Identification of a radical formed in the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using HPLC-EPR and HPLC-EPR-MS

Identification of a free radical is performed for the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using EPR, high performance liquid chromatography-electron paramagnetic resonance spectrometry (HPLC-EPR) and high performance liquid chromatography-electron paramagnetic resonance-mass spectrometry (HPLC-EPR-MS). EPR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT). No EPR spectrum was detectable without rat brain homogenate, suggesting that the radical is derived from rat brain homogenate. An HPLC-EPR analysis of the reaction mixture showed a peak with retention time of 33.7 min. An HPLC-EPR-MS analysis of the peak gave two ions at m/z 224 and 137, suggesting that alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)/ethyl radical adduct forms in the reaction mixture.     

Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethyl-porphyrinatoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using (19)F NMR and the O(2) binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in alpha- and beta- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O(2) affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O(2) affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O(2) affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.     

Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.     

Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624

Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 × 10⁷ protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.     

Enhanced iturin A production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on suppression of the plant pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The behavior of Streptomyces peucetius var. caesius N47 was studied in a glucose limited chemostat with a complex cultivation medium. The steady-state study yielded the characteristic constants μ _max over 0.10 h⁻¹, Y _XS 0.536 g g⁻¹, and m_S 0.54 mg g⁻¹ h⁻¹. The product of secondary metabolism, ɛ-rhodomycinone, was produced with characteristics Y _PX 12.99 mg g⁻¹ and m _P 1.20 mg g⁻¹ h⁻¹. Significant correlations were found for phosphate and glucose consumption with biomass and ɛ-rhodomycinone production. Metabolic flux analysis was conducted to estimate intracellular fluxes at different dilution rates. TCA, PPP, and shikimate pathway fluxes exhibited bigger values with production than with growth. Environmental perturbation experiments with temperature, airflow, and pH changes on a steady-state chemostat implied that an elevation of pH could be the most effective way to shift the cells from growing to producing, as the pH change induced the biggest transient increase to the calculated ɛ-rhodomycinone flux.     

Abscinazole-E2B, a practical and selective inhibitor of ABA 8'-hydroxylase CYP707A

We developed abscinazole-E2B (Abz-E2B), a practical and specific inhibitor of abscisic acid (ABA) 8'-hydroxylase (CYP707A), by structural modification of abscinazole-E1 (Abz-E1), another compound we developed. A butoxy group was introduced to Abz-E2B instead of the tosylate group of Abz-E1, in expectation of better water solubility, because the calculated logP value of Abz-E2B is 3.47, which is smaller than that of Abz-E1 (4.02). The water solubility of Abz-E2B was greater than 90% at a concentration of 100 μM, at which the solubility of Abz-E1 was 20%. The enzyme specificity was improved significantly. In in vitro assays constructed using recombinant enzymes, (±)-Abz-E2B was a considerably weaker inhibitor than (±)-Abz-E1 for CYP701A, a GA biosynthetic enzyme, which is a target of S-uniconazole (S-UNI), a lead compound of Abz-E1. (±)-Abz-E2B application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA, with little retardation of growth. We also prepared optically pure Abz-E2B and determined its absolute configuration. The R-enantiomer of Abz-E2B was the more potent inhibitor of CYP707A, unlike UNI, whereas both enantiomers were markedly less effective than S-UNI in inhibiting CYP701A. Because S-Abz-E2B arrested the growth of rice seedlings at 100 μM, probably because of off-target effects, R-Abz-E2B should be used as a chemical tool for research focusing on CYP707A when 100 μM or higher concentration is required, although (±)-Abz-E2B may be useful as an alternative option at a lower concentration.     

Participation in Sports Organizations and the Prevention of Functional Disability in Older Japanese: The AGES Cohort Study

Background

We sought to examine prospectively the difference in the association between incident functional disability and exercise with or without sports organization participation.

Methods

The study was based on the Aichi Gerontological Evaluation Study (AGES) Cohort Study data. In October 2003, self-reported questionnaires were mailed to 29,374 non-disabled Japanese individuals aged 65 years or older. Of these, 13,310 individuals were introduced to the Study, and they were followed for 4 years. Analysis was carried out on 11,581 subjects who provided all necessary information for the analysis.

Results

Analysis was carried out on incident functional disability by 4 groups of different combinations of performance of exercise and participation in a sports organization Active Participant (AP), Exercise Alone (EA), Passive Participant (PP) and Sedentary (S). Compared to the AP group, the EA group had a hazard ratio (HR) of 1.29 (1.02–1.64) for incident functional disability. No significant difference was seen with the PP group, with an HR of 1.16 (0.76–1.77). When a measure of social networks was added to the covariates, the HR of the EA group dropped to 1.27 (1.00–1.61), and significant differences disappeared. In contrast, it showed hardly any change when social support was added.

Conclusion

The results suggested that, even with a regular exercise habit, incident functional disability may be better prevented when a person participates in a sports organization than when he/she does not. In addition, participation in a sports organization correlates positively with social networks, which may lead to a small decrease in incident functional disability.     

Fungi are involved in the effects of litter mixtures on consumption by shredders

1. Decomposition of litter mixtures in both terrestrial and aquatic ecosystems often shows non‐additive diversity effects on decomposition rate, generally interpreted in streams as a result of the feeding activity of macroinvertebrates. The extent to which fungal assemblages on mixed litter may influence consumption by macroinvertebrates remains unknown. 2. We assessed the effect of litter mixing on all possible three‐species combinations drawn from four tree species (Alnus glutinosa, Betula pendula, Juglans regia and Quercus robur) on both fungal assemblages and the rate of litter consumption by a common shredder, Gammarus fossarum. After a 9‐week inoculation in a stream, batches of leaf discs were taken from all leaf species within litter mixture combinations. Ergosterol, an indicator of fungal biomass, and the composition of fungal assemblages, assessed from the conidia released, were determined, and incubated litter offered to G. fossarum in a laboratory‐feeding experiment. 3. Mixing leaf litter species enhanced both the Simpson’s index of the fungal assemblage and the consumption of litter by G. fossarum, but had no clear effect on mycelial biomass. Specifically, consumption rates of J. regia were consistently higher for mixed‐species litter packs than for single‐species litter. In contrast, the consumption rates of B. pendula were not affected by litter mixing, because of the occurrence of both positive and negative litter‐mixing effects in different litter species combinations that counteracted each other. 4. In some litter combinations, the greater development of some fungal species (e.g. Clavariopsis aquatica) as shown by higher sporulation rates coincided with increased leaf consumption, which may have resulted from feeding preferences by G. fossarum for these fungi. 5. Where litter mixture effects on decomposition rate are mediated via shredder feeding, this could be due to indirect effects of the fungal assemblage.     

Positive selection of Toll-like receptor 2 polymorphisms in two closely related old world monkey species, rhesus and Japanese macaques

Toll-like receptor 2 (TLR2) plays an important role in the recognition of a variety of pathogenic microbes. In the present study, we compared polymorphisms of TLR2 locus in two closely related old world monkey species, rhesus monkey (Macaca mulatta) and Japanese monkey (Macaca fuscata). By nucleotide sequencing of the third exon of TLR2 gene from 21 to 35 respective individuals, we could assign 17 haplotype combinations of 17 coding SNPs of ten non-synonymous and seven synonymous substitutions. A non-synonymous substitution at codon position 326 appeared to be differentially fixed in each species, asparagine for M. mulatta whereas tyrosine for M. fuscata, and may contribute to certain functional properties because it locates in the region contributing to ligand binding and interaction with dimerization partner of TLR2-TLR1 heterodimeric complex. Although TLR2 alleles have diverged to similar extent in both species, they have evolved in significantly different ways; TLR2 of M. fuscata has undergone purifying selection while the membrane-proximal part of the extracellular domain of M. mulatta TLR2 exhibits higher rates of non-synonymous substitutions, indicating a trace of Darwinian positive selection.