Intense visible light can damage retinal photoreceptor cells by photochemical or thermal processes, leading to cell death. The precise mechanism of light-induced damage is unknown; however, oxidative stress is thought to be involved, based on the protective effect of antioxidants on the light-exposed retina. To explore the in vivo effects of light on retinal DNA, rats were exposed to intense visible light for up to 24 h and the time courses of single-strand breaks in restriction fragments containing the opsin, insulin 1 and interleukin-6 genes were measured. All three gene fragments displayed increasing single-strand modifications with increasing light exposure. Treatment with the antioxidant dimethylthiourea prior to light exposure delayed the development of net damage. The time course of double-strand DNA damage was also examined in specific genes and in repetitive DNA. The appearance of discrete 140-200 base-pair DNA fragments after 20 h of light exposure implicated a nonrandom, possibly enzymatic damaging mechanism. The generation of nucleosome core-sized DNA fragments, in conjunction with single-strand breaks, suggests two phases of light-induced retinal damage, with random attack on DNA by activated oxygen species preceding enzymatic degradation.
Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro- and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so-called Prx6-type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady-state kinetics with tert-butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four- to six-fold in vitro. Stopped-flow kinetics with reduced PfPrx6 , PfPrx6 , and PfPrx6 revealed a preference for H O as an oxidant with second order rate constants for H O and tBuOOH around 2.5 × 10 M s and 3 × 10 M s , respectively. Differences between the oxidation kinetics of PfPrx6 , PfPrx6 , and PfPrx6 were observed during a slower second-reaction phase. Our kinetic data support the interpretation that the reductive half-reaction is the rate-limiting step for PfPrx6 catalysis in steady-state measurements. Whether the increased activity of PfPrx6 is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6-type enzymes is non-essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain-of-function mutant PfPrx6 might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.
Mia40/CHCHD4 and Erv1/ALR are essential for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals. In contrast, many protists, including important apicomplexan and kinetoplastid parasites, lack Mia40. Furthermore, the Erv homolog of the model parasite Leishmania tarentolae (LtErv) was shown to be incompatible with Saccharomyces cerevisiae Mia40 (ScMia40). Here we addressed structure-function relationships of ScErv1 and LtErv as well as their compatibility with the oxidative protein folding system in yeast using chimeric, truncated, and mutant Erv constructs. Chimeras between the N-terminal arm of ScErv1 and a variety of truncated LtErv constructs were able to rescue yeast cells that lack ScErv1. Yeast cells were also viable when only a single cysteine residue was replaced in LtErvC17S. Thus, the presence and position of the C-terminal arm and the kinetoplastida-specific second (KISS) domain of LtErv did not interfere with its functionality in the yeast system, whereas a relatively conserved cysteine residue before the flavodomain rendered LtErv incompatible with ScMia40. The question whether parasite Erv homologs might also exert the function of Mia40 was addressed in another set of complementation assays. However, neither the KISS domain nor other truncated or mutant LtErv constructs were able to rescue yeast cells that lack ScMia40. The general relevance of Erv and its candidate substrate small Tim1 was analyzed for the related parasite L. infantum. Repeated unsuccessful knockout attempts suggest that both genes are essential in this human pathogen and underline the potential of mitochondrial protein import pathways for future intervention strategies.
Psychosocial issues have been recognized as important factors in children's health for decades. This study documents the relation among several important psychosocial variables (e.g., mothers' depressive symptoms) and a new instrument that assesses parents' perception of their communities' social capital. Mothers were recruited from their children's primary care (PC) pediatricians' offices within the Southwestern Ohio Ambulatory Research Network or from a children's hospital developmental clinic (DC). Mothers completed a questionnaire that included the Social Capital Scale (SCS), Children with Special Health Care Needs Screener (CSHCNS), Pediatric Quality of Life Inventory, Maternal Social Support Index and the Center for Epidemiologic Studies Depression Scale (CES-D). Mothers were sorted into three subgroups based on site of recruitment (PC or DC) and results of the CSHCNS. The sample (N = 620) was also sorted into terciles based on SCS scores. Mean SCS was about 73 for each of the three subgroups. Compared to mothers in the highest SCS tercile, mothers in the lowest SCS tercile reported lower education, lower income and higher CES-D median scores. The SCS subscale "sense of belonging" had an inverse correlation with CES-D scores (r = -.248, p < 0.001). Mothers from primary care and sub-specialty clinics had similar perceptions about their communities' social capital. Compared to mothers in the highest one third of SCS scores, mothers in the lowest one third were more likely to report less education and income as well as more depressive symptoms. A decreased sense of belonging in their communities was also correlated with more depressive symptoms. The SCS is a new useful tool for investigators and clinicians who work with children and their families.
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