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.     

Analysis of SDF-1/CXCR4 signaling in primordial germ cell migration and survival or differentiation in Xenopus laevis

Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus.     

Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c(+) dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-alpha) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-alpha by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.     

An isozyme of earthworm serine proteases acts on hydrolysis of triacylglycerol

An enzyme catalyzing the hydrolysis of triacylglycerol was purified from an earthworm. The N-terminal amino acid sequence and the catalytic function of the purified enzyme were identical to those of Isozyme C, an isozyme of the earthworm-serine proteases. No other lipase proteins were found in the earthworm cells. The isozyme might act on the hydrolysis of triacylglycerol as well as the protein decomposition.     

Oxygen and hydrogen peroxide enhance light-induced carotenoid synthesis in Neurospora crassa

Previously, we found that intracellular reactive oxygen species (ROS) affect photomorphogenesis in Neurospora crassa. In this study, we investigated the physiological roles of ROS in the response to light and found that the exposure of mycelia to air was important for the light-induced carotenogenesis. Mycelia treated with a high concentration of O(2) gas and H(2)O(2) to release ROS showed an enhancement of light-induced carotenoid accumulation and the expression of gene related to light-inducible carotenogenesis. These results suggested that stimuli caused by the exposure of the mycelia to air containing O(2) gas triggered the light-induced carotenoid synthesis.     

Rapid degradation of Cdt1 upon UV-induced DNA damage is mediated by SCFSkp2 complex

Cdt1 is a licensing factor for DNA replication, the function of which is tightly controlled to maintain genome integrity. Previous studies have indicated that the cell cycle-dependent degradation of Cdt1 is triggered at S phase to prevent re-replication. In this study, we found that Cdt1 is degraded upon DNA damage induced by either UV treatment or gamma-irradiation (IR). Although the IR-triggered degradation of Cdt1 was caffeine-insensitive, the UV-triggered degradation of Cdt1 was caffeine-sensitive. This indicates that the cells treated with UV utilize the checkpoint pathway, which differs from that triggered by IR. A recent study has suggested that Cdt1 is phosphorylated, ubiquitylated, and degraded at the G(1)/S boundary in the normal cell cycle. Treatment with MG132, a proteasome inhibitor, inhibited the degradation of Cdt1 and resulted in the accumulation of the phosphorylated form of Cdt1 after UV treatment. In the case of UV treatment, phosphorylation of Cdt1 induced the recruitment of Cdt1 to a SCF(Skp2) complex. Moreover, ectopic overexpression of Cdt1 after UV treatment interfered the inhibition of DNA synthesis. These results indicate that Cdt1 is a target molecule of the cell cycle checkpoint in UV-induced DNA damage.     

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.     

Light-dependent subcellular localization of nucleoside diphosphate kinase-1 in Neurospora crassa

Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme localized in cellular organelles and distributed in various organs in prokaryotes and eukaryotes. In Neurospora crassa, NDK-1 is suggested to control catalases in response to heat, oxidative stress and light. In this study, we identified the presence of NDK-1 during most developmental stages in submerged mycelia, aerial hyphae, asexual conidia and perithecia, and the localization of it in soluble, mitochondrial, nuclear and membrane fractions in the mycelial cell. A light-dependent localization of NDK-1 was shown by Western blotting and immunohistochemical analysis using anti-NDK-1 antibody. In the mycelia, NDK-1 was compartmentalized on the plasma membrane in darkness, while it was relocated in the cytoplasm under light. These results suggest that NDK-1 protein was translocated from the plasma membrane to cytoplasm in response to light, and may interact with catalase.     

Stereoselective reduction of carbonyl compounds using the cell-free extract of the earthworm, Lumbricus rubellus, as a novel source of biocatalyst

We found the reducing activity toward carbonyl compounds in the cell-free extract of the earthworm, Lumbricus rubellus. The earthworm extract had a reducing activity for keto esters in the presence of NADH or NADPH as a coenzyme. The earthworm extract reduced ethyl 3-methyl-2-oxobutanoate to the corresponding alcohol with a high enantiomeric excess (91%, R-form) at 50 degrees C in the presence of NADH. In particular, ethyl 2-oxoheptanoate was exclusively reduced to the corresponding (R)-hydroxyl ester with a high enantiomeric excess (>99%).