Dehydroepiandrosterone increased oxidative stress in a human cell line during differentiation

Dehydroepiandrosterone (DHEA), a reversible inhibitor of glucose-6-phosphate dehydrogenase (G6PD), is increasingly taken as an antioxidative and anti-ageing supplement. This study investigated the effects of DHEA on the expression of G6PD and on the state of oxidative stress in a human promyelocytic leukaemia cell line, HL60, during the differentiation to neutrophil-like cell. This study differentiated HL60 with dimethyl sulfoxide (DMSO) in the presence (DMSO-HL60/DHEA) or absence (DMSO-HL60) of DHEA. During the differentiation, activity, mRNA and protein levels of G6PD were increased. DHEA increased these levels further. DHEA by itself suppressed the production of superoxide from DMSO-HL60 upon stimulation with phorbol myristate acetate (PMA). However, DMSO-HL60/DHEA stimulated with PMA in the absence of DHEA produced superoxide and 8-oxo-deoxyguanosine more than PMA-stimulated DMSO-HL60. After addition of H₂O₂, the ratio of reduced glutathione to oxidized glutathione was lower in DMSO-HL60/DHEA than in DMSO-HL60. These findings indicate that DHEA acts both as an antioxidant and as a pro-oxidant.     

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.     

Kinetic analysis of enhanced thermal stability of an alkaline protease with engineered twin disulfide bridges and calcium-dependent stability

The thermal stability of a cysteine-free alkaline protease (Alp) secreted by the eukaryote Aspergillus oryzae was improved both by the introduction of engineered twin disulfide bridges (Cys-69/Cys-101 and Cys-169/Cys-200), newly constructed as part of this study, and by the addition of calcium ions. We performed an extensive kinetic analysis of the increased thermal stability of the mutants as well as the role of calcium dependence. The thermodynamic activation parameters for irreversible thermal inactivation, the activation free energy (deltaG), the activation enthalpy (deltaH), and the activation entropy (deltaS) were determined from absolute reaction rate theory. The values of deltaH and deltaS were significantly and concomitantly increased as a result of introducing the twin disulfide bridges, for which the increase in the value of deltaH outweighed that of deltaS, resulting in significant increases in the value of deltaG. The enhancement of the thermal stability obtained by introducing the twin disulfide bridges is an example of the so-called low-temperature stabilization of enzymes. The stabilizing effect of calcium ions on wild-type Alp is similar to the results we obtained by introducing the engineered twin disulfide bridges.     

Requirement of domain-domain interaction for conformational change and functional ATP hydrolysis in myosin

Coordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis.     

Optical bio-sniffer for ethanol vapor using an oxygen-sensitive optical fiber

An optical bio-sniffer for ethanol was constructed by immobilizing alcohol oxidase (AOD) onto a tip of a fiber optic oxygen sensor with a tube-ring, using an oxygen sensitive ruthenium organic complex (excitation, 470 nm; fluorescent, 600 nm). A reaction unit for circulating buffer solution was applied to the tip of the device. After the experiment in the liquid phase, the sniffer-device was applied for gas analysis using a gas flow measurement system with a gas generator. The optical device was applied to detect the oxygen consumption induced by AOD enzymatic reaction with alcohol application. The sensor in the liquid phase was used to measure ethanol solution from 0.50 to 9.09 mmol/l. Then, the bio-sniffer was calibrated against ethanol vapor from 0.71 to 51.49 ppm with good gas-selectivity based on the AOD substrate specificity. The bio-sniffer with the reaction unit was also used to monitor the concentration change of gaseous ethanol by rinsing and cleaning the fiber tip and the enzyme membrane with buffer solution.     

Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.     

Phospholipids promote dissociation of ADP from the Mycobacterium avium DnaA protein

The biochemical aspects of the initiation of DNA replication in Mycobacterium avium are unknown. As a first step towards understanding this process, M. avium DnaA protein, the counterpart of Escherichia coli replication initiator protein, was overproduced in E. coli with an N-terminal histidine tag and purified to homogeneity on a nickel affinity column. The recombinant DnaA protein bound both ATP and ADP with high affinity and showed a weak ATPase activity. ADP, following the hydrolysis of ATP, remained bound to the protein strongly and the exchange of ATP for bound ADP was found to be weak. Acidic phospholipids such as phosphatidylinositol, phosphatidylglycerol, and cardiolipin, promoted the dissociation of ADP from the DnaA protein, whereas the neutral phospholipid, phosphatidylethanolamine, did not. The phospholipid promoted dissociation of ADP from DnaA protein was stimulated in the presence of the M. avium origin of replication. We suggest that the initiation of DNA replication in M. avium involves an interplay among DnaA, adenine nucleotides and phospholipids.     

Immunogenicity and safety of an inactivated quadrivalent influenza vaccine in healthy adults: a phase II,open‐label,uncontrolled trial in Japan

Two antigenically distinct B strain lineages of influenza virus have co‐circulated since the mid‐1980s; however, inactivated trivalent influenza vaccines contain only one B lineage. The mismatch between the circulating and vaccine lineages has been a worldwide issue. In this study, an inactivated quadrivalent influenza vaccine (QIV) candidate containing two B lineages was manufactured and its immunogenicity and safety evaluated in an open‐label, uncontrolled trial. In this phase II trial, 50 subjects aged 20–64 years received two doses of QIV s.c. 1 to 4 weeks apart. Sera were collected pre‐ and post‐vaccination and safety assessed from the first vaccination to 21 ± 7 days after the second vaccination. After the first vaccination, hemagglutination inhibition titers against each strain increased markedly; the seroconversion rate, geometric mean titer ratio and seroprotection rate being 94.0%, 24.93, and 100.0%, respectively, for the A/H1N1pdm09 strain; 94.0%, 12.47, and 98.0%, respectively, for the A/H3N2 strain; 54.0%, 4.99, and 66.0%, respectively, for B/Yamagata strain, and 72.0%, 6.23 and 80.0%, respectively, for the B/Victoria strain, thus fulfilling the criteria of the European Medical Agency's Committee for Medicinal Products for Human Use. Also, the QIV induced sufficient single radial hemolysis and neutralizing antibodies against all four vaccine strains. No noteworthy adverse events were noted. The results of this trial demonstrate that QIV is well tolerated and immunogenic for each strain, suggesting that QIV potentially improves protection against influenza B by resolving the issue of B lineage mismatch.     

PtdIns4KIIα generates endosomal PtdIns(4)P and is required for receptor sorting at early endosomes

Phosphatidylinositol 4-kinase IIα (PtdIns4KIIα) localizes to the trans-Golgi network and endosomal compartments and has been implicated in the regulation of endosomal traffic, but the roles of both its enzymatic activity and the site of its action have not been elucidated. This study shows that PtdIns4KIIα is required for production of endosomal phosphatidylinositol 4-phosphate (PtdIns(4)P) on early endosomes and for the sorting of transferrin and epidermal growth factor receptor into recycling and degradative pathways. Depletion of PtdIns4KIIα with small interfering RNA significantly reduced the amount of vesicular PtdIns(4)P on early endosomes but not on Golgi membranes. Cells depleted of PtdIns4KIIα had an impaired ability to sort molecules destined for recycling from early endosomes. We further identify the Eps15 homology domain–containing protein 3 (EHD3) as a possible endosomal effector of PtdIns4KIIα. Tubular endosomes containing EHD3 were shortened and became more vesicular in PtdIns4KIIα-depleted cells. Endosomal PtdIns(4,5)P₂ was also significantly reduced in PtdIns4KIIα-depleted cells. These results show that PtdIns4KIIα regulates receptor sorting at early endosomes through a PtdIns(4)P-dependent pathway and contributes substrate for the synthesis of endosomal PtdIns(4,5)P₂.