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The yeast protein Mpo1 belongs to a protein family that is widely conserved in bacteria, fungi, protozoa, and plants, and is the only protein of this family whose function has so far been elucidated. Mpo1 is an Fe2+-dependent dioxygenase that catalyzes the alpha-oxidation reaction of 2-hydroxy (2-OH) long-chain FAs (LCFAs) produced in the degradation pathway of the long-chain base phytosphingosine. However, several biochemical characteristics of Mpo1, such as its catalytic residues, membrane topology, and substrate specificity, remain unclear. Here, we report that yeast Mpo1 contains two transmembrane domains and that both its N- and C-terminal regions are exposed to the cytosol. Mutational analyses revealed that three histidine residues conserved in the Mpo1 family are especially important for Mpo1 activity, suggesting that they may be responsible for the formation of coordinate bonds with Fe2+. We found that, in addition to activity toward 2-OH LCFAs, Mpo1 also exhibits activity toward 2-OH very-long-chain FAs derived from the FA moiety of sphingolipids. These results indicate that Mpo1 is involved in the metabolism of long-chain to very-long-chain 2-OH FAs produced in different pathways. We noted that the growth ofmpo1 Delta cells is delayed upon carbon deprivation, suggesting that the Mpo1-mediated conversion of 2-OH FAs to nonhydroxy FAs is important for utilizing 2-OH FAs as a carbon source under carbon starvation. Our findings help to elucidate the as yet unknown functions and activities of other Mpo1 family members.

Sjögren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder whose causative gene encodes the fatty aldehyde dehydrogenase ALDH3A2. To date, the detailed molecular mechanism of the skin pathology of SLS has remained largely unclear. We generated double-knockout (DKO) mice for Aldh3a2 and its homolog Aldh3b2 (a pseudogene in humans). These mice showed hyperkeratosis and reduced fatty aldehyde dehydrogenase activity and skin barrier function. The levels of ω-O-acylceramides (acylceramides), which are specialized ceramides essential for skin barrier function, in the epidermis of DKO mice were about 60% of those in wild-type mice. In the DKO mice, levels of acylceramide precursors (ω-hydroxy ceramides and triglycerides) were increased, suggesting that the final step of acylceramide production was inhibited. A decrease in acylceramide levels was also observed in human immortalized keratinocytes lacking ALDH3A2. Differentiated keratinocytes prepared from the DKO mice exhibited impaired long-chain base metabolism. Based on these results, we propose that the long-chain-base-derived fatty aldehydes that accumulate in DKO mice and SLS patients attack and inhibit the enzyme involved in the final step of acylceramide production. Our findings provide insight into the pathogenesis of the skin symptoms of SLS, i.e., decreased acylceramide production, and its molecular mechanism.

Phytosphingosine (PHS) is the major long-chain base component of sphingolipids in Saccharomyces cerevisiae The PHS metabolic pathway includes a fatty acid (FA) α-oxidation reaction. Recently, we identified the novel protein Mpo1, which is involved in PHS metabolism. However, the details of the FA α-oxidation reaction and the role of Mpo1 in PHS metabolism remained unclear. In the present study, we revealed that Mpo1 is involved in the α-oxidation of 2-hydroxy (2-OH) palmitic acid (C16:0-COOH) in the PHS metabolic pathway. Our in vitro assay revealed that not only the Mpo1-containing membrane fraction but also the soluble fraction was required for the α-oxidation of 2-OH C16:0-COOH. The addition of Fe2+ eliminated the need for the soluble fraction. Purified Mpo1 converted 2-OH C16:0-COOH to C15:0-COOH in the presence of Fe2+, indicating that Mpo1 is the enzyme body responsible for catalyzing the FA α-oxidation reaction. This reaction was also found to require an oxygen molecule. Our findings indicate that Mpo1 catalyzes the FA α-oxidation reaction as 2-OH fatty acid dioxygenase, mediated by iron(IV) peroxide. Although numerous Mpo1 homologs exist in bacteria, fungi, protozoa, and plants, their functions had not yet been clarified. However, our findings suggest that these family members function as dioxygenases.

The fatty aldehyde dehydrogenase (FALDH) ALDH3A2 is the causative gene of Sjogren Larsson syndrome (SLS). To date, the molecular mechanism underlying the symptoms characterizing SLS has been poorly understood. Using Aldh3a2(-/-) mice, we found here that Aldh3a2 was the major FALDH active in undifferentiated keratinocytes. Long-chain base metabolism was greatly impaired in Aldh3a2(-/-) keratinocytes. Phenotypically, the intercellular spaces were widened in the basal layer of the Aldh3a2(-/-) epidermis due to hyperproliferation of keratinocytes. Furthermore, oxidative stress-induced genes were upregulated in Aldh3a2(-/-) keratinocytes. Upon keratinocyte differentiation, the activity of another FALDH, Aldh3b2, surpassed that of Aldh3a2. As a result, Aldh3a2(-/-) mice were indistinguishable from wild-type mice in terms of their whole epidermis FALDH activity, and their skin barrier function was uncompromised under normal conditions. However, perturbation of the stratum corneum caused increased transepidermal water loss and delayed barrier recovery in Aldh3a2(-/-) mice. In conclusion, Aldh3a2(-/-) mice replicated some aspects of SLS symptoms, especially at the basal layer of the epidermis. Our results suggest that hyperproliferation of keratinocytes via oxidative stress responses may partly contribute to the ichthyosis symptoms of SLS.

The applicability of an electrochemical Fenton-type process (EF-HOCl-ReFe) to the treatment of three actual wastewaters, namely wastewater from an automobile factory (automobile wastewater), metal scrap-cleansing wastewater, and municipal wastewater, is discussed in this research. The EF-HOCl-ReFe successfully removed the chemical oxygen demand (COD) from automobile wastewater pre-treated by a coagulation process without any inhibition. The apparent current efficiency reached 86%, 46% of which was ascribed to the electrochemical Fenton-type mechanism. The metal scrap-cleansing wastewater had a yellow colour and high concentrations of COD (6550 mg/L) and Cl− (1560 mM). The EF-HOCl-ReFe could achieve almost complete COD removal and decolourization after 48 h of treatment, although a temporary intensification of colour was observed before the decolourization. The EF-HOCl-ReFe was also effective in the removal of 1,4-dioxane from municipal wastewater pre-treated by activated sludge and coagulation processes, which were unable to remove 1,4-dioxane. The 1,4-dioxane removal efficiency after 30 min of treatment reached 68.5%. Thus, the EF-HOCl-ReFe was applicable to the treatment of these actual wastewaters.

Although normal fatty acids (FAs) are degraded via β-oxidation, unusual FAs such as 2-hydroxy (2-OH) FAs and 3-methyl-branched FAs are degraded via α-oxidation. Phytosphingosine (PHS) is one of the long-chain bases (the sphingolipid components) and exists in specific tissues, including the epidermis and small intestine in mammals. In the degradation pathway, PHS is converted to 2-OH palmitic acid and then to pentadecanoic acid (C15:0-COOH) via FA α-oxidation. However, the detailed reactions and genes involved in the α-oxidation reactions of the PHS degradation pathway have yet to be determined. In the present study, we reveal the entire PHS degradation pathway: PHS is converted to C15:0-COOH via six reactions [phosphorylation, cleavage, oxidation, CoA addition, cleavage (C1 removal), and oxidation], in which the last three reactions correspond to the α-oxidation. The aldehyde dehydrogenase ALDH3A2 catalyzes both the first and second oxidation reactions (fatty aldehydes to FAs). In Aldh3a2-deficient cells, the unmetabolized fatty aldehydes are reduced to fatty alcohols and are incorporated into ether-linked glycerolipids. We also identify HACL2 (2-hydroxyacyl-CoA lyase 2) [previous name, ILVBL; ilvB (bacterial acetolactate synthase)-like] as the major 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in the FA α-oxidation of the PHS degradation pathway. HACL2 is localized in the endoplasmic reticulum. Thus, in addition to the already-known FA α-oxidation in the peroxisomes, we have revealed the existence of FA α-oxidation in the endoplasmic reticulum in mammals.

Although very few studies have addressed the molecular and cellular mechanisms underlying the development of biliary tract cancer (BTC), several lines of evidence suggest a role for the erbB receptor family. Overexpression and activation of erbB2 has been reported in a significant percentage of human BTC. Further, we previously reported that overexpression of erbB2 basal epithelial cells of the biliary tract (BK5.erbB2 mouse) led to the development of BTC. However, the mechanisms by which erbB2 overexpression led to the spontaneous development of tumors specifically in the biliary tract are not completely understood. The goals of the current study were to (1) determine whether a cooperative relationship between bile acid exposure and erbB2 activation exists during biliary tract carcinogenesis and (2) to characterize the mechanism(s) underlying bile acid-mediated biliary tract carcinogenesis in cells with activated erbB2. In this study, we demonstrated that the secondary conjugated bile acid, taurochenodeoxycholic acid (TCDC), increased proliferation of primary cultured gallbladder epithelial cells from BK5.erbB2 mice and human BTC cells. TCDC treatment activated EGFR/erbB2 and downstream signaling molecules in both primary cultured cells and human BTC cells. TCDC also increased the expression of epidermal growth factor receptor (EGFR) ligands and TACE activity in human BTC cells. Inhibition of src activation led to attenuation of bile-induced upregulation of TACE activity as well as signaling through the EGFR/erbB2, suggesting that during the development of BTC erbB2 overexpression/activation accelerates the bile acid-induced signaling cascade: bile acid src TACE EGFR/erbB2 downstream signaling. We also provide direct evidence that bile acids possess tumor promoting capacity in epithelial cells overexpressing erbB2 using the two-stage skin carcinogenesis model. Collectively these findings suggest cooperative roles for bile acid and erbB2 activation in epithelial cell proliferation; bile acid appears to accelerate erbB2-induced pro-tumorigenic activities in the biliary tract and skin. © 2013 Wiley Periodicals, Inc.

The accumulation of reactive aldehydes is implicated in the development of several disorders. Aldehyde dehydrogenases (ALDHs) detoxify aldehydes by oxidizing them to the corresponding carboxylic acids. Among the 19 human ALDHs, ALDH3A2 is the only known ALDH that catalyzes the oxidation of long-chain fatty aldehydes including C16 aldehydes (hexadecanal and trans-2-hexadecenal) generated through sphingolipid metabolism. In the present study, we have identified that ALDH3B1 is also active in vitro toward C16 aldehydes and demonstrated that overexpression of ALDH3B1 restores the sphingolipid metabolism in the ALDH3A2-deficient cells. In addition, we have determined that ALDH3B1 is localized in the plasma membrane through its C-terminal dual lipidation (palmitoylation and prenylation) and shown that the prenylation is required particularly for the activity toward hexadecanal. Since knockdown of ALDH3B1 does not cause further impairment of the sphingolipid metabolism in the ALDH3A2-deficient cells, the likely physiological function of ALDH3B1 is to oxidize lipid-derived aldehydes generated in the plasma membrane and not to be involved in the sphingolipid metabolism in the endoplasmic reticulum. (C) 2013 Elsevier B.V. All rights reserved.

This paper reports on the reusability and the optimal reuse of iron-rich sludge for an electrochemical Fenton-type process using sequencing batch mode and separation batch mode reuse models. In the sequencing batch mode, processes of electrochemical treatment, neutralization, sedimentation, and re-dissolution of iron sludge were all performed in the same reactor, whereas the neutralization and sedimentation of iron sludge in the separation batch mode was carried out in an iron recovery tank separated from the electrochemical reactor. The effects of iron speciation at different pH levels were discussed. It was found that ferric ions at a pH ≤2.5 were suitable for this electrochemical Fenton-type process where ferrous ions acted as hydroxyl radical scavengers, generating brownish deposits on the cathode at pH 3. When the sequencing batch mode was applied, the current efficiency in the Fenton-type process after the iron recovery declined due to the formation of insoluble deposits on the electrodes, which decreased at lower pH. The deposits were mainly formed during the neutralization and sedimentation steps after the electrochemical process. Fortunately, iron from the sludge could be reused for the electrochemical process when the separation batch mode was used, with 100% iron recovery and no decline in current efficiency.

Sphingosine 1-phosphate (S1P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD1 and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S1P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K1 mutant cells caused abnormal metabolism of S1P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa1 and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in Δfaa1 Δfaa4 mutant cells. These results unveil the entire S1P metabolic pathway: S1P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S1P metabolite hexadecenal contributes to the pathogenesis of SLS.

過酸化水素を次亜塩素酸に置き換えたFenton型反応を用いる電解促進酸化処理の運転操作条件を検討した。本処理法では塩化物イオン（Cl-）のアノード酸化により次亜塩素酸を供給するとともに，鉄（III）イオン（Fe3+）のカソード還元により鉄（II）イオン（Fe2+）を供給することが可能となる。OHラジカルプローブには1，4－ジオキサンを用いた。その結果，pH 3.0以下，電流密度2.8 mA・cm-2以下，Cl-濃度10～200 mM，Fe2+濃度1.0～4.0 mMの条件下において比較的高い電流効率を観測した。また初期に投入する鉄イオン形態はFe2+よりもFe3+の方が効果的であり，その原因として過剰に存在するFe2+がラジカルスカベンジャーとして働くことが考えられた。初期にFe3+を1 mM投入し，pH 2.5，電流密度2.8 mA・cm-2，Cl-濃度100 mMとしたときの電流効率は74.2%であった。

Idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease of unknown etiology, is characterized by the expansion of myofibroblasts and abnormal deposition of extracellular matrix in the lung parenchyma. To elucidate the molecular mechanisms that lead to IPF, we analyzed myofibroblasts established from patients with IPF by oligonucleotide microarrays. Gene expression profiles clearly suggested that lipid peroxidation is enhanced in myofibroblasts, which was confirmed by measuring cellular lipid hydroperoxides. One of the most highly up-regulated proteins in myofibroblasts was selenoprotein P, an antioxidant protein not previously associated with IPF. Subsequent analysis demonstrated that selenoprotein P reduces lipid hydroperoxides and maintains the viability of myofibroblasts. Thus, our results reveal a novel pathophysiologic function of myofibroblasts as a generator of lipid hydroperoxides, and a self-defense mechanism against self-generated oxidative stress.

Corrigendum: Identification of the phytosphingosine metabolic pathway leading to odd-numbered fatty acids

The long-chain base phytosphingosine is a component of sphingolipids and exists in yeast, plants and some mammalian tissues. Phytosphingosine is unique in that it possesses an additional hydroxyl group compared with other long-chain bases. However, its metabolism is unknown. Here we show that phytosphingosine is metabolized to odd-numbered fatty acids and is incorporated into glycerophospholipids both in yeast and mammalian cells. Disruption of the yeast gene encoding long-chain base 1-phosphate lyase, which catalyzes the committed step in the metabolism of phytosphingosine to glycerophospholipids, causes an similar to 40% reduction in the level of phosphatidylcholines that contain a C15 fatty acid. We also find that 2-hydroxypalmitic acid is an intermediate of the phytosphingosine metabolic pathway. Furthermore, we show that the yeast MPO1 gene, whose product belongs to a large, conserved protein family of unknown function, is involved in phytosphingosine metabolism. Our findings provide insights into fatty acid diversity and identify a pathway by which hydroxyl group-containing lipids are metabolized.

Self-limiting monolayer adsorption of Ge has been demonstrated on Si(100) at 520°C using dimethylgermane as a self-limiting molecular precursor. This opens a way for hetero-ALE of Ge on the Si surface.

Atomic layer epitaxy of germanium has been demonstrated successfully by alternating exposures of atomic hydrogen, which acts as a reactant for extracting hydrocarbon from the surface, and of dimethylgermane as a self-limiting molecular precursor. The ideal growth rate of one monolayer per cycle has been achieved with a wide ALE temperature window between 420°C and 528°C.

Background & Aims Gallbladder carcinoma (GBCa), a type of biliary tract cancer (BTC), has proven challenging to treat, demonstrating the need for more effective therapeutic strategies. In our current study, we examined the therapeutic effects of the histone deacetylase (HDAC) inhibitor PCI-24781 against GBCa that developed in BK5.erbB2 mice. Methods PCI-24781 [50mg/kg/day] and control solutions were administered to BK5.erbB2 mice for 4 weeks. The therapeutic effect of PCI-24781 was evaluated by ultrasound biomicroscopy (USBM) throughout the experiment and histological analyses at the end of the experiment. To investigate potential mechanisms underlining the therapeutic effects of PCI-24781 on GBCa in BK5.erbB2 mice, PCI-24781-treated gallbladders were subjected to Western blot and RT-PCR analysis. The inhibitory effect of PCI-24781 on the growth of BTC cells was compared to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and gemcitabine. To study the role of miRNAs in GBCa tumorigenesis, the expression profile of 368 miRNAs in GBCas from BK5.erbB2 (both treated and untreated) and wild type mice was analyzed. Results Treatment of BK5.erbB2 mice with PCI-24781 for 1 month prevented 79% of GBCa cases from progression and showed a clinical effect in 47% of cases. We also confirmed a potent inhibitory effect on tumor cell growth in human BTC cell lines treated with PCI-24781. This effect was associated with downregulation of ErbB2 mRNA and ErbB2 protein/activity and upregulation of acetylated histone and acetylated tubulin. Treatment with PCI-24781 resulted in decreased expression of Muc4, an intramembrane ligand for ErbB2, in BTC cells. PCI-24781 had more effects on growth inhibition of BTC cells than SAHA. In addition, PCI-24781 effectively inhibited the growth of gemcitabine-resistant cells. miRNA profiling revealed that the expression of several miRNAs was significantly altered in GBCa in the BK5.erbB2 mouse compared to normal gallbladder, including upregulated miR21, which was downregulated by PCI-24781. Conclusions These results indicate that PCI-24781 potently inhibits the growth of BTC cells by decreasing ErbB2 expression and activity as well as regulating altered miRNA expression. PCI-24781 may have a potential value as a novel chemotherapeutic agent against human BTC in which ErbB2 is overexpressed.

Novel atomic layer epitaxy (ALE) methods have been proposed for Ge, where the elemental kinetics of the chemical vapor deposition method are divided into two sequential phases, i.e., a monolayer adsorption phase and a subsequent desorption phase for the surface-terminating species. Ge-ALE has been achieved, for the first time, with an ideal growth rate of one monolayer per cycle, under constant substrate temperature conditions. The detailed ALE method and growth characteristics have been presented. Furthermore, atomic hydrogen exposure is shown to be effective for widening the Ge-ALE window.

Rheumatoid arthritis (RA), a chronic and systemic disease of unknown etiology, is characterized by hyperplasia of synovial cells, which ultimately lead to the destruction of cartilage and bone. To elucidate the molecular mechanisms that lead to RA, we analyzed synovial cells established from patients with RA by oligonucleotide microarrays. Gene expression profiles clearly suggested that oxidative stress is enhanced in RA synovial cells, which was confirmed by measuring cellular levels of reactive oxygen species. One of the highly up-regulated proteins in RA synovial cells was thioredoxin reductase 1 (TRXR1), a protein that plays an important role in antioxidant defense system. Subsequent analysis demonstrated that TRXR1 suppresses hydrogen peroxide and inhibits apoptosis of RA synovial cells. Thus, our results reveal a novel pathophysiologic function of RA synovial cells as a generator of oxidative stress, and a self-defense mechanism against self-generated oxidative stress.

A zinc porphyrin dimer, in which two porphyrin moieties were linked with an optically active (1R,2R)-trans-1,2-bis(aklylamide)cyclohexane unit, was incorporated into nanoscale fibers made of organogelators and the fluorescence quenching of zinc porphyrin moieties was diminished by the incorporation.

Chiral-twisted iron(III) porphyrin dimers were self-assembled to high-molecular-weight polynuclear species through the intermolecular formation of μ-oxo dimers.

Glycine-conjugated bile acids serve as new hosts for multimolecular inclusion compounds; a novel three-leaved assembly is found in the crystal structure of a 1 : 1 complex between glycodeoxycholic acid and THF.