Micro-quantitation of lens myo-inositol by anion exchange chromatography.

A method capable of the determination of pmole quantities of myo-inositol in mgr amounts of tissue by anion exchange chromatography is detailed for use in lens and potentially other tissues. Samples were rendered protein-free through ZnSO4/Ba(OH)2 precipitation, lyophilized and reconstituted in water just prior to analysis. An aliquot of sample was injected onto an anion exchange column and eluted with a 0.045 M sodium hydroxide mobile phase. Each analysis requires 30 minutes to complete. Average recovery of myoinositol added to lens sample prior to injection was 100.6%. The coefficient of variation for repeated sample injections was 2.9%. Rabbit lens averaged 11.4 mumol/gr wet weight with epithelium containing 8.5 mumol/gr wet weight while human lens contained 20.1 mumol/gr wet weight and human epithelial cells had 17.5 nmol/mgr protein.

Glutathione levels in human lens: regional distribution in different forms of cataract.

The content of glutathione is high in the anterior lens cortex (plus epithelium) and the posterior lens cortex, whereas it is substantially lower in the lens nucleus. A decrease of glutathione in the lens cortex does not occur with lenses from adults up to the highest age. The glutathione content is highest in the cortex of clear fresh lenses and shows a decrease with deep supranuclear cataract, primary nuclear cataract (cataracta brunescens nigra) and clear lenses post-mortem (approximately 20 hr). The subcapsular cataract, especially with additional secondary nuclear cataract, with cataracta matura or intumescens, shows a rapid and pronounced progressive decrease in the glutathione content.

Distribution of phospholipid-malondialdehyde-adduct in the human lens.

The distribution of phospholipid-malondialdehyde adduct was measured in human lenses pooled by age over a range of 13 to 68 years. Lipid extracts from sections of the central core and from the equatorial region of the lens were probed for lipid adduct using laser induced fluorescence. For an excitation of 351.1/363.8 nm the fluorescence maximum occurred at 473 nm for all ages and regions of the lens. The adduct concentration was always highest in the anterior nuclear section of the central core and decreased toward the anterior and posterior surfaces. The adduct concentration was lowest in the equatorial section for all samples. The regional distribution observed suggests that the formation of lipid-malondialdehyde adduct in the lens could be influenced by the cumulative amount of UV radiation absorbed. No changes with age were observed in the concentration of the adduct in the whole lens nor in the cortical and nuclear regions suggesting the possibility of an equilibrium between the formation and degradation of the adduct.

Distribution of two metabolically related fluorophors in human lens measured by laser microprobe.

Automated Raman microprobe spectrometry revealed the distribution of a major fluorophor, 3-OH-L-kynurenine-O-beta-glucoside, in human lenses from 0.38 to 71 yr. A three-dimensional perspective grid map with fluorescence intensity as the third dimension shows maximum fluorescence in the infant lens nucleus. At 12 yr the fluorescence peak is broadened and a toroid-shaped maximum occurs also in the outer cortex, creating a toroid-shaped minimum between the two maxima. By 71 yr the nuclear maximum is lower but a new (green) fluorophor (excitation 488 nm: emission 530 nm) has appeared as a toroidal maximum in the same location as the blue minimum, suggesting the conversion of the blue fluorophor to the unidentified green fluorophor.

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...

Local variation in absolute water content of human and rabbit eye lenses measured by Raman microspectroscopy.

Raman spectra were obtained from fresh, fixed and sliced rabbit lenses and from human lens slices. For all lenses and lens slices the ratio R, defined as the Raman intensity at 3390 cm-1 divided by the Raman intensity at 2935 cm-1, was measured at different locations along the visual and equatorial axis. The ratios R were transformed to absolute water mass percentages by measuring solutions with known protein concentrations. It was shown that fixation and slicing have very little effect on the absolute water content of the lenses. The values obtained for the absolute water content are comparable to values given in literature. It was also shown that the water content in rabbit and human lenses rapidly decreases from the immediate anterior and posterior subsurface region to the deep superficial cortex and is relatively constant in the nucleus. Raman microspectroscopy appears to be a reliable method for the measurement of the absolute water content of small volumes on defined positions in the lens. This can be very useful when analyzing the possible relation between local variations in water content and the occurrence of opacities in the lens.

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.

Age-related changes in a fiber cell-specific extrinsic membrane protein.

Western blot analysis using a monoclonal antibody raised against a lens fiber cell-specific, extrinsic membrane protein reveals several immunologically related bands in fractions derived from bovine lens. Previous work suggests that the parent molecule is the Mr 115 species, and that lower molecular weight bands represent the products of a progressive, step-wise, post-translational degradation. In this report we compare the extent of proteolytic degradation in extracts prepared from the lens cortex and lens nucleus, using both protease-suppressive and protease-permissive isolation protocols. The results suggest that the observed degradation is a result of in vivo post-translational modification of the Mr 115 antigen, and thus represents physiologic aging of this protein. This analysis also suggests that degradation alters the solubility and/or membrane affinity of this antigen, resulting in a progressive shift to the insoluble phase.

Age-related variations in the distribution of crystallins within the bovine lens.

The native water-soluble proteins of equator, anterior cortex, posterior cortex and nucleus from bovine lenses in the age range 0.3-33.7 years were analyzed by high-pressure gel-permeation chromatography and high-pressure ion-exchange chromatography. Unlike the equator and cortices, the nucleus shows a gradual decrease in alpha-crystallin proportion with age which is not compensated for by an increase in HM-crystallin. The beta 6H-crystallin species, almost the only beta H-component in the youngest lens, is largely replaced by at least four fractions with higher and lower molecular weights in the older lenses. In the nucleus a beta L-component (39,000 MW) increasingly seems to replace the major beta L-crystallin (beta 2L, 50,000 MW). Moreover, a switch in the synthesis of monomeric crystallins is demonstrated. This study clearly reveals an age-related increase in the size heterogeneity of the native soluble crystallins with age.

Membrane interlocking domains in the lens.

"Ball and socket"-like membrane processes interlock fiber cells in the sheep lens cortex, but appear reduced deeper in the lens. Wheat germ agglutinin (WGA) binds preferentially to these ball and socket structures, and more weakly to other membrane regions. On protein blots, 125I WGA binds to glycoproteins with 140,000 and 32,000 apparent molecular weight, the smaller protein also binding 125I fibronectin. In two animal cataract models, the intense WGA labeling of globular bodies replaces the spotty WGA staining pattern associated with the ball and sockets in the normal lens.

Distribution of the total and non-freezable water in rat lenses.

Twenty Sprague-Dawley rats of ages from 7 days to 29 weeks were studied. The cortical and nuclear samples from rat lenses were investigated by differential scanning calorimetry (freezable water content) and thermogravimetric analysis (total water content). Both cortical and nuclear regions showed a decrease in total water content with aging. Only the nuclear parts of the rat lenses showed a statistically significant increase in the non-freezable portion of the total water with aging. The cortical parts showed a slight but statistically non-significant increase in this parameter. It is concluded that in rat lenses aging is primarily an aggregation process and syneresis does not play a significant role.

Spatial and temporal mapping of the age-related changes in human lens crystallins.

Using the techniques of high performance liquid chromatography (HPLC), gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), and immunoblotting, we have analyzed the age-related changes in soluble crystallins of the human lens. A 3 mm core along the optical axis of each lens was frozen-sectioned and the sections were biochemically analyzed for distribution and quantity of the various soluble protein species. Both cortical and nuclear samples show a monotonic decrease in the concentration of the 19 000 and 21 000 MW proteins with age. We find that these proteins behave anomalously on SDS-polyacrylamide gels, running near the top of the gel when the samples are not boiled before loading; this permitted us to observe the gradual, age-related loss of these bands from the gels of both nuclear and cortical samples. The high molecular weight, or TSK-3000 void volume, fraction (greater than 350,000) of the cortex contained alpha crystallin at all ages. However, in the nucleus, while this fraction is primarily composed of alpha crystallin early in life (i.e. before 15 years of age), there is a gradual incorporation of other crystallins into the void volume. This change in the composition of the high-molecular-weight, soluble protein fraction is reflected in: a change in the subunit mobility on SDS-polyacrylamide gels; reactivity of the fraction to crystallin antibodies, i.e. in the young nucleus there is reactivity to anti-alpha crystallin only, with a gradual increase in reactivity to anti-beta and anti-gamma crystallins. The void volume fraction of the nucleus persists as a major component of the soluble protein pool until 42-44 years of age, at which time the proportion of the total soluble protein represented by this void volume fraction decreases precipitously. These changes in the soluble protein profile are discussed in terms of their potential influence on the functioning of the lens.

The influence of isolation conditions on the molecular weight of bovine alpha-crystallin.

The molecular weight of bovine alpha-crystallin, isolated at 37 degrees C, was studied and found to be about 800,000. This contrasts with the results of Thomson and Augusteyn (Thomson, J. A., and Augusteyn, R. C. (1983) Exp. Eye Res. 37, 367-377) who isolated a species of about half this molecular weight. We show here that this form of alpha-crystallin can only be isolated under unphysiological conditions with regard to buffer pH and ionic strength.

The photolysis of lens fiber membranes.

Calf lens fiber membranes were photolyzed in the presence and absence of sensitizers and scavengers. Photolytic damage was assessed by SDS-polyacrylamide gel electrophoresis, UV and fluorescence spectra and amino acid analyses. With irradiation, there is an apparent polymerization of the major membrane polypeptide (MP26) and the formation of material which does not enter SDS-polyacrylamide gels. Some degradation was also observed. These changes are accompanied by losses of histidine and tryptophan and changes in the UV spectra. The rate of photolysis is enhanced in the presence of the glucoside of 3-hydroxykynurenine (3-OH-KYN), a compound endogenous to the lens. The reaction is retarded in the presence of sulfhydryl-containing compounds such as glutathione.

Membrane cholesterol and phospholipid in consecutive concentric sections of human lenses.

Lens membrane preparations have been shown to have a remarkable rigidity which increases in the inner nuclear region of the lens and has been correlated with the cholesterol (C)/phospholipid (PL) ratio. However, the distribution of these lipids in single lenses had not been determined. Utilizing a new technique for isolating consecutive layers of a human lens, lipid composition and contents of seven pairs of normal lenses from subjects ranging from 54 to 77 years old have been analyzed. It was found that the PL content remains relatively constant at 22-24 micrograms/mg through all but the nuclear 10-15% of the lens dry weight where it drops precipitously to about 7 micrograms/mg. The C distribution is more complex; the C content is at a low level of 14 micrograms/mg in the outer cortical 15-20%, rises to 25 micrograms/mg in the inner cortical 40-60% of the total lens weight, and drops to 12 micrograms/mg upon reaching the nucleus. Thus, the continuous increase in the lens C/PL ratio is due to the increase in C in the cortex and the large decrease in PL in the nucleus. Analyses of phospholipid and fatty acid composition in the different regions of the lens indicate significant differences. However, the abundance of mono-unsaturated fatty acids contributing to the rigidity of the membrane has only minor variation. The lens has a remarkably low overall lipid content of 4% and only 2% in the nuclear region. Calculation of the surface area of the nuclear fiber cell suggests that less than one-third of the membrane is made of PL bilayer. Thus, a mosaic of PL and C patches or some other type of structure involving membrane fusion must be present. Conversion of the % dry weight occupied by the concentric fiber fractions to their location on the lens axis in mm indicates that the nuclear 15% dry weight of the tissue occupies more than 50% of the axial length. This region contains the embryonic lens and the primary lens fibers. Similarly, the metabolically active outer 20% of the dry weight accounts for less than 10% of the visual axial length and contains cells undergoing terminal differentiation. Cataractous lenses have lipid distributions similar to those of the normal lenses suggesting that membrane lipid is either not involved in cataract formation or that the primary insult is localized in an undetectable small number of fiber cells.

A quantitative microprobe analysis of elements in cortical and nuclear cells of the calf lens.

The outer cortical cells in the calf lens remain transparent under conditions that produce opacity in central nuclear cells. The nuclear cells opacify by a mechanism of cellular restructuring that is associated with a cytoplasmic phase separation while cortical cells do not opacify by this mechanism. In this study the differences in elemental composition of nuclear and cortical cells were analyzed using X-ray emission spectroscopy (XES) of tissue that was prepared for scanning electron microscopy. It was necessary to develop special methods of fixation and dehydration to prevent significant distortion of lens tissue and minimize solubilization and redistribution of elements during the histological processing of the tissue. We calibrated the microprobe for the quantitative analysis using gelatin standards which contained known concentrations of sulfur, potassium, phosphorus, chlorine, and cesium. The standard curves were used to determine proportionality constants, which related the intensity of X-ray emission to the molar concentration of each element, and to determine the minimum detectable levels of each element. An important finding is that the intensity of the X-ray emission is dependent on sample density only at low protein concentration. At the high protein concentrations that exist in lens, the intensity is not affected by sample density.(ABSTRACT TRUNCATED AT 250 WORDS)

Opacification of gamma-crystallin solutions from calf lens in relation to cold cataract formation.

To determine the molecular mechanisms for cold cataract formation in the nucleus of the young mammalian lens, we have investigated the thermally reversible opacification of gamma-crystallin solutions isolated from calf lens. Coexistence curves (plots of opacification temperature Tc versus protein concentration) were determined for the individual gamma-crystallin fractions II, III, and IV as well as for the unfractionated gamma-crystallin mixtures isolated from the nucleus and cortex. The coexistence curve of gamma IV-crystallin is remarkably elevated above those of gamma II- and gamma III-crystallin and the gamma-crystallin mixtures. The gamma IV-crystallin fraction is the major determinant of the opacification temperature within the whole lens or isolated cytoplasm. Quasielastic light-scattering spectroscopy of gamma IV-crystallin solutions indicates that above Tc there are two populations of protein aggregates of distinctly different mean size. As the temperature is lowered towards Tc, both populations increase in size. Opacification occurs when the population of large scatterers, which is composed of less than 0.1% protein by weight, reaches an average radius of about 20,000 A.

[Comparative study of crystallins from the nucleus and cortex of the bovine ocular lens by the gel filtration and x-ray diffraction methods]. / Sravnitel'noe issledovanie kristallinov iadra i korteksa glaza byka metodami gel'-filtratsii i difraktsii rentgenovskikh luchei.

Water--soluble proteins (alpha-, beta H-, beta L- and gamma-crystallins) from the bovine lens nucleus and cortex were fractionated and compared by gel filtration on Sephadex G-200. X-ray diffraction patterns from concentrated gels of these proteins were obtained. It allowed to compare qualitatively the structures of different crystallins and also to identify the maxima on X-ray diffraction patterns of the lens intact tissue.

Electron microscopic study of water-insoluble fractions in normal and cataractous human lens fibers.

Water-insoluble fractions of fiber cells from human transparent normal and opacified single lenses were compared ultrastructurally and by gel electrophoresis. Intermediate-sized filaments which had been clearly shown in aged transparent normal cortices, virtually vanished in the opacified nuclei in contrast to microfilaments.