Crystallization and preliminary crystallographic analysis of the C-terminal domain of MamM, a magnetosome-associated protein from Magnetospirillum gryphiswaldense MSR-1.

MamM is a unique magnetosome-associated protein that shares substantial homology with cation diffusion facilitator (CDF) proteins, a group of heavy-metal-ion efflux transporters that participate in metal-ion homeostasis in all domains of life. Magnetotactic bacteria utilize CDF proteins in iron-oxide biomineralization and in magnetosome formation. Here, the crystallization and preliminary X-ray analysis of recombinant Magnetospirillum gryphiswaldense MamM is reported. The C-terminal domain of MamM was crystallized in the orthorhombic space group C222(1), with unit-cell parameters a = 37.1, b = 94.0, c = 53.3 Å. X-ray diffraction data were collected to a resolution of 2.0 Å.

5-Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA.

The known functions of folate are to support one-carbon metabolism and to serve as photoreceptors for cryptochromes and photolyases. We demonstrate that 5-methyltetrahydrofolate (5-MTHF, the predominant folate in plasma) is also a potent, near diffusion limited, scavenger of singlet oxygen and quencher of excited photosensitizers. Both pathways result in decomposition of 5-MTHF, although ascorbate can protect against this loss. In the absence of photosensitizers, 5-MTHF is directly decomposed only very slowly by UVA or UVB. Although synthetic folic acid can promote DNA damage by UVA, submicromolar 5-MTHF inhibits photosensitization-induced strand breaks. These observations suggest a new role for reduced folate in protection from ultraviolet damage and have bearing on the hypothesis that folate photodegradation influenced the evolution of human skin color.

A simple assay for frequency of chromosome breaks and loss (micronuclei) by flow cytometry of human reticulocytes.

Exposure to environmental stress, such as radiation, poor nutrition, or smoking, can cause hazardous lesions in DNA, including double-strand breaks. In red blood cells, a DNA fragment or lagging chromosome forms a micronucleus when left behind after the main nucleus is extruded to form the mature reticulocyte during erythropoiesis. Reticulocytes with micronuclei in human peripheral blood are not generally available for analysis because the spleen removes aberrant cells. We have developed a simple and rapid method to isolate and analyze immature reticulocytes in the peripheral blood for the presence of micronuclei before these cells are removed by the spleen. This method applies single-laser flow-cytometry to measure micronuclei in an enriched transferrin-positive reticulocyte population. Abramsson-Zetterberg et al. (Abramsson-Zetterberg, L., Zetterberg, G., Bergqvist, M., and Grawe, J. Environ. Mol. Mutagen. 36, 22-31, 2000) have described a method to measure micronuclei in an enriched reticulocyte population using a dual-laser flow cytometry. Unlike the beads used in their magnetic-immunoseparation procedures, the beads used in this study do not require a prelabeling step and are compatible with the flow cell, sparing the need to release the cells from the beads and avoiding the potentially confounding DNase-treatment step. Dertinger et al. (Dertinger, S. D., Torous, D. K., Hall, N. E., Murante, F. G., Gleason, S. E., Miller, R. K., and Tometsko, C. R. Mutat. Res. 515, 3-14, 2002; Dertinger, S. D., Chen, Y., Miller, R. K., Brewer, K. J., Smudzin, T., Torous, D. K., Hall, N. E., Olvany, K. A., Murante, F. G., and Tometsko, C. R. (2003) Mutat. Res. 542, 77-87, 2003) further improved the scoring of micronuclei to enable the use of bench-top instruments in analyzing samples of unenriched reticulocyte-populations. The present method is distinct from flow cytometric assays, such as reported by Dertinger et al., which enable scoring of limited numbers of reticulocytes per sample and require lengthy data acquisition times. We assessed DNA damage in smokers using this novel flow-cytometry based micronuclei-assay. The results show that this assay can effectively detect micronuclei in human blood samples. This method, unlike available micronuclei assays, allows rapid evaluation of a large number of cells and therefore should prove to be useful in monitoring of human populations for genetic damage.

Measuring chromosome breaks in patients with thalassemia.

Iron-mediated oxidative stress plays an important role in the pathophysiology of thalassemia. Oxidative stress can cause lesions in DNA, including double-strand breaks. DNA damage, which is a cause of cancer (although not the only one), is recognized as deleterious. Unlike cancer, DNA damage can be assayed easily and relatively inexpensively in humans. In this study, a sensitive micronucleus assay was used to measure the frequency of chromosomal breaks in patients with alpha- and beta-thalassemia. The micronucleus test is based on the observation that a secondary nucleus (micronucleus) is formed around a chromosomal fragment, outside the main nucleus of a dividing cell. Micronuclei are readily apparent in red blood cells (RBCs), which otherwise lack DNA. We combined an immunomagnetic separation technique with single-laser flow cytometry to isolate and analyze reticulocytes in peripheral blood for the presence of micronuclei before these cells are removed by the spleen. Blood samples were obtained from patients with thalassemia and healthy volunteers. After immunomagnetic enrichment of CD71-positive reticulocytes, the cells were stained for micronuclei using the DNA dye 7-aminoactinomycin D (7-AAD) and evaluated by flow cytometry. Our findings indicate that higher levels of micronuclei frequencies are present in thalassemic RBCs.

Nitroxides inhibit peroxyl radical-mediated DNA scission and enzyme inactivation.

Nitroxides are cell-permeable stable radicals that protect biomolecules from oxidative damage in several ways. The mechanisms of protection studied to date include removal of superoxide radicals as SOD-mimics, oxidation of transition metal ions to preempt the Fenton reaction, and scavenging carbon-centered radicals. However, there is no agreement regarding the reaction of piperidine nitroxides with peroxyl radicals. The question of whether they can protect by scavenging peroxyl radicals is important because these radicals are formed in the presence of oxygen abundant in biological tissues. To further our understanding of the antioxidative behavior of piperidine nitroxides, we studied their effect on biochemical systems exposed to the water soluble radical initiator 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). AAPH thermally decomposes to yield tert-amidinopropane radicals (t-AP(*)) that readily react with oxygen to form peroxyl radicals (t-APOO(*)). It has recently been reported that piperidine nitroxides protect plasmid DNA from t-AP(*) though not from t-APOO(*). The present study was directed at the question of whether these nitroxides can protect biological systems from damage inflicted by peroxyl radicals. The reaction of nitroxides with AAPH-derived radicals was followed by cyclic voltammetry and electron paramagnetic resonance spectroscopy, whereas the accumulation of peroxide was iodometrically assayed. Assaying DNA damage in vitro, we demonstrate that piperidine nitroxides protect from both t-AP(*) and t-APOO(*). Similarly, nitroxides inhibit AAPH-induced enzyme inactivation. The results indicate that piperidine nitroxides protect the target molecule by reacting with and detoxifying peroxyl radicals.

Do stable nitroxide radicals catalyze or inhibit the degradation of hyaluronic acid?

Reactive oxygen-derived species and particularly OH radicals can degrade hyaluronic acid (HA), resulting in a loss of viscosity and a subsequent decrease in its effectiveness as a joint-lubricating agent. The production of OH in the vicinity of HA can be catalyzed by bound redox-active metals, which participate in the Haber-Weiss reaction. Damage to HA can also occur as a result of hypochlorite formed by myeloperoxidase (MPO). The protective reagents commonly used to inhibit oxidative stress-induced degradation of HA include antioxidative enzymes, such as SOD and catalase, chelators that coordinate metal ions rendering them redox-inactive, and scavengers of radicals, such as OH, as well as nonradical reactive species. In recent years, stable cyclic nitroxides have also been widely used as effective antioxidants. In many cases, nitroxide antioxidants operate catalytically and mediate their protective effect through an exchange between their oxidized and reduced forms. It was anticipated, therefore, that nitroxides would protect HA from oxidative degradation as well. On the other hand, nitroxides serve as catalysts in many oxidation reactions of alcohols, sugars and polysaccharides, including hyalouronan. Such opposite effects of nitroxides on oxidative degradation are particularly intriguing and the aim of the present study was to examine their effect on HA when subjected to diverse forms of oxidative stress. The results indicate that nitroxides protect HA from OH radicals generated enzymatically or radiolytically. The protective effect is attributable neither to the scavenging of OH nor to the oxidation of reduced metal, but to the reaction of nitroxides with secondary carbohydrate radicals-most likely peroxyl radicals.