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Elucidation of intermediate (mobile) and slow (solidlike) protein motions in bovine lens homogenates by carbon-13 NMR spectroscopy.

The motional dynamics of lens cytoplasmic proteins present in calf lens homogenates were investigated by two 13C nuclear magnetic resonance (NMR) techniques sensitive to molecular motion to further define the organizational differences between the cortex and nucleus. For the study of intermediate (mobile) protein rotational reorientation motion time scales [rotational correlation time (tau 0) range of 1-500 ns], we employed 13C off-resonance rotating frame spin-lattice relaxation, whereas for the study of slow (solidlike) motions (tau 0 greater than or equal to 10 microseconds) we used the solid-state NMR techniques of dipolar decoupling and cross-polarization. The frequency dependence of the peptide bond carbonyl off-resonance rotating frame spectral intensity ratio of the lens proteins present in native calf nuclear homogenate (42% protein) at 35 degrees C indicates the presence of a polydisperse mobile protein fraction with a tau 0,eff (mean) value of 57 ns. This mean value is consistent with the average value calculated from the known water-soluble nuclear lens protein polydispersity assuming a cytoplasmic viscosity 3 times that of pure water. Lowering the temperature to 1 degree C, a temperature which produces the cold cataract, results in an overall decrease in tau 0,eff to 43 ns, suggesting a selective removal of beta H-, LM-, and possibly gamma s-crystallins from the mobile lens protein population. The presence of solidlike or motionally restricted protein species was established by dipolar decoupling and cross-polarization. The fraction of motionally restricted protein in the nuclear region varied from 0.35 to 0.45 in the temperature range of 35-1 degree C. For native cortical homogenate (25% protein), the off-resonances rotating frame spectral intensity ratio frequency-dependent curves for the protein carbonyl resonance yielded tau 0,eff values of 34 and 80 ns at 35 and 1 degree C, respectively. Both values were reconciled with the known lens cortex soluble protein polydispersity using an assumed cytoplasmic viscosity 1.5 times that of pure water at the same temperature. Comparison of proton dipolar-decoupled and nondecoupled 13C NMR spectra of native cortical homogenate at 20 degrees C indicates the absence of significant contributions from slowly tumbling, motionally restricted species. This interpretation was confirmed by the failure to detect significant lens protein 13C-1H cross-polarization at this temperature. However, at 1 degree C, the fraction of solidlike protein was 0.15. Concentrated cortical homogenates at 20 degrees C (42% protein), by contrast, gave cross-polarization spectra with maximum absolute signal intensities 50-70% of native nuclear homogenates, but with similar magnetization parameters...

Animals

Relative abundance of aldose reductase mRNA in rat lens undergoing development of osmotic cataracts.

Aldose reductase (AR) messenger RNA concentration was determined in normal rat lens and in lens from rats fed a 50% galactose diet over a period of 20 days. The AR mRNA was detected by using a previously described AR cDNA clone. The relative concentration of the AR mRNA was estimated by cpm of 35S-UTP labeled antisense RNA hybridized to dot-blots prepared from cytosols isolated from single lens, decapsulated lens (cortex) and its respective capsule (epithelia). The results demonstrated that the concentration of the AR mRNA in the epithelium doubled over the 20 day period. Correspondingly, an increase in the concentration of the DNA was also observed, suggesting that the increase in epithelial cytosolic mRNA might be partially due to the increase in the number of epithelial cells occurring in lens undergoing cataractogenesis. The increase in AR mRNA in the epithelia was gradual, and it doubled by day 12 on galactose, while the increase in DNA was rapid and reached an optimum level by about day 4. By day 4 the cortex AR mRNA concentration increased, then rapidly decreased to insignificant levels by day 20. Changes in AR mRNA and in DNA following a high influx of galactose in the lens might suggest a heightened gene response to changes in the cellular environment for the lens epithelium.

Aldehyde Reductase

Bendazac lysine in selected types of human senile cataract. A long-term double-masked placebo-controlled clinical trial with multilinear densitometric image analysis of Scheimpflug photographs.

A double-masked placebo-controlled clinical trial with hard data evaluation by image analysis of Scheimpflug photographs taken at baseline and 6, 12 and 18 months after starting treatment was performed to assess the efficacy of bendazac lysine in four different types of senile cataract. The study had a classical split-plot design. For statistical evaluation, the analysis of variance and covariance for repeated measures were used for three different lens sections: anterior capsule and superficial layer, anterior cortex and nucleus. In the entire group of 53 evaluable patients (without separation into cataract-type subgroups), there was a significantly less increase over time in light scattering (i.e. film blackening) of the anterior cortex and nucleus with bendazac lysine than with placebo. There was also a strong trend in favour of the active drug at the anterior capsular level. Patients with water clefts and spokes showed a significantly less light scattering of the anterior capsule and cortex when treated with bendazac lysine. Those with nuclear changes also showed significantly less light scattering of the anterior cortex and nuclear region with the active drug than with placebo. The number of patients with subcapsular and wedge-shaped (cuneiform) cataracts was too small to be adequately assessed by statistical procedures. Nevertheless, there were indications of a beneficial effect of bendazac lysine on all the lens sections in patients with subcapsular cataracts and on the anterior cortical region in those with wedge-shaped cataracts. In conclusion, this study showed that the increase in light scattering over time, i.e. the progression of cataract, is less in bendazac lysine-treated patients than in those treated with placebo.

Adult

Lesion and regeneration of the anterior and posterior lens capsule and cortex in rabbits Nd:YAG laser.

With a mode-locked Nd:YAG laser set at various energy levels, anterior and posterior capsular and cortical lesions were produced in the lens of rabbits. Biomicroscopic and Scheimpflug examinations showed the anterior lesion to be healing in a whitish scar that was restricted to the area of the original lesion. Histology and transmission electron microscopy (TEM) demonstrated the proliferation of epithelial cells that produced a new, continuous, basement membrane-like capsule. In the area of the lesion collagenous material, myofibroblast-like cells and macrophages were found. Laser-induced lesions in the posterior lens capsule and lens protein resulted in damage to the capsule and grey opacity of the lenticular proteins, which remained unchanged for 6 weeks. Neither inflammatory nor epithelial cells appeared within the locally damaged lens. Macrophages migrated from the vitreous into the lesion; they were observed either in intercellular clefts or within a lens fibre, almost completely taking up their cross section.

Animals

Excimer laser ablation of the human lens at 308 nm with a fiber delivery system.

A 308 nm excimer laser has been used with a fiber delivery system to perform ablation of the human lens. Preliminary results demonstrate the system's ability to ablate lens nucleus and cortex selectively, preserving the anterior and posterior capsules. The total delivered energy necessary to ablate a human lens ranged from 35 to 63 joules. Laser tissue interaction and ablation rates were computed for the different components of the human lens (capsule, cortex, nucleus) for the operatively useful energy densities (fluences). Operative experience suggests that cortex and nucleus can be ablated while preserving the capsule if an adequate irrigation and aspiration system is developed. These results also suggest that this modality may be adequate for performing endocapsular cataract extraction. Laser tissue interactions were also computed at variable distances between the fiber tip and tissue. As this distance increased, the spread of the beam increased and a significant increase in energy was necessary to induce tissue ablation. This was due to the decrease in fluence with increasing distance to the target tissue and/or the absorption and scattering of the delivered energy within a short distance from the fiber tip by the ablated material. Evidence of a sonic effect was also present.

Aged

Cigarette smoking and risk of nuclear cataracts.

The current study was undertaken to assess the relationship of smoking to the risk of lens opacities. The risk was evaluated separately for nuclear and cortical opacities and for both types together. We studied 838 watermen in Maryland by detailed ocular examination for the presence and severity of each cataract type. All subjects were interviewed regarding smoking history, and a cumulative smoking dose was calculated. The results suggest a significantly increased risk of pure nuclear opacities associated with cigarette smoking. The increase in smoking dose was associated with increasing severity of nuclear opacity. The risk of nuclear opacities increased with increasing cigarette dose and decreased if the subject had quit smoking. The effect of smoking was most striking in those less than 80 years old. No increased risk of nuclear opacities was observed with earlier age when smoking started, after adjusting for dose and cessation of smoking. Further investigations are warranted on the biochemical and physical damage to the nucleus of the lens from smoking cigarettes.

Adult

The effect of early cataracts on glare and contrast sensitivity. A pilot study.

To establish the effect of cataracts on glare and contrast sensitivity, we graded type and amount of lens opacity in 110 subjects who underwent two glare tests (Brightness Acuity Tester and Berkeley glare test) and two contrast sensitivity tests (a sine-wave test and Pelli-Robson chart). Twenty-seven subjects (25%) had clear lenses (mean visual acuity of 20/20) and 83 subjects (75%) had early lens opacities (mean visual acuity of 20/40) in otherwise normal eyes. Multiple regression techniques were used to control for the effects of age and visual acuity. Glare test scores were significantly lower for nearly all patients with lens opacities than for patients with clear lenses and were the lowest for patients with lenses with posterior subcapsular opacity. Contrast sensitivity scores were lower for all patients with lens opacities than for patients with clear lenses at high frequencies only; all lens opacity groups scores similarly with each other. These results indicate reduced visual function among patients with cataracts whose visual acuity is only minimally impaired.

Cataract

Continuous-tear capsulotomy and insertion of a silicone bag lens.

Opacification of the posterior capsule and lack of intraocular lens fixation have been recurring problems and have resulted in a gradual shift from sulcus-fixated silicone lenses to bag-fixated silicone lenses. Postoperative capsular contraction, however, can cause capsular tearing in the periphery and intraocular lens decentration. A new method of combining a small circular capsulotomy, cataract removal without anterior capsular rim tearing, and meticulous bag placement of the silicone lens seems to allow centering and secure fixation of the silicone lens.

Cataract Extraction

Objective measurement of cortical and subcapsular opacification in retroillumination photographs.

An image analysis system has been developed for the objective analysis of cortical opacification. Lens photographs are obtained using a Neitz CTR retroillumination camera. The images are digitized using an IBM-PC-based imaging system. We have developed a program, called OPAC, through which the computer analyzes the image using a circular grid consisting of 93 sectors. The program outputs the percent of lens area which is opaque. Ninety-eight photographs, previously classified by the Lens Opacities Classification System (LOCS II), were analyzed using OPAC as well as a manual, square-grid counting system (GRID). The results show a linear correlation between OPAC and GRID with a correlation coefficient of 0.94. The results obtained with OPAC are also linearly correlated with the LOCS II classifications.

Cataract

Subjective (LOCS II) versus objective (BGS) measures of cortical and subcapsular cataracts in retroillumination photographs.

96 black-and-white Neitz retroillumination photographs were graded subjectively using the Lens Opacities Classification System Version II (LOCS II). They were also measured objectively using the background subtraction (BGS) analysis. Subjective and objective BGS gradings were compared and showed good agreement (kappa = 0.78 for cortical and 0.78 for posterior subcapsular cataracts) and good correlation (correlation coefficient = 0.82 for cortical and 0.84 for posterior subcapsular cataracts; Spearman rank analysis).

Cataract

[Histological characteristics of ocular structures after YAG-laser exposure of various sections of the crystalline lens in experimental studies].

This work was aimed at analysis of all the aspects, pathomorphologic included, of short-pulsed YAG laser exposure of the lens. Experiments were carried out with 79 rabbit eyes. After laser exposure the eyes were enucleated in 1-24 h and in 7-10 days. Thirty-one eyes were subjected to YAG laser ++capsule puncture. Fragmentation of intracapsular structures was carried out in 23 eyes. Combined operations were performed on 25 eyes. Every series comprised two groups, differing by the energy of laser exposure: 3-5 mJ. Histologic analysis has lead the authors to the following conclusions. The pattern and severity of pathomorphologic changes were directly related to the energy and time of laser exposure and site of the focal plane of irradiation. The major destructive shifts after YAG laser fragmentation of the cortical layer and capsulotomy are focussed at the site of exposure and involve the adjacent sections of the lens. Laser exposure of 3-5 mJ may be used to open the anterior capsule of the lens and to facilitate the cataract mass discharge in extracapsular cataract extraction. Use of combined method helps enhance induration and fragmentation of the cortical layers of the lens, rules out surgical discission of the anterior capsule of the lens, and facilitates removal of the nucleus and wash out of the lens mass.

Aluminum

Risk factors for age-related cortical, nuclear, and posterior subcapsular cataracts. The Italian-American Cataract Study Group.

A total of 1,008 cases and 469 controls, aged 45-79 years, were included in a clinic-based case-control study of age-related cataract in Parma, Italy, from 1987 to 1989. Associations were examined between specific types of cataract and physiologic, behavioral, environmental, and biochemical factors. Cases included subjects with pure cortical (n = 489), pure nuclear (n = 110), pure posterior subcapsular (n = 28), and mixed (n = 381) cataracts. With polychotomous logistic regression, an increased risk of cataract was found for females (cortical; odds ratio (OR) = 2.20) and persons with less than a high school education (all types; OR = 1.53), brown irises (nuclear, mixed; OR = 1.43), job locations in the sunlight (cortical, mixed; OR = 1.75), leisure time activities in the sunlight (cortical, mixed; OR = 1.45), a history of wearing a hat in summertime (posterior subcapsular, cortical, mixed; OR = 1.80), a positive family history of cataract (posterior subcapsular, cortical, mixed; OR = 1.88), a history of cortisone use (posterior subcapsular; OR = 8.39), increased red blood cell glucose-6-phosphate dehydrogenase activity (cortical, nuclear, mixed; OR = 1.36), and increased serum levels of uric acid (posterior subcapsular; OR = 1.62), lactic dehydrogenase (posterior subcapsular; OR = 1.76) and gamma-glutamyl transpeptidase (mixed; OR = 1.22). A decreased risk of cataract was found for persons with a positive history of arthritis (posterior subcapsular, nuclear, mixed; OR = 0.56) and increased handgrip strength (mixed; OR = 0.68). Findings from our study, combined with previously reported evidence, suggest that associations with educational status, cortisone use, sunlight exposure, and handgrip strength are real. Other findings require further evaluation.

Adult

[Absence of small-angle maximums on the x-ray images of ocular lens tissue].

X-ray small-angle scattering study of bovine lens tissue was carried out. It was shown that X-ray patterns of lens cortical and nuclear native tissues did not contain the small-angle maxima. The maximum in the range of 15-20 nm Bragg distance appeared as a result of the lens tissue partial dehydration. Earlier such maximum was considered by some authors as the evidence of crystallin proteins short-range order in the native lens. Now it is confirmed to be a preparative artefact of dehydration. It was shown also that similar maximum in 15-20 range existed in the case of concentrated crystallin solutions. This indicates that supramolecular organization of crystallins in the native lens is not similar to that in the concentrated solution.

Animals

Ageing and changes in protein conformation in the human lens: a Raman microspectroscopic study.

Using confocal Raman microspectroscopy with laser light of 660 nm the secondary and tertiary conformation of crystallins was studied in human lenses of varying age (20-75 years). Differences in cortical and nuclear proteins in individual lenses and among lenses of different age and differences between small equatorial opacities and adjacent clear sites were analysed using a difference spectrum approach. Intensity calibration allows assessment of local variations in protein content. The main findings and conclusions are as follows. (1) Irrespective of the age of the lens nuclear proteins proved to contain more aromatic amino acids, i.e. tryptophan, tyrosine and phenylalanine. This change most probably reflects differences in crystallin composition between nucleus and cortex as described in biochemical studies. (2) Changes in the amide bands indicate a more pronounced beta-sheet conformation of the nuclear proteins. Taking into account available biochemical evidence this most probably reflects a true post-translational change in the secondary conformation of the crystallins. (3) Cortical proteins in 'old' and 'young' lenses are largely identical indicating that ageing is not accompanied by gross alterations in the transcription/translation of the crystallin genes. (4) 'Old' and 'young' nuclear proteins deviate with respect to the amount of aromatic amino acids, being more abundant in 'young' nuclear proteins. (5) Proteins in small early opacities do not exhibit alterations in conformation. (6) A pronounced peak in the difference spectra in the region 1425-1435 cm-1 for nuclear proteins especially when compared with equatorial cortical proteins may be considered as evidence for the advanced photooxidation of tryptophan and/or deamidation of asparagine from superficial to deep regions of the lens.

Adult

Quantitation of high molecular weight protein aggregates in opaque and transparent parts from the same human cataractous lens.

Water soluble proteins from clear and opaque parts of the cortex of the same human cataractous lens were analyzed by high pressure liquid chromatography using a TSK 3000 SW gel filtration column. Studies of the 42 different microdissected parts from 7 different lenses demonstrated that the opaque parts possessed a higher percentage of high molecular weight material eluting with the void volume than did the corresponding clear part from the same lens. These results suggest that increased amounts of high molecular weight protein aggregates accompany the process of opacification in the cortex of the human cataractous lens.

Aged

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.

Aged