrolytes, amino acids, and myo-inositol becomes grossly altered. These changes affect lens viability resulting in opacification. That aldose reductase triggers the process is shown by the fact that several structurally unrelated aldose reductase inhibitors prevent cataracts from occurring. Aldose reductase is also implicated in diabetic retinopathy and keratopathy. Aldose reductase functions in the retinal capillary pericytes, the cells first affected in microvascular abnormalities in diabetes. Additionally, retinal capillary basement membrane thickening can be prevented by aldose reductase inhibitors. Clinical trials are underway to determine the efficacy and safety of aldose reductase inhibitors in treatment of diabetic retinopathy.

The ocular lens has high concentrations of free myoinositol. The ability of the lens to actively concentrate inositol may explain the high levels of the cyclic polyol. However, no detectable amounts of inositol are present in the aqueous humor. The biosynthesis of inositol may contribute to the inositol stores in the lens. This paper describes a study on the various factors involved in explaining the presence of inositol in the lens. During the course of this study an active phosphatase of inositol-1-phosphate was uncovered. Consequently, this paper deals primarily with the phosphatase and also gives some features of the biosynthesis of inositol from glucose-6-phosphate in the lens.

The beta-crystallin basic principal polypeptide (beta Bp) appears to be altered in the lens of the Philly mouse and may be the main defect in this hereditary cataract. Northern blot analysis showed that an mRNA encoding for beta Bp is present in the Philly mouse lens, but normal beta Bp could not be detected. Instead, a different protein related to beta Bp has been observed. Western blot analysis with antibodies against specific beta Bp peptide sequences showed that the Philly protein shares the same amino-terminal residue as beta Bp but lacks a part of the carboxyl-terminal half of normal beta Bp. The altered protein is slightly smaller than beta Bp and has a more acidic isoelectric point by two-dimensional gel electrophoresis. It also lacks the property of heat stability characteristic of normal beta Bp. The mapping of the alteration in beta Bp may give insight into the nature of the heat stability of this protein as well as some indication of the structural components that are necessary to maintain optical clarity in the lens.

A monoclonal antibody to a bovine lens 27K beta-crystallin polypeptide has been produced from a rat x mouse hybridoma. The antibody reacts with the 27K polypeptide of bovine, mouse, rat, and human lenses, but does not react to the 27K polypeptide of monkey lens nor does it react with any component in the Philly mouse lens which is missing the 27K polypeptide. The antibody does not recognize any of the other major bovine beta-crystallin polypeptides but does recognize a large number of native beta-crystallin proteins. The antigenic specificity and species cross-reactivity of this antibody provides an excellent opportunity to study many aspects of lens development and cataractogenesis.

During aging and particularly during cataractogenesis, crystallin polypeptides in the human lens become cross-linked by non-disulfide covalent bonds. Recently it has been hypothesized that singlet oxygen generated photodynamically within the lens oxidized certain amino acid residues in lens crystallins leading to cross-link formation. That singlet oxygen can produce cross-links in lens protein solutions has been established. In the present work we demonstrate by electrophoresis in sodium dodecyl sulfate that photodynamic cross-linking of crystallins can occur within the intact rat lens in organ culture. The cross-linking was most pronounced in the urea insoluble fraction of the peripheral lens cortex. Urea insoluble protein was increased in the irradiated lenses relative to non-irradiated controls. The process can be supported by either the photosensitizing dye rose bengal or kynurenine, an oxidation product of tryptophan which is endogenous to human lens. Based on inhibition by scavengers we conclude that the cross-linking is mediated by singlet oxygen. Cross-linking within the intact lens appeared to have greater specificity with respect to the polypeptides involved than did cross-linking in protein solutions.

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH-dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40–50% homology with ϱ-crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and ϱ-crystallin are members of a superfamily of related proteins.

Aldose reductase (AR) appears to initiate the cataractous process in galactosemic and diabetic animals. Sugars in excess are converted to polyols by lens AR. In sugar cataracts, polyols accumulate to levels substantial enough to cause a hypertonicity leading to lens fiber swelling. All other changes appear secondary to polyol accumulation and lens swelling. The development of sugar cataracts can be duplicated in organ culture. In culture, the various changes that occur were minimized or did not occur when inhibitors of AR were included in the medium. Moreover, AR inhibitors were shown to effectively delay the onset of sugar cataract development in animals. A defect in the corneal epithelium of diabetics became apparent in vitrectomy. One manifestation of this problem was the delay in the reepithelialization of denuded corneas. In examining this problem experimentally, the epithelium was removed from the corneas of diabetic and normal rats. The regeneration of epithelium in corneas of diabetic rats required a longer period than in the normal. The possibility that AR, active in the epithelium, was involved in this phenomenon was investigated. The corneal epithelium was removed from both eyes of a diabetic rat. One eye was treated topically with the AR inhibitor CP-45,634 while the other served as control. The eye treated with CP-45,635 regenerated epithelium much more quickly than the untreated eye. Other AR inhibitors had similar beneficial effects.

Laurell ‘rocket’ immunoelectrophoresis has been applied to the quantitation of lens crystallins during development of hereditary cataract in the Philly mouse. A marked decrease in γ crystallin and sma

It has been suggested that the presence of high molecular weight protein aggregates in the lens can lead to light scattering and a consequent loss of transparency. We have measured the concentration of aggregates having a molecular weight greater than approximately 150 × 106 g/mole present in the soluble fraction of both aging normal and cataractous human lenses. This protein population is approximately 5% of the total soluble protein in lenses up to age 75 and increases to 10–15% in lenses aged greater than 75 years and in cataracts. The amino acid composition of the heavy molecular weight soluble aggregates of normal lenses is different in leucine content from alpha, beta and gamma crystallins and different in tyrosine content from beta and gamma crystallins. The aggregates from cataractous lenses show a greater increase in leucine and greater decrease in tyrosine. An unidentified component is present in the cataractous aggregates. Furthermore, calcium, an ion which increases in concentration in cataractogenesis, induces alpha crystallin to aggregate and induces the heavy molecular weight fraction to aggregate. This aggregation is irreversible by dialysis or chelation.

It appears that gamma-crystallin of calf lens has more bound sugar than either alpha- or beta-crystallins. The various fractions of gamma-crystallin obtained from three different methods of the preparation all were found to contain some carbohydrate. In the major fractions, the carbohydrate was composed of xylose, mannose, galactose, glucose and hexosamine, which were present in the same proportions. Under certain procedures of separating gamma-crystallin from the other two crystallins, a minor protein fraction was isolated that contained a large carbohydrate component made up principally of glucose.

Hexokinase activity in calf, rabbit and rat lens represents the summated activities of two distinct forms of hexokinase. These are not unique “lens hexokinases”, but possess the same electrophoretic characteristics as Types I and II of other tissues. Lens is devoid of Types III and IV and IIb. The Michaelis constants of these lens heoxkinases are; Kml: 7·4 × 10−5M, and KmII: 1·0 × 10−3 m . In the calf lens eapsule-epithelium, 24% of the hexokinase is Type I; 33% of the nuclear hexokinase is Type I. The total hexokinase activity of the rat lens increases with age or lens size. The total activity of each member of a pair of lenses is essentially identical and lenses of similar size from rats of different sex have similar activities. The specific activity of lens heoxkinase decreases with increasing lens size and is probably a reflection of proportionally greater synthesis of non-enzymic structural protein. The capsule and epithelium contain 10% of the total lens hexokinase. The equatorial cortex contains approximately 3 times the hexokinase activity of the anterior or posterior cortex, and the nucleus is practically devoid of measureable hexokinase activity. Hexokinase and phosphofructokinase have been shown to be the rate limiting steps in lens glycolysis. Our data suggest that rather than a single form of hexokinase serving as pacemaker, there may actually be two—Type II hexokinase pacing glycolysis in areas of relatively high ambient glucose concentration (epithelium and cortex), and Type I in regions of low ambient glucose concentration (nucleus).

Paired lenses, from young rabbits, were incubated for several days in a medium containing 35·5 m m glucose. One lens was incubated with the vitreous intact and attached to the posterior lens capsule; the fellow lens was separated from and incubated in the absence of the vitreous in the usual manner. Ultimately, a mature sugar cataract formed in each lens preparation. There was, however, a significant delay in the appearance of the posterior cortical opacity in the lens incubated with the vitreous attached; there was also less of an increase in wet weight of the lens. Less glucose entered the lens with the attached vitreous and subsequently less sorbitol and fructose were formed. Also in this preparation the gain in sodium and loss in potassium were delayed. The vitreous delayed, but did not prevent, the formation of the mature sugar cataract.

1. 1. The distribution of aldose-reducing activities was studied in several rabbit organs. Although all the organs studied had the ability to use NADPH as a cofactor for the reduction of xylose, the different substrate specificities observed suggest that the activity may be due to the presence of at least two different enzymes. These were aldose reductase (polyol:NADP+ oxidoreductase, EC 1.1.1.21), in lens, adrenal and skeletal muscle and NADP+-L-hexonate dehydrogenase (L-gulonate:NADP+ oxidoreductase, EC 1.1.1.19) in liver, kidney, heart, spinal cord and brain. 2. 2. Calf lenses were dissected into three portions: capsule (including the epithelium), cortex and nucleus, and the concentrations of aldose reductase, galactokinase, (ATP:D-galactose 1-phosphotransferase, EC 2.7.1.6) and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) were determined in the soluble fractions of the extracts. The distribution patterns were different for the three enzymesl the ratio cortex:epithelium:nucleus was 1:21:1.1 for aldose reductase; 1:3:0.02 for galactokinase and 1:7:0.04 for glucose-6-phosphate dehydrogenase. 3. 3. When calf lenses were incubated in media containing galactose, the accumulation of dulcitol was greatest in the epithelial region and least in the nucleus with an intermediate amount in the cortex. 4. 4. No biosynthesis of ascorbate from either D-[6-14C]glucuronate or D-[6-14C]-glucuronolactone could be demonstrated in incubated rabbit lens. However, there was conversion of the labeled carbon to CO2 and some accumulation of labeled L-gulonate. Aldose reductase, although it reduces glucuronate to L-gulonate, does not appear to be involved in ascorbate biosynthesis.

When calf lenses are incubated in tissue culture with actinomycin D (10 −6 M), adenine incorporation into ribosomal and albuminoid RNA is almost completely inhibited within 4 h. Little inhibition can be observed upon adenine incorporation into soluble RNA even after 24-h exposure to actinomycin D. In contrast to these results only after prolonged preincubation with actinomycin can an inhibition of histidine incorporation into lens protein be observed. After a 30-h incubation with actinomycin, the incorporation of histidine is only 50 % inhibited and during the following 16 h a further 25 % inhibition is observed. Thus the results suggest that a relatively stable messenger RNA is present in lens. Actinomycin D inhibits the incorporation of [ 14 C]histidine into α, β, γ, and HL protein to approximately the same extent. Puromycin was found to almost completely inhibit incorporation of [ 14 C]adenine into ribosomal and albuminoid RNA while only a slight inhibition of incorporation into soluble RNA was observed. The incorporation of labeled histidine into lens protein was almost completely inhibited. Neither antibiotic was shown to have a significant effect upon the transport of [ 14 C]adenine into the lens or upon the synthesis of nucleotides from adenine. A major fraction of the adenine taken up by the lens is converted to ATP.

Methyl phenyldiazenecarboxylate (azoester) has been widely used for the intracellular oxidation of glutathione in the red blood cell. However, the reactions of azoester are complex, and azoester is unstable in aqueous solution. Using 86 Rb we have studied the effects of azoester on the cation transport and permeability of rat lenses. Lenses were exposed to both freshly prepared solutions of azoester (rapid delivery) and solutions which were allowed to stand for 35 min before lens exposure (delayed delivery). Rapid delivery of azoester effects a rapid oxidation of lens glutathione. In the presence of glucose this effect is diminished, presumably because the glutathione level is maintained by the NADPH generated by the pentose shunt mechanism. In contrast, delayed delivery of azoester does not affect lens glutathione content. Lenses pretreated with azoester (rapid delivery) without glucose for 15 min manifest a 68–73% decrease in 86 Rb uptake. Delayed delivery of azoester without glucose causes a similar decrease in 86 Rb uptake. Glucose medium improves the 86 Rb uptake of similarly treated lenses (both rapid and delayed delivery), but does not restore the uptake to normal values. In addition, azoester-treated lenses (rapid delivery) incubated without glucose manifest a 29% increase in 86 Rb run-out. In contrast, delayed delivery of azoester without glucose causes a 32% decrease in 86 Rb run-out. In the presence of glucose both rapid and delayed delivery of azoester effect a normal rate of 86 Rb run-out. Both rapid and delayed delivery of azoester result in a marked decrease in lens ATP content.

The reactivity of the SH groups in lens homogenates was studied by the amperometric titration procedure and by the rate of oxidation of these groups. The glutathione in the lens homogenates seems to be much more reactive than the protein SH groups. Apparently there exist varying degrees of reactivity of protein SH groups. There are those which are only observable when measured in the presence of 8 M urea. The age of the lens also influences the state of reactivity of these groups. In the mature cattle lens almost all of the protein SH groups are masked in some manner, while in the younger calf lens they appear much more reactive.