An abietane diterpenoid is a potent activator of systemic acquired resistance.

Abietane diterpenoids are major constituents of conifer resins that have important industrial and medicinal applications. However, their function in plants is poorly understood. Here we show that dehydroabietinal (DA), an abietane diterpenoid, is an activator of systemic acquired resistance (SAR), which is an inducible defense mechanism that is activated in the distal, non-colonized, organs of a plant that has experienced a local foliar infection. DA was purified as a SAR-activating factor from vascular sap of Arabidopsis thaliana leaves treated with a SAR-inducing microbe. Locally applied DA is translocated through the plant and systemically induces the accumulation of salicylic acid (SA), an important activator of defense, thus leading to enhanced resistance against subsequent infections. The NPR1 (NON-EXPRESSOR OF PR GENES1), FMO1 (FLAVIN-DEPENDENT MONOOXYGENASE1) and DIR1 (DEFECTIVE IN INDUCED RESISTANCE1) genes, which are critical for biologically induced SAR, are also required for the DA-induced SAR, which is further enhanced by azelaic acid, a defense priming molecule. In response to the biological induction of SAR, DA in vascular sap is redistributed into a SAR-inducing 'signaling DA' pool that is associated with a trypsin-sensitive high molecular weight fraction, a finding that suggests that DA-orchestrated SAR involves a vascular sap protein(s).

Protein-protein interactions and lens transparency.

Past studies have identified posttranslational modifications of human lens proteins occurring during cataract formation, and have also demonstrated that protein-protein interactions exist between different lens crystallins. Based upon current theories of lens transparency, these posttranslational modifications and their possible effects upon crystallin interactions may be the key to understanding why the lens is able to transmit light, and why transmission is decreased during cataractogenesis. This review will summarize current knowledge of posttranslational modifications during human cataractogenesis, and will propose their possible role in protein-protein interactions that are thought to be necessary for lens transparency. Based upon this premise, model systems will be described that will test the validity of the theory.

Age-dependent association of gamma-crystallins with aged alpha-crystallins from old bovine lens.

PURPOSE: Previous theoretical and experimental studies have predicted that the loss of weak protein interactions between alpha- and gamma-crystallins could result in a decrease in the transparent properties of the aging lens. METHODS: alpha-Crystallins were prepared from the nucleus of old bovine lens, and gamma-crystallins were prepared from whole fetal bovine lens or from the nucleus of old bovine lens. The possible interactions of old alpha-crystallins with either old gamma-crystallins or fetal gamma-crystallins were quantitated at equilibrium using microequilibrium dialysis. The amount of each gamma-crystallin species in the "full" versus "empty" chambers was determined by reverse phase chromatography to obtain a binding ratio (full/empty). RESULTS: A binding ratio greater than 1.00 is indicative of a alpha-crystallin/gamma-crystallin interaction. Within experimental error (+/-2X standard deviation), there were no interactions between aged gamma-crystallins with aged alpha-crystallins while there were significant interactions between some of the fetal gamma-crystallins with aged alpha-crystallins. CONCLUSIONS: In the aged bovine lens, when transparency is known to decrease, there is no detectable interaction of gamma-crystallins with alpha-crystallins as measured by microequilibrium dialysis. The results are consistent with the hypothesis that short-range, weak, attractive interactions between alpha- and gamma-crystallins are necessary for maximum transparency of the lens.

Differential binding of mutant (R116C) and wildtype alphaA crystallin to actin.

PURPOSE: Quantitate the interaction of mutant (R116C) and wildtype human alphaA crystallins with actin. METHODS: AlphaA crystallins, expressed in a recombinant system, were purified, followed by passage through an actin affinity column. RESULTS: Binding of mutant alphaA crystallin was significantly less than binding of wildtype alphaA crystallin. CONCLUSIONS: The R116C mutation of alphaA crystallin found in human cataracts binds less to the cytoskeletal component actin. Since both alphaA crystallin and actin are necessary for proper development of the lens, decreased binding of the mutant protein to actin may perturb normal differentiation processes of lens cells which are necessary for transparency.

Transcytotic passage of albumin through lens epithelial cells.

PURPOSE: To characterize the transcytotic passage of albumin through lens epithelial cells. METHODS: N/N 1003A rabbit lens epithelial cells were grown to a confluent monolayer on porous filter supports (Transwell Corning, Inc., Corning, NY). Monolayers were exposed apically to Alexa 488-labeled albumin (Alexa 488-BSA) in the absence and presence of endocytic inhibitors (filipin; dansylcadaverine [DCV]). Transcytotic passage of albumin was monitored for 4 hours by quantitating fluorescence in the basolateral compartment. The mechanism of albumin passage was studied by labeling cell monolayers and cryosections of whole rat lenses for clathrin or caveolin. RESULTS: The monolayer of cells formed a barrier to the passage of albumin, as shown by the 44% reduction in albumin passage in comparison to nonseeded membranes. Treatment with filipin or DCV reduced the passage of Alexa 488-BSA through lens epithelial cells by 73% and 66%, respectively. Confocal microscopy showed that albumin passage was predominantly transcellular and demonstrated colocalization of albumin with caveolin-1 and clathrin in lens epithelial and fiber cells. CONCLUSIONS: The Transwell apparatus is an excellent system to monitor transport systems across cell monolayers. In this study, rabbit lens epithelial cells formed a confluent monolayer that acted as a barrier to the passive diffusion of albumin. The kinetics of albumin movement across the monolayer and the inhibitor pharmacology suggests that lens cells actively transport albumin from the apical to the basolateral compartment. The inhibitory profile suggests the involvement of caveolae and clathrin-coated vesicles in the transcytotic process.

Role of short-range protein interactions in lens opacifications.

At high protein concentrations found in the lens, short-range order of lens proteins results in a medium of relatively constant protein density and refractive index that minimizes scattering of light. During aging and cataractogenesis of the lens, formation of high molecular weight aggregates causes fluctuations in this protein density, resulting in light scattering and a concomitant decrease in transparency, with eventual lens opacification. This review summarizes what is known about the molecular nature of short-range order, both in the normal and cataractous lens, then hypothesizes that part of this order involves heterologous crystallin interactions that may be necessary for the maintenance of lens transparency. A summary of past and possible future experimental approaches will be reviewed that can be used to ascertain the existence of these interactions and their possible changes during lens opacification.

Decreased association of aged alpha crystallins with gamma crystallins.

Previous studies have demonstrated non-covalent interactions of alpha crystallins with gamma crystallins, under true equilibrium conditions. These interactions could affect short-range interactions of lens crystallins that are necessary for the transparent properties of the lens. Since the transparent properties of the lens decrease during aging, it is possible that there are corresponding changes in the ability of aged alpha crystallins to interact with gamma crystallins. In the following study, alpha crystallins were prepared from fetal and aged bovine lenses, then tested for binding to gamma crystallins using microequilibrium dialysis. The results demonstrate that during aging of the normal bovine lens, there is a decrease in the ability of alpha crystallins to bind to gamma crystallins, consistent with the involvement of this interaction in the transparent properties of the lens.

Expression of superoxide dismutase in whole lens prevents cataract formation.

PURPOSE: Oxidative damage is a major factor causing cataracts, which account for almost half of human blindness cases worldwide. In this study, we wished to determine if overexpression of superoxide dismutase (SOD) in intact lenses could prevent cataract formation induced by oxidative stress. METHODS: Fresh, intact lenses from 6-week-old male/female Sprague Dawley rats were incubated with plasmid DNA encoding the human SOD1 (Cu/Zn-SOD) gene at 37 degrees C in a CO2 cell culture chamber with 95% air and 5% CO2. SOD1 expression was determined by western blotting and SOD enzyme activity. Lenses with or without overexpression of SOD1 were treated with H2O2 and cataract formation was examined. SOD1 regulation of protein kinase Cgamma (PKCgamma) was determined by PKCgamma enzyme activity assay. Intact lens gap junctions were determined by dye transfer assay. RESULTS: In the lens overexpression system, SOD1 cDNA was fused to EYFP to generate EYFP:SOD1 fusion proteins which allow detection from endogenous SOD1. Incubation of intact lenses with plasmid DNA produced EYFP:SOD1 fusion proteins as determined by western blot using anti-GFP or anti-SOD1 antibodies. This caused significant increases in SOD enzyme activity. Data indicated that SOD1 plasmid DNA can be expressed as a functional enzyme in intact lenses in culture. Lenses overexpressing SOD1 remained clear after H2O2 treatment at 100 muM for 24 h, similar to control. Overexpression of SOD1 diminished the effect of H2O2 on PKCgamma activation and subsequent inhibition of gap junctions, indicating that overexpression of SOD1 may reduce reactive oxygen species (ROS) production, and this would prevent the normal H2O2 effect on cataract formation. CONCLUSIONS: Overexpression of SOD1 in whole lens prevents H2O2-induced oxidative damage (cataract formation) to the lens and subsequent control of gap junctions by protein kinase Cgamma.

Screening of crystallin-crystallin interactions using microequilibrium dialysis.

PURPOSE: It has been hypothesized that short-range, protein-protein interactions of crystallin are necessary for the maintenance of lens transparency. Because of their probable weak nature, it has been difficult to both detect and quantitate the nature of these interactions. To determine if interactions exist between alpha-crystallin and gamma-crystallin under true equilibrium conditions, we have used microequilibrium dialysis. METHODS: Total alpha-crystallin and gamma-crystallin were prepared from soluble proteins of fetal bovine lenses by HPLC and gel filtration chromatography. The proteins were added to one side of a microequilibrium dialysis cell, comprised of two chambers separated by a membrane with 100 kDa molecular weight cut-off. After reaching equilibrium, the amount of free gamma-crystallin and the amount of gamma-crystallin bound to alpha-crystallin was determined by HPLC and reverse phase analysis of both chambers. Selected gamma-crystallin that bound to alpha-crystallin was further purified by ion exchange chromatography, and then incubated with alpha-crystallin, to verify the specificity of their binding. RESULTS: Analysis of both microequilibrium dialysis chambers incubated at different times at 37 degrees C indicated that equilibrium was reached at 4 days. When total alpha-crystallin and gamma-crystallin were incubated for this time period, significant binding was observed between alpha-crystallin and the IIIA, II, and IVA species of gamma-crystallin. These interactions were confirmed by microequilibrium dialysis determinations containing alpha-crystallin and purified gamma-crystallin species. CONCLUSIONS: These results show that microequilibrium dialysis can be used to demonstrate significant noncovalent interactions of alpha-crystallin and gamma-crystallin under true equilibrium conditions.

Interaction of lens alpha and gamma crystallins during aging of the bovine lens.

Heterologous, noncovalent interactions of lens crystallins, such as between alpha and gamma crystallin, are thought to play a key role in the transparent properties of the lens. To determine possible interactions between these two types of crystallins, bovine gamma B crystallin in its native state was purified from whole fetal lenses or from the nucleus of aged bovine lenses, and the purified protein was passed over immobilized alpha crystallin, using a surface plasmon resonance instrument (BIAcore 3000) to obtain refractive units (RU) of gamma B binding at equilibrium. The results demonstrate low binding of gamma B crystallin purified from fetal lenses, but higher binding of the same gamma species purified from aged lenses. Together, these results demonstrate that under equilibrium conditions, gamma B crystallin from the aging bovine lens shows increased noncovalent associations with alpha crystallins, consistent with the possibility that such interactions play an important role in the transparent properties of the aged lens.

Probing alpha-crystallin structure using chemical cross-linkers and mass spectrometry.

PURPOSE: Alternatives to X-ray crystallographic techniques are needed to probe the structure of the hetero-oligomeric lens protein alpha-crystallin. We utilized mass spectrometry for 3 dimensional analysis (MS3D) to study the quaternary structural characteristics of this important lens protein and molecular chaperone. METHODS: We have employed two types of chemical cross-linkers to probe key protein-protein and protein-solvent interactions of alpha-crystallin using MS3D. Native alpha-crystallin was exposed to 3,3'-dithiobis[sulfosuccinimidyl propionate] (DTSSP) and the common fixative, formaldehyde. The reaction products were denatured and enriched in cross-linked and modified species using size exclusion chromatography. Tryptic digests of these fractions were purified using reverse phase HPLC and analyzed by both electrospray and matrix assisted laser desorption mass spectrometry. Comprehensive spectra for each C18 fraction were screened for ions with mass unique to each chemical treatment and candidate sequences matching the experimental data were assigned using MS3D "Links" and "ASAP" software. Selected ions were sequenced by collision induced dissociation. RESULTS: Peptides including residues 164-175 of alphaB-crystallin and residues 1-99 of alphaA-crystallin were modified by formaldehyde and partially hydrolyzed DTSSP. Peptides containing modified lysines 11, 78, and 99 of alphaA-crystallin were sequenced and the modified amino acids identified. In addition, ions corresponding to intramolecular and/or intermolecular cross-links were assigned a sequence based on two criteria. First, the mass values observed were unique to a single cross-linking experiment and were not present in a control where no cross-linker was utilized. Second, two unique ions detected from different cross-linking experiments were correlated in that the structures assigned to the masses were equivalent apart from the structure of the cross-linker. One such correlation was found involving lysine121, within the "highly conserved alpha-crystallin domain" of alphaB-crystallin, cross-linked to either lysine11 or lysine99 of alphaA-crystallin. Another two independent correlations involving lysine72 of alphaB-crystallin were found that indicate cross-linking of two subunits of alphaB-crystallin through this same residue. CONCLUSIONS: Sequences of peptides modified by partially hydrolyzed DTSSP and formaldehyde provide experimental evidence for models of alpha-crystallin quaternary structure that suggest a similar tertiary fold for both alphaA-crystallin and alphaB-crystallin. Analogous to multiple phosphorylations along the N-terminus of alphaB-crystallin, our data indicate that the same region of alphaA-crystallin, up to and including lysine99 is also relatively accessible to modification despite its hydrophobicity. Mass correlation between experiments using different reagents suggests that cross-linking occurred between N-termini of adjacent subunits of alphaB-crystallin in the native complex in support of the amphiphilic, toroidal, or "open micelle" models. In addition, multiple cross-links involving lysine121 of the so called "dimer interface" region within the "highly conserved alpha-crystallin domain" indicate that this region is a site of inter-subunit contacts in the native context.

Inhibition of gap junction activity through the release of the C1B domain of protein kinase Cgamma (PKCgamma) from 14-3-3: identification of PKCgamma-binding sites.

We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cgamma (PKCgamma)to the membrane and the phosphorylation of Cx43 by PKCgamma and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D. L., Wagner, L. M., Shinohara, T., and Takemoto, D. J. (2003) Exp. Eye Res. 76, 565-572; Lin, D., Boyle, D. L., and Takemoto, D. J. (2003) Investig. Ophthalmol. Vis. Sci. 44, 1160-1168). Gap junction activity in lens epithelial cells is regulated by PKCgamma-mediated phosphorylation of Cx43. PKCgamma activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKCgamma-C1B domain that are responsible for its interaction with 14-3-3epsilon. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKCgamma. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3epsilon, resulting in the release of endogenous cellular PKCgamma from 14-3-3epsilon, in vivo or in vitro, in activation of PKCgamma enzyme activity, phosphorylation of PKCgamma, in the subsequent translocation of PKCgamma to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 microM of C1B1 or C1B5 peptides also caused a 10- or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKCgamma are regulated by its binding to 14-3-3epsilon at the C1B domain of PKCgamma. Synthetic peptides corresponding to these regions of PKCgamma successfully competed for the binding of 14-3-3epsilon to endogenous PKCgamma, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.

Differentially expressed genes in the lens of mimecan-null mice.

PURPOSE: Members of the small leucine-rich proteoglycans (SLRP) gene family are essential for normal collagen fibrillogenesis in various connective tissues and important regulators of cellular growth, differentiation, and tissue repair. Mimecan is a member of this gene family and is expressed in many connective tissues. We have previously reported that knockout of the mouse mimecan gene results in abnormal collagen fibrillogenesis, mainly in the cornea and skin. During the course of our studies on biological roles of mimecan in the eye, we found that this gene is expressed in the mouse lens. Here, we sought to identify gene expression changes in the lens that are associated with the absence of mimecan. METHODS: Reverse transcription-polymerase chain reaction amplification (RT-PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used to determine mimecan expression in human and mouse eyes. Microarray hybridization was used to determine gene expression differences between lenses isolated from mimecan-null and wild type mice. Relative quantitative RT-PCR was used to verify the expression levels of a subset of the identified genes. RESULTS: By ISH and IHC, mimecan mRNA was detected in cornea and lens at embryonic day 16.5 (E16.5) and postnatal day 10 (P10) mouse eyes. By RT-PCR, mimecan mRNA was detected in human cornea, lens, iris, and retina. In mimecan-null mice lenses, microarray analysis of 5,002 mouse genes demonstrated a more than two fold increase in expression of 65 genes and a more than two fold decrease in expression of 76 genes. Among genes with increased expression were cell adhesion molecules, G-protein coupled receptors, intracellular signaling molecules, genes involved in protein biosynthesis and degradation, and genes involved in immune function. Decreased expression was found in extracellular matrix molecules, calcium binding and transporting proteins, and genes known for their roles in regulating cellular motility. Intriguingly, decreased gene expression was observed with two SLRP family members, biglycan and condroadherin, as well as with several stress-response proteins, including gammaA-crystallin, hemoglobin alpha 1, and metallothionein 1. Quantitative RT-PCR confirmed changes in expression of 12 genes selected from the arrays. CONCLUSIONS: In this report we present the first demonstration that mimecan is constitutively expressed in the vertebrate lens. The results from gene expression profiling reveal the ability of mimecan to influence expression of biglycan and chondroadherin, thereby indicating possible novel regulatory interactions between these SLRP family members. As with mimecan, the expression of chondroadrein in vertebrate lens has not been reported previously. Our results provide insight into the function of mimecan in the lens and enable further characterization of molecular mechanisms by which this protein exerts its biological roles.

In vivo passage of albumin from the aqueous humor into the lens.

PURPOSE: To determine if albumin, the major protein component of the aqueous humor, passes into the lens in vivo. METHODS: Rat albumin was covalently-labeled with Alexa 488 fluorophore, purified by gel permeation chromatography, then injected into the aqueous chamber of living rats. At 5 min postinjection, lenses were removed and analyzed by HPLC gel permeation chromatography, confocal microscopy, and immunogold electron microscopy. RESULTS: At 5 min postinjection, HPLC analysis detected measurable amounts of Alexa-labeled albumin in the lens. The results were confirmed by confocal microscopy, which showed passage into epithelial and cortical fiber cells. Immunogold electron microscopy using antibody to the Alexa fluorophore demonstrated intracellular location of the Alexa-albumin complex. CONCLUSIONS: In vivo, significant amounts of albumin pass from the aqueous chamber into cells of the lens, consistent with a possible physiological role for this process involving passage of metabolites into the lens.

Characterization of alphaA-crystallin from high molecular weight aggregates in the normal human lens.

PURPOSE: Lens alpha-crystallins, composed of two subunits of alphaA- and alphaB-crystallin, form low molecular weight (LMW) water soluble aggregates with an average molecular mass of approximately 800 kDa. In the intact lens, some of the alpha-crystallins are associated with even larger high-molecular-weight (HMW) aggregates which are thought to be precursors of components found in the water insoluble fraction. Although the mechanism of HMW aggregation and insolubilization are not known, the process is considered to be related to cataract formation. The purpose of the present study is to compare alphaA-crystallins from HMW and LMW forms in order to help understand the mechanism of insolubilization. METHODS: HMW and LMW alphaA-crystallins were isolated from lenses of 50 year old and 2 year old human donors and compared with respect to chaperone activity and fluorescence. We also analyzed isomerization and racemization of Asp-58 and Asp-151 residues in alphaA-crystallin from HMW and LMW fractions. RESULTS: The chaperone activity of HMW alphaA-crystallin was lower than that of LMW alphaA-crystallin. Fluorescence spectra indicated that HMW alphaA-crystallin was more hydrophobic than that of LMW. Isomerization of normal alpha-linkage to abnormal beta-linkage at both Asp-58 and Asp-151 residues markedly increased in HMW alphaA-crystallin compared with that of LMW alphaA-crystallin. The D/L ratio of beta-Asp-58 of either HMW or LMW forms were higher than 1.0, showing that inversion occurred in this site. In addition, the D/L ratio of the Asp-151 residue from HMW alphaA-crystallin was significantly lower than that of the LMW form. CONCLUSIONS: These results were qualitatively the same as those previously found in alphaA-crystallin from total proteins of cataractous versus normal lenses, suggesting that changes in the native structure of alphaA-crystallin associated with the HMW fraction of normal lenses reflect the same changes that occur to a greater degree in total proteins of human cataractous lens.

Morphological characterization of the Alpha A- and Alpha B-crystallin double knockout mouse lens.

BACKGROUND: One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alpha A and alpha B are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alpha A- and alpha B-crystallin genes (alpha A/BKO). METHODS: Lenses from 129SvEvTac mice and alpha A/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. RESULTS: Equatorial and axial (sagittal) dimensions of lenses for alpha A/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alpha A/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alpha A/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alpha A/BKO mice as compared to wild mice. CONCLUSION: These results indicated that both alpha A- and alpha B-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.

Translocation of macromolecules into whole rat lenses in culture.

PURPOSE: Little is known about the endocytosis and transcytosis of macromolecules into lens epithelium and fiber cells. The objective of this study was to determine if proteins (alpha-crystallins, beta-crystallins, and gamma-crystallins), carbohydrate (dextran), and plasmid DNA translocate from culture medium into these parts of the lens, with and without prior encapsulation into liposomes. METHODS: alpha-Crystallins, beta-crystallins, gamma-crystallins, and dextran were coupled with the fluorochrome Texas red, and plasmid DNA was labeled with propidium iodide. Adult rat lenses were incubated in medium containing one of these components with and without prior encapsulation of the macromolecule in commercially available liposomes (BioPORTER for alpha-crystallins, beta-crystallins, gamma-crystallins, and dextran; GenePORTER for plasmid DNA). Translocation of fluorescent macromolecule from the medium into the lens capsule, epithelium and fiber cells was monitored by confocal microscopy. RESULTS: alpha-Crystallins, beta-crystallins, gamma-crystallins, and dextran were present in the capsule, epithelium, and fiber cells after 5 h of incubation. Translocation of fluorescent protein macromolecules into the epithelium was greatly facilitated by encapsulation in BioPORTER liposomes. These macromolecules were localized within the cytoplasm of epithelium and fiber cells. Plasmid DNA was localized to the epithelium, but not the fiber cells. Prior encapsulation of plasmid DNA into GenePORTER liposomes did not increase the intensity of fluorescence localized in epithelium. Without encapsulation, plasmid DNA preferentially localized to the nuclei of epithelial cells, while after encapsulation, plasmid DNA preferentially localized to the cytoplasm. CONCLUSIONS: After incubation with cultured lenses, large macromolecules comprised of proteins and carbohydrates were localized within the cytoplasm of epithelial cells and fiber cells. Prior encapsulation of protein macromolecules into BioPORTER liposomes facilitated the translocation of macromolecules into the cytoplasm of epithelium. Incubation of lenses with plasmid DNA resulted in localization to the epithelium, but not fiber cells. Localization of plasmid DNA was not facilitated by prior encapsulation in GenePORTER. Encapsulated DNA preferentially localized to the cytoplasm of epithelial cells, while without encapsulation, plasmid DNA localizes to the nuclei of epithelial cells. Together, these studies demonstrate that macromolecules of potential biological importance can readily pass through the lens capsule into epithelial cells and in some cases transcytose through the epithelium into fiber cells of the cortex. Furthermore, these studies suggest that prior encapsulation of protein macromolecules may be a possible therapeutic delivery system of physiologically important macromolecules into the epithelium and/or fiber cells of the intact lens.