Donita Garland

Role of site-specific, metal-catalyzed oxidation in lens aging and cataract: A hypothesis

The evidence reviewed here supports the hypothesis that metal catalyzed oxidation reactions occur in the lens and may make a significant contribution to the changes seen in the lens with age and in cataract formation. The major support for this hypothesis is as follows. (1) All of the components of the non-enzymic metal catalyzed oxidation systems are present in the lens normally. Ascorbate, glutathione and oxygen are present in much lower concentrations. Although, even at low concentrations, the reactions could occur over many years with significant consequences. Components of some of the enzymic systems are also present, although primarily in the epithelial layer and outer cortical region. Copper and iron levels may be increased in some cataracts. (2) Protein carbonyl derivatives are increased in both aging and cataractous lenses. Amino acid-derived protein carbonyl derivatives have only been demonstrated in oxidative reactions derived from oxygen radical generation, particularly those catalyzed by metal-catalyzed oxidation systems. (3) Treatment of isolated bovine crystallins with metal catalyzed oxidation systems generates modifications similar to those found in vivo. The proposed mechanism of site-specific metal catalyzed oxidation appears to be a feasible mechanism of oxidation in the lens, and verification of the mechanism requires further study. Although the focus of this manuscript has been on the oxidative modification induced in proteins,m oxidative damage to DNA or membrane resulting from similar mechanisms may also play an important role in alteration of lens function during aging and cataractogenesis.

Structural changes in bovine lens crystallins induced by ascorbate, metal, and oxygen

Ascorbate, Fe3+, or Cu2+ and oxygen induced the oxidation of bovine lens crystallins. The modifications mimicked those that occur in the lens with aging. The modifications included the formation of nondisulfide crosslinks in alpha- and beta H-crystallin and the cleavage of alpha-, beta H-, and the low molecular weight crystallin fractions. In all three fractions, there was a loss of the more basic protein species and an increase in the more acidic species. Nontryptophan fluorescence with emission spectra between 400 and 500 nm was produced in beta H-crystallin. Cu2+ was less effective than Fe3+ in catalyzing the modification of beta H- and gamma-crystallin. Both metal ions were equally effective in catalyzing the modification of alpha-crystallin.

The effects of "oxygen radicals" generated in the medium on lenses in organ culture: inhibition of damage by chelated iron.

Rat lenses in organ culture were exposed to activated species of oxygen generated in the culture medium either by xanthine oxidase and hypoxanthine or by riboflavin and visible light, two systems which have been shown to produce superoxide and H2O2. In each case there was marked damage to carrier-mediated transport systems of the lens. Under standard culture conditions this damage was strongly inhibited by catalase, but not by superoxide dismutase (SOD). By the addition to the medium of chelated iron, hydroxyl radicals were produced in a Fenton reaction with a concomitant decrease in H2O2 levels. With both oxygen radical-generating systems, the addition of chelated iron strongly inhibited lens damage. This inhibitory effect could be reversed by the addition of SOD with the chelated iron. Under such conditions SOD converts superoxide anion to H2O2, thereby preventing reduction of the chelated iron and thus stopping the generation of hydroxyl radicals. Increased lens damage following addition of SOD to the iron-containing systems correlated with higher H2O2 concentrations, and was inhibited by catalase. These findings suggest that, when generated in the fluids surrounding the lens, H2O2 poses a much greater oxidative stress for the lens than do the superoxide or hydroxyl free radicals.

Guidelines for reporting the use of gel electrophoresis in proteomics

Gibson, Frank Anderson, Leigh Babnigg, Gyorgy Baker, Mark Berth, Matthias Binz, Pierre-Alain Borthwick, Andy Cash, Phil Day, Billy W. Friedman, David B. Garland, Donita Gutstein, Howard B. Hoogland, Christine Jones, Neil A. Khan, Alamgir Klose, Joachim Lamond, Angus I. Lemkin, Peter F. Lilley, Kathryn S. Minden, Jonathan Morris, Nicholas J. Paton, Norman W. Pisano, Michael R. Prime, John E. Rabilloud, Thierry Stead, David A. Taylor, Chris F. Voshol, Hans Wipat, Anil Jones, Andrew R. 2 NATURE PUBLISHING GROUP NEW YORK 335WX

ζ-Crystallin is a major protein in the lens of Camelus dromedarius

Camel (Camelus dromedarius) lenses contain a protein with an apparent subunit Mr 38,000 that constitutes approximately 8-13% of the total protein. The protein has been purified and has a native Mr 140,000 as determined by gel filtration. This is consistent with its being a tetramer. The protein reacts with antibodies raised against both guinea pig zeta-crystallin and peptides corresponding to amino acids 1-10 and 295-308, but not to antibodies raised against amino acids 320-328 of zeta-crystallin. Based on these criteria it is concluded that this protein, which is a major constituent of camel lens, is zeta-crystallin. This may be the first example of a protein (enzyme) being independently utilized as a crystallin in the lens of species from two mammalian orders.

Tumor specific phage particles promote tumor regression in a mouse melanoma model

Within cancer research, phage display libraries have been widely used for the identification of tumor targeting peptides and antibodies. Additionally, phages are known to be highly immunogenic; therefore we evaluated the immunotherapeutic potential of tumor specific phages to treat established solid tumors in a mouse model of melanoma. We developed two tumor specific phages, one derived from a peptide phage display library and one Fab expressing phage with known specificity, for the treatment of mice bearing palpable B16-F10 or B16/A2Kb tumors. Therapy in B16-F10 tumor bearing mice with tumor specific phages was superior to treatment with non-tumor specific phages and lead to delayed tumor growth and increased survival. In B16/A2Kb tumor bearing mice, therapy with tumor specific phages resulted in complete tumor regression and long-term survival in 50% of the mice. Histological analysis of tumors undergoing treatment with tumor specific phages revealed that phage administration induced a massive infiltration of polymorphonuclear neutrophils. Furthermore, phages induced secretion of IL-12 (p70) and IFN-γ as measured in mouse splenocyte culture supernatants. These results demonstrate a novel, immunotherapeutic cancer treatment showing that tumor specific phages can promote regression of established tumors by recruitment of inflammatory cells and induction of Th1 cytokines.

The Oxidative Modification of Lens Proteins

In summary, in vitro oxidation of lens crystallins mimics many of the post-translational modifications observed with age and in cataracts These results lend further support to earlier proposals that oxidation is a key factor in cataract formation. The extent to which ascorbate contributes to the oxidation reactions in vivo is not known. In addition to the data presented here that ascorbate can produce these effects in vitro, other observations support the possibility that under certain conditions ascorbate may be involved in the generation of reactive oxygen species in the lens. Using electron spin resonance the ascorbyl radical can be detected early in nuclear cataract formation, and there appears to be a decrease in total ascorbic acid (reduced and oxidized), suggesting further oxidation of ascorbate. Iron and copper are both present in mammalian lenses, and there are reports of increased copper levels in the lens with age and in cataracts. Increased metal ion concentrations would facilitate these oxidative processes.

Betaine-homocysteine Methyltransferase Is a Developmentally Regulated Enzyme Crystallin in Rhesus Monkey Lens

We describe herein the characterization of a major 45-kDa protein from the soluble βH-crystallin fraction of rhesus monkey (Macaca mulatta) lens. Based on partial peptide sequence, immunoreactivity, and enzymatic activity, this protein has been identified as betaine-homocysteineS-methyltransferase (BHMT: EC 2.1.1.5), an enzyme that catalyzes the methylation of homocysteine using either betaine or thetins as methyl donors. This protein was found to be expressed abundantly in the nuclear region of the monkey lens, reaching ∼10% of the total nuclear protein, but was barely detectable in the epithelium and cortex regions of the lens. Because the nucleus represents the early embryonic and fetal stages of lens development, we infer that BHMT expression in the lens of the eye is developmentally regulated. By virtue of its high abundance, BHMT can be considered an enzyme crystallin (ψ-crystallin). This is the first enzyme crystallin to be found in primate lenses.

Two-dimensional gel electrophoretic analysis of human lens proteins

Human lens proteins from clear lenses were separated and identified using two-dimensional polyacrylamide electrophoresis. Isoelectric focusing, both equilibrium and non-equilibrium, was performed in the first dimension and SDS electrophoresis in the second dimension. Proteins were identified by Western blotting and sequencing techniques and by comparison with patterns obtained with purified crystallin fractions. Analyses were performed on total urea soluble proteins of lenses varying in age from fetal to 73 yr. Several hundred protein spots representing crystallins, cytoskeletal proteins and enzymes were resolved in the fetal lens. In the older lenses there was a dramatic increase in the number of protein species in the molecular weight range of the crystallins and a reduced number of discrete protein species visible at molecular weights greater than 50,000. Conversely, a number of proteins below approximately 15 kDa were visible even in the fetal lens. The number and amount of polypeptides in this molecular weight range were increased in the older lenses. Many of these low molecular weight species could be assigned to either the alpha-, beta- or gamma-crystallin fractions. An age dependent increase in the number of acidic species of both crystallins and other proteins, such as, glyceraldehyde 3-phosphate dehydrogenase was observed as well as the loss or mobility change of gamma-crystallin. Two-dimensional gel electrophoresis provides a sensitive and practical technique for characterizing all of the proteins of the human lens.

Mouse genetics and proteomic analyses demonstrate a critical role for complement in a model of DHRD/ML, an inherited macular degeneration

Macular degenerations, inherited and age related, are important causes of vision loss. Human genetic studies have suggested perturbation of the complement system is important in the pathogenesis of age-related macular degeneration. The mechanisms underlying the involvement of the complement system are not understood, although complement and inflammation have been implicated in drusen formation. Drusen are an early clinical hallmark of inherited and age-related forms of macular degeneration. We studied one of the earliest stages of macular degeneration which precedes and leads to the formation of drusen, i.e. the formation of basal deposits. The studies were done using a mouse model of the inherited macular dystrophy Doyne Honeycomb Retinal Dystrophy/Malattia Leventinese (DHRD/ML) which is caused by a p.Arg345Trp mutation in EFEMP1. The hallmark of DHRD/ML is the formation of drusen at an early age, and gene targeted Efemp1(R345W/R345W) mice develop extensive basal deposits. Proteomic analyses of Bruchs membrane/choroid and Bruchs membrane in the Efemp1(R345W/R345W) mice indicate that the basal deposits comprise normal extracellular matrix (ECM) components present in abnormal amounts. The proteomic analyses also identified significant changes in proteins with immune-related function, including complement components, in the diseased tissue samples. Genetic ablation of the complement response via generation of Efemp1(R345W/R345W):C3(-/-) double-mutant mice inhibited the formation of basal deposits. The results demonstrate a critical role for the complement system in basal deposit formation, and suggest that complement-mediated recognition of abnormal ECM may participate in basal deposit formation in DHRD/ML and perhaps other macular degenerations.