Identification of the scattering elements responsible for lens opacification in cold cataracts.

Using both quasi-elastic light scattering spectroscopy and angular dissymmetry in the intensity of the scattered light, we examined the onset of turbidity for intact calf lenses and for isolated nuclear cytoplasm. In the case of the nuclear cytoplasm these measurements demonstrate the presence of two kinds of scatterers: small units of approximately 100-A radius and larger elements whose size is distributed around 1,500 A. As the temperature is decreased towards the cold cataract temperature, the intensity of light scattered by the small units stays almost constant while the intensity scattered by the large elements increase very strongly. The opacification of the lens cytoplasm produced by decreasing the temperature results principally from an increase in the concentration of the large scattering elements. For the intact nucleus the situation is qualitatively similar, but the mean size of the large scattering elements shows a more substantial increase than in the isolated cytoplasm as temperature is lowered towards the cold cataract temperature.     

Permanent suppression of phase separation cataract in calf lens using amine modification agents

Low temperature induced opacification (cold cataract) of the nucleus of young mammalian lenses is associated with a phase separation of proteins in the lens cell cytoplasm. Calf lenses were treated with a variety of imido-esters and N-hydroxysuccinimide-esters, which react specifically with amino groups. Many potent inhibitors of phase separation cataract were identified which lower the opacification temperature by 6 degrees C or more. Lenses generally remain clear, colorless and soft. Furthermore, suppression of the cold cataract temperature is permanent upon removal of excess reagent.     

The in vitro retardation of porcine cataractogenesis by the calpain inhibitor,SJA6017

Calpain inhibitors show the potential to serve as non-surgical alternatives in treating diabetic cataract and other types of these disorders. Here, we have tested the recently developed calpain inhibitor, SJA6017, for its ability to inhibit cataractogenesis in porcine lenses. These lenses were incubated in increasing levels of extralenticular calcium (Ca²⁺; 5–30 mM). Atomic absorption spectroscopy was used to determine total internal lens Ca²⁺ and a correlation between porcine lens Ca²⁺ uptake and levels of lens opacification were found with a total internal lens Ca²⁺ level of 5.8 M Ca²⁺ g^–1 wet lens weight corresponding to the onset of catarctogenesis. A total internal lens Ca²⁺ level of 8.0 M Ca²⁺ g^–1 wet lens weight corresponded to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when SJA6017 (final concentration 0.8 M) was included in the extralenticular medium, suggesting that the Ca²⁺-mediated activation of calpains may be involved in the observed opacification. Supporting this suggestion atomic absorption spectroscopy showed that the effect of SJA6017 (final concentration 0.8 M) on lens opacification was not due to the compound restricting porcine lens Ca²⁺ uptake. The results indicate that calpain-induced cataractogenesis is dependent on extracellular Ca²⁺ and the calpain inhibitor SJA6017 (0.8 M) had no significant effect on Ca²⁺ uptake by lens. Its inhibitory effect on lens opacification may be due to a direct action on the activity of calpain. (Mol Cell Biochem 261: 169–173, 2004)     

Lens crystallins and oxidation: the special case of gammaS

Among lens crystallins, gamma-crystallins are particularly sensitive to oxidation, because of their high amount of Cys and Met residues. They have the reputation to induce, upon ageing, lens structural modifications leading to opacities. A combination of small angle X-ray scattering and chromatography was used to study the oxidation of gamma-crystallins. At pH 7.0, all the gamma-crystallins under study were checked to have the same structure in solution. Under gentle oxidation conditions at pH 8.0, human gammaS (hgammaS) and bovine gammaS (bgammaS) formed disulfide-linked dimers, whereas the other bgamma-crystallins did not. Cys20 was shown to be responsible for dimer formation since the C20S mutant only formed monomers. The hgammaS dimers were stable for weeks and did not form higher oligomers. In contrast, monomeric gammaS-crystallins freshly prepared at pH 8.0, and submitted to more drastic oxidation by X-ray induced free radicals, were rapidly transformed into higher oligomers. So, only extensive oxidation causing partial unfolding could be detrimental to the lens and linked to cataract formation. The gammaS-crystallins lack the temperature-induced opacification observed with the other gamma-crystallins and known as cold cataract. The oxidation-induced associative behaviour and cold cataract are therefore demonstrated to be uncoupled.     

大白鼠糖致白内障的激光喇曼光谱研究

作者用激光喇曼光谱法分析半乳糖导致大白鼠晶状体混浊过程中构象的变化。通过SPEX 1403型激光喇曼光谱仪得到了正常及不同混浊度晶状体的喇曼光谱。结果表明晶状体可溶性蛋白质二级结构的光谱未见异常,其残基酪氨酸及色氨酸微环境起了变化。随着晶状体混浊度的增加,SH谱峰强度变小而S-S键谱峰增强,同时观察到荧光背景逐渐加强。经分析认为晶状体混浊是与蛋白质分子的聚集有关。     

Integrin αVβ5-mediated Removal of Apoptotic Cell Debris by the Eye Lens and Its Inhibition by UV Light Exposure

Accumulation of apoptotic material is toxic and associated with cataract and other disease states. Identification of mechanisms that prevent accumulation of apoptotic debris is important for establishing the etiology of these diseases. The ocular lens is routinely assaulted by UV light that causes lens cell apoptosis and is associated with cataract formation. To date, no molecular mechanism for removal of toxic apoptotic debris has been identified in the lens. Vesicular debris within lens cells exposed to UV light has been observed raising speculation that lens cells themselves could act as phagocytes to remove toxic apoptotic debris. However, phagocytosis has not been confirmed as a function of the intact eye lens, and no mechanism for lens phagocytosis has been established. Here, we demonstrate that the eye lens is capable of phagocytizing extracellular lens cell debris. Using high throughput RNA sequencing and bioinformatics analysis, we establish that lens epithelial cells express members of the integrin αVβ5-mediated phagocytosis pathway and that internalized cell debris co-localizes with αVβ5 and with RAB7 and Rab-interacting lysosomal protein that are required for phagosome maturation and fusion with lysosomes. We demonstrate that the αVβ5 receptor is required for lens epithelial cell phagocytosis and that UV light treatment of lens epithelial cells results in damage to the αVβ5 receptor with concomitant loss of phagocytosis. These data suggest that loss of αVβ5-mediated phagocytosis by the eye lens could result in accumulation of toxic cell debris that could contribute to UV light-induced cataract formation.     

Quercetin metabolism in the lens: role in inhibition of hydrogen peroxide induced cataract

Oxidative stress is implicated in the initiation of maturity onset cataract. Quercetin, a major flavonol in the diet, inhibits lens opacification in a lens organ culture oxidative model of cataract. The aim of this research was to investigate the metabolism of quercetin in the lens and show how its metabolism affects the ability to prevent oxidation-induced opacity. The LOCH model (Free Radical Biology & Medicine 26:639; 1999) was employed, using rat lenses to investigate the effects of quercetin and metabolites on hydrogen peroxide-induced opacification. High-performance liquid chromatography analysis showed that the intact rat lens is capable of converting quercetin aglycone to 3'-O-methyl quercetin (isorhamnetin). Over a 6 h culture period no further metabolism of the 3'-O-methyl quercetin occurred. Loss of quercetin in the lens was accounted for by the increase in 3'-O-methyl quercetin. Incubation with 3,5-dinitrocatechol (10 microM), a catechol-O-methyltransferase (COMT) inhibitor, prevented the conversion of quercetin to 3'-O-methyl quercetin. The presence of both membrane-bound and soluble COMT was confirmed by immunoblotting. The results demonstrate that in the rat lens COMT methylates quercetin and that the product accumulates within the lens. Quercetin (10 microM) and 3'-O-methyl quercetin (10 microM) both inhibited hydrogen peroxide- (500 microM) induced sodium and calcium influx and lens opacification. Incubation of lenses with quercetin in the presence of COMT inhibitor revealed that the efficacy of quercetin is not dependent on its metabolism to 3'-O-methyl quercetin. The results indicate dietary quercetin and metabolites are active in inhibiting oxidative damage in the lens and thus could play a role in prevention of cataract formation.     

Determination of bilayer thickness and lipid surface area in unilamellar dimyristoylphosphatidylcholine vesicles from small-angle neutron scattering curves: a comparison of evaluation methods

Small-angle neutron scattering (SANS) experiments were performed on unilamellar 1,2-dimyristoylphosphatidylcholine (DMPC) vesicles prepared in heavy water by extrusion through polycarbonate filters with 500 Å pores. The data obtained at 30±0.1 °C were evaluated using a five-strip function model of the bilayer coherent neutron scattering length density, three different approximate form factors describing scattering from vesicles, and different methods of evaluation of the experimental data. It is shown that the results obtained from the SANS data in the range of scattering vector values 0.0316 Å^–1<q<0.0775 Å^–1 are not sensitive to the vesicle form factor, nor to the evaluation method. Using the hollow sphere model of vesicles convoluted with the Gaussian distribution of their sizes, a constrained bilayer polar region thickness of 9 Å and a DMPC headgroup volume of 325.5 ų, it was possible to obtain from the experimental data the DMPC surface area as 58.9±0.8 Ų, the bilayer thickness as 44.5±0.3 Å and the number of water molecules as 6.8±0.2 per DMPC located in the bilayer polar region.     

A specific chromosome element,the telomere of Drosophila polytene chromosomes

The submicroscopic organization of terminal chromosome regions of Drosophila hydei polytene chromosomes is described. A compact region composed of tightly packed fibrils of 100 to 125 Å diameter embedded in an amorphous material is located at each of the chromosome ends of the 5 long chromosome arms. From this compact region, sometimes containing cavities, fibrils extend onto the nearest normal band region. The diameter of the extending fibrils is 100–125 Å, 200–250 Å or 400 Å. Pronase digestion of fixed and squashed chromosomes reduced the electron density of the amorphous matrix in the compact regions but failed to affect the diameter of the fibrils. The extending fibrils, however, showed a decrease in diameter after pronase digestion. The most frequently observed diameter values were 100–125 Å. — The volume of the terminal structures, including the compact region as well as the extending fibrils, is characteristically different for the various elements of the karyotype. Chromosome 2 displays the largest terminal structure, whereas chromosome 4 only occasionally shows the presence of compact regions. — End to end association of the long chromosome arms involves the fusion of the compact terminal structures. The non-random distribution of end to end association seems to be correlated with the volume of the terminal structures. Chromosome 2 which contains the largest compact terminal region is more frequently involved in end to end associations than any other chromosome arm. — The terminal regions show replication of DNA. They belong to the group of regions which display a discontinuous labeling pattern along the chromosomes, representing a late phase of the replication cycle. — The unique structural organization of the terminal chromosome regions, which is never observed at any other location of the genome supports the idea that they are morphological manifestations of the postulated telomeres.     

不同剂量白藜芦醇对糖尿病性白内障大鼠晶状体抗氧化酶活力的影响

目的:探究不同剂量白藜芦醇对糖尿病性白内障大鼠晶状体抗氧化酶活力的影响。方法:75只5周龄健康SPF级雄性SD大鼠按照随机数字表法分为正常对照组、模型组,白藜芦醇低剂量组,白藜芦醇中剂量组和白藜芦醇高剂量组,每组各15只。五组大鼠均给予常规适应性喂养,模型组和白藜芦醇低、中、高剂量组大鼠采用链脲佐菌素(STZ)以60 mg/kg的给药剂量制作糖尿病大鼠模型,成模后白藜芦醇低剂量组按20 mg/kg、白藜芦醇中剂量组按50 mg/kg、白藜芦醇高剂量组按100 mg/kg的给药剂量每日给予白藜芦醇灌胃。观察12周后5组大鼠晶状体的混浊程度,检测血糖、体重后处死大鼠,检测晶状体内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)以及过氧化氢酶(CAT)的活性改变。结果:随着白藜芦醇剂量的升高,白藜芦醇低、中、高剂量组大鼠的血糖逐渐降低而体重逐渐升高,且组间比较均具有统计学差异(P0.05)。三组不同剂量白藜芦醇干预组大鼠晶状体的浑浊程度均低于模型组,且白藜芦醇高剂量组大鼠晶状体的浑浊程度最低,差异均具有统计学意义(P0.05)。白藜芦醇低、中、高剂量组大鼠SOD、GSH-PX和CAT酶活力与模型组大鼠相比均明显升高,而与正常对照组相比均明显降低(均P0.05)。随着白藜芦醇剂量的升高,白藜芦醇低、中、高剂量组大鼠SOD、GSH-PX和CAT酶活力逐渐升高,且组间比较均具有统计学差异(P0.05)。结论:高剂量白藜芦醇可更为明显地降低血糖浓度,提高晶状体SOD、GSH-Px及CAT酶活力,改善糖尿病性白内障晶状体的浑浊程度。     

Tempol-H inhibits opacification of lenses in organ culture

Cataract is the world's leading cause of blindness and a disease for which no efficacious medical therapy is available. To screen potential anti-cataract agents, a lens organ culture model system was used. Opacification of lenses maintained in culture was induced by specific insults including H(2)O(2) or the cataractogenic sugar xylose. Potential anti-cataract agents were added to the culture medium and their ability to inhibit opacification and certain biochemical changes associated with the opacification were assessed. Among the compounds tested, Tempol-H, the hydroxylamine of the nitroxide Tempol, gave the most promising results. It significantly inhibited opacification of rat lenses in an H(2)O(2)-induced cataract system as well as opacification of rhesus monkey lenses induced by xylose. Tempol-H inhibited the loss of glutathione, the leakage of protein, and decreases in the ability of cultured lenses to accumulate (3)H-choline from the medium, all of which were associated with the development of lens opacification. The antioxidative activity of Tempol-H and its ability to re-dox cycle make it an attractive candidate as a therapeutic agent for the prevention of aging-related cataract.     

A new interpretation of plasmodesmatal ultrastructure

Summary It is shown that simple, unbranched, plasmodesmata between young xylem ray cells of willow have no direct intercellular continuity apart from the plasmalemma which limits the cytoplasm and lines the plasmodesmatal canal. Each plasmodesma is traversed by a 200 Å diameter tubule (the desmotubule) which has a wall with probably 11 subunits arranged around a central cavity through which runs a 40 Å diameter rod. This rod is connected to the inside of the tubule wall, by fine filaments. At the ends of each plasmodesma the plasmalemma and cell wall are closely appressed to the tubule, thus precluding direct continuity between the cytoplasm of adjacent cells. Through the central part of the plasmodesmata the tubule is separated from the plasmalemma by a 90–100 Å wide gap. Cytoplasmic microtubules in the same tissue have a diameter of approximately 250 Å and a wall probably composed of 13 subunits: both desmotubules and cytoplasmic microtubules therefore have a centre-to-centre subunit spacing of about 47 Å. It is suggested that the desmotubules are not microtubules but may be nuclear spindle fibres which become trapped in the wall during cell plate formation. The endoplasmic reticulum, while closely approaching the plasmodesmata, is not continuous across them. It is thought most unlikely that the endoplasmic reticulum traverses plasmodesmata, as the dimensions of the central tubule — found here as well as by other workers — are smaller than those which would be expected to allow a stable molecular configuration in a unit membrane. The plasmalemma, where it lines the plasmodesmatal canal, appears to have particulate subunits in the outer opaque layers and the presence of these subunits may be attributable to the need for stability in membranes arranged about so small a radius.     

Comparative Analysis of Human γD-Crystallin Aggregation under Physiological and Low pH Conditions

Cataract, a major cause of visual impairment worldwide, is the opacification of the eye’s crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human γD-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.     

Role of long non‐coding RNA MIAT in proliferation,apoptosis and migration of lens epithelial cells: a clinical and in vitro study

Age‐related cataract is among the most common chronic disorders of ageing and is the world's leading blinding disorder. Long non‐coding RNAs play important roles in several biological processes and complicated diseases. However, the role of lncRNAs in the setting of cataract is still unknown. Here, we extracted total RNAs from the transparent and age‐matched cataractous human lenses, and determined lncRNA expression profiles using microarray analysis. We found that 38 lncRNAs were differentially expressed between transparent and cataractous lenses. 17 of 20 differentially expressed lncRNAs were further verified by quantitative RT‐PCRs. One top abundant lncRNA, MIAT, was specifically up‐regulated both in the plasma fraction of whole blood and aqueous humor of cataract patients. MIAT knockdown could affect the proliferation, apoptosis and migration of Human lens epithelial cells (HLECs) upon oxidative stress. Posterior capsule opacification (PCO) is a common complication of cataract surgery, which is associated with abnormal production of inflammatory factors. MIAT knockdown could repress tumour necrosis factor‐α‐induced abnormal proliferation and migration of HLECs, suggesting a potential role of MIAT in PCO‐related pathological process. Moreover, we found that MIAT acted as a ceRNA, and formed a feedback loop with Akt and miR‐150‐5p to regulate HLEC function. Collectively, this study provides a novel insight into the pathogenesis of age‐related cataract.     

Increased content of zinc and iron in human cataractous lenses

The purpose of the study was to examine the zinc and iron content of human lenses in different types of cataract and to investigate the possible influence of diabetes on the zinc and iron content of the lens. Iron and zinc of 57 human lenses (28 corticonuclear cataracts and 29 mature cataracts with a mean age of 70.6±16.1 and 74.7±11.1 yr, 41 nondiabetics and 16 diabetics) were determined by atomic absorption spectroscopy. The zinc content of human lenses was significantly increased in mature cataracts compared to corticonuclear cataracts (0.51±0.33 vs 0.32±0.20 μmol/g dry mass, p=0.012). The iron content of mature cataracts was also higher than in corticonuclear cataracts (0.11±0.09 vs 0.07±0.05 μmol/g dry mass, p=0.071). Furthermore, a significant increase of the lens zinc content could be observed with increasing lens coloration (light brown 0.33±0.17 vs dark brown 0.52±0.35 μmol/g dry mass, p=0.032). Diabetic patients seem to have both increased zinc and iron contents in the lens compared to nondiabetic subjects (zinc: 0.45±0.42 vs 0.40±0.22 μmol/g dry mass; iron: 0.12±0.10 vs 0.08±0.05 μmol/g dry mass). These data suggest a possible influence of the lens zinc and iron content on the development of lens opacification. Especially advanced forms of cataract and dark brown colored lenses show significantly increased zinc and iron content.     

先天性与后天性大鼠白内障晶体中游离氨基酸含量变化的研究

用药物诱发大鼠先天性白内障动物模型,检测了晶体中１８种游离氨基酸（ＦＡＡ）含量,并与大鼠硒性和半乳糖性白内障晶体中ＦＦＡ含量进行了比较,发现：仔一代和仔二代先天性白内障晶体中各ＦＡＡ含量接近;先天性白内障晶体内,酪氨酸和羟脯氨酸含量明显高于正常对照组（Ｐ＜０．０５）;谷氨酸,甘氨酸等９种ＦＡＡ明显低于硒性白内障（Ｐ＜０．０５）;而酪氨酸高于半乳糖性白内障组,半胱氨酸等５种ＦＡＡ含量均低于半乳糖性白内障组。同时还发现：硒性和半乳糖性白内障晶体内一些ＦＡＡ变化与以往报道不同,这些现象提示先天性与后天性白内障病变过程可能不同。     

The in vitro retardation of porcine cataractogenesis by the calpain inhibitor, SJA6017

Calpain inhibitors show the potential to serve as non-surgical alternatives in treating diabetic cataract and other types of these disorders. Here, we have tested the recently developed calpain inhibitor, SJA6017, for its ability to inhibit cataractogenesis in porcine lenses. These lenses were incubated in increasing levels of extralenticular calcium (Ca2+; 5-30 mM). Atomic absorption spectroscopy was used to determine total internal lens Ca2+ and a correlation between porcine lens Ca2+ uptake and levels of lens opacification were found with a total internal lens Ca2+ level of 5.8 microM Ca2+ g(-1) wet lens weight corresponding to the onset of catarctogenesis. A total internal lens Ca2+ level of 8.0 microM Ca2+ g(-1) wet lens weight corresponded to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when SJA6017 (final concentration 0.8 microM) was included in the extralenticular medium, suggesting that the Ca2+-mediated activation of calpains may be involved in the observed opacification. Supporting this suggestion atomic absorption spectroscopy showed that the effect of SJA6017 (final concentration 0.8 microM) on lens opacification was not due to the compound restricting porcine lens Ca2+ uptake. The results indicate that calpain-induced cataractogenesis is dependent on extracellular Ca2+ and the calpain inhibitor SJA6017 (0.8 microM) had no significant effect on Ca2+ uptake by lens. Its inhibitory effect on lens opacification may be due to a direct action on the activity of calpain.     

Prevention of cataract by pyruvate in experimentally diabetic mice

Previous studies have demonstrated that administration of pyruvate prevents cataract formation in diabetic rats. It is known that the induction of cataractous process in this case is initiated by aldose reductase (AR) catalyzed synthesis and accumulation of excessive sorbitol in the lens fibres and epithelium and their consequent osmotic hydration. Synthesis of this and other polyols is competitively inhibited by pyruvate. The objective of the present investigations was hence to determine whether pyruvate would have a similar protective effect in species where cataract formation is relatively independent of sorbitol synthesis such as in humans where the lens AR activity is extremely low, especially with glucose as a substrate. The Km of AR for glucose is known to be very high. The possible protective effect of pyruvate in the low AR models was conceived on the basis of our previous findings suggesting that it can also exert substantial antiglycating as well as antioxidant effects. The present studies have hence been conducted with mice, a species known to be low in lens AR, similar to that in humans. As stipulated, pyruvate administration has indeed been found to offer a significant protection against development of diabetic cataract in this model also. The effect correlated with the inhibition of protein glycation as well as of oxidative stress. The latter was apparent by the prevention of the loss of glutathione known to be associated with diabetes. Although there was a small but noticeable increment in the sorbitol content of the diabetic lenses, this was osmotically insignificant. Even this increase was prevented by pyruvate. The magnitude of the elevation in the contents of glycated proteins and the depression in the level of glutathione were, on the contrary, highly pronounced, suggesting a more prominent role of the latter factors. In addition, the possibility of a direct metabolic support it could offer to the tissue is also imminent by its effect on the maintenance of ATP, as shown earlier. The present studies are therefore considered more relevant to the pathogenesis of cataract in human diabetics and its possible prevention by endogenous compounds with antiglycating and antioxidant properties. Inhibition of cataract formation by pyruvate in an animal model with low lens AR, similar to that in humans, has been shown for the first time. (Mol Cell Biochem 269: 115–120, 2005)     

Spermidine Delays Eye Lens Opacification in vitro by Suppressing Transglutaminase-Catalyzed Crystallin Cross-Linking

A Ca²⁺-dependent TG activity, identified in the eye lens of several mammalian species, has long been implicated in cataract formation. The precise mechanism of the involvement of this enzyme in this process remains unclear. The purpose of this work was to investigate the modulatory effect of polyamines on TG activity during rabbit eye lens in vitro opacification. We observed, in an in vitro Ca²⁺-induced cataract model, a rapid decrease of the endogenous levels of SPD with the progression of opacification, paralleled by an increase of crystallin cross-linking by bis(γ-glutamyl)SPD. This pattern was reversed adding exogenous SPD to the incubation medium. Indeed, endogenous SPD levels were restored and cross-linking by bis(γ-glutamyl)SPD were drastically reduced. Surprisingly, under this experimental condition, the loss of transparency of lens was delayed. We found that exogenous SPD incubation led to a remarkable increase of mono(γ-glutamyl)SPD, likely responsible of the inhibition of cross-linking of lens crystallins and of the transparency persistence.