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• W2160653669 abstract "Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner. Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner. Plants produce a variety of secondary metabolites, and some of these are potentially toxic to animals (1Fowler M.E. J. Wildl. Dis. 1983; 19: 34-43Crossref PubMed Scopus (53) Google Scholar). Herbivora have developed behavioral and physiological strategies to avoid specific plants and to detoxify any toxins ingested. Detoxification can occur in the mouth and the gut rumen with or without the help of microbes (2Owens J.B. J. Agric. Sci. (Camb.). 1992; 119: 151-155Crossref Scopus (20) Google Scholar). The absorbed toxins must be detoxified in the intestine and liver, but studies on these mechanisms are limited because to date most animal experiments have been carried out using laboratory diets. Recently we found that a nuclear receptor, peroxisome proliferator-activated receptor α (PPARα), 2The abbreviations used are: PPARα, peroxisome proliferator-activated receptor α; FALDH, fatty aldehyde dehydrogenase; ALDH, aldehyde dehydrogenase; GFP, green fluorescent protein; RT, reverse transcription; PBS, phosphate-buffered saline; GC-MS, gas chromatography-mass spectrometry; HEK, human embryonic kidney. is involved in the detoxification by using plant seeds as a diet for mice (3Motojima M. Hirai T. FEBS J. 2006; 273: 292-300Crossref PubMed Scopus (26) Google Scholar). PPARα is activated by fatty acid ligands and is an important regulator of lipid metabolism in animals (4Barbier O. Villeneuve L. Bocher V. Fontaine C. Torra I.P. Duhem C. Kosykh V. Fruchart J.C. Guillemette C. Staels B. J. Biol. Chem. 2003; 278: 13975-13983Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Despite the claimed essential role of this receptor in the liver, the PPARα-null mouse shows little phenotypic change when fed a normal laboratory diet (5Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1501) Google Scholar, 6Kersten S. Seydoux J. Peters J.M. Gonzalez F.J. Desvergne B. Wahli W. J. Clin. Investig. 1999; 103: 1489-1498Crossref PubMed Scopus (1362) Google Scholar). We have examined its extrahepatic roles and found that PPARα induces the expression of 17β-hydroxysteroid dehydrogenase type 11 in the intestine (7Motojima K. Eur. J. Biochem. 2004; 271: 4141-4146Crossref PubMed Scopus (25) Google Scholar). Recent studies on the substrates of 17β-hydroxysteroid dehydrogenase type 11 showed that they include not only glucocorticoids and sex steroids but also bile acids, fatty acids, and branched amino acids (8Mindnich R. Moller G. Adamski J. Mol. Cell. Endocrinol. 2004; 218: 7-20Crossref PubMed Scopus (297) Google Scholar, 9Chai Z. Brereton P. Suzuki T. Sasano H. Obeyesekere V. Escher G. Saffery R. Fuller P. Enriquez C. Krozowski Z. Endocrinology. 2003; 144: 2084-2091Crossref PubMed Scopus (54) Google Scholar). So we examined the possibility that PPARα plays a vital role in inducing enzymes for metabolizing secondary metabolites of plants in normal and PPARα knock-out mice fed various plant seeds and found that sesame often killed PPARα knock-out mice but not normal mice (3Motojima M. Hirai T. FEBS J. 2006; 273: 292-300Crossref PubMed Scopus (26) Google Scholar). A DNA microarray analysis revealed that sesame induces the expression of 17β-hydroxysteroid dehydrogenase type 11 and also various detoxifying enzymes in the intestine and liver in a PPARα-dependent and -independent manner (3Motojima M. Hirai T. FEBS J. 2006; 273: 292-300Crossref PubMed Scopus (26) Google Scholar). As the enzyme most significantly induced in a PPARα-dependent manner, we identified a fatty aldehyde dehydrogenase, FALDH or ALDH, encoded by the mouse Aldh3A2 gene (this study). 3B. Ashibe and K. Motojima, unpublished data. ALDHs comprise a superfamily of NAD(P)+-dependent enzymes that catalyze the oxidation of a wide variety of endogenous and exogenous aliphatic and aromatic aldehydes (10Vasiliou V. Pappa A. Pharmacology. 2000; 61: 192-198Crossref PubMed Scopus (134) Google Scholar). The ALDH3 subfamily enzymes efficiently oxidize middle and long chain aldehydes (10Vasiliou V. Pappa A. Pharmacology. 2000; 61: 192-198Crossref PubMed Scopus (134) Google Scholar), and one member of this subfamily, FALDH encoded by ALDH3A2, has a distinct role from the dehydrogenase encoded by ALDH3A1 (11Miyauchi K. Masaki R. Taketani S. Yamamoto A. Akayama M. Tashiro Y. J. Biol. Chem. 1991; 266: 19536-19542Abstract Full Text PDF PubMed Google Scholar, 12Kelson T.L. McVoy Jr., S. Rizzo W.B. Biochim. Biophys. Acta. 1997; 1335: 99-110Crossref PubMed Scopus (118) Google Scholar). FALDH is essential for the complete breakdown of phytanic acid, a branched fatty acid derived from the chlorophyll molecule (13Verhoeven N.M. Jakobs C. Carney G. Somers M.P. Wanders R.J. Rizzo W.B. FEBS Lett. 1998; 429: 225-228Crossref PubMed Scopus (58) Google Scholar), and loss of its activity has been proved to be the cause of Sjögren-Larsson syndrome (14De Laurenzi V. Rogers G.R. Hamrock D.J. Marekov L.N. Steinert P.M. Compton J.G. Markova N. Rizzo W.B. Nat. Genet. 1996; 12: 52-57Crossref PubMed Scopus (231) Google Scholar). It has been suggested that FALDH protects cells against oxidative stress associated with lipid peroxidation and plays an important role in insulin action (15Demozay D. Rocchi S. Mas J.C. Grillo S. Pirola L. Chavey C. Van Obberghen E. J. Biol. Chem. 2004; 279: 6261-6270Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Because of these potential roles in protection against oxidative stress and PPARα-regulated expression (16Gloerich J. van den Brink D.M. Ruiter J.P. van Vlies N. Vaz F.M. Wanders R.J. Ferdinandusse S. J. Lipid Res. 2007; 48: 77-85Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), we speculate that PPARα-dependent induction of FALDH expression plays an important role in the detoxification of the toxic compound(s) directly or indirectly derived from sesame seeds. However, FALDH has not been sufficiently characterized at the protein level. Phytanic acid, a metabolite of phytol, has a methyl group at the carbon 3 position and must first undergo α-oxidation and then β-oxidation (17van den Brink D.M. van Miert J.N. Dacremont G. Rontani J.F. Wanders R.J. J. Biol. Chem. 2005; 280: 26838-26844Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 18van den Brink D.M. Wanders R.J. Cell. Mol. Life Sci. 2006; 63: 1752-1765Crossref PubMed Scopus (103) Google Scholar). It is well established that α-oxidation occurs exclusively in peroxisomes (18van den Brink D.M. Wanders R.J. Cell. Mol. Life Sci. 2006; 63: 1752-1765Crossref PubMed Scopus (103) Google Scholar, 19Wanders R.J.A. Waterham H.R. Annu. Rev. Biochem. 2006; 75: 295-332Crossref PubMed Scopus (710) Google Scholar). However, opinions differ on the identity and subcellular localization of the key enzyme converting pristanal to a β-oxidizable pristanic acid. Human genetic analyses using FALDH-deficient fibroblasts have shown that the product of the ALDH3A2 gene is responsible for most, if not all, of the activity behind the conversion (13Verhoeven N.M. Jakobs C. Carney G. Somers M.P. Wanders R.J. Rizzo W.B. FEBS Lett. 1998; 429: 225-228Crossref PubMed Scopus (58) Google Scholar). However, van den Brink and Wanders (18van den Brink D.M. Wanders R.J. Cell. Mol. Life Sci. 2006; 63: 1752-1765Crossref PubMed Scopus (103) Google Scholar) and Jansen et al. (20Jansen G.A. van den Brink D.M. Ofman R. Draghici O. Dacremont G. Wanders R.J. Biochem. Biophys. Res. Commun. 2001; 283: 674-679Crossref PubMed Scopus (39) Google Scholar) claimed the possibility of the existence of one or more additional aldehyde dehydrogenases reacting with pristanal in peroxisomes, and Masaki et al. (21Masaki R. Yamamoto A. Tashiro Y. J. Cell Biol. 1994; 126: 1407-1420Crossref PubMed Scopus (68) Google Scholar) reported that FALDH expressed from cDNA in COS-1 cells is exclusively detected in the endoplasmic reticulum (ER). The organization of the mouse and human ALDH3A2 genes is well conserved, and similar alternative splicing patterns of transcripts have been reported (22Lin Z. Carney G. Rizzo W.B. Mol. Genet. Metab. 2000; 71: 496-505Crossref PubMed Scopus (20) Google Scholar, 23Rizzo W.B. Lin Z. Carney G. Chem.-Biol. Interact. 2001; 130-132: 297-307Crossref PubMed Scopus (26) Google Scholar). The insertion of an additional exon produces a minor variant of FALDH with a distinct carboxyl-terminal domain of unknown function (22Lin Z. Carney G. Rizzo W.B. Mol. Genet. Metab. 2000; 71: 496-505Crossref PubMed Scopus (20) Google Scholar). The existence of alternative splicing and variants present in different amounts at various subcellular locations have made it difficult to characterize FALDH at the protein level. In this study, we analyzed the subcellular distribution and function of the major and various variant forms of mouse FALDH using a mouse FALDH-specific antibody after overexpressing each cDNA in human HEK293 cells under the control of an inducible promoter, thus avoiding problems originating from the extremely similar structures and incomplete separation by subcellular fractionation of various forms of FALDH with large differences in expression levels. Our data suggest that only one specific variant of FALDH is expressed exclusively in peroxisomes and plays an essential role in the efficient degradation of branched chain fatty acids in the peroxisomal α-oxidation system and that it protects cells from the damage induced by lipid peroxidation. Animals and Treatments—All procedures involving animals were approved by the Meiji Pharmaceutical University Committee for Ethics of Experimentation and Animal Care. Male C57/BL6J and PPARα-null mice (around 6 weeks old) were maintained under a 12-h light-dark cycle with free access to food and water. After being fed a diet containing the PPARα agonist Wy14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) (Tokyo-Kasei, Tokyo, Japan) at 0.05% (w/w) or a normal laboratory diet, the mice were killed by cervical dislocation, and portions of the intestine and liver were removed for homogenization. Subcellular Fractionation of Mouse Liver Homogenate—Subcellular fractionation was performed as described previously (24Ghosh M.K. Hajra A.K. Anal. Biochem. 1986; 159: 169-174Crossref PubMed Scopus (67) Google Scholar) with some modifications. A homogenate was prepared by one stroke with a Teflon-glass homogenizer at 1000 rpm in 3 volumes of ice-cold homogenization buffer (0.25 m sucrose, 1 mm EDTA, and 10 mm Tris-HCl, pH 7.4). A postnuclear supernatant fraction was prepared by centrifugation of the homogenate for 5 min at 1200 × g (3Motojima M. Hirai T. FEBS J. 2006; 273: 292-300Crossref PubMed Scopus (26) Google Scholar). Homogenization buffer was added to the supernatant at up to 10 volumes of the original tissue and recentrifuged for 5 min at 6600 × g. The pellet was suspended in 2 volumes of ice-cold homogenization buffer (mitochondrial fraction). The supernatant was recentrifuged for 20 min at 12,500 × g to obtain the supernatant (microsomal and cytosolic fraction). The pellet was suspended in 2 volumes of ice-cold homogenization buffer (lysosomal fraction). For isolation of the peroxisome-enriched fraction, 1 ml of the lysosomal fraction was layered onto 7.5 ml of Nycodenz (Sigma-Aldrich) solution (30% Nycodenz in 1 mm EDTA, pH 7.3) and centrifuged for 1 h at 131,000 × g. The pellet obtained was suspended in 200 μl of Nycodenz solution and used as the peroxisome-enriched fraction. To disassemble peroxisomes, the peroxisome-enriched fraction was diluted with 4 volumes of 12.5 mm sodium pyrophosphate, pH 9.0 or with Triton X-100 to a final concentration of 0.1% as described previously (25Alexson S.E.H. Fujiki Y. Shio H. Lazarow P.B. J. Cell Biol. 1985; 101: 294-305Crossref PubMed Scopus (90) Google Scholar) and centrifuged for 1 h at 131,000 × g. Preparation of an Antibody against Mouse FALDH and Western Blot Analysis—A rabbit polyclonal antibody against mouse FALDH was prepared using the peptide corresponding to Cys425-Arg439 as the antigen. This sequence is unique to mouse FALDH and common to all FADLH variants (22Lin Z. Carney G. Rizzo W.B. Mol. Genet. Metab. 2000; 71: 496-505Crossref PubMed Scopus (20) Google Scholar). The antibody used for immunofluorescence microscopy was purified by affinity purification using a peptide-coupled Sepharose 4B column (EAH-Sepharose 4B, GE Healthcare). The specificity of the antibody was confirmed by an enzyme-linked immunosorbent assay method using the synthesized peptide and then by Western blotting (3Motojima M. Hirai T. FEBS J. 2006; 273: 292-300Crossref PubMed Scopus (26) Google Scholar). Protein concentration was determined using the Protein Assay Rapid Kit wako (Wako, Tokyo, Japan), and the same amounts of protein samples were analyzed by SDS-PAGE. The separated proteins were blotted to polyvinylidene difluoride membranes (Immobilon, Millipore), and FALDH was detected by the antibody followed by horseradish peroxidase-conjugated anti-rabbit IgG antibody (MP Biochemicals) with SuperSignal West Pico Chemiluminescent Substrate (Pierce). The antibody against the ER membrane protein α-calnexin was purchased from StressGen, and that against the rat peroxisomal membrane protein PMP22 was a gift from Dr. T. Imanaka (Toyama University, Toyama, Japan). Isolation of RNA and RT-PCR Analysis—Total RNA from mouse tissue was prepared by the acid guanidinium thiocyanate-phenol-chloroform method as described previously (26Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar, 27Motojima K. Passilly P. Peters J.M. Gonzalez F.J. Latruffe N. J. Biol. Chem. 1998; 273: 16710-16714Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Total RNA from the cultured cells was obtained using Quick-Gene (Fujifilm, Tokyo, Japan) and QuickGene RNA cultured cell kit S (Fujifilm). Reverse transcription was performed using an ExScript RT reagent kit (Takara Bio, Kyoto, Japan). Real time PCR was performed with a LightCycler 1.5 instrument (Roche Diagnostics) and SYBR ExScript RT-PCR kit (Takara Bio) as directed by the manufacturer. The primers for real time PCR were as follows: 5′-CGGCTACCACATCCAAGGAA and 5′-GCTGGAATTACCGCGGCT for 18 S rRNA, 5′-TGACCTTGATTTATTTTGCATACC and 5′-CGAGCAAGACGTTCAGTCCT for human hypoxanthine phosphoribosyltransferase, 5′-GTCAGCTGGGCCAAGTTCTTC and 5′-TCATTACAGCTGATCCTTGACAATC for FALDH-N, 5′-GTCAGCTGGGCCAAGTTCTTC and 5′-GAAGCCAACAGGGCTTTTCC for FALDH-V, 5′-CAGCTGTGATTGTCAAGTTTGT and 5′-TCCGTATCACCAGGACGACTTC for FALDH-V2, 5′-CTCTGCCCTTTGGAGGTGTG and 5′-AGGGTCAGAAGGACTGGTTTGTC for FALDH-V3 (22Lin Z. Carney G. Rizzo W.B. Mol. Genet. Metab. 2000; 71: 496-505Crossref PubMed Scopus (20) Google Scholar), 5′-TGCACTTCACGCTCAACTCT and 5′-GATAAGCTCCCATCCCACTG for human FALDH-N, and 5′-ATTGTAGCCGCTGTGCTT and 5′-TGAACTACCAGAAAAATCAACAGG for human FALDH-V (23Rizzo W.B. Lin Z. Carney G. Chem.-Biol. Interact. 2001; 130-132: 297-307Crossref PubMed Scopus (26) Google Scholar). Isolation of Splice Variants and DNA Sequencing—The PCR primers used for the isolation of splice variants of mouse FALDH were the exon 8 primer 5′-CTCTGCCTTTGGAGGTGTG and exon 9′ primer 5′-GAAGCCAACAGGGCTTTTCC. Purified RT-PCR products were ligated into pGEM-T Easy (Promega). The cycle sequencing was performed with T7 and SP6 primers in both directions, and the sequences were analyzed using the LIC-4200L-2G sequencer (LI-COR). To annotate each sequence, National Center for Biotechnology Information (NCBI) Blast (www.ncbi.nlm.nih.gov/BLAST/) was used. To confirm that the variants obtained are not splicing intermediates, RT-PCR was performed using primers annealing to 5′-untranslated region (5′-AAGTGGCAGTGAGCTGTGGCATC) and to each variant-specific region (5′-TCCGTATCACCAGGACGACTTC or 5′-AGGGTCAGAAGGACTGGTTTGTC). cDNA Cloning and Construction of Tetracycline-inducible T-REx On Expression Plasmids—A full-length cDNA clone of the major mouse FALDH was obtained from a fetal mouse cDNA library using the vector pCMV6-XL3 (Origen) by colony hybridization with a 32P-labeled cDNA probe obtained by PCR using the mouse FALDH primers (5′-GCTCCTTGGCCATTCATTTTCCTC and 5′-CATCATGTTGCCTAGGCTGGCTTC). cDNA clones of other variants were constructed by swapping the cDNA fragments obtained by PCR of cDNA synthesized from mouse liver total RNA using primers (5′-CATCAGCGCCCCTGCTTGTTAAA and 5′-TGCTCTCAATTGCGGAGATTTGG for FALDH-V and 5′-CTCTGCCTTTGGAGGTGTG and 5′-GAAGCCAACAGGGCTTTTCC for FALDH-V2 and -V3) with restriction sites for KpnI (for V, V2, and V3) and HpaI (for V), BstXI (blunted after PCR) (for V2), or PstI (blunted after PCR) (for V3) into KpnI and HpaI restriction sites of the FALDH full-length plasmid. To construct expression plasmids under tetracycline control (T-REx system, Invitrogen), the FALDH cDNA inserts in pCMV6-XL3 were excised with NotI and SmaI and cloned into the NotI and blunted ApaI sites of pcDNA5/FRT/TO (Invitrogen). A full-length cDNA clone for human ALDH3A2 isoform 1 (human FALDH-V) (GenBank™ accession number NM_001031806) was obtained by RT-PCR of cDNA synthesized from total RNA isolated from human HEK293 cells using primers 5′-CGGACCGTGCAGTTCTCTG and 5′-AGACAGGGCTGGGTTTTGAA followed by cloning into pGEM-T Easy (Promega). After verification by sequencing, the cDNA insert was cloned into pCMV-tag3A (Stratagene) to construct an expression plasmid for amino-terminally Myc-tagged human FALDH-V. A human FALDH-N (GenBank accession number NM_00382) expression vector was constructed by swapping the RT-PCR fragment obtained by using primers 5′-TTCTCACCATTCAGCCACTG and 5′-CAACCCCGCAGTTTGTATTT after digestion with HindIII in the cDNA fragment and ApaI in the vector region of the FALDH-V expression plasmid above. Generation of Tetracycline-inducible Flp-In T-REx HEK293 Cells—Human HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum at 37 °C in a humidified atmosphere of air, 5% CO2. To cause Flp-In T-REx HEK293 cells to express FALDH-N, -V, -V2, or -V3 under the control of tetracycline, the cells were transfected with a mixture of DNA of one of the pcDNA5/FRT/TO plasmids and pOG44 (Invitrogen) using Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. After 48 h of transfection, the medium was supplemented with hygromycin B (50 μg/ml) to initiate selection for stably transfected cells. The selected cells were cloned and cultured in medium supplemented with hygromycin B (50 μg/ml) and blasticidin (5 μg/ml). For expression of FALDH, tetracycline (1 μg/ml) was added 24 h before the treatment of the cells with phytol, dodecanal, or phytanic acid. Immunofluorescence Localization—Stably or transiently transfected HEK293 cells cultured on polylysine-coated cover-slips were washed with PBS and then fixed with 4% paraformaldehyde for 10 min at 4 °C. After being washed with PBS, cells were permeabilized with ice-cold methanol for 10 min at 4 °C or with a mixture of acetone and methanol (1:1) for 10 min at 4 °C. The cells preincubated with 5% bovine serum albumin in PBS for 30 min at room temperature to block nonspecific binding were then incubated with the antibodies for 30 min at room temperature. To detect the Myc-tagged proteins, an anti-c-Myc mouse monoclonal antibody (Nacalai Tesque, Kyoto, Japan) was used. After a rinse with wash buffer (0.4% Triton X-100 in PBS), the cells were incubated with secondary goat anti-rabbit or anti-mouse IgGs conjugated with fluorescein isothiocyanate (MP Biochemicals) or with Alexa Fluor 594-labeled goat anti-rabbit IgG (Invitrogen). After being washed with wash buffer and rinsed with PBS, the cells were fixed in Mowiol (Sigma-Aldrich), and the specimens were subjected to confocal fluorescence microscopy with a Fluoview FV500 microscope (Olympus, Tokyo, Japan). DsRed containing peroxisome-targeting signal-1 (DsRed-SKL) (28Vizeacoumar F.J. Vreden W.N. Aitchison J.D. Rachubinski R.A. J. Biol. Chem. 2006; 281: 14805-14812Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 29Legakis J.E. Koepke J.I. Jedeszko C. Barlaskar B. Terlecky L.J. Edwards H.J. Walton P.A. Terlecky S.R. Mol. Biol. Cell. 2002; 13: 4243-4255Crossref PubMed Scopus (130) Google Scholar) was used to locate peroxisomes in HEK293 cells. GC-MS Analysis—GC-MS was performed according to a published method (30Komen J.C. Duran M. Wanders R.J. J. Lipid Res. 2004; 45: 1341-1346Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) with modifications on an Agilent GC6890 gas chromatographer coupled to a JMS-AM150 mass spectrometer (Jeol, Tokyo, Japan). A 30-m HP-5 column (0.32-mm inner diameter with 0.25-μm filter, Agilent) was used with helium as the carrier gas. Samples (2 μl) were injected in the splitless mode. The gas chromatographer oven temperature was programmed as follows: 2 min at 70 °C followed by a rise to 120 °C at 5 °C/min, a rise to 260 °C at 7 °C/min, a pause at 260 °C for 3.5 min, a rise to 300 °C at 15 °C/min, and then 10 min at 300 °C. The identities of pristanic acid and stearic acid were determined by comparing their retention times and mass spectra with those of the tert-butyldimethylsilyl derivatives of purchased materials (stearic acid from Wako and pristanic acid from Sigma-Aldrich) as standards. The single ion monitoring mode was used for the detection of the (M - 57)+ ions for both pristanic acid and stearic acid. The amount of pristanic acid and stearic acid was quantified by integration of the respective peaks. Pristanic Acid Assay—The cells were cultured with 750 μm phytol with or without tetracycline for 24 h. Pristanic acid was extracted from the cells according to a procedure described previously (30Komen J.C. Duran M. Wanders R.J. J. Lipid Res. 2004; 45: 1341-1346Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) with a slight modification. After being washed with PBS, the cells were homogenized with a Multi-beads shocker (Yasui Kikai, Osaka, Japan) in PBS. Then 0.1 volumes of 12 m HCl was added and mixed by vortexing. Six volumes of ether was added, and the mixture was stored at 4 °C for 48 h after mixing. The organic layer was collected, and the solvents were evaporated to 2 ml at 40 °C and dried with MgSO4. The solution was transferred to reaction vials, and the solvent was evaporated completely. The extracted compounds were converted to their corresponding tert-butyldimethylsilyl derivatives using 10 μl of N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (Pierce) and 10 μl of pyridine at 80 °C for 1 h. After the derivatization, the solution was directly used as a sample for GC-MS. Cell Viability Assay—The viability of the HEK293 cells was determined using the Cell Counting Kit-F (Dojindo, Kumamoto, Japan). The assay is based on the activity of living cells to hydrolyze calcein-AM to produce fluorescent calcein (31Mount A.S. Wheeler A.P. Paradkar R.P. Snider D. Science. 2004; 304: 297-300Crossref PubMed Scopus (346) Google Scholar). The excitation and emission wavelengths of calcein are 485 and 535 nm, respectively, and the intensity of fluorescence is directly proportional to the number of living cells in culture. The fluorescence was quantified with a Typhoon 9410 variable mode imager (GE Healthcare). ER Stress Response Assay—ER stress was quantified by measuring the amount of variant XBP1 mRNA as described previously (32Hirota M. Kitagaki M. Itagaki H. Aiba S. J. Toxicol. Sci. 2006; 31: 149-156Crossref PubMed Scopus (89) Google Scholar). Briefly total RNA was extracted from the test cells with Quick-Gene 810 (Fujifilm). The mRNA of the XBP1 variant was quantified by real time PCR using specific primers (5′-GCTGAGTCCGCAGCAGGT and 5′-TTGTCCAGAATGCCCAACAG). For an internal standard, the amount of human glyceraldehyde-3-phosphate dehydrogenase mRNA was measured using the primers 5′-AGCCACATCGCTCAGACAC and 5′-GCCCAATACGACCAAATCC. Statistical Analysis—Data are means ± S.D. Significance was examined by the unpaired t test. FALDH Was Induced in the Liver of Normal Mice Fed Sesame but Was Far Less Abundant in PPARα-null Mice—Our preliminary analysis to detect the PPARα-dependently induced mRNAs in the liver of normal mice fed with sesame showed that FALDH, in addition to several P450s, was induced severalfold. 3B. Ashibe and K. Motojima, unpublished data. To confirm this, we prepared a mouse FALDH-specific anti-body (see Fig. 4A) and analyzed the changes in the amount of FALDH in the liver of wild-type or PPARα-null mice fed a normal laboratory diet or sesame seeds by Western blotting. As shown in Fig. 1, sesame induced FALDH expression by severalfold in the liver of wild-type mice. In the liver of PPARα-null mice, the basal expression levels of FALDH were low, about a fifth to a tenth of that in the corresponding tissue of wild-type mice, and a small but significant induction by sesame was observed. These results indicated that the induction of FALDH expression by sesame is dependent on the expression of PPARα, which is consistent with a recent publication (16Gloerich J. van den Brink D.M. Ruiter J.P. van Vlies N. Vaz F.M. Wanders R.J. Ferdinandusse S. J. Lipid Res. 2007; 48: 77-85Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The sesame seeds may contain a natural ligand for PPARα (3Motojima M. Hirai T. FEBS J. 2006; 273: 292-300Crossref PubMed Scopus (26) Google Scholar). Identification of this ligand and elucidation of the mechanism for PPARα-independent induction of FALDH expression by sesame are important issues needing to be solved. However, we are first interested in the physiological significance of the induction of FALDH expression and started the characterization of FALDH at the protein level.FIGURE 1FALDH expression was induced in the liver of normal mice fed sesame but was far less expressed in PPARα-null mice. The proteins (20 μg) in post nuclear fractions of the liver of male C57/BL6J (+/+) or PPARα-null (-/-) mice fed a normal laboratory diet (-), sesame seeds (S), or a diet containing Wy14,643 (Wy) for 3 days were separated by SDS-PAGE, and the expression levels of FALDH were compared by Western blotting using anti-mouse FALDH antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mouse FALDH mRNA Has Several Variants Generated by Alternative Splicing—FALDH is known to have variant forms produced by alternative splicing of the primary transcript (22Lin" @default.
• W2160653669 created "2016-06-24" @default.
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• W2160653669 date "2007-07-01" @default.
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• W2160653669 title "Dual Subcellular Localization in the Endoplasmic Reticulum and Peroxisomes and a Vital Role in Protecting against Oxidative Stress of Fatty Aldehyde Dehydrogenase Are Achieved by Alternative Splicing" @default.
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