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Purpose The present study examined the effect of ingestion of Koji extract containing 14-dehydroergosterol (14-DHE), prepared from Aspergillus kawachii NBRC4308, on improvement of skin conditions among healthy volunteers. Subjects and methods In a randomized, double-blind, placebo-controlled, parallel-group study, 70 healthy adult women who felt that their skin was dry ingested either a placebo dietary supplement or Koji extract (200 mg/day) supplement containing 0.1% 14-DHE for 12 weeks. Throughout the treatment period and for 4 weeks afterward, objective indicators - including moisture content of the stratum corneum, trans-epidermal water loss (TEWL), and skin wrinkles - were evaluated; in addition, the subjects answered a questionnaire on their skin conditions with ratings on a visual analog scale. Statistical analysis was conducted on the basis of differences from baseline scores. Results Compared with the placebo group, the Koji extract group showed significantly increased forearm moisture at 4, 8, and 16 weeks (p < 0.05 on unpaired t-test). The questionnaire survey showed a marked improvement in skin conditions, particularly crow's feet, in the Koji extract group versus the placebo group at 8 weeks (p < 0.05 by unpaired t-test). Furthermore, the Koji extract group showed a trend (p < 0.10) toward improvement in skin moisture (at 4 weeks), dryness around the eyes/mouth (at 4 weeks), and overall skin condition (at 8 weeks) versus the placebo group. Conclusion Ingestion of Koji extract containing 14-DHE was demonstrated to have positive effects toward improving skin conditions - in particular, on increasing skin moisture in the stratum corneum.

Purpose The present study examined the effect of ingestion of Koji extract containing 14-dehydroergosterol (14-DHE), prepared from Aspergillus kawachii NBRC4308, on improvement of skin conditions among healthy volunteers. Subjects and methods In a randomized, double-blind, placebo-controlled, parallel-group study, 70 healthy adult women who felt that their skin was dry ingested either a placebo dietary supplement or Koji extract (200 mg/day) supplement containing 0.1% 14-DHE for 12 weeks. Throughout the treatment period and for 4 weeks afterward, objective indicators – including moisture content of the stratum corneum, trans-epidermal water loss (TEWL), and skin wrinkles – were evaluated; in addition, the subjects answered a questionnaire on their skin conditions with ratings on a visual analog scale. Statistical analysis was conducted on the basis of differences from baseline scores. Results Compared with the placebo group, the Koji extract group showed significantly increased forearm moisture at 4, 8, and 16 weeks (p < 0.05 on unpaired t-test). The questionnaire survey showed a marked improvement in skin conditions, particularly crow’s feet, in the Koji extract group versus the placebo group at 8 weeks (p < 0.05 by unpaired t-test). Furthermore, the Koji extract group showed a trend (p < 0.10) toward improvement in skin moisture (at 4 weeks), dryness around the eyes/mouth (at 4 weeks), and overall skin condition (at 8 weeks) versus the placebo group. Conclusion Ingestion of Koji extract containing 14-DHE was demonstrated to have positive effects toward improving skin conditions – in particular, on increasing skin moisture in the stratum corneum.

A non-protein amino acid, L-ornithine (Orn), has been shown to stimulate the urea cycle and tissue protein synthesis in the liver. The purpose of the current study was to assess whether Orn affects the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway, which is involved in protein synthesis. Primary cultured cells isolated from Wistar rat liver were incubated in an amino acid-free medium, followed by addition of Orn for 3 h. The cell lysate was subjected to immunoblotting to evaluate the phosphorylation of downstream targets of mTORC1, including p70S6K, S6, and 4EBP1. To assess the involvement of mTORC1 for the effect of Orn, the cells were pretreated with the mTOR inhibitor rapamycin before the addition of Orn and the cell lysate was subjected to immunoblotting. We next examined whether the effects of Orn were exerted in vivo. Orn was orally administered to 18 h food-deprived rats, the blood and the livers were collected at 1 and 3 h after administration for immunoblotting. Orn treatment for primary cultured cells for 3 h enhanced the phosphorylation of p70S6K, S6, and 4EBP1. In addition, rapamycin blocked the effects of Orn completely (p70S6K and S6) or partially (4EBP1). The oral administration of Orn to the rat also augmented the phosphorylation of mTORC1 downstream targets notably in S6 at 1 h. Our findings demonstrate that Orn has the potential to induce the phosphorylation of downstream targets of mTORC1 in the rat liver. This may be mediated by the augmentation of mTORC1 activity.

The importance of the ubiquitin system largely depends on ubiquitin ligases, E3s, as they determine the specificity of the system. Rbx1/ROC1/Hrt1, a RING finger protein, functions as an important component of the cullin-containing SCF and VBC-Cul2 ligases. Modification of cullins by NEDD8 (NEDDylation), has been shown to be essential for the E3 activity of both SCF and VBC-Cul2, and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation. RING finger is composed of eight cysteine and histidine residues that bind to zinc ions. Rbx1 is a highly evolutionarily conserved protein; however, the eighth coordination residue in its RING finger is aspartate (D97) rather than cysteine. Substitution of D97 with each of the other 19 amino acids demonstrates that aspartate is superior to cysteine in cullin NEDDylation. Interestingly, however, different D97 mutants demonstrate different activities towards 6 cullins tested. Importantly, we were able to discriminate between the NEDDylating activity of Rbx1 and its involvement in the ubiquitylation reaction within the context of VBC-Cul2. Moreover, while Rbx1 is not involved in governing the stability of SCF, Rbx1 mutants destabilize VBC-Cul2. Taken together, these results indicate that various mechanisms regulate both the activities and the stability of cullin-based ligases.

Macroautophagy is a bulk degradation process induced by starvation in eukaryotic cells. In yeast, 15 Apg proteins coordinate the formation of autophagosomes. Several key reactions performed by these proteins have been described, but a comprehensive understanding of the overall network is still lacking. Based on Apg protein localization, we have identified a novel structure that functions in autophagosome formation. This pre-autophagosomal structure, containing at least five Apg proteins, i.e. Apg1p, Apg2p, Apg5p, Aut7p/Apg8p and Apg16p, is localized in the vicinity of the vacuole. Analysis of apg mutants revealed that the formation of both a phosphatidylethanolamine-conjugated Aut7p and an Apg12p– Apg5p conjugate is essential for the localization of Aut7p to the pre-autophagosomal structure. Vps30p/Apg6p and Apg14p, components of an autophagy- specific phosphatidylinositol 3-kinase complex, Apg9p and Apg16p are all required for the localization of Apg5p and Aut7p to the structure. The Apg1p protein kinase complex functions in the late stage of autophagosome formation. Here, we present the classification of Apg proteins into three groups that reflect each step of autophagosome formation.

Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole1,2. Apg8/Aut7 is an essential factor for autophagy in yeast3,4,5. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus6. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane6. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy6.

We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation. Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM. Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures. These structures would represent intermediate structures during autophagosome formation. Here, we also showed that microtubule does not play an essential role in the autophagy in yeast. The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant. Apg8p would play an important role in the autophagosome formation.

The ubiquitin system plays important roles in the regulation of numerous cellular processes by conjugating ubiquitin to target proteins. In most cases, conjugation of polyubiquitin to target proteins regulates their function. In the polyubiquitin chains reported to date, ubiquitin monomers are linked via isopeptide bonds between an internal Lys and a C-terminal Gly. Here, we report that a protein complex consisting of two RING finger proteins, HOIL-1L and HOIP, exhibits ubiquitin polymerization activity by recognizing ubiquitin moieties of proteins. The polyubiquitin chain generated by the complex is not formed by Lys linkages, but by linkages between the C- and N-termini of ubiquitin, indicating that the ligase complex possesses a unique feature to assemble a novel head-to-tail linear polyubiquitin chain. Moreover, the complex regulates the stability of Ub-GFP (a GFP fusion protein with an N-terminal ubiquitin). The linear polyubiquitin chain generated post-translationally may function as a new modulator of proteins.

The importance of the ubiquitin system largely depends on ubiquitin ligases, E3s, as they determine the specificity of the system. Rbx1/ROC1/Hrt1, a RING finger protein, functions as an important component of the cullin-containing SCF and VBC-Cul2 ligases. Modification of cullins by NEDD8 (NEDDylation), has been shown to be essential for the E3 activity of both SCF and VBC-Cul2, and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation. RING finger is composed of eight cysteine and histidine residues that bind to zinc ions. Rbx1 is a highly evolutionarily conserved protein; however, the eighth coordination residue in its RING finger is aspartate (D97) rather than cysteine. Substitution of D97 with each of the other 19 amino acids demonstrates that aspartate is superior to cysteine in cullin NEDDylation. Interestingly, however, different D97 mutants demonstrate different activities towards 6 cullins tested. Importantly, we were able to discriminate between the NEDDylating activity of Rbx1 and its involvement in the ubiquitylation reaction within the context of VBC-Cul2. Moreover, while Rbx1 is not involved in governing the stability of SCF, Rbx1 mutants destabilize VBC-Cul2. Taken together, these results indicate that various mechanisms regulate both the activities and the stability of cullin-based ligases.

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.

準備号, 取材構成：由利伸子 / 米沢美由樹

Autophagy is the nonselective bulk protein degradation system in the lytic organelle, the lysosome/vacuole, and an example of non-classical vesicular transport from the cytoplasm to the compartment via double-membrane vesicles, autophagosomes. So far, 15 APG genes essential for autophagy have been cloned in the yeast Saccharomyces cerevisiae, and some APG gene products have been characterised. However, the molecular mechanism of membrane dynamics of autophagy, in particular the formation of autophagosomes, is still unclear. The WIGS gene encodes a small hydrophilic protein with about 15 kD in mass. The gene product, Apg8p, drastically increases in abundance by induction of autophagy. The elevation of amount of Apg8p is regulated transcriptionally. In spite of the hydrophilic nature, Apg8p is mainly bound to membrane both growth and starvation conditions. Many homologs of Apg8p exist in various eukatyotic organisms. One of the homologs, LC3, binds to microtubule in vitro. Thus, I examined requirement of microtubule for autophagy morphologically and biochemically using a microtubule-depolymerizing drug, and clarified that microtubule is not necessary for autophagy. After confirming that Apg8p functions in the formation of autophagosome, intracellular localization of Apg8p was assessed by immunofluorescence and immunoelectron microscopic analyses. The analyses revealed that Apg8p resides on the membrane structures at the various stages in autophagosome formation, autophagosomes and autophagic bodies. Apg8p is the first identified molecule that traces the whole process of membrane dynamics in autophagy. Immunoelectron microscopy also revealed that Apg8p is more enriched on the membranes of the intermediate structures of autophagosome than on those of mature autophagosomes. The result indicated that Apg8p should directly function at the step of autophagosome formation and suggested that it may be detached from the membranes before autophagosome is formed up completely. Furthermore, the observation of the formation process of autophagosomes traced with Apg8p provided a new proposal that autophagosomal membrane may be generated de novo but not from pre-existing membrane cistema. As described above, Apg8p seemed to be a key molecule for elucidating the molecular mechanism of autophagosome formation. Thus, I performed further characterization of Apg8p and discovered that Apg8p undergoes a novel reversible modification. Apg8p is produced as the precursor form in cells. The newly synthesized Apg8p is cleaved by a novel cysteine protease, Apg4p, to remove the original carboxy-terminal arginine residue, and thereby a Gly residue becomes the carboxy-terminal residue of the protein. Subsequently, Apg8p forms a covalent conjugate with a general glycerophospholipid, phosphatidylethanolamine (PH), which is a major component of biological membrane. Apg8p binds to PE via amide bond between carboxyl group of the carboxy-terminal glycine of Apg8p and amino group of PE. The formation of the adduct, designated Apg8p-PE, requires Apg7p and Apg3p. Finally, Apg8p-PE is deconjugated to Apg8p and PH by cleavage action of Apg4p. Apg7p and Apg3p were already identified as the modification enzymes for Apg8p, which are equivalent to E1 and E2 enzymes for ubiquitination, respectively. Furthermore, the mode of action of Apg4p, which cleaves both newly synthesized Apg8p and Apg8p-PE, well resembles that of some deubiquitinating enzymes which cleave both the precursor of ubiquitin and the adducts of ubiquitin with substrate. The overall similarity demonstrates that ubiquitination-like system involves in the formation of Apg8p-PB. So far, there had been no report that ubiquitination and the related systems are utilized to protein- lipidation. This is the first evidence that ubiquitination-like modification mediates protein-lipidation. At the point of protein-lipidation, this study showed the first example that protein is covalently conjugated to phosphatidylethanolamine. The formation and deconjugation of Apg8p-PE is essential for formation of autophagosomes. Free Apg8p is originally loosely membrane bound or soluble. The reversible modification changes the membrane-binding state of Apg8p. Apg8p turns to be bound to membrane tightly by the conjugation with PE, and it is returned to loosely-membrane bound or soluble by the deconjugation. These features are consistent with morphological evidence that Apg8p is well localized along the membrane of autophagosome intermediates but little detected on mature autophagosome. The regulation of the membrane-binding state of Apg8p by the reversible modification is essential for autophagosome formation.

Background Residual alcohol effects on physiological and psychological symptoms are commonly experienced the morning after alcohol consumption. The purpose of this study was to assess the effects of L-ornithine on subjective feelings and salivary stress markers the morning after alcohol consumption and to investigate whether L-ornithine acutely accelerates ethanol metabolism. Methods This study had a randomized, placebo-controlled, double-masked crossover design. Subjects were all healthy Japanese adults with the ‘flusher’ phenotype for alcohol tolerance. In experiment 1, 11 subjects drank 0.4 g/kg body weight alcohol 1.5 h before their usual bedtime. Half an hour after drinking, they ingested either a placebo or 400 mg ornithine. The next morning on awakening, subjects completed a questionnaire containing a visual analog scale (VAS), the Oguri-Shirakawa-Azumi sleep inventory MA version (OSA-MA), and a profile of mood states (POMS) and collected a saliva sample for measurement of salivary stress markers (cortisol, secretory immunoglobulin A, and α-amylase). In experiment 2, placebo or 400 mg ornithine were administrated to 16 subjects both before and after drinking, and the feeling of drunkenness, breath ethanol concentration and one-leg standing time were repeatedly investigated until 180 min after alcohol consumption. Results There were significant decreases in “awareness”, “feeling of fatigue” and “lassitude” VAS scores and in “anger-hostility” and “confusion” POMS scores and a significant increase in “sleep length” in the OSA-MA test. Salivary cortisol concentrations on awakening were reduced after ornithine supplementation. There were no differences between ornithine and placebo in any of the subjective or physiological parameters of acute alcohol metabolism. Conclusions Taking 400 mg ornithine after alcohol consumption improved various negative feelings and decreased the salivary stress marker cortisol the next morning. These effects were not caused by an increase in acute alcohol metabolism.