Iron release analyses from ferritin by visible light irradiation

We investigated the iron release from ferritin by irradiation from a white fluorescent light in the absence or presence of ADP. Irradiation of a ferritin solution at 17,000 lx in the absence of ADP slightly induces iron release from ferritin but only at acidic pH conditions (pH 5.0 or pH 6.0). Irradiation in the presence of ADP markedly enhances iron release from ferritin under the same conditions. In the absence of irradiation, the iron release from ferritin was low even in the presence of ADP. The induction of the iron release by irradiation in the presence of ADP was also affected by various factors such as irradiation dose and acidity, but not temperature (4-47°C), oxygen concentration, or free radical generations during the irradiation. The iron release during the irradiation ceased to increase by turning off the light and was found to increase again after additional irradiation. These results suggest that visible light directly induces iron release from ferritin via the photoreduction of iron stored inside ferritin.     

Morphometric deformations of small airways and alveoli under quasi-static inflation process

Localized morphometric deformations of small airways and alveoli during respiration have many biomedical and physiological implications. We developed fast synchrotron radiation CT system to visualize the small airways and alveoli of an intact mouse lung without fixation and dehydration, and analyzed their localized morphometric deformations between functional residual capacity (FRC) and total lung capacity (TLC). In the diameter behavior, the averaged and range values were significantly larger for smaller airways (68.8%, range: 0.36-0.89) than larger airways (45.2%, range: 0.40-0.57). These results indicated that the airway did not deformed in same manner and that these morphological differences characterized the heterogeneous lung function.     

Benzimidazole derivatives as novel nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 1: Benzimidazole-5-sulfonamides

A new class of benzimidazole-5-sulfonamides has been identified as nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Initial structure-activity relationships are presented resulting in compounds 19 and 28 with submicromolar dual functional activity on human and rat receptors.     

Visualization of conformational distribution of short to medium size segments in globular proteins and identification of local structural motifs

Analysis of the conformational distribution of polypeptide segments in a conformational space is the first step for understanding a principle of structural diversity of proteins. Here, we present a statistical analysis of protein local structures based on interatomic C(alpha) distances. Using principal component analysis (PCA) on the intrasegment C(alpha)-C(alpha) atomic distances, the conformational space of protein segments, which we call the protein segment universe, has been visualized, and three essential coordinate axes, suitable for describing the universe, have been identified. Three essential axes specified radius of gyration, structural symmetry, and separation of hairpin structures from other structures. Among the segments of arbitrary length, 6-22 residues long, the conservation of those axes was uncovered. Further application of PCA to the two largest clusters in the universe revealed local structural motifs. Although some of motifs have already been reported, we identified a possibly novel strand motif. We also showed that a capping box, which is one of the helix capping motifs, was separated into independent subclusters based on the C(alpha) geometry. Implications of the strand motif, which may play a role for protein-protein interaction, are discussed. The currently proposed method is useful for not only mapping the immense universe of protein structures but also identification of structural motifs.     

Carbon cycling and budget in a forested basin of southwestern Hokkaido,northern Japan

Quantification of annual carbon sequestration is very important in order to assess the function of forest ecosystems in combatting global climate change and the ecosystem responses to those changes. Annual cycling and budget of carbon in a forested basin was investigated to quantify the carbon sequestration of a cool-temperate deciduous forest ecosystem in the Horonai stream basin, Tomakomai Experimental Forest, northern Japan. Net ecosystem exchange, soil respiration, biomass increment, litterfall, soil-solution chemistry, and stream export were observed in the basin from 1999–2001 as a part of IGBP-TEMA project. We found that 258 g C m^–2 year^–1 was sequestered annually as net ecosystem exchange (NEE) in the forested basin. Discharge of carbon to the stream was 4 g C m^–2 year^–1 (about 2% of NEE) and consisted mainly of dissolved inorganic carbon (DIC). About 43% of net ecosystem productivity (NEP) was retained in the vegetation, while about 57% of NEP was sequestered in soil, suggesting that the movement of sequestered carbon from aboveground to belowground vegetation was an important process for net carbon accumulation in soil. The derived organic carbon from aboveground vegetation that moved to the soil mainly accumulated in the solid phase of the soil, with the result that the export of dissolved organic carbon to the stream was smaller than that of dissolved inorganic carbon. Our results indicated that the aboveground and belowground interaction of carbon fluxes was an important process for determining the rate and retention time of the carbon sequestration in a cool-temperate deciduous forest ecosystem in the southwestern part of Hokkaido, northern Japan.     

Glucosamine induces lipid accumulation and adipogenic change in C2C12 myoblasts

Hyperglycemia-induced activation of hexosamine biosynthesis pathway (HBP) has been implicated in the development of insulin resistance in skeletal muscles. In the present study, the content of uridine-5'-diphospho-N-acetylglucosamine, the end product of the HBP, was elevated in skeletal muscle of obese diabetic KKA(y) mice, compared with control mice. To elucidate the effect of elevated HBP in the skeletal muscle, we treated C2C12 myoblasts with glucosamine, an intermediate metabolite of the HBP. Glucosamine induced lipid accumulation and significantly increased the mRNA expression levels of peroxisome proliferator-activated receptor gamma, adiponectin, and aP2 in C2C12 myoblasts. Similar mRNA changes were observed in skeletal muscles of Sprague-Dawley rats treated with glucosamine infusion. Our results provide a possible explanation of hyperglycemia-induced insulin resistance in skeletal muscle.     

Hyperthermophilic α-L-arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein

A putative -L-arabinofuranosidase (AFase) gene belonging to family 51 of glycosyl hydrolases of a hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein (Tm-AFase) was purified to apparent homogeneity by heat treatment (80°C, 30 min), followed by hydrophobic interaction, anion-exchange, and gel permeation column chromatography. Tm-AFase had a molecular mass of 55,284 Da on matrix assisted laser desorption ionization time-of-flight mass spectrometry and ~332 kDa on gel permeation column chromatography. Therefore, Tm-AFase comprised six identical subunits as in the case of homologous AFase from Geobacillus stearothermophilus. Regarding substrate specificity, Tm-AFase was active with p-nitrophenyl -L-arabinofuranoside but not with p-nitrophenyl -L-arabinopyranoside. Regarding polysaccharides, Tm-AFase hydrolyzed arabinan and debranched arabinan but not arabinoxylan, arabinogalactan, and carboxymethyl cellulose. Tm-AFase was extremely thermophilic, displaying an optimal reaction temperature of 90°C in a 10 min assay. When Tm-AFase was heated at 90°C, no loss of activity was observed for at least 24 h. At 100°C, the activity dropped to ~50% in 20 min; thereafter, inactivation occurred very slowly exhibiting a half-life of ~2.7 h, characterizing the enzyme to be the most thermophilic AFase reported thus far.     

Stretched-Exponential Analysis of Heat-Induced Aggregation of Apo-Concanavalin A

The kinetics of heat-induced aggregation of apo-concanavalin A (aConA) was investigated as a function of temperature and protein concentration by circular dichroism and turbidity. Heat-induced aggregation, as well as conformational change, of aConA was fitted to stretched-exponential equations. The exponent of the conformational change maintained 0.5 despite the protein concentration and temperature, indicating the presence of a common intermediate during the conformational change. After the process, aggregates grew with increasing temperature and initial protein concentration. The reaction order of aggregation was 1.5, indicating that the rate-limiting steps of aConA aggregation involve both conformational change and aggregation.     

Amino Acid esters prevent thermal inactivation and aggregation of lysozyme

Small potent inhibitors of aggregation are eagerly demanded for preventing the inactivation of proteins. This paper shows that amino acid esters (AAEs) prevent heat-induced aggregation and inactivation of hen egg lysozyme. Lysozyme was completely inactivated (<1% original activity) during heat treatment at 98 degrees C for 30 min in a solution containing 0.2 mg/mL lysozyme in 50 mM Na-phosphate buffer (pH 6.5). The residual activities only slightly increased (<5%) in the presence of 100 mM commonly used additives such as arginine, guanidine, urea, and sugars. However, in the presence of 100 mM AAEs, the residual activities were >60% and no aggregates were observed during the heat treatment at 98 degrees C for 30 min. This fact provides new information on the scaffold for designing additives to prevent heat-induced aggregation.