Phospholipid meets all-trans-retinal: the making of RPE bisretinoids

The lipid phase of the photoreceptor outer segment membrane is essential to the photon capturing and signaling functions of rhodopsin. Rearrangement of phospholipids in the bilayer accompanies the formation of the active intermediates of rhodopsin following photon absorption. Furthermore, evidence for the formation of a condensation product between the photolyzed chromophore all-trans-retinal and phosphatidylethanolamine indicates that phospholipid may also participate in the movement of the retinoid in the membrane. The downside of these interactions is the formation of bisretinoid-phosphatidylethanolamine compounds that accumulate in retinal pigment epithelial cells with age and that are particularly abundant in some retinal disorders. The propensity of these compounds to negatively impact on the cells has been linked to the pathogenesis of some retinal disorders including juvenile onset recessive Stargardt disease and age-related macular degeneration.     

Human enhancement and sexual dimorphism

I argue that the existence of sexual dimorphism poses a profound challenge to those philosophers who wish to deny the moral significance of the idea of 'normal human capacities' in debates about the ethics of human enhancement. The biological sex of a child will make a much greater difference to their life prospects than many of the genetic variations that the philosophical and bioethical literature has previously been concerned with. It seems, then, that bioethicists should have something to say about the choice between a male and a female embryo. Either, 1) parents have reason to choose boys over girls; (2) parents have reason to choose girls over boys; or, (3) parents have neither reason to choose girls over boys nor reason to choose boys over girls. Embracing either of the first two alternatives has strongly counterintuitive--and arguably morally repugnant--consequences. To motivate the third option we must either make reference to the idea of 'normal human capacities' or argue that parents should consider the interests of society when thinking about what sort of children they should bring into the world - an implication that should be extremely controversial in debates about the 'new eugenics'. I conclude, then, that the idea of 'normal human capacities' is properly crucial to reasoning about the ethics of shaping future persons.     

Taking down the FLAG! How insect cell expression challenges an established tag-system

In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system.     

A novel source of methylglyoxal and glyoxal in retina: implications for age-related macular degeneration

Aging of retinal pigment epithelial (RPE) cells of the eye is marked by accumulations of bisretinoid fluorophores; two of the compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer. These pigments are implicated in pathological mechanisms involved in some vision-threatening disorders including age-related macular degeneration (AMD). Studies have shown that bisretinoids are photosensitive compounds that undergo photooxidation and photodegradation when irradiated with short wavelength visible light. Utilizing ultra performance liquid chromatography (UPLC) with electrospray ionization mass spectrometry (ESI-MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyls glyoxal (GO) and methylglyoxal (MG), that are known to modify proteins by advanced glycation endproduct (AGE) formation. By extracellular trapping with aminoguanidine, we established that these oxo-aldehydes are released from irradiated A2E-containing RPE cells. Enzyme-linked immunosorbant assays (ELISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation. This AGE deposition was suppressed by prior treatment of the cells with aminoguanidine. AGE-modification causes structural and functional impairment of proteins. In chronic diseases such as diabetes and atherosclerosis, MG and GO modify proteins by non-enzymatic glycation and oxidation reactions. AGE-modified proteins are also components of drusen, the sub-RPE deposits that confer increased risk of AMD onset. These results indicate that photodegraded RPE bisretinoid is likely to be a previously unknown source of MG and GO in the eye.     

Aging and epigenetics: longitudinal changes in gene-specific DNA methylation

DNA methylation has been associated with age-related disease. Intra-individual changes in gene-specific DNA methylation over time in a community-based cohort has not been well described. We estimated the change in DNA methylation due to aging for nine genes in an elderly, community-dwelling cohort of men. Seven hundred and eighty four men from the Veterans Administration Normative Aging Study who were living in metropolitan Boston from 1999-2009 donated a blood sample for DNA methylation analysis at clinical examinations repeated at approximately 3-5 year intervals. We used mixed effects regression models. Aging was significantly associated with decreased methylation of GCR, iNOS and TLR2 and with increased methylation of IFNγ, F3, CRAT and OGG. Obstructive pulmonary disease at baseline modified the effect of aging on methylation of IFNγ (interaction p = 0.04). For participants who had obstructive pulmonary disease at their baseline visit, the rate of change of methylation of IFNγ was -0.05% 5-methyl-cytosine (5-mC) per year (95% CI: -0.22, 0.13), but was 0.14% 5-mC per year (95% CI: 0.05, 0.24) for those without this condition. Models with random slopes indicated significant heterogeneity in the effect of aging on methylation of GCR, iNOS and OGG. These findings suggest that DNA methylation may reflect differential biological aging.     

Gene promoter methylation is associated with lung function in the elderly: The normative aging study

Lung function is a strong predictor of mortality. While inflammatory markers have been associated with lung function decrease, pathways are still poorly understood and epigenetic changes may participate in lung function decline mechanisms. We studied the cross-sectional association between DNA methylation in nine inflammatory genes and lung function in a cohort of 756 elderly men living in the metropolitan area of Boston. Participants donated a blood sample for DNA methylation analysis and underwent spirometry at each visit every 3 to 5 y from 1999–2006. We used separate multivariate mixed effects regression models to study the association between each lung function measurement and DNA methylation within each gene. Decreased CRAT, F3 and TLR2 methylation was significantly associated with lower lung function. One interquartile range (IQR) decrease in DNA methylation was associated with lower forced vital capacity (FVC) and forced expiratory volume in one second (FEV₁), respectively by 2.94% (p < 10⁻⁴) and 2.47% (p < 10⁻³) for F3 and by 2.10% (p < 10⁻²) and 2.42% (p < 10⁻³) for TLR2. Decreased IFNγ and IL6 methylation was significantly associated with better lung function. One IQR decrease in DNA methylation was associated with higher FEV₁ by 1.75% (p = 0.02) and 1.67% (p = 0.05) for IFNγ and IL6, respectively. These data demonstrate that DNA methylation may be part of the biological processes underlying the lung function decline and that IFNγ and IL6 may have ambivalent roles through activation of negative feedback.Key words: DNA methylation, genes, spirometry, FEV₁, lungs, TLR2, F3, INOS, GCR, OGG1     

Post-translational processing of the amino terminus affects actin function

We have studied the importance of N-terminal processing for normal actin function using the Drosophila Act88F actin gene transcribed and translated in vitro. Despite having different charges as determined by two-dimensional (2D) gel electrophoresis, Act88F expressed in vivo and in vitro in rabbit reticulocyte lysate bind to DNase I with equal affinity and are able to copolymerise with bulk rabbit actin equally well. Using peptide mapping and thin-layer electrophoresis we have shown that bestatin [( 3-amino-2-hydroxy-4-phenyl-butanoyl]-L-leucine), an inhibitor of aminopeptidases, can inhibit actin N-terminal processing in rabbit reticulocyte lysate. Although processed and unprocessed actins translated in vitro are able to bind to DNase I equally well, unprocessed actins are less able to copolymerise with bulk actins. This effect is more pronounced when bulk rabbit actin is used but is still seen with bulk Lethocerus actin. Also, the unprocessed actins reduce the polymerisation of the processed actin translated in vitro with the bulk rabbit actin. This suggests that individual actins do interact, even in non-polymerising conditions. The reduced ability of unprocessed actin to polymerise shows that correct post-translational modification of the N terminus is required for normal actin function.     

Apolipoprotein E: phospholipid binding studies with synthetic peptides containing the putative receptor binding region

To define the lipid and receptor binding regions of apolipoprotein E (apoE), we have synthesized four peptides beginning at residue 169 and continuing through the putative receptor binding region and ending at residue 129 so as to include a proposed lipid binding domain. The peptides were synthesized by solid-phase techniques, cleaved with anhydrous HF, and purified by ion-exchange and semipreparative reversed-phase high-performance liquid chromatography (HPLC). The peptides had the correct amino acid composition and were greater than 99% pure by analytical reversed-phase HPLC. The circular dichroic spectrum of each peptide was recorded before and after mixing with dimyristoylphosphatidylcholine. With apoE (148-169), apoE (144-169), and apoE (139-169), no changes were observed in the ellipticity at 222 nm. However, with apoE (129-169), an increase in alpha-helicity to approximately 42% was observed. Density gradient ultracentrifugation of the lipid-peptide mixture permitted isolation of a complex with apoE (129-169) with a molar ratio of lipid to peptide of 125:1, which was stable to recentrifugation. The alpha-helicity of the peptide in the complex was estimated to be 56%. No complexes were isolated from the gradients of the shorter peptides. Therefore, we conclude that the amphipathic helix formed by residues 130-150 contains one of the lipid binding regions of apoE.