Intact Lens Research Articles

Interactions between Small Heat Shock Protein α-Crystallin and Galectin-Related Interfiber Protein (GRIFIN) in the Ocular Lens

As a member of the small heat shock protein superfamily, alpha-crystallin has a chaperone-like ability to recognize and bind denatured or unfolded proteins and prevent their aggregation. Recent studies suggest that alpha-crystallin may also interact with a variety of proteins under native conditions in vitro. To identify potential binding partners for alpha-crystallin in the intact ocular lens, we conducted cross-linking studies in transgenic mouse lenses designed for overexpression of His-tagged human alphaA-crystallin. Interacting proteins were copurified with the epitope-tagged crystallin complexes and were identified by tandem mass spectrometry. This approach identified GRIFIN (galectin-related interfiber protein) as a novel binding partner. Consistent with results from cross-linking, GRIFIN subunits copurified with alpha-crystallin complexes during size exclusion chromatography of nontransgenic mouse lens extracts prepared without chemical cross-linking. Equilibrium binding to GRIFIN was studied using native alpha-crystallin isolated from calf lenses as well as oligomeric complexes reconstituted from recombinant alphaA- and alphaB-crystallin subunits. Calf lens alpha-crystallin binds GRIFIN with relatively high affinity (K(d) = 6.5 +/- 0.8 microM) at a stoichiometry of 0.25 +/- 0.01 GRIFIN monomer/alpha-crystallin subunit. The binding interaction between alpha-crystallin and GRIFIN is enhanced up to 5-fold in the presence of 3 mM ATP. These binding data support the hypothesis that GRIFIN is a novel binding partner of alpha-crystallin in the lens.

Structure, Biomass, and Productivity of a Late Paleocene Arctic Forest

ABSTRACT. Abundant fossil plant remains are preserved in the high-latitude late Paleocene Iceberg Bay Formation on Ellesmere Island, Nunavut, Canada. Intact leaf litter lenses and permineralized, in situ logs and stumps offer for the first time an opportunity to determine the structure, biomass, and productivity of a redwood-dominated forest that grew in the polar regions of Nunavut (paleolatitude 75–80° N). Well-preserved fossil tree trunks were excavated to develop equations that describe the height, structure, and mass of the aboveground components of late Paleocene-age (approximately 55.8 to 58.7 million years old) Metasequoia (redwoods) trees. We then combined those data with measurements of the in situ stumps to determine the structure, biomass, and productivity of this polar fossil forest. The height of the canopy trees in the forest was calculated to be 32 ± 2 m. Abundant branch stubs that represent the remnants of living branches were found on a wide range of stem sizes including larger logs from...

The Effects of GPX-1 Knockout on Membrane Transport and Intracellular Homeostasis in the Lens

Glutathione peroxidase-1 (GPX-1) is an enzyme that protects the lens against H2O2-mediated oxidative damage. The purpose of the present study was to determine the effects of GPX-1 knockout (KO) on lens transport and intracellular homeostasis. To investigate these lenses we used (1) whole lens impedance studies to measure membrane conductance, resting voltage and fiber cell gap junction coupling conductance; (2) osmotic swelling of fiber cell membrane vesicles to determine water permeability; and (3) injection of Fura2 and Na+-binding benzofuran isophthalate (SBFI) into fiber cells to measure [Ca2+]i and [Na+]i, respectively, in intact lenses. These approaches were used to compare wild-type (WT) and GPX-1 KO lenses from mice around 2 months of age. There were no significant differences in clarity, size, resting voltage, membrane conductance or fiber cell membrane water permeability between WT and GPX-1 KO lenses. However, in GPX-1 KO lenses, coupling conductance was 72% of normal in the outer shell of differentiating fibers and 45% of normal in the inner core of mature fibers. Quantitative Western blots showed that GPX-1 KO lenses had about 50% as much labeled Cx46 and Cx50 protein as WT, whereas they had equivalent labeled AQP0 protein as WT. Both Ca2+ and Na+ accumulated significantly in the core of GPX-1 KO lenses. In summary, the major effect on lens transport of GPX-1 KO was a reduction in gap junction coupling conductance. This reduction affected the lens normal circulation by causing [Na+]i and [Ca2+]i to increase, which could increase cataract susceptibility in GPX-1 KO lenses.

Understanding the dynamics of biological colloids to elucidate cataract formation and develop a methodology for its early diagnosis

The eye lens is the most notable example of mammalian tissues exhibiting complex colloidal behavior. In this paper, we briefly describe how dynamics in colloidal suspensions can address selected aspects of cataracts, which is ultimately related to protein self-assembly under pathological conditions. Results from dynamic light-scattering of eye lens homogenates over a wide protein concentration were analyzed and the various relaxation modes were identified in terms of collective and self-diffusion processes. Using this information as an input, the complex relaxation pattern of the intact lens nucleus was rationalized. The model of cold cataract–a phase separation effect of the lens cytoplasm with cooling–was used to simulate a lens cataract under in vitro conditions to determine the correlation function parameters, which could be used as reliable indicators of cataract onset. The applicability of dynamic light scattering as a non-invasive, early diagnostic tool for ocular diseases is also demonstrated in light of these findings.

Contributions of Mouse Genetic Background and Age on Anterior Lens Capsule Thickness

Accurate lens capsule thickness measurements are necessary for studies investigating mechanical characteristics of the capsule. Confocal Z-axis imaging was used to measure the anterior lens capsule thickness of living intact lenses with minimal tissue manipulation. Measurements of the anterior capsule thickness is reported for the first time in young and old mice from four inbred strains, BALB/c, FVB/N, C57BL/6, and 129X1, and the outbred strain ICR. Our data demonstrates that the mouse anterior lens capsule continues to grow postnatally similar to that described in other mammals. It is also shown there is a significant difference in anterior lens capsule thickness between unrelated mouse strains, suggesting that capsule thickness is a quantitative trait shared by strains with common ancestry. Measurements, taken from other regions of FVB/N capsules revealed the anterior pole to be the thickest, followed by the equatorial region and posterior pole. In addition to mouse, anterior capsule measurements taken from intact cattle, rabbit, rat lenses, and human capsulotomy specimens correlated with the overall size of the animal.

A Case of Polymicrobial Keratitis Violating an Intact Lens Capsule

To report a case of polymicrobial keratitis resistant to topical and intraocular antibiotics with recurrence after penetrating keratoplasty. Case report. We report a case of polymicrobial keratitis caused by Phialophora verrucosa, Candida tropicalis, and Propionibacterium acnes. Initial treatment included topical vancomycin, tobramycin, amphotericin B, voriconazole, and oral fluconazole, as well as subconjunctival amphotericin B. Penetrating keratoplasty was performed, and the infection seemed to have resolved until 4 weeks after keratoplasty. A second penetrating keratoplasty was performed, followed by 4 weekly intracameral injections of voriconazole. Two weeks after the fourth intracameral injection, the infection manifested as an active anterior-chamber reaction. The patient's eye was subsequently enucleated. Histopathologic evaluation showed penetration of the crystalline lens by fungus at the site of synechiae between the intact lens capsule and iris. To our knowledge, this is the first documented case of a polymicrobial keratitis caused by a bacterium, yeast, and a fungus. It is the first histopathologic demonstration of fungal penetration of intact lens capsule from infected iris.

Free and Bound Water in Normal and Cataractous Human Lenses

To analyze free and total water in human normal and cataractous lenses. Thermogravimetric analysis was used to determine total water, and differential scanning calorimetry was used for free water. In normal human lenses, the total water content of the nucleus remained unchanged with age, but the state of the water altered. The ratio of free to bound water increased steadily throughout adult life. In a 20-year-old person, there was approximately one bound water molecule for each free water molecule in the lens center, whereas in a 70- to 80-year-old person, there were two free water molecules for each bound water molecule. This conversion of bound to free water does not appear to be simply a consequence of the aggregation of soluble crystallins into high molecular weight aggregates because studies with intact pig lenses, in which such processes were facilitated by heat, did not show similar changes. The region of the lens in which the barrier to diffusion develops at middle age corresponds to a transition zone in which the protein concentration is intermediate between that of the cortex and the nucleus. In cataractous lenses, the free-to-bound water ratio was not significantly different from that of age-matched normal lenses; however, total water content in the center of advanced nuclear cataractous lenses was slightly lower than in normal lenses. As the human lens ages, bound water is progressively changed to free water. Advanced nuclear cataract may be associated with lower total hydration of the lens nucleus.

Dual Roles for Prox1 in the Regulation of the Chicken βB1-Crystallin Promoter

Lens fiber cell differentiation is marked by the onset of betaB1-crystallin expression and is controlled by the cooperative action of a set of transcription factors including Prox1, an atypical homeodomain protein. Previously, the authors reported that Prox1 directly interacts with the OL2 element found in the chicken betaB1-crystallin basal promoter to activate the expression of this gene. Here they mapped the location of activating and repressing sequences of the full-length chicken betaB1-crystallin promoter (-432/+30) in lens epithelial cells, annular pad cells, and intact lens and characterized Prox1-binding sites found in this region. Transfection analysis and transgenic mice were used to characterize upstream regions of the chicken betaB1-crystallin gene. DNaseI footprinting and chromatin immunoprecipitation was performed to identify Prox1-binding sites, and transfection analyses were used to characterize these sites functionally. Sequences between -152 and -432 of the chicken betaB1-crystallin promoter mediated either promoter activation or repression, depending on the stage of lens differentiation tested. Two new Prox1-binding sites were found in this region that bound Prox1 more avidly than the OL2 element. However, neither binding site conferred Prox1-mediated activation on a heterologous promoter; instead, each allowed Prox1 to repress promoter function. The function of the upstream region of the chicken betaB1-crystallin promoter changes depending on cellular context. These data suggest that Prox1 function as a transcriptional activator could be regulated at the DNA level based on the characteristics of the responsive elements.

Differentiation-dependent changes in the membrane properties of fiber cells isolated from the rat lens

Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level, we analyzed the membrane properties of fiber cells isolated in the presence of the nonselective cation channel inhibitor Gd3+. Isolated fiber cells were viable in physiological [Ca2+] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a subgroup of cells whose conductivity matched values measured in the whole lens (1). In this group of cells, membrane resistance, conductivity, and reversal potential all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments, we showed that newly differentiated fiber cells (<150 microm) contained variable combinations of Ba2+-and tetraethylammonium-sensitive K+ currents. Longer fiber cells (150-650 microm) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens. Longer cells also exhibited a low-level, nonselective conductance that was eliminated by the replacement of extracellular Na+ with N-methyl-d-glucamine, indicating that the lens contains both Gd3+-sensitive and -insensitive nonselective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant K+ conductance(s) toward larger contributions from anion and nonselective cation conductances as fiber cells elongate.

Mutation of Solute Carrier SLC16A12 Associates with a Syndrome Combining Juvenile Cataract with Microcornea and Renal Glucosuria

Unobstructed vision requires a particular refractive index of the lens, a measure based on the organization of the structural proteins within the differentiated lens cells. To ensure an intact lens structure, homeostasis within the lens cells is indispensable. Alterations of the lens structure result in opacity and lead to cataract. Renal glucosuria is defined by elevated glucose level in the urine without hyperglycemia and without evidence of morphological renal anomalies. In a Swiss family with autosomal dominant juvenile cataract, microcornea, and renal glucosuria, we have identified a nonsense mutation in a member of the carboxylic acid transporter family SLC16. The underlying gene defect in SLC16A12 resides within a 3 cM region on chromosome 10q23.13 defined by linkage mapping of this phenotype. We found tissue-specific variability of SLC16A12 transcript levels in control samples, with high expression in the eye and kidney, the two organs affected by this syndrome. This report demonstrates biological relevance of this solute carrier. We hypothesize that SLC16A12 is important for lens and kidney homeostasis and discuss its potential role in age-related cataract.

Perfluorodecaline residue in the anterior chamber of a patient with an intact crystalline lens: a case report

BackgroundPerfluorocarbon liquids are frequently used as intraoperative tools in vitreoretinal surgery and may occasionally be retained in the vitreous cavity. We report a patient who underwent pars plana vitrectomy for a giant tear after receiving blunt trauma to his right eye and sustained postoperative perfluorocarbon liquid residue in the anterior chamber in spite of an intact crystalline lens.Case presentationPerfluorodecaline was used as a temporary retinal tamponade. Three weeks after the surgery, a residue of heavy liquid was observed in the anterior chamber, even though the patient had an intact crystalline lens without any tilt or dislocation. The remnant of the heavy liquid was taken out of the anterior chamber immediately to avoid secondary complications.ConclusionPresence of heavy liquids in the anterior chamber may be associated with zonular defects even though the patient has an intact crystalline lens.

Identification of the Primary Targets of Carbamylation in Bovine Lens Proteins by Mass Spectrometry

Purpose: Carbamylation, an important post-translational modification of proteins, inevitably causes conformational changes of lens proteins. It may increase aggregation between crystallin molecules and disrupt the close packing required for transparency thus leading to cataract. The aim of this study was to isolate the primary targets of carbamylation in the lens and identify them by mass spectrometry. Materials and Methods: Fresh intact bovine lenses were incubated with 100 mM potassium cyanate for 7 days. The proteins in the water-soluble fractions from the normal control and the cyanate-modified lens proteins were separated by two-dimensional (2-D) gel electrophoresis with identification after silver staining. Protein spots that differed between the normal and carbamylated groups were selected for further analysis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Results: The 2-D gel results showed that the major lens proteins were in the section of pI 5–8, with relative molecular masses of 20–35 kDa, and changes in the carbamylated fraction like strings of beads indicating modification. The mass spectrometry analysis and a database search identified carbamylated proteins originating from αA-crystallin, βB2- and γS-(βS)-crystallins. Conclusions: These crystallins may be vulnerable proteins targeted by carbamylation. The accumulated aggregation and loss of chaperone activity may contribute to cataract formation.

Different factors promote axonal regeneration of adult rat retinal ganglion cells after lens injury and intravitreal peripheral nerve grafting

We have investigated the differential mediators of the neurotrophic effects of intravitreal peripheral nerve grafting and lens injury on adult rat retinal ganglion cells (RGC). Lens injury and intravitreal peripheral nerve grafting both stimulated RGC neurite growth in vitro and axon regeneration past the optic nerve lesion site in vivo concomitant with activation of retinal glia and invasion of macrophages into the eye. These observations, together with the results of coculture studies using a macrophage-free intact peripheral nerve segment, a macrophage-free intact lens, a macrophage-rich peripheral nerve segment, or a macrophage-rich injured lens in retinal cultures suggest that the stimulation of RGC axon regeneration by lens injury and intravitreal peripheral nerve grafting share a common macrophage-derived component overlain by distinct lens-derived and peripheral nerve-derived neurotrophic factors, respectively. RGC axon regeneration following lens injury and intravitreal peripheral nerve grafting was similar in vivo, correlating with similar retinal glia activation whereas, in vitro, the level of RGC neurite outgrowth was significantly higher following intravitreal peripheral nerve grafting compared with lens injury, concomitant with the presence of increased numbers of activated retinal glia. This suggests that in vivo RGC axon regeneration induced by lens injury and peripheral nerve grafting may be limited, in part, by factors derived from activated retinal glia.

Diffusion of nanoparticles into the capsule and cortex of a crystalline lens

The purpose of this study is to determine the ability of fluorescent nanoparticles to diffuseinto a crystalline lens. Intact porcine lenses from five-month-old pigs, intact human lensesobtained from three donors aged 41, 42 and 45 years, and sections of human lens cortexobtained from four donors aged 11, 19, 32, and 34 years were incubated for 72 h at7 °C in aqueous solutions of green (566 nm) and red (652 nm) fluorescent water soluble cadmiumtellurium (CdTe) nanoparticles. As demonstrated by fluorescent and confocalmicroscopy, the CdTe nanoparticles diffused into the porcine and human lenscapsule and into human cortical lens fibres; however, the nanoparticles did not passthrough the intact lens capsule. Nanoparticles can be used as a method for studyingintracellular structure and biochemical pathways within the lens capsule and corticallens fibres to further understand cataractogenesis and may serve as a carrier forchemotherapeutic agents for the potential treatment of primary and secondarycataracts.

Dynamics of proteins: Light scattering study of dilute and dense colloidal suspensions of eye lens homogenates

We report a dynamic light scattering study on protein suspensions of bovine lens homogenates at conditions (pH and ionic strength) similar to the physiological ones. Light scattering data were collected at two temperatures, 20 and 37 degrees C, over a wide range of concentrations from the very dilute limit up to the dense regime approaching the physiological lens concentration. A comparison with experimental data from intact bovine lenses was advanced, revealing differences between dispersions and lenses at similar concentrations. In the dilute regime, two scattering entities were detected and identified with the long-time self-diffusion modes of alpha-crystallins and their aggregates, which naturally exist in lens nucleus. Upon increasing protein concentration, significant changes in time correlation function were observed starting at approximately 75 mg ml(-1), where a new mode originating from collective diffusive motions becomes visible. Self-diffusion coefficients are temperature insensitive, whereas the collective diffusion coefficient depends strongly on temperature revealing a reduction of the net repulsive interparticle forces with decreasing temperature. While there are no rigorous theoretical approaches on particle diffusion properties for multicomponent, nonideal hard sphere polydispersed systems, as the suspensions studied here, a discussion of the volume fraction dependence of the long-time self-diffusion coefficient in the context of existing theoretical approaches was undertaken. This study is purported to provide some insight into the complex light scattering pattern of intact lenses and the interactions between the constituent proteins that are responsible for lens transparency. This would lead to understand basic mechanisms of specific protein interactions that lead to lens opacification (cataract) under pathological conditions.

Presbyopia and heat: changes associated with aging of the human lens suggest a functional role for the small heat shock protein, α‐crystallin, in maintaining lens flexibility

Presbyopia, the inability to focus up close, affects everyone by age 50 and is the most common eye condition. It is thought to result from changes to the lens over time making it less flexible. We present evidence that presbyopia may be the result of age-related changes to the proteins of the lens fibre cells. Specifically, we show that there is a progressive decrease in the concentration of the chaperone, alpha-crystallin, in human lens nuclei with age, as it becomes incorporated into high molecular weight aggregates and insoluble protein. This is accompanied by a large increase in lens stiffness. Stiffness increases even more dramatically after middle age following the disappearance of free soluble alpha-crystallin from the centre of the lens. These alterations in alpha-crystallin and aggregated protein in human lenses can be reproduced simply by exposing intact pig lenses to elevated temperatures, for example, 50 degrees C. In this model system, the same protein changes are also associated with a progressive increase in lens stiffness. These data suggest a functional role for alpha-crystallin in the human lens acting as a small heat shock protein and helping to maintain lens flexibility. Presbyopia may be the result of a loss of alpha-crystallin coupled with progressive heat-induced denaturation of structural proteins in the lens during the first five decades of life.

Purinergic Receptor–Mediated Regulation of Lens Connexin43

To determine whether the purinergic receptor-mediated, delayed transient inhibition of lens cell-to-cell communication is due to the protein kinase C (PKC)-catalyzed phosphorylation of connexin (Cx)43. The functional activity of gap junctions was determined in the presence of various pharmacologic agents by injecting fluorescent dye into a single cell in a confluent monolayer of HeLa cells that had been stably transfected with either wild-type or mutant Cx43, and the number of cells taking up dye was determined. Application of adenosine triphosphate (ATP) to Cx43-transfected HeLa cells resulted in a delayed, transient decrease in cell-to-cell transfer of fluorescent dye similar to the authors' previous report in sheep lens epithelial cell cultures. The ATP-mediated, delayed, transient decrease in dye transfer was prevented by the inhibition of PKC or phospholipase C, but not by calmodulin inhibition or by preloading the cells with BAPTA (bis-(o-aminophenoxy)-N,N'N'-tetraacetic acid). This functional inhibition of Cx43 cell-to-cell dye transfer was sustained in the presence of the nonhydrolyzable ATP analogue AMP-PNP (adenyl-5'-yl imidophosphate), the ectonucleotidase inhibitor ARL 67156, or the protein phosphatase inhibitor okadaic acid. In experiments in HeLa cells transfected with Cx43(Delta 257), a Cx43 C terminus truncation mutant, or Cx43(S368A), a Cx43 point mutant, cell-to-cell coupling was unaffected by the addition of ATP. The results indicate the essential role of serine 368 in the ATP-dependent inhibition of Cx43. This novel mechanism of regulating Cx43 most likely plays an important role in maintaining the microcirculation that is essential for the movement of water and solutes in the intact lens.

Redox regulation of the proteasome in human lens epithelial cells and intact mouse lens

Abstract Purpose: This study examined the proteasome susceptibility to oxidative stress and changes in the redox system after proteasome inhibition. Results: Oxidative stress by H2O2 significantly decreased all three main peptidase activities of the proteasome, the chymotrypsin‐like, the trypsin‐like and the peptidylglutamyl peptidase activities of the proteasome in human lens epithelial cells (HLEC). Oxidatively stressed mouse lenses showed trends of decreased chymotrypsin‐like proteasome activity as well as decreased GSH‐levels.In HLEC lysate, the chymotrypsin‐like and the peptidylglutamyl peptidase activities were stimulated by GSH and all three peptidase activities were inhibited by GSSG addition.Inhibition of the proteasome in HLEC by MG‐132 and lactacystin caused increased apoptosis as revealed by Hoechst 33342 staining and caspase‐3 determination. Proteasome inhibition increased superoxide and peroxide production, depolarised the mitochondria and decreased the level of glutathione. However apoptosis could be prevented by addition of GSH. Non‐stressed HLEC exposed to GSH, did not present any change in apoptotic respons. Conclusions: The present data indicate that the protesasome may regulate apoptosis through oxidative mechanisms.

Do not try this at home: trash can intracapsular cataract extraction

The authors present a 39-year-old woman who sustained blunt trauma from a metal garbage can to her left eye and presented with her intact crystalline lens luxated subconjunctivally after expulsion through a scleral wound. External photographs show the lens found under intact conjunctiva and a computed tomography scan demonstrates the extrascleral lens. The patient had a ruptured globe repair and intraoperative photographs showed removal of the lens.

Purinergic agonists stimulate lens Na-K-ATPase-mediated transport via a Src tyrosine kinase-dependent pathway

The Na-K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na-K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na-K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na-K-ATPase function and SFK activation was examined in the rabbit lens. Na-K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium ((86)Rb) uptake by the intact lens, and Na-K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium ((86)Rb) uptake. Na-K-ATPase activity was increased in the epithelium of lenses pretreated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased Western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the COOH terminal). A single PY416-Src immunoreactive band at approximately 60 kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased, while active Yes did not change. Preincubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium ((86)Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na-K-ATPase-mediated transport in the organ-cultured lens.