Characterization of three iron ferredoxins by microwave power saturation

We have measured the electronic spin lattice relaxation time T1 in the temperature range 4 K-10 K, by microwave power saturation on the 3Fe ferredoxins from Desulfovibrio gigas and Azotobacter vinelandii. The comparison with the results previously obtained on other iron sulfur proteins emphasizes the particularly fast relaxing properties of the E.P.R. signal in 3Fe ferredoxins. These results support the models of the active site which predict very low lying excited levels.     

Sulfide induces phosphate release from polyphosphate in cultures of a marine Beggiatoa strain

Sulfur bacteria such as Beggiatoa or Thiomargarita have a particularly high capacity for storage because of their large size. In addition to sulfur and nitrate, these bacteria also store phosphorus in the form of polyphosphate. Thiomargarita namibiensis has been shown to release phosphate from internally stored polyphosphate in pulses creating steep peaks of phosphate in the sediment and thereby inducing the precipitation of phosphorus-rich minerals. Large sulfur bacteria populate sediments at the sites of recent phosphorite formation and are found as fossils in ancient phosphorite deposits. Therefore, it can be assumed that this physiology contributes to the removal of bioavailable phosphorus from the marine system and thus is important for the global phosphorus cycle. We investigated under defined laboratory conditions which parameters stimulate the decomposition of polyphosphate and the release of phosphate in a marine Beggiatoa strain. Initially, we tested phosphate release in response to anoxia and high concentrations of acetate, because acetate is described as the relevant stimulus for phosphate release in activated sludge. To our surprise, the Beggiatoa strain did not release phosphate in response to this treatment. Instead, we could clearly show that increasing sulfide concentrations and anoxia resulted in a decomposition of polyphosphate. This physiological reaction is a yet unknown mode of bacterial polyphosphate usage and provides a new explanation for high phosphate concentrations in sulfidic marine sediments.     

Dimorphism in methane seep-dwelling ecotypes of the largest known bacteria

We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization.     

Apolipoprotein F concentration,activity, and the properties of LDL controlling ApoF activation in hyperlipidemic plasma

Apolipoprotein F (ApoF) modulates lipoprotein metabolism by selectively inhibiting cholesteryl ester transfer protein activity on LDL. This ApoF activity requires that it is bound to LDL. How hyperlipidemia alters total plasma ApoF and its binding to LDL are poorly understood. In this study, total plasma ApoF and LDL-bound ApoF were quantified by ELISA (n = 200). Plasma ApoF was increased 31% in hypercholesterolemic plasma but decreased 20% in hypertriglyceridemia. However, in donors with combined hypercholesterolemia and hypertriglyceridemia, the elevated triglyceride ameliorated the rise in ApoF caused by hypercholesterolemia alone. Compared with normolipidemic LDL, hypercholesterolemic LDL contained ～2-fold more ApoF per LDL particle, whereas ApoF bound to LDL in hypertriglyceridemia plasma was <20% of control. To understand the basis for altered association of ApoF with hyperlipidemic LDL, the physiochemical properties of LDL were modified in vitro by cholesteryl ester transfer protein ± LCAT activities. The time-dependent change in LDL lipid composition, proteome, core and surface lipid packing, LDL surface charge, and LDL size caused by these factors were compared with the ApoF binding capacity of these LDLs. Only LDL particle size correlated with ApoF binding capacity. This positive association between LDL size and ApoF content was confirmed in hyperlipidemic plasmas. Similarly, when in vitro produced and enlarged LDLs with elevated ApoF binding capacity were incubated with LPL to reduce their size, ApoF binding was reduced by 90%. Thus, plasma ApoF levels and the activation status of this ApoF are differentially altered by hypercholesterolemia and hypertriglyceridemia. LDL size is a key determinate of ApoF binding and activation.     

Effect of hyperoxidized guanine on DNA primer-template structures: Spiroiminodihydantoin leads to strand slippage

Oxidation of guanine in DNA can lead to mutagenic lesions such as 7-hydro-8-oxoguanine (oG). Upon further oxidation, a more mutagenic lesion, spirominodihydantoin (Sp), can occur. In this study, nuclear magnetic resonance (NMR) investigations were performed to determine the structural features of DNA primer-template models with 5′-GG, 5′-G(oG), 5′-G(Sp) and 5′-T(Sp) templates, that mimic the situation in which the downstream G of the template has been oxidized to oG or hyperoxidized to Sp. Our results show that misalignment occurs only in the 5′-G(Sp) and 5′-T(Sp) templates, providing structural insights into the observed differences in mutagenicity of Sp and oG during DNA replication.     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

Long-term stability of parameters of antioxidant status in human serum

Abstract

The antioxidant status of serum or plasma can be determined using several commercially available assays. Here, four different assays, total antioxidant status (TAS), its second-generation assay (TAS2), biological antioxidant potential (BAP), and enzymatic assay using horseradish peroxidase (EAOC), were applied on human serum samples to test the temperature stability of antioxidants, upon storage of serum for 12 months. The two or three most commonly used temperatures for storage, that is, ? 20, ? 70 (or ? 80), and ? 196°C, were selected. The general conclusion is that all assays were stable at the temperatures tested. In addition, there were almost no statistically significant differences between the samples stored at different temperatures. Only the rank order of the EAOC assay was not very good in samples stored at ? 20°C. Also three components contributing to the total antioxidant capacity, uric acid, creatinine and bilirubin, showed no statistically significant differences between the temperatures. Therefore, storage at ? 20°C is sufficient to maintain a proper assay outcome of most of the total antioxidant assays, although storage at ? 70/80°C is to be preferred for longer storage times.     

Soil and plant tests for available sulfur in wetland rice soils

Summary Soil tests, plant performance, and plant tissue analyses were used to study the availability of sulfur to wetland rice in 30 Philippine soils. The critical concentrations of available sulfur by the calcium phosphate, lithium chloride, ammonium acetate, and hydrochloric acid extractions were 9, 25, 30, and 5 mg/kg, respectively. The critical total sulfur limits were 0.11% in the shoot at maximum tillering 0.055% in the straw at maturity, and 0.065% in the grain. The critical N:S ratio was 15 in the shoot at maximum tillering, 14 in the straw at maturity, and 26 in the grain. The critical sulfate-sulfur limit was 150 mg/kg in the shoot at maximum tillering and 100 mg/kg in the straw at maturity. The critical sulfate-sulfur/total sulfur percentage ratio was 15% in the shoot at maximum tillering and the straw at maturity. Plant performance, judged by appearance and yield of dry matter, straw, and grain, was generally poorer in the sulfur deficient soils than in the other soils. Although the calcium phosphate and ammonium acetate methods gave a better correlation between plant performance and available sulfur than the others, all four methods separated sulfur-deficient soils from non-deficient ones. The hydrochloric acid method merits further study because it is simple and versatile.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> FtnA acts as an iron buffer for re-assembly of iron–sulfur clusters in response to hydrogen peroxide stress

Iron–sulfur clusters are one of the most ubiquitous redox centers in biology. Ironically, iron-sulfur clusters are highly sensitive to reactive oxygen species. Disruption of iron-sulfur clusters will not only change the activity of proteins that host iron–sulfur clusters, the iron released from the disrupted iron–sulfur clusters will further promote the production of deleterious hydroxyl free radicals via the Fenton reaction. Here, we report that ferritin A (FtnA), a major iron-storage protein in Escherichia coli, is able to scavenge the iron released from the disrupted iron–sulfur clusters and alleviates the production of hydroxyl free radicals. Furthermore, we find that the iron stored in FtnA can be retrieved by an iron chaperon IscA for the re-assembly of the iron–sulfur cluster in a proposed scaffold IscU in the presence of the thioredoxin reductase system which emulates normal intracellular redox potential. The results suggest that E. coli FtnA may act as an iron buffer to sequester the iron released from the disrupted iron–sulfur clusters under oxidative stress conditions and to facilitate the re-assembly of the disrupted iron–sulfur clusters under normal physiological conditions.