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• W2068085464 abstract "Multiple retinoic acid responsive cDNAs were isolated from a high density cDNA microarray membrane, which was developed from a cDNA library of human tracheobronchial epithelial cells. Five selected cDNA clones encoded the sequence of the same novel gene. The predicted open reading frame of the novel gene encoded a protein of 319 amino acids. The deduced amino acid sequence contains four motifs that are conserved in the short-chain alcohol dehydrogenase/reductase (SDR) family of proteins. The novel gene shows the greatest homology to a group of dehydrogenases that can oxidize retinol (retinol dehydrogenases). The mRNA of the novel gene was found in trachea, colon, tongue, and esophagus. In situhybridization of airway tissue sections demonstrated epithelial cell-specific gene expression, especially in the ciliated cell type. Both all-trans-retinoic acid and 9-cis-retinoic acid were able to elevate the expression of the novel gene in primary human tracheobronchial epithelial cells in vitro. This elevation coincided with an enhanced retinol metabolism in these cultures. COS cells transfected with an expression construct of the novel gene were also elevated in the metabolism of retinol. The results suggested that the novel gene represents a new member of the SDR family that may play a critical role in retinol metabolism in airway epithelia as well as in other epithelia of colon, tongue, and esophagus. Multiple retinoic acid responsive cDNAs were isolated from a high density cDNA microarray membrane, which was developed from a cDNA library of human tracheobronchial epithelial cells. Five selected cDNA clones encoded the sequence of the same novel gene. The predicted open reading frame of the novel gene encoded a protein of 319 amino acids. The deduced amino acid sequence contains four motifs that are conserved in the short-chain alcohol dehydrogenase/reductase (SDR) family of proteins. The novel gene shows the greatest homology to a group of dehydrogenases that can oxidize retinol (retinol dehydrogenases). The mRNA of the novel gene was found in trachea, colon, tongue, and esophagus. In situhybridization of airway tissue sections demonstrated epithelial cell-specific gene expression, especially in the ciliated cell type. Both all-trans-retinoic acid and 9-cis-retinoic acid were able to elevate the expression of the novel gene in primary human tracheobronchial epithelial cells in vitro. This elevation coincided with an enhanced retinol metabolism in these cultures. COS cells transfected with an expression construct of the novel gene were also elevated in the metabolism of retinol. The results suggested that the novel gene represents a new member of the SDR family that may play a critical role in retinol metabolism in airway epithelia as well as in other epithelia of colon, tongue, and esophagus. retinoic acid retinoic acid receptor retinoid X receptor retinol dehydrogenase SDR, short chain (alcohol) dehydrogenase/reductase TBE, cell-specific human retinol dehydrogenase polymerase chain reaction untranslated region high performance liquid chromatography base pair(s) Vitamin A (retinol) and its metabolites (retinoids) are essential to the development and maintenance of the airway epithelial phenotype (1Huang T.H. Ann D.K. Zhang Y.J. Chang A.T. Crabb J.W. Wu R. Am. J. Respir. Cell Mol. Biol. 1994; 10: 192-201Crossref PubMed Scopus (26) Google Scholar, 2Jetten A.M. Rearick J.I. Smits H.L. Biochem. Soc Trans. 1986; 14: 930-933Crossref PubMed Scopus (33) Google Scholar, 3Wu, R. (ed). (1997) in Growth and Differentiation of Tracheobronchial Epithelial Cells. Lung Growth and Development (McDonald, J. A., ed) pp. 211-241,Marcel Dekker, Inc., New YorkGoogle Scholar, 4Jetten A.M. Nervi C. Vollberg T.M. J. Natl. Cancer Inst. Monogr. 1992; 13: 93-100PubMed Google Scholar). Epithelial tissues, including the airway epithelia, are vitamin A target tissues that require retinoids (5Wolbach S.B. Howe P.R. J. Exp. Med. 1925; 42: 753-781Crossref PubMed Scopus (1094) Google Scholar, 6Fell H.B. Mellanby E. J. Physiol. 1953; 119: 470-488Crossref PubMed Scopus (185) Google Scholar, 7De Luca L.M. Roop D. Huang F.L. Acta Vitamin. Enzymol. 1985; 7 (suppl.): 13-20PubMed Google Scholar). Vitamin A metabolites, such as all-trans-retinoic acid (RA)1 and 9-cis-RA, are important regulators for gene transcription as the ligands for various transcriptional factors of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families (for review, see Ref. 8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2605) Google Scholar). Extensive progress has been made in determining the specificity of these RA metabolites with the interactions with these RARs and RXRs (8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2605) Google Scholar, 9Allenby G. Bocquel M.T. Saunders M. Kazmer S. Speck J. Rosenberger M. Lovey A. Kastner P. Grippo J.F. Chambon P. Levin A.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 30-34Crossref PubMed Scopus (671) Google Scholar). As compared with these RA metabolites, retinol is not as potent as those RAs in terms of the interactions with these receptors and in terms of transcriptional regulation (10Connor M.J. Smit M.H. Biochem. Pharmacol. 1987; 36: 919-924Crossref PubMed Scopus (39) Google Scholar, 11Kurlandsky S.B. Xiao J.H. Duell E.A. Voorhees J.J. Fisher G.J. J. Biol. Chem. 1994; 269: 32821-32827Abstract Full Text PDF PubMed Google Scholar). Thus, it was suggested that retinol has to be metabolized to various RAs to exert its biological activity (11Kurlandsky S.B. Xiao J.H. Duell E.A. Voorhees J.J. Fisher G.J. J. Biol. Chem. 1994; 269: 32821-32827Abstract Full Text PDF PubMed Google Scholar). However, the mechanism by which vitamin A and its metabolites exert this activity to regulate airway development and the maintenance of mucociliary functions in epithelium is unknown. As lipid soluble compounds, retinoids can readily cross the membrane into any cell in the body from the plasma circulation. The major form of retinoid in plasma circulation is all-trans-retinol at about 1.5–2.0 μm (12Ribaya-Mercado J.D. Mazariegos M. Tang G. Romero-Abal M.E. Mena I. Solomons N.W. Russell R.M. Am. J. Clin. Nutr. 1999; 69: 278-284Crossref PubMed Scopus (39) Google Scholar, 13Booth S.L. Tucker K.L. McKeown N.M. Davidson K.W. Dallal G.E. Sadowski J.A. J. Nutr. 1997; 127: 587-592Crossref PubMed Scopus (88) Google Scholar). However, the level of RA in the plasma is around 4–14 nm (14De Leenheer A.P. Lambert W.E. Claeys I. J. Lipid Res. 1982; 23: 1362-1367Abstract Full Text PDF PubMed Google Scholar, 15Eckhoff C. Nau H. J. Lipid Res. 1990; 31: 1445-1454Abstract Full Text PDF PubMed Google Scholar), which may be insufficient to supply the needed cellular RA level for its biological activity in vitamin A target tissues and cells. In addition, in some vitamin A target tissues and cells, such as skin and keratinocytes (16Jurukovski V. Markova N.G. Karaman-Jurukovska N. Randolph R.K. Su J. Napoli J.L. Simon M. Mol. Genet. Metab. 1999; 67: 62-73Crossref PubMed Scopus (51) Google Scholar), the access to blood vessels can be quite limited. Thus, there is a need in these vitamin A target cells for an efficient machinery to transport and to metabolize plasma retinol into various RA metabolites. The production of RA from retinol can take place within the cell if the cell contains enzymes that can sequentially oxidize retinol to retinaldehyde and retinaldehyde to RA. The reversible oxidation of retinol to retinaldehyde has been suggested to be the rate-limiting step in the metabolism of retinol to retinoic acid and therefore the step most likely to be tightly regulated by the cell (17Chai X. Boerman M.H. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 18Napoli J.L. Posch K.P. Fiorella P.D. Boerman M.H. Biomed. Pharmacother. 1991; 45: 131-143Crossref PubMed Scopus (102) Google Scholar). The second step, the oxidation of retinaldehyde to retinoic acid, seems to be irreversible (reviewed in Ref. 19Duester G. Biochemistry. 1996; 35: 12221-12227Crossref PubMed Scopus (234) Google Scholar). Consistent with the first step in this model of retinol metabolism in vitamin A target cells, there have been reports of multiple enzymes with retinol dehydrogenase activities that are isoform-specific or cell type-specific. These enzymes include (but are not limited to) RODH-I (17Chai X. Boerman M.H. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), RODH-II (20Chai X. Zhai Y. Popescu G. Napoli J.L. J. Biol. Chem. 1995; 270: 28408-28412Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), RODH-III (21Chai X. Zhai Y. Napoli J.L. Gene ( Amst. ). 1996; 169: 219-222Crossref PubMed Scopus (63) Google Scholar), RDH-4 (22Gough W.H. VanOoteghem S. Sint T. Kedishvili N.Y. J. Biol. Chem. 1998; 273: 19778-19785Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), hRDH-E (16Jurukovski V. Markova N.G. Karaman-Jurukovska N. Randolph R.K. Su J. Napoli J.L. Simon M. Mol. Genet. Metab. 1999; 67: 62-73Crossref PubMed Scopus (51) Google Scholar), 9- and 11-cRDH (23Mertz J.R. Shang E. Piantedosi R. Wei S. Wolgemuth D.J. Blaner W.S. J. Biol. Chem. 1997; 272: 11744-11749Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 24Gamble M.V. Shang E. Zott R.P. Mertz J.R. Wolgemuth D.J. Blaner W.S. J. Lipid Res. 1999; 40: 2279-2292Abstract Full Text Full Text PDF PubMed Google Scholar, 25Gamble M.V. Mata N.L. Tsin A.T. Mertz J.R. Blaner W.S. Biochim. Biophys. Acta. 2000; 1476: 3-8Crossref PubMed Scopus (32) Google Scholar), RDH-5 (26Simon A. Hellman U. Wernstedt C. Eriksson U. J. Biol. Chem. 1995; 270: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 27Driessen C.A. Janssen B.P. Winkens H.J. van Vugt A.H. de Leeuw T.L. Janssen J.J. Invest. Ophthalmol. Vis. Sci. 1995; 36: 1988-1996PubMed Google Scholar, 28Simon A. Lagercrantz J. Bajalica-Lagercrantz S. Eriksson U. Genomics. 1996; 36: 424-430Crossref PubMed Scopus (66) Google Scholar, 29Wang J. Chai X. Eriksson U. Napoli J.L. Biochem. J. 1999; 338: 23-27Crossref PubMed Scopus (75) Google Scholar), RDH-6 (30Chai X. Zhai Y. Napoli J.L. J. Biol. Chem. 1997; 272: 33125-33131Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and RDH-7 (31Su J. Chai X. Kahn B. Napoli J.L. J. Biol. Chem. 1998; 273: 17910-17916Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). All of these enzymes are members of the short chain (alcohol) dehydrogenase/reductase (SDR) gene superfamily. Enzymes in the alcohol dehydrogenase and cytochrome P450 families have been implicated in the oxidation of retinaldehyde to retinoic acid, which is the second step in the metabolism of retinol to retinoic acid. There are reports of retinaldehyde-specific dehydrogenases that catalyze this step (32Bhat P.V. Bader T. Nettesheim P. Jetten A.M. Biochem. Cell Biol. 1998; 76: 59-62Crossref PubMed Scopus (9) Google Scholar, 33Haselbeck R.J. Hoffmann I. Duester G. Dev. Genet. 1999; 25: 353-364Crossref PubMed Google Scholar, 34Niederreither K. McCaffery P. Drager U.C. Chambon P. Dolle P. Mech. Dev. 1997; 62: 67-78Crossref PubMed Scopus (435) Google Scholar, 35Penzes P. Wang X. Napoli J.L. Biochim. Biophys. Acta. 1997; 1342: 175-181Crossref PubMed Scopus (38) Google Scholar, 36Penzes P. Wang X. Sperkova Z. Napoli J.L. Gene ( Amst. ). 1997; 191: 167-172Crossref PubMed Scopus (47) Google Scholar, 37Ulven S.M. Gundersen T.E. Weedon M.S. Landaas V.O. Sakhi A.K. Fromm S.H. Geronimo B.A. Moskaug J.O. Blomhoff R. Dev. Biol. 2000; 220: 379-391Crossref PubMed Scopus (101) Google Scholar, 38Zhao D. McCaffery P. Ivins K.J. Neve R.L. Hogan P. Chin W.W. Drager U.C. Eur. J. Biochem. 1996; 240: 15-22Crossref PubMed Scopus (256) Google Scholar). Of particular interest, at least one of these retinaldehyde dehydrogenases is specifically expressed in tracheal epithelial cells in rat, with a putative role in the conversion of retinaldehyde to retinoic acid in these cells. However, the critical enzyme that converts retinol to retinaldehyde is still unknown in airway epithelial cells. In this communication, we report the cloning and characterization of a novel airway epithelial cell-specific SDR gene from a cDNA library derived from primary human tracheobronchial epithelial (TBE) cells, using a differential hybridization approach on a high density cDNA microarray membrane. Five independently selected RA responsive cDNA clones encoded the same novel gene. The conceptual translation of the novel cDNA sequence encodes a protein that contains amino acid motifs that are conserved in the SDR family of genes. Within the SDR superfamily, the novel gene is most homologous to the retinol dehydrogenases. We provide evidence suggesting that this novel gene represents a new member of the SDR family that may play a critical role in retinoid metabolism in airway epithelia. Human airway tissues were obtained from the University of California at Davis Medical Center with donor consent. The protocol for obtaining human tissues has been approved and periodically reviewed by the campus Human Subject Review Committee. TBE cells were isolated from these tissues by a protease dissociation procedure and cultured in a serum-free hormone-supplemented medium as previously described (39Robinson C.B. Wu R. J. Tissue Culture Methods. 1991; 13: 95-102Crossref Scopus (39) Google Scholar). Briefly, cells were grown in 6-well tissue culture plates either without additional substratum (TC) or with collagen gel substratum (Cg) or in air-liquid biphasic culture insert chambers (Transwell™ chamber, Corning/Costar 3450, Corning, NY) without (Bi) or with collagen gel substratum (Bi-Cg). The serum-free hormone-supplemented medium was slightly modified from that described in Ref. 39Robinson C.B. Wu R. J. Tissue Culture Methods. 1991; 13: 95-102Crossref Scopus (39) Google Scholar; the modified medium contained the supplements insulin (5 μg/ml), transferrin (5 μg/ml), EGF (10 ng/ml), cholera toxin (20 ng/ml), dexamethasone (0.1 μm), bovine hypothalamus extract (15 μg/ml), bovine serum albumin (1 mg/ml), and all-trans-RA (30 nm, Fluka, Milwaukee, WI) in a nutrient medium containing 1:1 of Ham's F12/Dulbecco's modified Eagle's medium. This modified culture medium allows airway epithelial cells to better grow and express mucociliary differentiation than before in culture, especially under the Bi-Cg condition. For both Bi and Bi-Cg culture conditions, cells were immersed in the culture medium for 7 days and then the Transwell™ chambers were lifted up in between the air and liquid interface for the remaining days in culture. For dose response and time course experiments, cultures were maintained for 7 to 10 days without all-trans-RA supplementation and then RA at 30 nm or at various concentrations (1–1000 nm) was added accordingly as described in the experiments. After various lengths of time in culture, total RNA was isolated from cultured cells by a single step acid guanidium thiocyanate extraction method (40Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63225) Google Scholar). COS cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Life Technology, Grand Island, NY). Thirty thousand cDNA clones were derived from primary human TBE cells that had been cultured for more than 30 days under an air-liquid interface culture condition in the completed medium containing both the hormonal supplements and all-trans-RA. Under such an in vitro culture system, the human TBE cells differentiated into a mucociliary epithelium resembling that seen in vivo. The cDNA clones were packaged using a pBK CMV phagemid packaging system (Stratagene, La Jolla, CA). From the 30,000 cDNA clones, we developed a high density microarray membrane (3.1 × 4.6 cm, Nytran N+ nylon membrane, Schleicher&Schuell, Keene, NH). This 30,000 cDNA high density microarray membrane was hybridized simultaneously with two-color cDNA probes, magenta- and cyan-based, derived from all-trans-RA-treated and untreated cultures of primary human TBE cells, respectively, as described before (41Chen J.J.W. Wu R. Yang P.C. Huang J.Y. Sher Y.P. Han M.H. Kao W.C. Lee P.J. Chiu T.F. Chang F. Chu Y.W. Wu C.W. Peck K. Genomics. 1998; 51: 313-324Crossref PubMed Scopus (203) Google Scholar). Based on a quantitative ratio of cyan/magenta either greater than 5 or less than 0.2, clones were selected for further characterization. These characterizations included the use of Northern blot hybridization to further confirm the differentially expressed nature of the corresponding messages in these clones, DNA sequencing to allow searches for homologous genes of known function to provide clues as to the function of a novel gene, and, in some cases, in situhybridization to elucidate cell type-specific gene expression. DNA sequencing was carried out at the DBS Automated DNA Sequencing Facility, UC-Davis. DNA sequence data was analyzed with Lasergene software (DNASTAR Inc., Madison, WI) and with the online GCG Software package SeqWeb (Madison, WI). Sequence homology to published sequences in public databases was determined by the BLASTn or BLASTp program at the National Center for Biotechnology Information (NCBI) through Internet services. The phylogenetic tree was compiled by the GCG program GrowTree with the following parameters: Kimura distance correction method, UPGMA tree construction method, scoring matrix blossum 62, 8-residue-gap creation penalty, 2-residue-gap extension penalty, 1000-residue maximum sequence input range, 6000-residue maximum gap character insertion allowed, with all branches of the same length (a cladogram). All five cDNAs that represent the novel gene are in the vector pBK-CMV (Stratagene) with the 5′-end at the T3 side of the multiple cloning site. PCR using any of these cDNAs as template, with insert-specific primers (forward primer, 5′-TGAGCAAGTCCACCAACAGT-3′ and reverse primer, 5′-GCACGATCTAAATGAGTCCA-3′) generated a fragment of the cDNA containing mostly coding sequence; this PCR product was used to probe Northern blots. For FLAG tagging, the entire coding region of the novel cDNA was fused in frame 3′ to a FLAG tag in the vector pFLAG CMV2 (Sigma, St. Louis, MO). This was done by introducing a unique EcoRI site just upstream of the presumed translational start codon (see Fig.1 C). The EcoRI site was introduced during PCR; the forward primer (5′-GGGAATTCAATGCTCTTTTGGGTGCTAG) altered five bases of the novel gene's 5′-UTR (the underlined bases), whereas the reverse primer was a commercially available T7 primer. The resulting amplicon was digested with EcoRI andKpnI and inserted into pFLAG-CMV2, which had been cut with the same enzymes. The vector/insert junction was verified by nucleotide sequencing. Twenty micrograms of total RNA were fractionated on a 0.66 m formaldehyde, 1.0% agarose gel and transferred overnight to a Nytran N+ nylon membrane. RNA was fixed to the membrane by UV cross-linking (Stratalinker, Stratagene). The PCR product derived from the novel cDNA was labeled with [α-32P]dCTP (ICN, Costa Mesa, CA) to a specific activity of ∼2 × 109 dpm/μg with a Ready-To-Go random primer labeling kit (Amersham Pharmacia Biotech Life Sciences, Arlington Heights, IL). The membranes were prehybridized in hybridization solution (6× SSC, 0.5% SDS, 0.01m EDTA, 0.5% disodium pyrophosphate, 5× Denhardt's solution) at 68 °C for a minimum of 4 h followed by hybridization in the same solution plus specific probe at 68 °C for 16–20 h (overnight). Hybridized membranes were washed once with 2× SSC, 0.1% SDS for 10 min at room temperature and twice with 1.0× SSC, 0.1% SDS for 30 min at 68 °C. Following the second wash, membranes were checked for excessive radioactivity, and, if necessary, washed in 0.1× SSC and 0.1% SDS for various times at 68 °C. PhosphorImager screens (Molecular Dynamics, Amersham Pharmacia Biotech Life Sciences) and/or X-Omat film (Kodak, Rochester, NY) were exposed to hybridized membranes for various times. To study the novel gene's expression patterns in normal tissues, Northern blots containing 20 μg of total RNA from various adult primate tissues were prepared, and Northern analysis was carried out as described above. Monkey tissues were obtained from necropsies done at the California Regional Primate Research Center of UC Davis. The sequence of the full-length cDNA was determined by 5′ extension on RNA from human TBE cultured under the Bi-Cg condition with 30 nm all-trans-RA, using an endlabeled antisense primer (5′-AGGAGGGTGAGGCTGGTGATAGAG-3′) and the dideoxynucleotide chain termination method according to the manufacturer's recommendations (Promega, Madison, WI). A genomic PCR chromosome walking fragment from the promoter region of the gene was sequenced in parallel with the 5′extension product using the same endlabeled primer and the TaqtrackTM sequencing kit (Promega). Clones obtained from the original phage library screening were converted to phagemids according to the manufacturer's protocol (Strategene). The recombinant plasmids were linearized with EcoRI or XhoI to generate antisense and sense templates, respectively. The linearized templates were transcribed in vitro with T7 and T3 RNA polymerases using MAXIscriptTM according to the manufacturer's recommendations (Ambion Inc., Austin, TX) to produce35S-UTP-labeled antisense and sense probes, respectively.In situ hybridization was carried out as described previously (42Simmons D.M. Arriza J.L. Swanson L.W. J. Histotechnol. 1989; 12: 169-181Crossref Google Scholar). A 15-mer oligopeptide antigen was synthesized (Research Genetics, Inc., Huntsville, AL) using deduced amino acid sequence 244–258 of hRDH-TBE (see Fig. 1 C). The peptide was conjugated to multiple antigen peptide to increase its antigenicity, and rabbit-based polyclonal antibodies were generated as described before (1Huang T.H. Ann D.K. Zhang Y.J. Chang A.T. Crabb J.W. Wu R. Am. J. Respir. Cell Mol. Biol. 1994; 10: 192-201Crossref PubMed Scopus (26) Google Scholar). The specificity of the polyclonal antiserum was determined by ELISA and Western blot analysis. For Western blot analysis, cultured cells were harvested as described (43Huang T.H. St. George J.A. Plopper C.G. Wu R. Differentiation. 1989; 41: 78-86Crossref PubMed Scopus (18) Google Scholar, 44Wu Y.J. Parker L.M. Binder N.E. Beckett M.A. Sinard J.H. Griffiths C.T. Rheinwald J.G. Cell. 1982; 31: 693-703Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 45Wu R. Wu M.M. J. Cell. Physiol. 1986; 127: 73-82Crossref PubMed Scopus (39) Google Scholar). Supernatant protein concentrations were determined by the method of Lowry using the Bio-Rad Dc assay (Bio-Rad, Hercules, CA). Equal protein amounts were subjected to discontinous SDS-polyacrylamide gel electrophoresis according to Laemmli (46Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207472) Google Scholar). Proteins were blotted onto PVDF or Nytran membranes according to the manufacturer's recommendations with a semi-dry blotting apparatus (Schleicher&Schuell) at 120 mA/45 min/10 cm2 gel surface area. Western hybridization was done using a Vectastain ABC kit (Vector Labs, Inc., Burlingame, CA) and the appropriate primary and secondary antibodies. TBE cells were grown in air-liquid interface (Bi) without or with 100 nmall-trans-RA. After 21 days, some of the cells were harvested for total RNA and protein isolation. The remaining cells were incubated with either 3 μm all-trans retinol (Fluka) or the equivalent volume of the Me2SO vehicle for 2 h. Alternatively, COS cells were transiently transfected with vector alone or FLAG·hRDH-TBE fusion constructs using FuGENE 6 reagent, following the manufacturer's instructions (Roche Molecular Biochemicals, Basel, Switzerland). Two days post-transfection, one dish from each transfection group was harvested for protein isolation. The remaining COS cells were exposed to 2 μmall-trans retinol or an equivalent volume of the Me2SO vehicle for 5 h. After incubation, cells were rinsed twice in ice-cold phosphate-buffered saline, then harvested by scraping into 1–2 ml of 0.002% (v/v) SDS. Cell suspensions were stored frozen at −80 °C. Cell suspensions were thawed in a 37 °C bath. The suspensions were sonicated on ice to complete cell lysis. A small aliquot of each lysate was reserved for protein concentration determination by the Bio-Rad Dc assay. An internal standard was added to the remaining lysate. Retinoids were then extracted by sequential additions of 1.5 volumes of acetonitrile/butanol (1:1 v/v), 0.5 volumes of hexane/chloroform (2:1 v/v), followed by 0.2 volumes of saturated K2HPO4 and were mixed vigorously. Following centrifugation at 13,000 × g for 10 min, the organic phase was transferred to an amber tube and dried in the dark, under vacuum. The resultant sample residue was reconstituted in 150 μl of methanol/acetonitrile/isopropyl alcohol (3:1:1 v/v) and analyzed using HPLC for the identification and quantitation of retinoids. Retinoid analysis and quantitation were performed using a reverse phase Nova-Pak C18 (8.0 cm × 10 cm) 4-μm pore size analytical column (Waters Associates, Milford, MA). Reconstituted cell extracts were analyzed for retinoids under isocratic conditions using an elution solvent mobile phase of methanol/tetrahydrofuran-acetonitrile-isopropyl alcohol (TAI)/0.005 m ammonium acetate (67:10:23 v/v). The TAI solution was comprised of tetrahydrofuran/acetonitrile/2-propanol (3:1:0.02 v/v). Retinoids were detected by UV absorbance at 350 nm. Under these conditions, the system has a lower limit of detection for RA of ∼1.3 pmol at a signal-to-noise ratio of 2.5; detection limits for retinol are ∼3.1 pmol. Cell-associated retinoids are expressed as pmol per mg protein. The retinoid per sample was calculated from each peak area normalized to the peak area of the internal standard and from the amount of protein. All HPLC analyzed groups are from duplicate samples. The amount of all-trans-RA is an indirect measure of retinol dehydrogenase activity because the immediate product of dehydrogenase activity is retinaldehyde, which is irreversibly further oxidized to RA. For calibrations and standards, pure retinoids were made in methanol and their concentrations determined by UV absorbance using published maximal absorbance wavelengths and corresponding molar extinction coefficients (47Furr H.C. Barua A.B. Olson J.A. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine.in: Raven Press, Ltd., New York1994: 179-209Google Scholar). The elution position of matching retinoid standards was used to identify specific retinoid peaks that were then quantified by computer integration of the areas under the respective peaks. The purity of the retinoids was verified by determining the absorption spectra of isolated peaks. Accuracy of the method and calibration conditions were further verified using authenticated samples containing known quantities of retinoids (National Bureau of Standards). Based on differential hybridization results, 79 cDNA clones were selected as RA-responsive clones for further study. 2J. Chen, Y. P. Di, M. M. Chang, K. Chmiel, J. Zhou, M. Juarez, R. W. Harper, Y. Chen, P. C. Yang, C. M. Soref, K. Peck, and R. Wu, manuscript in preparation. Northern blot hybridization confirmed that the corresponding messages of 76 clones were differentially regulated by all-trans-RA. Of the remaining three clones, one was a false positive and the other two were unable to be characterized by Northern blot because of extremely low abundance message levels. Among the 76 differentially expressed clones, 14 encoded novel cDNA sequences. Five of the 14 novel clones, DD13, DD18, DD90, DD91, and DD95, had identical Northern profiles and identical cDNA sequences at the 3′-end of a novel gene. A complete nucleotide sequencing of these clones further confirmed their identical nature. The full-length cDNA sequence of this novel DD13/18/90/91/95 gene was determined by 5′ primer extension (Fig.1). The cDNA contains a 557 bp 5′-UTR, a predicted open reading frame of 960 bp, which is presumably the coding sequence, and a 403 bp 3′-UTR. The conceptual translation of the open reading frame encodes a peptide of 319 amino acids with a predicted molecular size of 35 kDa. All five selected cDNA clones have the same 3′-UTR, coding sequence, and partial 5′-UTR; the clones differ only in how far the 5′-UTR extends upstream of the translational start site (Fig.1 A). Primer extension experiments generated only one product band, indicating a single transcriptional start site (Fig.1 B). These observations suggest that the five clones are incomplete cDNAs generated from a unique template during the creation of the cDNA library. Searches of the GenBankTM non-redundant (nr) database using various incomplete fragments of the novel cDNA sequence as query revealed homology at the DNA level to members of the SDR gene family. A BLAST search of the GenBankTM nr database with the completed cDNA sequence revealed an almost complete identity to three clones identified as retinol dehydrogenase isoforms-1 and -2, and retinol dehydrogenase homolog (GenBankTM accession numbersAF240698, AF240697, and AF067174). No information other than the sequences has been provided for these clones. Alignment of the homologous sequences with the DD13/18/90/91/95 clones indicates that the clones were short in sequence at the 5′-end of the novel gene (Fig.1 A). There are gaps in the alignment of two of the homologous clones, which may represent alternative splicing events. In addition to the close homology to clones AF240698, AF240697, andAF067174, the newly i" @default.
• W2068085464 created "2016-06-24" @default.
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• W2068085464 date "2001-06-01" @default.
• W2068085464 modified "2023-10-16" @default.
• W2068085464 title "Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol" @default.
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