Search

The small heat shock protein superfamily, extending over all kingdoms, is characterized by a common core domain with variable N- and C-terminal extensions. The relatively hydrophobic N-terminus plays a critical role in promoting and controlling high-order aggregation, accounting for the high degree of structural variability within the superfamily. The effects of N-terminal volume on aggregation were studied using chimeric and truncated proteins. Proteins lacking the N-terminal region did not aggregate above the tetramers, whereas larger N-termini resulted in large aggregates, consistent with the N-termini packing inside the aggregates. Variation in an extended internal loop differentiates typical prokaryotic and plant superfamily members from their animal counterparts; this implies different geometry in the dimeric building block of high-order aggregates.

Primary open-angle glaucoma is a disease caused by an increase in intraocular pressure due to a decreased facility of outflow of aqueous humor through the trabecular meshwork. The etiology of primary open-angle glaucoma is currently unknown, but it has been suggested that one possible mechanism may be the obstruction of flow through the trabecular meshwork by small macromolecules, analogous to the effect of ‘fines’ in column chromatography. One such candidate is α-crystallin, a lens protein which may be released into the aqueous humor from lens fiber cells at concentrations below that necessary for the formation of the native aggregate. Results of in vitro binding experiments indicate that α-crystallin and serum albumin, which is secreted at the anterior root of the iris and is believed to act as a protein escort through the trabecular meshwork in mammalian eyes, will interact at concentrations of α-crystallin up to the critical micelle concentration for α-crystallin (3.5–5 mg/ml, or 0.18–0.25 mM). There is little interaction at or above this concentration. This binding could serve the necessary function of preventing interactions between α-crystallin monomers or small aggregates and hydrophobic surfaces within the trabecular meshwork. Since, however, the interaction between the two proteins is not extremely strong, the accumulation of unbound α-crystallin monomers and/or dimers could contribute to the development of primary open-angle glaucoma.

The mechanism by which alpha-crystallin subunits form the native 800 kD aggregate is currently unknown. Experiments were performed to investigate the mechanism for this process. Gel-filtration Fast Performance Liquid Chromatography (FPLC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), with and without cross-linking with glutaraldehyde, indicate that alpha-crystallin undergoes a concentration-dependent aggregation process. The denaturation of alpha-crystallin, and its subsequent renaturation and reaggregation, lead to the formation of several different species. At very low concentrations (0.5 microM), only monomeric and/or dimeric species exist. With a ten-fold increase in alpha-crystallin concentration from 0.05 microM to 0.5 microM, the amount of the monomeric/dimeric species increases to a plateau coincident with the appearance of a tetrameric species at 0.5 microM. With an additional ten-fold increase in concentration from 0.5 microM to 5 microM, the amount of the tetrameric species increases and levels off to its own plateau coincident with the appearance of the native 800 kD alpha-crystallin aggregate at 5 microM. The amount of the native species is extremely small at this concentration, but increases sharply and linearly with increasing concentration, while the concentrations of monomeric/dimeric and tetrameric species remain constant. The concentration at which the relative amount of the native species begins to increase sharply is within the range of the critical micelle concentration previously characterized.

Statistical analysis of Scheimpflug images from the crystalline lenses of 100 emmetropic human subjects ranging in age from 18 to 70 yr confirms that specific zones of discontinuity are a function of lens development and growth. At and beyond the age of 40 yr, as many as four sharply demarcated and complementary zones are seen within the anterior and posterior lens cortex. The locations of the inner edges of the anterior cortical zones of discontinuity were characterized relative to the central sulcus of the lens. Consecutively from the central sulcus, the distances were 1.094, 1.415, 1.695, and 1.994 (± 0.11 mm). Since nuclear thickness in the adult lens is age-independent and the rate of cortical growth has been characterized, the location of the inner margins of the zones are indicative of the age at which they originated; these ages were 4 (± 1 yr), 9 (± 2 yr), 19 (± 4 yr), and 46 (± 10 yr). All of the zones become broader along the outer margin and more dense upon aging, with specific zones appearing to merge in older presbyopic lenses. While lens fetal nuclear transparency decreases with age, it does not feature zones of discontinuity; instead, symmetrically amorphous regions appear centrally in the anterior and posterior nucleus. This demonstration of the onset of specific zones of discontinuity in emmetropic individuals, at defined periods of lens growth that are synchronous the production of successively more complex lens sutures, strongly suggests a causal relationship between lens sutures and the zones of discontinuity.

Changes in the unaccommodated human crystalline lens were characterized as a function of subject age for 100 normal emmetropes over the age range 18-70 yr by Scheimpflug slit-lamp photography. With increasing age, the lens becomes thicker sagittally, but since the distance from the cornea to the posterior lens surface remains unchanged, this indicates that the center of lens mass moves anteriorly and the anterior chamber becomes shallower. Sagittal nuclear thickness is independent of age, but both anterior and posterior cortical thicknesses increase with age, shifting the location of the nucleus and the central sulcus in the anterior direction. The amount of light scattered by the lens at high angles, as represented by normalized and integrated lens densities from the digitized images, increases with increasing age in an exponential fashion. Similar relationships to age are observed for the major anterior zone of discontinuity (maximum density) and the central sulcus (minimum density). The relationships of these results to accommodation and presbyopia are discussed.

In solutions of high ionic strength, native titin-2, a large extractable fragment of the sarcomere matrix protein titin, appears as extremely long, flexible, and slender beaded strings. We report here that in solutions of lower ionic strength near neutral pH, titin-2 assembles into higher-order aggregates with surface projections. Solid phase binding assays show that two myosin-binding proteins, C-protein and AMP-deaminase, are also titin-binding proteins. Both proteins decorate titin aggregates, producing filaments of more uniform appearance. Numerical Fourier transforms of these decorated aggregates show approximately 12-nm periodicities. The interaction of titin with myosin-associated proteins such as C-protein may take part in the anchoring mechanism that prevents the stretching and extension of titin filaments in the A band.

To accurately model lens-based functions such as accommodation and image formation on the retina, it is essential to know anterior chamber depth, anterior segment length, lens thickness, and, most importantly, lens curvature both on the surfaces and internally. With the exception of lens curvatures, all these data can be obtained with a high degree of precision by one or more techniques (i.e., A-scan ultrasonography and pachymetry). Lens curvatures can be collected by Scheimpflug slit lamp photography, but the curvature data must be extracted from these images, a problem complicated by both linear and nonlinear image distortion. Previous approaches have involved significant magnification of the image combined with major subjective input and judgment. We present here a computer-based application of the Hough technique for measurement of curvature of lens surfaces observed in Scheimpflug slit lamp photography and related evaluation of (and solutions for) the associated image distortion. Minimal user input is required for successful application of this method, but the time required to obtain a fit for each surface is >1 min. Results obtained by this technique on test images compare favorably with those obtained by independent methods.

The sequentially variable COOH-terminal region of small heat shock protein superfamily members usually contains a conserved IXI/V feature where X is typically a proline. When present in solved sHsp crystal structures (e.g. MjHsp16.5 and wheat Hsp16.9), this short sequence forms an isolated beta strand apparently involved in the alignment of dimers into larger oligomers. Because it is a common feature of many sHsp family members, it is possible that this triad has a similar role in alphaA-crystallin. This study was undertaken to determine the contribution of this conserved triad to the quaternary structure and function of alphaA-crystallin.A series of site-directed mutants was generated in both wild type alphaA and in an alphaA deletion mutant lacking the NH2-terminal residues 1-50. After overexpression and purification, each protein's oligomer size was characterized by size-exclusion fast protein liquid chromatography (FPLC), thermal transition temperature by non-denaturing composite gel electrophoresis, and chaperone activity by the inhibition of DL-dithiothreitol (DTT)-induced insulin aggregation.Using the alphaA-crystallin NH2-deletion mutant, the hydrophobic triad was changed from IPV to TPT, GPG, IGV, ITV, or GGG. All six D51 mutants associated into tetramers with small amounts of dimer and monomer also present. Chaperone-like activity was reduced but not eliminated in some of these triad mutants with GGG and ITV the most strongly affected. Similar modifications to wild type alphaA-crystallin (IPV to ITV, IGV, or GGG) restored oligomer sizes similar, but not identical to, native alphaA-crystallin, with additional small amounts of tetramer and dimer. Interestingly, equivalent mutants of wild type alphaA-crystallin did not have reduced chaperone-like activity but differed considerably in their thermal transition temperatures.The conserved COOH-terminal triad does not appear to have a strong effect on the steady-state aggregation of wild type alphaA-crystallin or its 50-residue deletion mutant at 25 degrees C. However, it can exert a considerable effect on chaperone-like activity in the absence of the NH2-terminal 50-residue sequence extension and can influence the thermal transition temperature in its presence. These results suggest that the conserved triad in alphaA-crystallin contributes to the stability of higher order oligomers but is not essential for the formation of tetramers.

Scheimpflug photography, high-resolution MRI, and other non-invasive methods have been used to characterize accommodation and presbyopia development in normal adult human eyes. The resultant model provides a framework for accommodating IOL design and testing.

To assess the role of the NH2-terminal in alphaA-crystallin folding and chaperone-like activity.Two NH2-terminal deletion mutants of alphaA-crystallin were generated by standard mutagenesis methods, one with and one without a leader sequence in place of the first 50 residues. Aggregate size of each before and after thermal stress was assessed by FPLC, and chaperone-like activity was assessed using DTT-induced insulin denaturation.Both mutants assemble primarily into tetramers, and both exhibit similar levels of chaperone-like activity, but are less protective than recombinant alphaA-crystallin. After a cycle of heat stress to 70 degrees C, tetramers of the mutant without the leader sequence dissociate into dimers and monomers and show severely reduced chaperone-like activity. In contrast, the mutant with the leader sequence retains its tetrameric form and its chaperone-like activity.The NH2-terminal region is an important determinant of alpha-crystallin aggregate size, but is not required for folding of the alpha-crystallin domain, since the aggregate size and chaperone-like activity of the two mutants at room temperature are essentially the same. The leader sequence appears to increase the thermal stability of the alpha-crystallin domain and/or to contribute to the reformation of the active form after cooling, suggesting that the native NH2-terminal also plays a role in alpha-crystallin's resistance to environmental stress.

Accommodation in humans refers to the ability of the lens to change shape in order to bring near objects into focus. Accommodative loss begins during childhood, with symptomatic presbyopia, or presbyopia that affects one's day to day activities, striking during midlife. While symptomatic presbyopia has traditionally been treated with reading glasses or contact lenses, a number of surgical interventions and devices are being actively developed in an attempt to restore at least some level of accommodation. This is occurring at a time when the underlying cause of presbyopia remains unknown, and even the mechanism of accommodation is occasionally debated. While Helmholtz' theory regarding the mechanism of accommodation is generally accepted with regard to broad issues, additional details continue to emerge. Age-related changes in anterior segment structures associated with accommodation have been documented, often through in vitro and/or rhesus monkey studies. A review of these findings suggests that presbyopia develops very differently in humans compared to non-human primates. Focusing on non-invasive in vivo human imaging technologies, including Scheimpflug photography and high-resolution magnetic resonance imaging (MRI), the data suggest that the human uveal tract acts as a unit in response to age-related increasing lens thickness and strongly implicates lifelong lens growth as the causal factor in the development of presbyopia.

Whole eye lens and alpha-crystallin gels and solutions were investigated using X-ray scattering techniques at temperatures ranging from 20 degrees C to 70 degrees C. In whole lens isolated in phosphate-buffered saline, the spacing of the dominant X-ray reflection seen with low-angle scattering was constant from 20 degrees C to 45 degrees C but increased at 50 degrees C from 15.2 nm to 16.5 nm. At room temperature, the small-angle X-ray diffraction pattern of the intact lens was very similar to the pattern of alpha-crystallin gels at near-physiological concentration (approximately 300 mg/ml), so it is reasonable to assume that the alpha-crystallin pattern dominates the pattern of the intact lens. Our results therefore indicate that in whole lens alpha-crystallin is capable of maintaining its structural properties over a wide range of temperature. This property would be useful in providing protection for other lens proteins super-aggregating. In the alpha-crystallin gels, a moderate increase in both the spacing and intensity of the reflection was observed from 20 degrees C to 45 degrees C, followed by an accelerated increase from 45 degrees C to 70 degrees C. Upon cooling, this effect was found to be irreversible over 11 hours. Qualitatively similar results were observed for alpha-crystallin solutions at a variety of lower concentrations.

Accommodation in the mammalian lens requires flexure of lens fibres and some redistribution of their contents involving limited viscous flow. Shear-dependent viscosity of bovine alpha-crystallin solutions was determined with the Contraves Low-Shear Rheometer between 4.4 and 347 mg ml(-1), and at 15.5, 25, 30, and 37 degrees C. All solutions showed significant shear thinning, with markedly higher viscosity at physiological levels of approximately 300 mg ml(-1). Viscosity-concentration graphs were similar at low (1.0 s(-1)) and high (94.5 s(-1)) shear rates, indicating low molecular interaction in solution. Arrhenius plots which might have indicated the size of the energy barrier to displacement of molecules or aggregates were inconclusive.

Accommodation in the human eye occurs through controlled changes in crystalline lens shape, thickness, and refractive surface placement relative to the cornea. The changes in lens curvatures, whether surface or internal, have been characterized as a function of accommodation and subject age by use of quantitative analysis of Scheimpflug slit-lamp photographic images. Radii of curvature of the major lens refractive surfaces--the external and nuclear boundaries--decrease linearly with increasing accommodation in all eyes that are capable of accommodation. The rates at which they change with accommodation are age dependent, decreasing steadily toward zero with increased age. For the curves visible in each lens half, arising from boundaries between adjacent zones of discontinuity, radius of curvature and location are linearly related over the entire accommodative range for a given lens and over the age range for the population. The slope of this relationship changes with both accommodation and age, decreasing linearly in both cases. The relationship between these geometric changes and the loss of accommodative amplitude is discussed.

The crystalline lens of the human visual system provides the variable refractive power needed for focus by the eye at all distances. Of the major refractive tissues, only the normal lens exhibits a uniquely specific and reproducible development path with increasing age. Optical modeling of the process of image formation on the retina thus relies on accurate and complete descriptions of the age dependence of lens shape, curvature, placement within the globe relative to the cornea and retina, and refractive index gradient. Other contexts in which an optical model of the lens may be important include studies of the posterior of the globe, and especially the retina, where changes in lens transparency, color, and refractive power could affect images and spectra in this region. Finally, the recent interest by both vision scientists and refractive surgeons in improving visual resolution to its theoretical limit (also known as “super-vision”), using the Shack-Hartmann method for collection of the wavefront and Zernike polynomials for its analysis, necessitates a more detailed examination of the changing contribution of the crystalline lens to overall image formation and image quality.

Biometric data describing the geometry and spacings of emmetropic human eyes were combined with lens shape and placement within the globe to generate paraxial models of image formation as a function of age. Three different representations of the shape of the internal refractive index gradient of the lens were evaluated—a Gullstrand-type model consisting of cortical and nuclear regions with different refractive indices, a power series model, and a linear-gradient model. All three refractive models satisfy the requirements for focus for all the data sets, indicating that lenticular refractive index gradient shape is essentially underdetermined in the paraxial limit. Lens refractive power decreases by almost 2 D during a 50-year period, and the concomitant decrease in system refractive power is due almost entirely to this effect. The reduction in spacing between the lens principal planes is a function of this, as is their anterior movement with age, and suggests that the compensatory processes maintaining far focus at the expense of near are not exactly balanced. Despite these changes in the lens contribution and their effect on the location of the system principal planes, which also move anteriorly, the spacing between the system principal planes remains constant. However, the trend toward reduced overall system power with age indicates the primary role of the lens in mediating image formation onto the retina over time.

Scheimpflug photographs of the zero-diopter-accommodated anterior segments of 100 human subjects, aged 18 to 70 yr and evenly spaced over this range, were digitized and analyzed to characterize lens and lens nucleus shape as a function of age by the Hough transform and other image analysis methods. Anterior and posterior lens surface curves exhibit a decrease in radius of curvature with increasing age, in qualitative but not quantitative agreement with the earlier observations of Brown [Exp. Eye Res. 19, 175 (1974)]. In contrast, the shape of the lens nuclear boundaries changes little with age. Overall lens volume at zero diopters increases with age, but the volume of the lens nucleus remains unchanged. The lens center of mass moves anteriorly with increasing age, as does the central clear region of the lens. Although these data sets were found to be more variable than those of Brown, the complementary variability of other factors, such as anterior chamber depth, for each subject leads to a very high statistical correlation between lens shape and lens location relative to the cornea. This supports the finding of previous work that image formation on the retina for a given individual results from the multifactorial balancing of related factors.

The normal crystalline lens of the human eye develops and ages in a programmed series of additions and modifications that provides a surprising degree of similarity interindividually for a given subject age.

Image formation by the human visual system depends specifically on the curvatures and refractive indices of the cornea and crystalline lens, as well as on the spacings between refractive surfaces within the system. For the adult emmetropic human eye, many of these spacings and curvatures appear to be confined to rather narrow ranges of magnitude (Koretz et al.,1989; Cook et al., 1994). These ranges are either age-independent (e.g., globe length) or linearly dependent on age (e.g., anterior chamber depth). In addition, the dependence of many of these spacings on accommodative amplitude (e.g., change in lens thickness per diopter of accommodation) is both linear and age-independent (Koretz et al., 1997). Taken together, these relationships with age and accommodative amplitude indicate a dynamic balance that leads to focus on the retina over the majority of an emmetropes lifetime (Koretz et al., 1995; Bron et al., 1999).

The ability of the young adult human eye to focus an image onto the retina over a range from infinity to a few centimenters is a function of the shape, thickness, and placement of the crystalline lens relative to the cornea. The cornea provides a fixed refractive contribution to image formation, while the lens provides a variable contribution controlled by coupling ciliary muscle contraction to lens elastic recovery. The precise mechanism by which this coupling occurs remains the subject of study, although most proposed mechanisms are based on a Helmholtzian model. The gradual reduction in accommodative range with age leading to presbyopia is characterized by the retreat of the nearest comfortable focal point toward the far point; because lens shape becomes more sharply curved with age, this maintenance of far focus at the expense of near must be addressed in terms of other compensatory age-related changes within the lens. The development of the lens and the factors leading to image formation during accommodation are described. The potential factors contributing to age-related accommodative loss are assessed, and models of the process evaluated for both human and non-human primates. The lens-based geometric model of accommodation and presbyopia appears to fit the human visual system optimally.

The human eye is programmed to achieve emmetropia in youth and to maintain emmetropia with advancing years. This is despite the changes in all eye dimensions during the period of growth and the continuing growth of the lens throughout life. The process of emmetropisation in the child's eye is indicated by a shift from the Gaussian distribution of refractive errors around a hypermetropic mean value at birth to the non-Gaussian leptokurtosis around an emmetropic mean value in the adult. Emmetropisation is the result of both passive and active processes. The passive process is that of proportional enlargement of the eye in the child. The proportional enlargement of the eye reduces the power of the dioptric system in proportion to the increasing axial length. The power of the cornea is reduced by lengthening of the radius of curvature. The power of the lens is reduced by lengthening radii of curvature and the effectivity of the lens is reduced by deepening of the anterior chamber. Ametropia results when these changes are not proportional. The active mechanism involves the feedback of image focus information from the retina and consequent adjustment of the axial length. Defective image formation interferes with this feedback and ametropia then results. Heredity determines the tendency to certain globe proportions and environment plays a part in influencing the action of active emmetropisation. The maintenance of emmetropia in the adult in spite of continuing lens growth with increasing lens thickness and increasing lens curvature, which is known as the lens paradox, is due to the refractive index changes balancing the effect of the increased curvature. These changes may be due to the differences between nucleus and cortex or to gradient changes within the cortex.

The effects of non-ionic and ionic denaturation and denaturation/renaturation on the native structure of alpha-crystallin at room temperature were examined. Native alpha-crystallin, at concentrations above and below the previously reported critical micelle concentration (CMC) range, was denatured by varying concentrations of urea and guanidine hydrochloride. The resulting denatured samples were examined by gel filtration fast performance liquid chromatography (FPLC), circular dichroism spectropolarimetry (CD), and transmission electron microscopy. Elution peak samples from gel filtration chromatography with sufficiently high concentrations were examined for subunit composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The studies presented herein demonstrate that the denaturation and renaturation of alpha-crystallin via non-ionic urea denaturation results in different renaturation species, depending upon the initial concentration of alpha-crystallin which is denatured and the concentration of urea, including certain species which, by gel filtration FPLC, have an apparent molecular weight greater than the native 800 kD aggregate. Transmission electron microscopy has also demonstrated the existence of a high molecular weight aggregate form for denatured samples. Ionic dissociation, in contrast, proceeds much in the same manner above and below the CMC range, the major difference occurring at 2 M guanidine hydrochloride. alpha B-crystallin is preferentially removed from the native alpha-crystallin aggregate upon treatment with 2 M guanidine hydrochloride indicating, once again, differences between the two subunits. Above and below the CMC range, dissociation with guanidine hydrochloride appears to plateau after 4 M guanidine hydrochloride as indicated by the presence of two apparent homotetrameric species and no further dissociation of these species with increasing guanidine hydrochloride concentrations. CD demonstrates that some secondary structure, which is lost with lower concentrations of alpha-crystallin, is still present when concentrations of alpha-crystallin, well above the critical micelle concentration range, are treated with high concentrations of urea at room temperature. In contrast, concentrations both above and below the CMC range demonstrate a significant loss of secondary structure upon treatment with 2 M guanidine hydrochloride. Finally, ionic denaturation and subsequent renaturation results in the formation of a species which is functionally incapable of protecting gamma-crystallin from heat-induced aggregation.

The concept that normal human visual development proceeds from the balanced and compensatory interaction of various contributory elements can be traced back to the seminal cross-sectional studies of Sorsby and coworkers four decades ago1-3.

The effects of mild environmental changes (e.g. the addition of divalent cations or EDTA, as well as variations of buffer pH) on the heat stability and chaperone-like activity of native alpha-crystallin, and denatured-renatured alpha-crystallin in the native molar isoform ratio, have been investigated using circular dichroism (CD) spectropolarimetry and functional assays. The presence or absence of divalent cations has little or no effect on the secondary structure of renatured samples, although chaperone-like activity levels can vary widely; the only relevant spectral difference observed is a loss of some alpha-helical content in all the renatured samples relative to the native protein, but this change has no impact on function. The range of concentration over which the inhibitory Mg2+ effect is observed is 10-fold higher for dialyzed fresh protein than for protein renatured into buffers containing Mg2+, but for both sets of samples, the full effect is established below physiological Mg2+ concentrations. Renaturing into various pH buffers, in contrast, affects both heat stability and chaperone-like activity below pH 7.0, with essentially no functionality observed at pH 6.0. CD spectra of these samples indicate that acidic conditions lead to some degree of unfolding, and that this unfolding correlates directly with functionality. Similar results are obtained for fresh protein dialyzed against these pH levels. Overall, these results suggest that heat stability is a function of the protein's secondary structure and folding state, while chaperone-like activity is primarily a function of factors at the tertiary and quaternary levels of organization.

Of the methods developed (e.g., phakometry, magnetic resonance imaging, etc.) for noninvasive measurement of the geometry of the anterior segment of the human eye, Scheimpflug photography offers the best resolution and the highest precision. The primary obstacle encountered with this or any other image-based method has been in obtaining quantitative measurements directly from the images. Image enhancement (gray-scale gradient analysis) and pattern recognition methods (Hough transform and recursive least-squares algorithms) are developed so that parametric representations of lens surfaces and zone boundaries can be obtained directly from the images. Methods to correct for nonlinear Scheimpflug camera reproduction ratios and provide error estimates for geometrical parameters are also developed and will be presented separately. Combined, these techniques yield representations of lens geometry having sufficient precision, to which paraxial ray tracing can be applied to determine lens optical properties by using well-posed optical models with one unknown.

The crystalline lens of the human eye acts, along with the cornea, to focus an image on the retina. The cornea provides most of the eye's overall power in a fixed refractive contribution, while the lens acts in a secondary capacity, providing a variable refractive contribution that allows focus from the eye's far point to a near point which is age-dependent. The lens's ability to provide this supplementary refractive capacity is due to changes in its axial thickness, surface and internal curvature, and to shallowing of anterior chamber depth; these changes are controlled by changes in the degree of contraction of the surrounding ciliary muscle, the change in force of which is transferred to the lens through the zonular apparatus. As humans age, the location of the nearest comfortable point of focus gradually recedes, leading eventually to a coincidence of the near and far point, disregarding depth of focus, which necessitates optical prostheses [1,2].

alpha-crystallin, the major protein component of the crystallin lens of mammalian eyes, is found in vivo as two separate gene products. Both isoforms are expressed in different major tissues of the body, with the lens the only location where both are found together. Both sequences can be phosphorylated, though at different locations. Both exhibit a high sequence homology to the small heat shock proteins, and it has been shown that alpha-crystallin also resists heat-induced denaturation. Horwitz [J. Horowitz (1992) Proc. Natl. Acad. Sci. USA 89, 10449-10453] demonstrated that alpha-crystallin can exhibit chaperone-like protection against heat-induced turbidity increases, and it has been suggested that this may be an in vivo function as well. However, neither isoform, when purified, shows the same overall level of chaperone-like activity as the native species, except for one phosphorylated species [M. A. M. van Boekel, S. E. A. Hoogakker, J. J. Harding, and W. W. de Jong (1996) Ophthalmic Res. 28(Suppl. 1), 32-38]. Experiments designed to determine the factors leading to loss of chaperone-like activity indicate that strong ionic conditions, such as those used in isoform separation and/or the presence of divalent cations reduce the efficiency of this function and that the presence of EDTA fully restores it irrespective of prior treatment or buffer conditions. Heat stability is essentially preserved under all conditions. These results suggest that alpha-crystallin may serve primarily as a heat shock protein in vivo and that the chaperone-like function may be inhibited under physiological conditions.

Accommodation, the process by which the human eye focuses on near objects, takes place through carefully controlled changes of the crystalline lens in shape, thickness, and anterior surface location relative to the cornea. These changes, which involve an elastic recovery of the lens upon accommodation from the maximally stressed and flattened non-accommodated lens shape, are coupled with the contraction of the ciliary muscle, which itself changes in shape and location relative to fixed points within the globe. The refractive range over which accommodation can occur is age-dependent. In youthful eyes, the range can be as great as 15 diopters, but the near point recedes steadily with increasing age in the adult eye until, by about 50 yr, it closely approaches the far point. This diminution in accommodative range is accompanied by other aging changes in the lens, ciliary muscle, and anterior segment geometry.

Accommodation, the mechanism by which the eye focuses on near objects, and the age-related loss of this function leading to presbyopia are inextricably linked to the development and aging of the anterior segment. Steady lens growth primarily in the sagittal direction throughout life and the concomitant shallowing of the anterior chamber are both highly correlated to reduced accommodative range (p≪0.001; Koretz et al., 1989), and other changes in the lens with age (e.g., increased scatter of light at high angles; Cook et al., 1994) are also observed. In addition to age-related changes in lens thickness, mass, and placement relative to the cornea and retina, the non-accommodated shape of the lens is itself age-dependent, becoming more sharply curved on both the anterior and posterior surfaces with increased age. This phenomenon was first characterized by Brown (1974), who noted that the human eye should lose far, not near, vision with aging based on these observations; this "lens paradox" can, of course, be resolved by assuming an overall reduction in lens refractive power with age (Koretz and Handelman, 1986; Cook and Koretz, 1995) without or with (Smith et al., 1992) a fundamental change in the shape of the refractive index gradient.

Of the many methods that have been developed (e.g., phakometry, NMI, etc.) for non-invasive measurement of the geometry of the anterior segment, at present Scheimpflug photography offers the best resolution and the highest accuracy. The primary obstacle encountered with this or any other image based method has been obtaining quantitative measurements of the position and curvature of lens surfaces and zone boundaries from the images directly. Image enhancement (Sobel gradient scanning), and pattern recognition methods (Hough transformation and recursive least squares algorithms) have been applied successfully to this problem. These techniques have been described previously [1] as well as the algorithms used to correct for nonuniform Scheimpflug camera scaling. Combined, these techniques yield representations of lens geometry having sufficient accuracy that ray tracing can be applied to determine lens optical properties from well poised models with one unknown. The image processing methods will be reviewed as an introduction.

The size of alpha-crystallin aggregates, as well as the structural organization of each particle's subunits, is currently unknown, although a number of different laboratories have suggested both structures and average molecular weights (Thomson, J.A. and Augusteyn, R.C. (1984) Proc. Int. Soc. Eye Res. 3, 152). One hypothesis, compatible with literature reports and consistent with what is known of subunit primary structure and physiological function, is that alpha-crystallin exists in vivo as a naturally occurring protein micelle (Sen, A.C. and Chakrabarti, B. (1991) Biophysical J. 59, 108a.) To test this hypothesis, experiments were performed on this protein to determine its behavior under increased hydrostatic pressure and the effect of its concentration on aqueous surface tension. With increasing hydrostatic pressure, the turbidity of an alpha-crystallin solution increases exponentially to a plateau at about 6000-8000 psi; upon release of pressure, the samples slowly return to their original turbidity level. Other naturally aggregating proteins, such as skeletal muscle myosin, demonstrate a decrease in turbidity under the same conditions. The surface tension of alpha-crystallin in aqueous solution decreases to a plateau with increasing protein subunit concentration, with an inflection point over the range 0.18-0.25 mM; cholate and other amphiphiles exhibit similar behavior. In contrast, plots of surface tension over the equivalent concentration range for other protein aggregates in the same buffer more closely approximate the types of curve obtained with short chain aliphatic acids. These results indicate that alpha-crystallin behaves like the protein version of a micelle.

Native thick filaments isolated from freshly prepared rabbit psoas muscle were found to be resistant to pressure-induced dissociation. With increasing pressure application and release, a bimodal distribution of filament lengths was observed. The shorter filament length is associated with filament breakage at the center of the bare zone, while the longer length is associated with relatively intact filaments. Intact filaments and filament halves decrease in length by no more than 20% after exposure to and release of 14 000 psi. Bimodal distributions were not observed in equivalent experiments performed on filaments isolated from muscle glycerinated and stored at −20°C for 6 months. Instead, filament dissociation proceeds linealy as a function of increasing pressure. Filaments prepared from muscle glycerinated and stored for 2 and 4 months exhibited pressure-induced behavior intermediate between the filaments prepared from fresh muscle and filaments prepared from muscle stored for 6 months. Since there appears to be no difference in the protein profiles of the various muscle samples, it is possible that stabilization of the native thick filament against hydrostatic pressure arises from trapped ions that are leached out over time.

The slit-lamp biomicroscope is essential in the clinical evaluation of the human ocular anterior segment. A narrow slit of intense white light is shone into the globe, illuminating a thin optical section of the anterior segment, and the illuminated section is examined at an angle through a icroscope. While qualitatively useful, the resultant image is quantitatively severely distorted by the angle of observation, which is a variable projection of the true spacings, and by the passage of the scattered light through the cornea, a strong and sharply curved refractor.

We dispute the claim made by Dubbelman and colleagues [J. Opt. Soc. Am. A22, 1216 (2005); this issue] that “incorrect statistical methods” were used to compare the MRI and Scheimpflug data. We clearly stated that we “analyzed the covariance of regression lines” in the results section. We believe the analysis of covariance of regression lines, as shown by Snedecor and Cochran [Statistical Methods (Iowa State U. Press, Ames, Iowa, 1989)] is as “correct” as the “straightforward statistical comparison” of confidence intervals employed by Dubbelman and colleagues; however; our statistical method has the benefit of being more precise and stringent than a simple comparison of confidence intervals.

The size and shape of native alpha crystallin aggregates extracted at either 4 degrees C (alpha c-crystallin) or 37 degrees C (alpha m-crystallin), were compared with each other, as well as with aggregates reconstituted from either pure alpha A or alpha B subunits using electron microscopy. The alpha B aggregates were the most uniform in size (about 9 nm in diameter) and the best stained. alpha A particles were about the same size as the alpha B, but the population distribution was broader and some indication of interparticle association was observed. alpha c particles exhibited a bimodal distribution, with one peak greater than the reconstituted particles and the other about the same size; alpha m was smaller than the reconstituted structures.

An explanation of the mechanism of visual accommodation depends heavily on understanding the mechanical properties of the lens, as well as the way in which its shape is altered in small accommodative changes. An initial attempt to relate these properties to a mechanism has already been performed (Koretz and Handelman, 1982) for the young (age 11 yr) human lens, using certain simplifying assumptions (spherical curvature on the anterior lens surface and elastic isotropy). However, since it has been shown that the lens behaves as an anisotropic body, the previous treatment has been extended to include the variation of lens elastic properties in the polar and radial directions. With this modified representation, it is found that only one combination of elastic constants is consistent with the generally accepted qualitative theory of accommodation and with clinical data on the accommodative range of the emmetropic age 11 human eye. For this unique solution of the equations, however, the general mechanism already suggested by us, which includes support by the vitreous and alteration of the magnitude and angle of application of zonular force with accommodation, remains little changed.

Slit-lamp photographic studies of 144 caged rhesus monkeys, aged 2 months to 35 years, show age-related changes in anterior-chamber depth, lens thickness, anterior and posterior curvatures of the lens, and location of the posterior lens surface relative to the anterior corneal surface. For these parameters, as well as for those measured by other techniques, a difference in slope magnitude and (or) slope sign was found between the growth phase which lasts for 5–6 years, and the adult phase (greater than 5–6 years). Age-related changes in the adult rhesus eye are qualitatively similar in almost all aspects to those observed in the human eye, indicating that the rhesus is a good animal model for the study of human loss of accommodative amplitude.

A simplified mathematical representation of accommodation—the process by which the eye focuses on near objects—is described. This model is based upon assumptions which limit its applicability to the young human eye, but it nevertheless leads to new insights about the physiological mechanism. A hypothesis about the underlying cause(s) of presbyopia—the loss of accommodative amplitude with age—is also presented here, which combines aspects of the expanded accommodative mechanism with the aging characteristics of the human lens. Using a similar mathematical strategy which incorporates new experimental data about lens shape and aging, a more general model of the accommodative mechanism can be developed and used to test the presbyopia hypothesis.

The effects of variations in temperature and protein concentration on the renaturation of bovine alpha-crystallin have been examined using gel permeation chromatography, sedimentation analysis, fluorescence spectroscopy and electron microscopy. High protein concentration (3-53 mg/ml) were found to generate heterogenous populations of aggregates. It was concluded that concentrations above 3 mg/ml were inappropriate for renaturation of alpha-crystallin. Aggregates with molecular masses gradually increasing from 461,000 to 695,000 Da were produced with increasing temperature over the range 6-39 degrees C. Electron microscopy demonstrated that the reaggregates were composed predominantly of particles with circular cross-sections and mean diameters of 13-14 nm. As the renaturation temperature increased, increasing amounts of sheet-like structures were observed. Tryptophan accessibility to acrylamide quenching decreased in these aggregates as the size increased. These observations are consistent with the concept that there is no unique quaternary structure, or set of structures, for alpha-crystallin but that the protein can exist in a variety of forms containing different numbers of subunits.

The rhesus monkey lens exhibits two zones of discontinuity, one anteriorly and one posteriorly. They are first discernible by slit-lamp photography as performed in this study at around age 7 years in iridectomized eyes, and become more distinct with increasing age. Their thickness and distance from each other along the polar axis are independent of lens age, but their distance from the lens surface increases with increasing age. Upon accommodation, the distance between the two zones increases while all other distances along the polar axis remain unchanged, indicating that, as in the human, overall alterations in rhesus lenticular shape and thickness with accommodation primarily reflect changes in the shape of the central region. The curvature of each zone becomes sharper in a linear fashion with accommodation, with the slope of the relationship being similar to those for lens surfaces.

Slit-lamp photographs from four human subjects, aged 11, 19, 29, and 45 were reanalyzed using computer-based digitization and curve-fitting methods in order to obtain more complete information on internal lens curvature changes during accommodation. All discernible curves (N = 742) could be fit to parabolas with χ2 less than or equal to 0.001 irrespective of lens age, accommodative state, or curve location within the lens. For each lens, the coefficients of the parabolas, when displayed in graphic form, exhibit a linear relationship between location within the lens and the coefficient of the χ2 term. The slope of this line remains unchanged over accommodation for a given lens, but is shifted in position. The slope changes as a function of age. The age 45 lens exhibits these characteristics to a limited extent only, the differences possibly related to the development of presbyopia. The further a given curve is located from the lens surface, the smaller the region of its arc that can be considered approximately circular. A roughly hourglass figure is generated by these circular bounds; the waist of the hourglass decreases with increasing accommodation, since changes in radius of curvature with accommodation are more pronounced internally. Calculations of arc lengths as a function of increasing accommodation indicate that these lengths change very little over the entire accommodative range.

Column-purified myosin at pH 7.0 will reproducibly aggregate into filaments of known average length and structure when dialyzed against a low ionic strength medium under controlled conditions. When exposed to increased hydrostatic pressure, followed by quick return to atmospheric pressure, the original filaments shorten linearly with increasing pressure; in addition, a second population of filaments is seen, presumably the result of reaggregation of myosin after release of pressure. This second population is about 0.5 μm long, bipolar, and about half the diameter of the original filaments. The number of these filaments, but not their physical characteristics, is a function of the shortening of the original filament population. Both the remnants of the original population and the new aggregates, once formed, are stable over these and at room temperature. The addition of C-protein to myosin solutions before filament preparation results in a filament population of slightly shorter length. When these filaments are exposed to increased hydrostatic pressure, they are more resistant to disaggregation than myosin filaments without C-protein. However, like the filaments prepared in the absence of C-protein, a second population of shorter, thinner filaments is visible after exposure to pressure.