Immuno-spin trapping of protein and DNA radicals: “Tagging” free radicals to locate and understand the redox process

Biomolecule-centered radicals are intermediate species produced during both reversible (redox modulation) and irreversible (oxidative stress) oxidative modification of biomolecules. These oxidative processes must be studied in situ and in real time to understand the molecular mechanism of cell adaptation or death in response to changes in the extracellular environment. In this regard, we have developed and validated immuno-spin trapping to tag the redox process, tracing the oxidatively generated modification of biomolecules, in situ and in real time, by detecting protein- and DNA-centered radicals. The purpose of this methods article is to introduce and update the basic methods and applications of immuno-spin trapping for the study of redox biochemistry in oxidative stress and redox regulation. We describe in detail the production, detection, and location of protein and DNA radicals in biochemical systems, cells, and tissues, and in the whole animal as well, by using immuno-spin trapping with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide.     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

8-Oxoguanine DNA glycosylase (Ogg1) causes a transcriptional inactivation of damaged DNA in the absence of functional Cockayne syndrome B (Csb) protein

We have analysed the effect of oxidative guanine lesions on the expression of a transfected reporter gene in mouse embryonic fibroblasts deficient in Cockayne syndrome B protein (Csb) and/or the 8-oxoguanine DNA glycosylase (Ogg1). We used a highly sensitive flow cytometry-based approach and quantitative real-time PCR to measure the changes in gene expression caused by the presence of oxidised guanine residues generated by photosensitisation in the vector DNA. In wild-type cells, small numbers (one or three) of oxidised guanines did not affect gene expression at short times after transfections, whereas progressive reduction of the transgene expression was observed at later time points. Although Ogg1 has a major contribution to the repair of oxidised guanine bases, its absence did not have a strong effect on the gene expression. In contrast, the lack of functional Csb protein caused a pronounced inactivation of the damaged reporter gene. Most strikingly, an additional Ogg1 deficiency significantly attenuated this effect. The results indicate that the processing of oxidative guanine modifications by Ogg1 can mediate host cell inactivation rather than reactivation of the damaged genes and that this effect is strongly enhanced in the absence of Csb.     

Defensin promoters as potential tools for engineering disease resistance in cereal grains

Engineering of plant protection in cereals requires well characterized tissue-specific and wounding/pathogen-inducible promoters for targeted expression of pathogen responsive and resistance genes. We describe the isolation of seven wheat and rice defensin genes expressed in early developing grain and during grain germination, two developmental stages that are particularly vulnerable to pathogens and insects. Comparison of three-dimensional (3D) models of these rice and wheat PRPI defensins indicated variations in spatial architectures that could reflect their functional diversities. Wheat and rice were stably transformed with promoter–GUS fusion constructs and the spatial and temporal activities of four promoters were studied using whole-mount and histological assays. PRPI promoters were active before and at anthesis in both transgenic wheat and rice with activity mainly in the ovary. In rice, GUS activity was also observed in vascular tissue of the lemma, palea and anthers. After fertilization, GUS was strongly expressed in the outer cell layers of the pericarp and in the main vascular bundle of the grain. During, and a short time after, seed germination, wheat promoters were active in transgenic rice embryos, roots and/or coleoptiles. All wheat and rice promoters were strongly induced by wounding in leaf, stem and grain of transgenic rice plants. These results suggest that PRPI promoters will be useful for specific targeting and accumulation of proteins conferring resistance to pathogens in vulnerable tissues of developing and germinating grain.     

Cytotoxicity of novel derivatives of the spin trap EMPO

Free radicals are involved in different regulatory and pathological processes. The formation of superoxide in living cells or whole organisms is of major interest. ESR spin trapping allows identification of the radicals if proper spin traps are available. Our study investigates the toxicity of novel derivatives of the spin trap EMPO to cultured human lung carcinoma cells (A549), breast carcinoma cells (SKBR3), colon carcinoma cells (SW480) as well as to human fibroblasts (F2000). A dose-dependent decrease of the cell number was observed for all spin traps. At 100mM BuMPO, t-BuMPO and s-BuMPO caused pronounced cell loss (>90%) and increased LDH-release, while DEPMPO, EMPO, PrMPO and i-PrMPO caused only moderate cell loss (<60%) without any effect on the LDH-release after 24h. At 10mM and 50mM the latter agents even decreased LDH-release. 10mM and 50mM of i-PrMPO as well as 10mM PrMPO increased intracellular GSH content acting like antioxidants, whereas 50mM s-BuMPO and PrMPO decreased GSH content by 67% and 38%, respectively. Staining for apoptotic nuclei did not reveal any differences between controls and treated cultures indicating necrotic cell death possibly due to membrane toxicity. The following toxicity ranking was obtained: t-BuMPO>BuMPO>s-BuMPO>PrMPO>i-PrMPO approximately DEPMPO approximately EMPO. The least toxic compounds were DEPMPO (LD(50)=143 mM for SW480, 117 mM for A549 or 277 mM for F2000) and i-PrMPO (LD(50)=114 mM for SKBR3), the most toxic one was t-BuMPO (LD(50)=5-6mM for all cell types). In conclusion, up to 50mM i-PrMPO (t(1/2)=18.8 min) and up to 10 mM s-BuMPO (t(1/2)=26.3 min) can be recommended for further investigation of superoxide in biological systems.     

Infection with the Lyme disease pathogen suppresses innate immunity in mice with diet‐induced obesity

Obesity is a major global public health concern. Immune responses implicated in obesity also control certain infections. We investigated the effects of high‐fat diet‐induced obesity (DIO) on infection with the Lyme disease bacterium Borrelia burgdorferi in mice. DIO was associated with systemic suppression of neutrophil‐ and macrophage‐based innate immune responses. These included bacterial uptake and cytokine production, and systemic, progressive impairment of bacterial clearance, and increased carditis severity. B. burgdorferi‐infected mice fed normal diet also gained weight at the same rate as uninfected mice fed high‐fat diet, toll‐like receptor 4 deficiency rescued bacterial clearance defects, which greater in female than male mice, and killing of an unrelated bacterium (Escherichia coli) by bone marrow‐derived macrophages from obese, B. burgdorferi‐infected mice was also affected. Importantly, innate immune suppression increased with infection duration and depended on cooperative and synergistic interactions between DIO and B. burgdorferi infection. Thus, obesity and B. burgdorferi infection cooperatively and progressively suppressed innate immunity in mice.     

Effect of age on high-fat diet-induced hypertension

Aging and obesity both have a significant impact on central blood pressure (BP) regulation, and previous studies indicated that changes in central redox signaling with age may affect high-fat (HF) diet-induced cardiovascular responses. Therefore, we investigated the effects of 60% HF feeding on BP regulation in young adult (5 mo) and old (26 mo) Fischer-344 × Brown-Norway rats. Radiotelemetric transmitters were implanted to measure BP, heart rate (HR), locomotor activity, and spontaneous baroreflex sensitivity. Expression and activity of NADPH oxidase and ANG II type 1 receptor were assessed in the hypothalamus and in the nucleus tractus solitarii. Old animals gained more weight on HF diet compared with young, whereas central NADPH oxidase expression and activity elevated similarly in the two age groups. After an initial hypotensive and tachycardic response during the first week of HF feeding, BP in young animals increased and became significantly elevated after 6 wk of HF feeding. In contrast, BP in old animals remained depressed. Nighttime HR and locomotor activity decreased in both young and old rats fed with HF diet, but these changes were more significant in young rats. As a result, amplitudes of circadian variation of BP, HR, and activity that were originally higher in young rats declined significantly and became similar in the two age groups. In conclusion, our experiments led to the surprising finding that HF diet has a more serious impact on cardiovascular regulation in young animals compared with old.     

Recognition and specificity determinants of the human cbx chromodomains

The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1-8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1-8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity.