Mouse genetic corneal disease resulting from transgenic insertional mutagenesis.

BACKGROUND/AIMS: To report the generation of a new mouse model for a genetically determined corneal abnormality that occurred in transgenesis experiments. METHODS: Transgenic mice expressing mutant forms of Rab27a, a GTPase that has been implicated in the pathogenesis of choroideremia, were generated. RESULTS: Only one transgenic line (T27aT15) exhibited an unexpected eye phenotype. T27aT15 mice developed corneal opacities, usually unilateral, and cataracts, resulting in some cases in phthisical eyes. Histologically, the corneal stroma was thickened and vacuolated, and both epithelium and endothelium were thinned. The posterior segment of the eye was also affected with abnormal pigmentation, vessel narrowing, and abnormal leakage of dye upon angiography but was histologically normal. CONCLUSION: Eye abnormality in T27aT15 mice results from random insertional mutagenesis of the transgene as it was only observed in one line. The corneal lesion observed in T27aT15 mice most closely resembles posterior polymorphous corneal dystrophy and might result from the disruption of the equivalent mouse locus.

Chromosomal mapping, gene structure and characterization of the human and murine RAB27B gene.

BACKGROUND: Rab GTPases are regulators of intracellular membrane traffic. The Rab27 subfamily consists of Rab27a and Rab27b. Rab27a has been recently implicated in Griscelli Disease, a disease combining partial albinism with severe immunodeficiency. Rab27a plays a key role in the function of lysosomal-like organelles such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Little is known about Rab27b. RESULTS: The human RAB27B gene is organised in six exons, spanning about 69 kb in the chromosome 18q21.1 region. Exon 1 is non-coding and is separated from the others by 49 kb of DNA and exon 6 contains a long 3' untranslated sequence (6.4 kb). The mouse Rab27b cDNA shows 95% identity with the human cDNA at the protein level and maps to mouse chromosome 18. The mouse mRNA was detected in stomach, large intestine, spleen and eye by RT-PCR, and in heart, brain, spleen and kidney by Northern blot. Transient over-expression of EGF-Rab27b fusion protein in cultured melanocytes revealed that Rab27b is associated with melanosomes, as observed for EGF-Rab27a. CONCLUSIONS: Our results indicate that the Rab27 subfamily of Ras-like GTPases is highly conserved in mammals. There is high degree of conservation in sequence and gene structure between RAB27A and RAB27B genes. Exogenous expression of Rab27b in melanocytes results in melanosomal association as observed for Rab27a, suggesting the two Rab27 proteins are functional homologues. As with RAB27A in Griscelli Disease, RAB27B may be also associated with human disease mapping to chromosome 18.

Cloning, mapping and characterization of the human RAB27A gene.

Choroideremia (CHM) is an X-linked retinal degenerative disease that results from mutations in Rab Escort Protein-1 (REP1). REP1 acts in the prenylation of Rab GTPases, regulators of intracellular protein trafficking. Rab27a is unique among Rabs in that it is selectively unprenylated in CHM cells, suggesting that the degenerative process in CHM may result from unprenylation and consequent loss-of-function of Rab27a. As a first step towards the analysis of the Rab27a protein in patients, we report here the characterization of the human RAB27A gene. The putative protein encoded by this gene shares 96% identity with the previously cloned rat homologue. The RAB27A gene comprises five coding exons and two non-coding exons, of which one is alternatively used, and spans approximately 65 kb of DNA. There are three alternative poly-A addition sites in the long 3' UTR and also six potential single-nucleotide polymorphisms. The gene is located on chromosome 15q15-21.1, as determined by fluorescent in situ hybridization, and between markers D15S209 and AFM321ZD5 by radiation hybrid mapping.

Role of glycation in human lens protein structure change.

PURPOSE: Protein glycation may be involved in cataract development, by altering protein structure, particularly amino acid composition, and formation of fluorophores through a Maillard reaction. This study was designed to evaluate major changes in early and advanced (fluorescent) glycation products, with special emphasis on glycation-induced changes in amino acid composition of lens proteins. METHODS: We analyzed 50 human cataractous lenses (25 diabetic and 25 non-diabetic). Glycated proteins were isolated by affinity chromatography. Glycated and non-glycated proteins were separated by molecular sieve chromatography and further analyzed by RP-HPLC to establish the amino acid content. Early glycation levels were determined as furosine content and advanced glycation products were quantified by the characteristic fluorescence. RESULTS: Specific lens fractions (HMW and LMW) present significant differences in fluorescence levels between glycated and non-glycated proteins, specially in cataractous lenses from diabetic patients in which all proteins analyzed presented higher glycation levels than in non-diabetic patients. The amino and analysis of glycated proteins also revealed some important differences in specific basic residues (namely Lys, Arg and His) compared to the non-glycated fraction. CONCLUSIONS: The results suggest that protein glycation may be involved in changes in amino acid composition and fluorophore formation. This process may well account for the increased risk factor that diabetes represents for cataract development.

An experimental model for the evaluation of lipid peroxidation in lens membranes.

Lipid peroxidation has been associated with a number of specific manifestations related both to lens aging and cataract development. The assessment of the effect of various naturally occurring prooxidants as well as the development of antioxidant strategies has often been limited by the lack of appropriate and simple experimental models. In this study we discuss the adaptation of a method based on the incorporation of a fluorescent probe (parinaric acid) into biological membranes to monitor early stages of lipid peroxidation. After establishing the appropriate conditions, the method can be successfully applied to study peroxidation in bovine lens membranes, allowing for the evaluation of the effect of several free radical generating systems, including the following metal-dependent initiators: ascorbate/ iron, hydrogen peroxide/copper and cumene hydroperoxide/ copper. The inhibitory effect of the chelating agent diethylene-triaminepenta-acetic acid and the competitive hydroxyl radical scavenger sorbitol, was consistently observed on parinaric acid degradation, on hydroxyl radical yield and on the amount of thiobarbituric acid reactive material produced. It could be shown that oxidative degradation of the probe gives direct information on lens membrane susceptibility to a specific peroxidation system. Parinaric acid can therefore be used as an efficient oxidation probe to evaluate oxidative damage inflicted to lens membranes by different systems, allowing also the evaluation of the antioxidant effect of various drugs including those with potential anticataractogenic effect.

A technical approach to the evaluation of glucose oxidation: implications for diabetic cataract.

Oxidative stress has recently been involved in a number of diseases including development of diabetic cataract. If hyperglycemia is the relevant factor in diabetes, then it is reasonable to assume that under physiological conditions glucose may be toxic. The mechanisms involved in such a type of glucose 'toxicity' are still poorly understood but may involve glucose autoxidation. In this study we discuss a new methodological approach to the evaluation of glucose-induced oxidative damage to bovine lens membranes. The method is based on the incorporation of a fluorescent probe (parinaric acid) into lens membranes. The oxidative degradation of the probe is evaluated by monitoring its fluorescence decrease. It was possible to show that glucose may induce oxidative damage in the presence of trace amounts of transition metals. Furthermore, the data obtained by monitoring oxidative degradation of parinaric acid could be related to the amount of thiobarbituric acid-reactive substances formed under identical periods of time. The technique was shown to be reproducible, straightforward and highly sensitive as compared to other classical methods. Moreover, this methodological approach allows not only the evaluation of the extension of oxidative stress inflicted upon lens membranes but also the evaluation of the antioxidant effect of various compounds including some drugs with a potential anticataractogenic effect.

Bendazac decreases in vitro glycation of human lens crystallins. Decrease of in vitro protein glycation by bendazac.

Bendazac has been used as an anti-cataractogenic drug. It has been reported that this acts by preventing protein denaturation. In this study the ability of bendazac to inhibit in vitro glycation of human lens crystallins was evaluated. Possible effects of bendazac were detected by incubation of WS crystallins with the reducing sugars glucose and fructose. The efficiency of bendazac was evaluated by means of selected parameters including: browning, glycation (measured as tyrosine content) and specific NTP-fluorescence. The results showed clearly that bendazac (bendazac L-lysine and sodium) inhibits the early stages of protein glycation, as well as the formation of fluorescent advanced glycation products. Bendazac lysine (20 mM) proved to be more effective in inhibiting fluorescence development (67% inhibition) that the corresponding sodium salt (35% inhibition). No significant differences were found with respect to furosine levels; about 40% inhibition was produced with either bendazac lysine or sodium salt bendazac clearly inhibits glycation of human lens crystallins, as can be efficiently monitored by following specific changes in lens protein fluorescence. These results may constitute a new and relevant therapeutic approach to monitoring cataract development.

Protein glycation and in vivo distribution of human lens fluorescence.

Glycated proteins formed by the Maillard reaction were measured by furosine determination in human normal lenses and in senile and diabetic cataracts. Furosine, an hydrolysis product of fructose-lysine adduct formed in the early stages of the Maillard reaction, was measured by high performance liquid chromatography (HPLC). Furosine levels in diabetic cataracts were found to be 3 to 4 times higher than those observed for senile cataracts. The increased glycation levels both in cortex and nucleus were related to the increase of fluorescence determined in vitro by fluorometry and in vivo by Scheimpflug photography. Lens proteins were incubated with glucose and it has been demonstrated that protein glycation occurred parallel with the increase in concentration of fluorescent chromophores that present similar characteristics as those observed in vivo. The results indicate that protein insolubilization seemed to involve preferentially glycated proteins and at least in diabetic cataracts, the process seems to be initiated in the cortical region.

Age-related changes in normal and cataractous human lens crystallins, separated by fast-performance liquid chromatography.

The water-soluble crystallins from normal human lenses (n = 32), cataractous lenses of diabetic patients (n = 9) and cataractous lenses of nondiabetic patients (n = 9) were analyzed with fast-performance liquid chromatography. Six different crystallin classes were separated reproducibly by chromatography on Superose 6. The fractions were identified as alpha H, alpha L, beta H, beta L1, beta L2 and low-molecular-weight (LMW) crystallins by their elution order and molecular mass. The results obtained show that during lens aging there is a progressive increase in alpha H-crystallin and a decrease in alpha L-crystallin content, while no significant age-related changes were observed in the LMW fraction. Analysis of changes in crystallin content in human cataractous lenses showed that apparently cataractogenesis can be described as an acceleration of the normal aging process. Important differences were found between the chromatographic profiles of cataracts from diabetic and nondiabetic patients mainly in the LMW fraction, suggesting that in cataract formation of diabetics alternative mechanisms may be superimposed on the normal aging process.

Monitoring in vivo lens changes. A comparative study with biochemical analysis of protein aggregation.

In this study the AA attempted to evaluate the relationship between lens optical density and lens fluorescence determined in vivo, with some specific (in vitro) biochemical changes occurring during cataract development. Special attention has been given to the comparison between diabetic and non diabetic cataracts. Prior to surgery all lenses were analysed by Scheimpflug photography to evaluate the topography of opacities and fluorescence distribution. Individual lenses were separated into cortex and nucleus and the amount of high molecular weight (HMW) protein aggregates was determined by FPLC (Fast Performance Liquid Chromatography). The results found in this study have shown that, as it would be expected, diabetic cataractous lenses present higher fluorescence levels than senile cataracts. It has also been shown that the increase in lens optical density, determined by Scheimpflug photography is clearly related to the increase in the amount of HMW-aggregates. Furthermore, in diabetic cataracts, a good correlation between protein aggregation and lens fluorescence determined in vivo has been found. Thus, it seems that in diabetic cataracts chemical or metabolic mechanisms leading to the production of fluorescent chromophores may be related to protein aggregation and therefore to the major processes involved in cataract development.