@article{reading_hiramatsu_schilling_molloy_glassbrook_mizuta_luo_baltzegar_williams_todo_et al._2014, title={Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes}, volume={55}, ISSN={["1539-7262"]}, DOI={10.1194/jlr.m050286}, abstractNote={Transcripts encoding a novel member of the lipoprotein receptor superfamily, termed LDL receptor-related protein (Lrp)13, were sequenced from striped bass (Morone saxatilis) and white perch (Morone americana) ovaries. Receptor proteins were purified from perch ovary membranes by protein-affinity chromatography employing an immobilized mixture of vitellogenins Aa and Ab. RT-PCR revealed lrp13 to be predominantly expressed in striped bass ovary, and in situ hybridization detected lrp13 transcripts in the ooplasm of early secondary growth oocytes. Quantitative RT-PCR confirmed peak lrp13 expression in the ovary during early secondary growth. Quantitative mass spectrometry revealed peak Lrp13 protein levels in striped bass ovary during late-vitellogenesis, and immunohistochemistry localized Lrp13 to the oolemma and zona radiata of vitellogenic oocytes. Previously unreported orthologs of lrp13 were identified in genome sequences of fishes, chicken (Gallus gallus), mouse (Mus musculus), and dog (Canis lupus familiaris). Zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) lrp13 loci are discrete and share genomic synteny. The Lrp13 appears to function as a vitellogenin receptor and may be an important mediator of yolk formation in fishes and other oviparous vertebrates. The presence of lrp13 orthologs in mammals suggests that this lipoprotein receptor is widely distributed among vertebrates, where it may generally play a role in lipoprotein metabolism. Transcripts encoding a novel member of the lipoprotein receptor superfamily, termed LDL receptor-related protein (Lrp)13, were sequenced from striped bass (Morone saxatilis) and white perch (Morone americana) ovaries. Receptor proteins were purified from perch ovary membranes by protein-affinity chromatography employing an immobilized mixture of vitellogenins Aa and Ab. RT-PCR revealed lrp13 to be predominantly expressed in striped bass ovary, and in situ hybridization detected lrp13 transcripts in the ooplasm of early secondary growth oocytes. Quantitative RT-PCR confirmed peak lrp13 expression in the ovary during early secondary growth. Quantitative mass spectrometry revealed peak Lrp13 protein levels in striped bass ovary during late-vitellogenesis, and immunohistochemistry localized Lrp13 to the oolemma and zona radiata of vitellogenic oocytes. Previously unreported orthologs of lrp13 were identified in genome sequences of fishes, chicken (Gallus gallus), mouse (Mus musculus), and dog (Canis lupus familiaris). Zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) lrp13 loci are discrete and share genomic synteny. The Lrp13 appears to function as a vitellogenin receptor and may be an important mediator of yolk formation in fishes and other oviparous vertebrates. The presence of lrp13 orthologs in mammals suggests that this lipoprotein receptor is widely distributed among vertebrates, where it may generally play a role in lipoprotein metabolism. The LDL receptor (LDLR) gene family is comprised of different genes encoding membrane receptors involved in endocytosis of a variety of ligands, most notably plasma lipoproteins. Characterized members of this family in vertebrates include LDLR (1Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russel D.W. 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Chicken oocyte growth is mediated by an eight ligand binding repeat member of the LDL receptor family.EMBO J. 1994; 13: 5165-5175Crossref PubMed Scopus (217) Google Scholar, 14Davail B. Pakdel F. Bujo H. Perazzolo L.M. Waclawek M. Schneider W.J. Le Menn F. Evolution of oogenesis: the receptor for vitellogenin from the rainbow trout.J. Lipid Res. 1998; 39: 1929-1937Abstract Full Text Full Text PDF PubMed Google Scholar). These receptors typically consist of unique configurations of epidermal growth factor precursor, class b YWxD (LDLb), and O-linked sugar domains, which define their identities, and class A ligand binding (LDLa) repeats, which determine their particular ligand specificities. Exceptional receptors also contain complement C1r/C1s, Uegf, Bmp1 (CUB) domains (i.e., LRP3, LRP10, LRP12) or motif at the N terminus with seven cysteines (MANEC) and polycystic kidney disease (PKD) domains (i.e., LRP11). With the exception of LRP4, LDLR family receptors are type I membrane proteins and all members contain transmembrane and cytoplasmic domains. Acanthomorph fishes express three distinct lipoprotein yolk precursors, vitellogenins (Vtgs) (VtgAa, VtgAb, and VtgC) (15Finn R.N. Kristoffersen B.A. Vertebrate vitellogenin gene duplication in relation to the "3R hypothesis": correlation to the pelagic egg and the oceanic radiation of teleosts.PLoS ONE. 2007; 2: e169Crossref PubMed Scopus (173) Google Scholar, 16Reading B.J. Hiramatsu N. Sawaguchi S. Matsubara T. Hara A. Lively M.O. Sullivan C.V. Conserved and variant molecular and functional features of multiple egg yolk precursor proteins (vitellogenins) in white perch (Morone americana) and other teleosts.Mar. Biotechnol. (NY). 2009; 11: 169-187Crossref PubMed Scopus (67) Google Scholar). These Vtgs are produced by the liver, released into the circulatory system, and taken up specifically by growing oocytes via receptor-mediated endocytosis. We discovered four Vtgr proteins in white perch (Morone americana) ovary: a receptor greater than 212 kDa that binds only VtgAa (VtgAar), two receptors (116 kDa and 110.5 kDa) that preferentially bind VtgAb (VtgAbr), and a 150 kDa putative LDLR (pLDLR) that weakly and indiscriminately binds both VtgAa and VtgAb (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar). The VtgC does not bind ovary Vtgr proteins in this species. We reported the molecular identity of a white perch Vtgr that is orthologous to mammalian VLDLR (18Hiramatsu N. Chapman R.W. Lindzey J.K. Haynes M.R. Sullivan C.V. Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana).Biol. Reprod. 2004; 70: 1720-1730Crossref PubMed Scopus (66) Google Scholar). This Vtgr is termed "Lr8−", because it is a spliced variant gene transcript of vldlr that does not encode the O-linked sugar domain, as is characteristic of this form of Vldlr in fishes and chickens (19Bujo H. Lindstedt K.A. Hermann M. Dalmau L.M. Nimpf J. Schneider W.J. Chicken oocytes and somatic cells express different splice variants of a multifunctional receptor.J. Biol. Chem. 1995; 270: 23546-23551Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 20Prat F. Coward K. Sumpter J.P. Tyler C.R. Molecular characterization and expression of two ovarian lipoprotein receptors in the rainbow trout, Oncorhynchus mykiss.Biol. Reprod. 1998; 58: 1146-1153Crossref PubMed Scopus (67) Google Scholar). Herein, we refer to these Vldlr forms as Lr8+ and Lr8−, based on the presence or absence of the O-linked sugar domain, respectively. We have suggested that white perch Lr8− corresponds to one or both VtgAbr proteins based on the predicted molecular mass of the protein and on prior reports of fish and chicken Lr8− (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar). However, the molecular identity of VtgAar remains unclear. Here, we describe the structure, expression, subcellular localization, and Vtg-binding properties of a novel lipoprotein receptor named 'Lrp13' that corresponds to VtgAar and show that the Lr8− corresponds to the VtgAbr. All experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (21National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsGuide for the Care and Use of Laboratory Animals. 8th edition. National Academies Press, Washington, DC2011Google Scholar) and the procedures were approved by the North Carolina State University (NCSU) Institutional Animal Care and Use Committee. White perch (weight 425 ± 113 g; total length 265 ± 25 mm; all values are reported as mean ± standard deviation) were reared at NCSU (Raleigh, NC). Blood plasma was sampled from male white perch injected with estradiol-17β, as described previously (22Heppell S.A. Jackson L.F. Weber G.M. Sullivan C.V. Enzyme-linked immunosorbent assay (ELISA) of vitellogenin in temperate basses (genus Morone): plasma and in vitro analyses.Trans. Am. Fish. Soc. 1999; 128: 532-541Crossref Scopus (22) Google Scholar), for purification of Vtgs. Ovaries were excised from vitellogenic white perch (n = 3; maximum oocyte diameter 536–552 μm) (23Jackson L.F. Sullivan C.V. Reproduction of white perch (Morone americana): the annual gametogenic cycle.Trans. Am. Fish. Soc. 1995; 124: 563-577Crossref Google Scholar), for preparation of ovary membranes. Striped bass (Morone saxatilis) were reared at the NCSU Pamlico Aquaculture Field Laboratory (Aurora, NC). Ovary tissues from striped bass (n = 3) were collected by dissection or biopsy using a plastic cannula inserted through the urogenital pore at four time points: August (weight 1.90 ± 0.61 kg; total length 512 ± 18.0 mm), November (3.80 ± 0.14 kg; 625 ± 6.0 mm), February (4.40 ± 0.94 kg; 626 ± 46.6 mm), and April (4.54 ± 1.37 kg; 663 ± 74.5 mm). The most advanced oocytes during these time points represented one of four oocyte growth stages (24Reading B.J. Williams V.N. Chapman R.W. Williams T.I. Sullivan C.V. Dynamics of the striped bass (Morone saxatilis) ovary proteome reveal a complex network of the translasome.J. Proteome Res. 2013; 12: 1691-1699Crossref PubMed Scopus (23) Google Scholar): early secondary growth (ESG, oocyte diameter 310 ± 21.8 μm), mid-vitellogenic growth (MVG, 503 ± 60.1 μm), late-vitellogenic growth (LVG, 831 ± 276 μm), and post-vitellogenic growth (PVG, 986 ± 33.0 μm), respectively. Ovary and liver samples were preserved in RNALater (Ambion, Austin, TX) for real-time quantitative RT-PCR. Ovary tissue was frozen in liquid nitrogen for quantitative tandem mass spectrometry. Ovary tissue from MVG and LVG striped bass was fixed for in situ hybridization in 0.2 M sodium phosphate buffer (pH 7.4) containing 4% paraformaldehyde, and for immunohistochemistry in Bouin's solution (Sigma, Saint Louis, MO). Brain, heart, liver, ovary, foregut, muscle, and adipose from MVG striped bass were preserved in RNALater for semi-quantitative RT-PCR. Primers (SB618 F1 and SB618 R1; see supplementary Table I for all primer sequences used in this study) were designed for striped bass contig 00618 (25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar). All DNA oligos were designed with Mac Vector (Accelrys Software, San Diego, CA) and obtained from Integrated DNA Technologies (Coralville, IA). Products were cloned from a Stratagene Uni-Zap XR cDNA library (La Jolla, CA) constructed from pooled white perch ovaries (18Hiramatsu N. Chapman R.W. Lindzey J.K. Haynes M.R. Sullivan C.V. Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana).Biol. Reprod. 2004; 70: 1720-1730Crossref PubMed Scopus (66) Google Scholar), and those from four colonies were bi-directionally sequenced according to our prior studies (16Reading B.J. Hiramatsu N. Sawaguchi S. Matsubara T. Hara A. Lively M.O. Sullivan C.V. Conserved and variant molecular and functional features of multiple egg yolk precursor proteins (vitellogenins) in white perch (Morone americana) and other teleosts.Mar. Biotechnol. (NY). 2009; 11: 169-187Crossref PubMed Scopus (67) Google Scholar). Total RNA was extracted from pooled white perch ovaries using TRIzol reagent (Invitrogen, Carlsbad, CA) (25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar) and 3′ and 5′ rapid amplification of cDNA ends (RACE) were performed using FirstChoice RLM-RACE kit (Ambion). Primers for 5′RACE (WP618 R1 O and WP618 R1 N) and 3′RACE (WP618 F1 O, SB618 F0 O, WP618 F1 N, WP618 F2 N, and WP618 F3 N) with the suffix "O" or "N" were paired with outer and inner RACE primers, respectively. Sequences were assembled using MacVector and polypeptide domains were characterized using SMART (26Letunic I. Doerks T. Bork P. SMART 7: recent updates to the protein domain annotation resource.Nucleic Acids Res. 2012; 40: D302-D305Crossref PubMed Scopus (1301) Google Scholar). Eukaryotic Linear Motif (27Gould C.M. Diella F. Via A. Puntervoll P. Gemünd C. Chabanis-Davidson S. Michael S. Sayadi A. Bryne J.C. Chica C. et al.ELM: the status of the eukaryotic linear motif resource.Nucleic Acids Res. 2010; 38: D167-D180Crossref PubMed Scopus (208) Google Scholar) was used to identify putative functional motifs within the polypeptide domains. The NCBI databases were queried for lrp13 orthologs and Lrp13 polypeptide sequences were collected from fishes, birds, and mammals along with sequences of other representative lipoprotein receptors. Sequences were aligned by ClustalW (28Thompson J.D. Higgins D.G. Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55720) Google Scholar) to generate a dendrogram. Synteny of lrp13 and vldlr (lr8) were compared using zebrafish (Danio rerio) chromosome 5_9,505,781-9,678,348 and chromosome 10_15,283,756-15,333,718 (v. Zv9) and Nile tilapia (Oreochromis niloticus) scaffold GL831141.1_3,169,873-3,288,125 (reverse complemented) and scaffold GL831199.1_1,600,394-1,676,338 (v. Orenil1.0) available from Ensembl (29Flicek P. Amode M.R. Barrell D. Beal K. Brent S. Carvalho-Silva D. Clapham P. Coates G. Fairley S. Fitzgerald S. et al.Ensembl 2012.Nucleic Acids Res. 2012; 40: D84-D90Crossref PubMed Scopus (768) Google Scholar). A mixture of VtgAa and VtgAb (VtgAa/b) was purified according to our studies (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar, 30Hiramatsu N. Matsubara T. Hara A. Donato D.M. Hiramatsu K. Denslow N.D. Sullivan C.V. Identification, purification and classification of three forms of vitellogenin from white perch (Morone americana).Fish Physiol. Biochem. 2002; 26: 355-370Crossref Scopus (63) Google Scholar) and 20 mg of VtgAa/b was coupled to an equal volume (12 ml) of Affi-Gel 15 activated immunoaffinity support (Bio-Rad, Hercules, CA) following procedures adapted from Roehrkasten et al. (31Roehrkasten A. Ferenz H. Buschmann-Gebhardt B. Hafer J. Isolation of the vitellogenin-binding protein from locust ovaries.Arch. Insect Biochem. Physiol. 1989; 10: 141-149Crossref Scopus (37) Google Scholar). Affinity medium was poured into a 25 mm internal diameter column (12 ml) and the chromatography system was initialized with 10 column volumes of coupling buffer [20 mM HEPES, 150 mM NaCl, 1 mM CaCl2 (pH 7.4)] and equilibrated with 10 column volumes of binding buffer [20 mM Tris-HCl, 2 mM CaCl2, and 150 mM NaCl (pH 8.0), containing 1 mM phenylmethyl-sulfonyl fluoride and 4 IU/l aprotinin] at 4°C. Solubilized membrane proteins prepared from 25 g of vitellogenic white perch ovaries, according to our studies (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar, 32Hiramatsu N. Hara A. Hiramatsu K. Fukada H. Weber G.M. Denslow N.D. Sullivan C.V. Vitellogenin-derived yolk proteins of white perch, Morone americana: purification, characterization and vitellogenin-receptor binding.Biol. Reprod. 2002; 67: 655-667Crossref PubMed Scopus (82) Google Scholar), were recycled through the affinity media for 4.5 h at 4°C (1.0 ml/min flow rate). The column was washed with 15 vol of binding buffer at 4°C and buffer in the top reservoir was decanted and replaced with elution buffer [20 mM Tris-HCl, 10 mM suramin, 5 mM EDTA, 150 mM NaCl (pH 6.0)]. The column was equilibrated for 30 min at 4°C before eluting proteins with 5 vol of elution buffer. Fractions were collected and assayed for protein concentration using a BCA protein assay kit (Pierce, Rockford, IL) and a Bio-Rad 3550 microplate reader. Peak fractions were pooled and concentrated to 100 μg protein/ml with a Centricon YM-30 (Millipore, Billerica, MA). Affinity purified Vtgrs diluted 1:1 in Laemmli buffer were electrophoresed through 5% acrylamide precast Tris-HCl Ready Gels, as we described (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar), and stained with Silver Stain Plus (Bio-Rad). Aliquots of solubilized ovary membrane proteins, affinity purified Vtgrs, and purified VtgAa/b were processed for LC-ESI-MS/MS using an LTQ linear ion trap mass spectrometer (Thermo, San Jose, CA) at the NCSU Genomic Sciences Laboratory (Raleigh, NC). Samples were analyzed in quadruplicate and ion fragmentation spectra were queried by MASCOT (Matrix Science, Boston, MA) against white perch VtgAa, VtgAb, VtgC (16Reading B.J. Hiramatsu N. Sawaguchi S. Matsubara T. Hara A. Lively M.O. Sullivan C.V. Conserved and variant molecular and functional features of multiple egg yolk precursor proteins (vitellogenins) in white perch (Morone americana) and other teleosts.Mar. Biotechnol. (NY). 2009; 11: 169-187Crossref PubMed Scopus (67) Google Scholar), white perch Lr8− (18Hiramatsu N. Chapman R.W. Lindzey J.K. Haynes M.R. Sullivan C.V. Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana).Biol. Reprod. 2004; 70: 1720-1730Crossref PubMed Scopus (66) Google Scholar), and the translated striped bass ovary transcriptome (24Reading B.J. Williams V.N. Chapman R.W. Williams T.I. Sullivan C.V. Dynamics of the striped bass (Morone saxatilis) ovary proteome reveal a complex network of the translasome.J. Proteome Res. 2013; 12: 1691-1699Crossref PubMed Scopus (23) Google Scholar, 25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar). A portion of cutthroat trout (Oncorhynchus clarki) CtLR13+1 (Lrp13) (33Hiramatsu N. Luo W. Reading B.J. Sullivan C.V. Mizuta H. Ryu Y-W. Nishimiya O. Todo T. Hara A. Multiple ovarian lipoprotein receptors in teleosts.Fish Physiol. Biochem. 2013; 39: 29-32Crossref PubMed Scopus (26) Google Scholar) was cloned into pET302/NT-His expression vector (Invitrogen) using In-Fusion Advantage PCR cloning kit (Clontech, Mountain View, CA) and primers CtLR13+1F and CtLR13+1R. Recombinant CtLR13+1 His-fusion proteins were expressed in Rosetta-gami B (DE3) pLysS (Novagen, Madison, WI) and recovered using BugBuster protein extraction reagent and Ni-charged His-Bind resin chromatography (Novagen). Superdex 200 gel purified recombinant CtLR13+1 was used to raise polyclonal α-CtLrp13 in rabbit as described by Hong et al. (34Hong L. Fujita T. Wada T. Amano H. Hiramatsu N. Zhang X. Todo T. Hara A. Choriogenin and vitellogenin in red lip mullet (Chelon haematocheilus): purification, characterization, and evaluation as potential biomarkers for detecting estrogenic activity.Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 2009; 149: 9-17Crossref PubMed Scopus (21) Google Scholar). Synthetic white perch Lrp13 peptides (CSLGYSGDSCQDHLLKT and TTLNESSQLRNLATQDC) and Lr8− peptides (CRPEANVSTSIQVDSTARGSA and CSVDLNGDNRKKVLQS) were used to raise polyclonal α-WpLrp13 and α-WpLr8− in chickens (GeneTel Laboratories, Madison, WI). Solubilized white perch ovary membrane proteins were separated by electrophoresis through 7.5 and 5% acrylamide precast Tris-HCl ready gels and subjected to ligand blotting with digoxigenin (DIG)-labeled VtgAa/b or Western blotting with α-WpLrp13 or α-WpLr8− at 1:10,000, as we previously described (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar, 35Mizuta H. Luo W. Ito Y. Mushiroba Y. Todo T. Hara A. Reading B.J. Sullivan C.V. Hiramatsu N. Ovarian expression and localization of vitellogenin receptor with eight ligand binding repeats in the cutthroat trout (Oncorhynchus clarki).Comp. Biochem. Physiol. B. 2013; 166: 81-90Crossref PubMed Scopus (40) Google Scholar). Primers were designed for striped bass lrp13 (SB618For1 and SB618Rev1) and ribosomal protein L9 (rpl9) [contig 10830 (25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar)] (RPl9For and RPl9Rev). The quality of total RNA extracted from brain, heart, liver, ovary, foregut, muscle, and adipose tissue was evaluated by NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE) OD260/OD280 and OD260/OD230 and agarose electrophoresis. Extracts were treated with DNA-Free (Applied Biosystems, Grand Island, NY) and cDNA was synthesized using SuperScript First-Strand synthesis system (Invitrogen). Amplifications were performed using PCR SuperMix (Invitrogen) and no template and no reverse transcription controls were incorporated into the assay. Primers for lr8− variant of vldlr (SBLR8For and SBLR8Rev) and lrp13 (SBLRX+1For and SBLRX+1Rev) were designed from striped bass contigs 04238 and 00618 (25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar). Priming sites of low complementarity between these striped bass contig sequences were chosen to ensure primer specificity. Total ovary and liver RNA was extracted and evaluated as described above and cDNA was synthesized with a high capacity cDNA synthesis kit (Applied Biosystems). Absolute real-time quantitative PCR assays were performed as previously described (36Tipsmark C.K. Baltzegar D.A. Ozden O. Grubb B.J. Borski R.J. Salinity regulates claudin mRNA and protein expression in the teleost gill.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008; 294: R1004-R1014Crossref PubMed Scopus (74) Google Scholar, 37Williams V.N. Reading B.J. Amano H. Hiramatsu N. Schilling J. Salger S.A. Islam Williams T. Gross K. Sullivan C.V. Proportional accumulation of yolk proteins derived from multiple vitellogenins is precisely regulated during vitellogenesis in striped bass (Morone saxatilis).J. Exp. Zool. A Ecol. Genet. Physiol. 2014; 321: 301-315Crossref PubMed Scopus (36) Google Scholar) using Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA). Samples were measured in triplicate using a 7300 real-time PCR system (Applied Biosystems) and gene expression was reported as copy number calculated from serially diluted plasmid DNA standard curves. Picha and colleagues (38Picha M.E. Silverstein J.T. Borski R.J. Discordant regulation of hepatic IGF-I mRNA and circulating IGF-I during compensatory growth in a teleost, the hybrid striped bass (Morone chrysops x Morone saxatilis).Gen. Comp. Endocrinol. 2006; 147: 196-205Crossref PubMed Scopus (56) Google Scholar, 39Picha M.E. Turano M.J. Tipsmark C.K. Borski R.J. Regulation of endocrine and paracrine sources of Igfs and Gh receptor during compensatory growth in hybrid striped bass (Morone chrysops X Morone saxatilis).J. Endocrinol. 2008; 199: 81-94Crossref PubMed Scopus (84) Google Scholar) show that normalization of target RNA to total RNA shows similar results to that for normalization to 18S RNA in hybrid striped bass, and we also show this trend when normalization of target RNA to total RNA is compared with normalization to ribosomal protein L9 in striped bass (37Williams V.N. Reading B.J. Amano H. Hiramatsu N. Schilling J. Salger S.A. Islam Williams T. Gross K. Sullivan C.V. Proportional accumulation of yolk proteins derived from multiple vitellogenins is precisely regulated during vitellogenesis in striped bass (Morone saxatilis).J. Exp. Zool. A Ecol. Genet. Physiol. 2014; 321: 301-315Crossref PubMed Scopus (36) Google Scholar). Therefore, we used total RNA for normalization of lr8− and lrp13 gene expression. Melting curve analysis and agarose gel electrophoresis were performed to verify primer specificity and no template and no reverse transcription controls were employed in the assay. DIG-labeled antisense and sense RNA probes were prepared by in vitro transcription of striped bass lrp13 using primers SB618ishF and SB618ishR (Roche, Indianapolis, IN). In situ hybridization of DIG-labeled probes was performed for 40 h at 65°C according to our previous report (40Luo W. Ito Y. Mizuta H. Massaki K. Hiramatsu N. Todo T. Reading B.J. Sullivan C.V. Hara A. Molecular cloning and partial characterization of an ovarian receptor with seven ligand binding repeats, an orthologue of low-density lipoprotein receptor, in the cutthroat trout (Oncorhynchus clarki).Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2013; 166: 263-271Crossref PubMed Scopus (18) Google Scholar). Sections were photographed using a DXM1200F camera cou}, number={11}, journal={JOURNAL OF LIPID RESEARCH}, author={Reading, Benjamin J. and Hiramatsu, Naoshi and Schilling, Justin and Molloy, Katelyn T. and Glassbrook, Norm and Mizuta, Hiroko and Luo, Wenshu and Baltzegar, David A. and Williams, Valerie N. and Todo, Takashi and et al.}, year={2014}, month={Nov}, pages={2287–2295} }
 @article{williams_reading_hiramatsu_amano_glassbrook_hara_sullivan_2014, title={Multiple vitellogenins and product yolk proteins in striped bass, Morone saxatilis: molecular characterization and processing during oocyte growth and maturation}, volume={40}, ISSN={["1573-5168"]}, DOI={10.1007/s10695-013-9852-0}, abstractNote={The multiple vitellogenin (Vtg) system of striped bass, a perciform species spawning nearly neutrally buoyant eggs in freshwater, was investigated. Vitellogenin cDNA cloning, Western blotting of yolk proteins (YPs) using Vtg and YP type-specific antisera, and tandem mass spectrometry (MS/MS) of the YPs revealed the complex mechanisms of yolk formation and maturation in this species. It was discovered that striped bass possesses a tripartite Vtg system (VtgAa, VtgAb, and VtgC) in which all three forms of Vtg make a substantial contribution to the yolk. The production of Vtg-derived YPs is generally similar to that described for other perciforms. However, novel amino-terminal labeling of oocyte YPs prior to MS/MS identified multiple alternative sites for cleavage of these proteins from their parent Vtg, revealing a YP mixture far more complex than reported previously. This approach also revealed that the major YP product of each form of striped bass Vtg, lipovitellin heavy chain (LvH), undergoes limited degradation to smaller polypeptides during oocyte maturation, unlike the case in marine fishes spawning buoyant eggs in which LvHAa undergoes extensive proteolysis to osmotically active free amino acids. These differences likely reflect the lesser need for hydration of pelagic eggs spawned in freshwater. The detailed characterization of Vtgs and their proteolytic fate(s) during oocyte growth and maturation establishes striped bass as a freshwater model for investigating teleost multiple Vtg systems.}, number={2}, journal={FISH PHYSIOLOGY AND BIOCHEMISTRY}, author={Williams, V. N. and Reading, B. J. and Hiramatsu, N. and Amano, H. and Glassbrook, N. and Hara, A. and Sullivan, C. V.}, year={2014}, month={Apr}, pages={395–415} }
 @article{reed_lee_zhang_rashid_poe_hsieh_deighton_glassbrook_bodmer_gibson_2014, title={Systems genomics of metabolic phenotypes in wild-type Drosophila melanogaster}, volume={197}, number={2}, journal={Genetics}, author={Reed, L. K. and Lee, K. and Zhang, Z. and Rashid, L. and Poe, A. and Hsieh, B. and Deighton, N. and Glassbrook, N. and Bodmer, R. and Gibson, G.}, year={2014}, pages={781–630} }
 @article{bartz_glassbrook_danehower_cubeta_2013, title={Modulation of the phenylacetic acid metabolic complex by quinic acid alters the disease-causing activity of Rhizoctonia solani on tomato}, volume={89}, ISSN={0031-9422}, url={http://dx.doi.org/10.1016/j.phytochem.2012.09.018}, DOI={10.1016/j.phytochem.2012.09.018}, abstractNote={The metabolic control of plant growth regulator production by the plant pathogenic fungus Rhizoctonia solani Kühn (teleomorph = Thanatephorus cucumeris (A.B. Frank) Donk) and consequences associated with the parasitic and saprobic activity of the fungus were investigated. Fourteen genetically distinct isolates of the fungus belonging to anastomosis groups (AG) AG-3, AG-4, and AG-1-IA were grown on Vogel’s minimal medium N with and without the addition of a 25 mM quinic acid (QA) source of carbon. The effect of QA on fungal biomass was determined by measuring the dry wt of mycelia produced under each growth condition. QA stimulated growth of 13 of 14 isolates of R. solani examined. The production of phenylacetic acid (PAA) and the chemically related derivatives 2-hydroxy-PAA, 3-hydroxy-PAA, 4-hydroxy-PAA, and 3-methoxy-PAA on the two different media was compared by gas chromatography coupled with mass spectrometry (GC–MS). The presence of QA in the growth medium of R. solani altered the PAA production profile, limiting the conversion of PAA to derivative forms. The effect of QA on the ability of R. solani to cause disease was examined by inoculating tomato (Solanum lycopersicum L.) plants with 11 isolates of R. solani AG-3 grown on media with and without the addition of 25 mM QA. Mean percent survival of tomato plants inoculated with R. solani was significantly higher when the fungal inoculum was generated on growth medium containing QA. The results of this study support the hypotheses that utilization of QA by R. solani leads to reduced production of the plant growth regulators belonging to the PAA metabolic complex which can suppress plant disease development.}, journal={Phytochemistry}, publisher={Elsevier BV}, author={Bartz, Faith E. and Glassbrook, Norman J. and Danehower, David A. and Cubeta, Marc A.}, year={2013}, month={May}, pages={47–52} }
 @article{bartz_glassbrook_danehower_cubeta_2012, title={Elucidating the role of the phenylacetic acid metabolic complex in the pathogenic activity of Rhizoctonia solani anastomosis group 3}, volume={104}, ISSN={["0027-5514"]}, DOI={10.3852/11-084}, abstractNote={The soil fungus Rhizoctonia solani produces phytotoxic phenylacetic acid (PAA) and hydroxy (OH-) and methoxy (MeO-) derivatives of PAA. However, limited information is available on the specific role that these compounds play in the development of Rhizoctonia disease symptoms and concentration(s) required to induce a host response. Reports that PAA inhibits the growth of R. solani conflict with the established ability of the fungus to produce and metabolize PAA. Experiments were conducted to clarify the role of the PAA metabolic complex in Rhizoctonia disease. In this study the concentration of PAA and derivatives required to induce tomato root necrosis and stem canker, in the absence of the fungus, and the concentration that inhibits mycelial growth of R. solani were determined. The effect of exogenous PAA and derivatives of PAA on tomato seedling growth also was investigated. Growth of tomato seedlings in medium containing 0.1-7.5 mM PAA and derivatives induced necrosis of up to 85% of root system. Canker development resulted from injection of tomato seedling stems with 7.5 mM PAA, 3-OH-PAA, or 3-MeO-PAA. PAA in the growth medium reduced R. solani biomass, with 50% reduction observed at 7.5 mM. PAA, and derivatives were quantified from the culture medium of 14 isolates of R. solani belonging to three distinct anastomosis groups by GC-MS. The quantities ranged from below the limit of detection to 678 nM, below the concentrations experimentally determined to be phytotoxic. Correlation analyses revealed that isolates of R. solani that produced high PAA and derivatives in vitro also caused high mortality on tomato seedlings. The results of this investigation add to the body of evidence that the PAA metabolic complex is involved in Rhizoctonia disease development but do not indicate that production of these compounds is the primary or the only determinant of pathogenicity.}, number={4}, journal={MYCOLOGIA}, author={Bartz, Faith E. and Glassbrook, Norman J. and Danehower, David A. and Cubeta, Marc A.}, year={2012}, pages={793–803} }
 @article{veleaz_glassbrook_daub_2008, title={Mannitol biosynthesis is required for plant pathogenicity by Alternaria alternata}, volume={285}, ISSN={["0378-1097"]}, DOI={10.1111/j.1574-6968.2008.01224.x}, abstractNote={Mannitol has been hypothesized to play a role in antioxidant defense. In previous work, we confirmed the presence of the two mannitol biosynthetic enzymes, mannitol dehydrogenase (MtDH) and mannitol 1-phosphate 5-dehydrogenase (MPDH), in the fungus Alternaria alternata and created disruption mutants for both enzymes. These mutants were used to investigate the role of mannitol in pathogenicity of A. alternata on its host, tobacco. Conidia of all mutants were viable and germinated normally. GC-MS analysis demonstrated elevated levels of trehalose in the mutants, suggesting that trehalose may substitute for mannitol as a storage compound for germination. Tobacco inoculation showed no reduction in lesion severity caused by the MtDH mutant as compared with wild type; however, the MPDH mutant and a mutant in both enzymes caused significantly less disease. Microscopy analysis indicated that the double mutant was unaffected in the ability to germinate and produce appressoria on tobacco leaves and elicited a defense response from the host, indicating that it was able to penetrate and infect the host. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for spore germination either in vitro or in planta or for initial infection.}, number={1}, journal={FEMS MICROBIOLOGY LETTERS}, author={Veleaz, Heriberto and Glassbrook, Norman J. and Daub, Margaret E.}, year={2008}, month={Aug}, pages={122–129} }
 @article{johnson_egner_obrian_glassbrook_roebuck_sutter_payne_kensler_groopman_2008, title={Quantification of urinary aflatoxin B-1 dialdehyde metabolites formed by aflatoxin aldehyde reductase using isotope dilution tandem mass spectrometry}, volume={21}, ISSN={["0893-228X"]}, DOI={10.1021/tx700397n}, abstractNote={The aflatoxin B1 aldehyde reductases (AFARs), inducible members of the aldo-keto reductase superfamily, convert aflatoxin B1 dialdehyde derived from the exo- and endo-8,9-epoxides into a number of reduced alcohol products that might be less capable of forming covalent adducts with proteins. An isotope dilution tandem mass spectrometry method for quantification of the metabolites, C-8 monoalcohol, dialcohol, and C-6a monoalcohol, was developed to ascertain their possible role as urinary biomarkers for application to chemoprevention investigations. This method uses a novel 13C17-aflatoxin B1 dialcohol internal standard, synthesized from 13C17-aflatoxin B1 biologically produced by Aspergillus flavus. Chromatographic standards of the alcohols were generated through sodium borohydride reduction of the aflatoxin B1 dialdehyde. This method was then explored for sensitivity and specificity in urine samples of aflatoxin B1-dosed rats that were pretreated with 3H-1,2-dithiole-3-thione to induce the expression of AKR7A1, a rat isoform of AFAR. One of the two known monoalcohols and the dialcohol metabolite were detected in all urine samples. The concentrations were 203.5 ± 39.0 ng of monoalcohol C-6a/mg of urinary creatinine and 10.0 ± 1.0 ng of dialcohol/mg of creatinine (mean ± standard error). These levels represented about 8.0 and 0.4% of the administered aflatoxin B1 dose that was found in the urine at 24 h, respectively. Thus, this highly sensitive and specific isotope dilution method is applicable to in vivo quantification of urinary alcohol products produced by AFAR. Heretofore, the metabolic fate of the 8,9-epoxides that are critical for aflatoxin toxicities has been measured by biomarkers of lysine-albumin adducts, hepatic and urinary DNA adducts, and urinary mercapturic acids. This urinary detection of the alcohol products directly contributes to the goal of mass balancing the fate of the bioreactive 8,9-epoxides of AFB1 in vivo.}, number={3}, journal={CHEMICAL RESEARCH IN TOXICOLOGY}, author={Johnson, Denise N. and Egner, Patricia A. and OBrian, Greg and Glassbrook, Norman and Roebuck, Bill D. and Sutter, Thomas R. and Payne, Gary A. and Kensler, Thomas W. and Groopman, John D.}, year={2008}, month={Mar}, pages={752–760} }
 @article{velez_glassbrook_daub_2007, title={Mannitol metabolism in the phytopathogenic fungus Alternaria alternata}, volume={44}, ISSN={["1096-0937"]}, DOI={10.1016/j.fgb.2006.09.008}, abstractNote={Mannitol metabolism in fungi is thought to occur through a mannitol cycle first described in 1978. In this cycle, mannitol 1-phosphate 5-dehydrogenase (EC 1.1.1.17) was proposed to reduce fructose 6-phosphate into mannitol 1-phosphate, followed by dephosphorylation by a mannitol 1-phosphatase (EC 3.1.3.22) resulting in inorganic phosphate and mannitol. Mannitol would be converted back to fructose by the enzyme mannitol dehydrogenase (EC 1.1.1.138). Although mannitol 1-phosphate 5-dehydrogenase was proposed as the major biosynthetic enzyme and mannitol dehydrogenase as a degradative enzyme, both enzymes catalyze their respective reverse reactions. To date the cycle has not been confirmed through genetic analysis. We conducted enzyme assays that confirmed the presence of these enzymes in a tobacco isolate of Alternaria alternata. Using a degenerate primer strategy, we isolated the genes encoding the enzymes and used targeted gene disruption to create mutants deficient in mannitol 1-phosphate 5-dehydrogenase, mannitol dehydrogenase, or both. PCR analysis confirmed gene disruption in the mutants, and enzyme assays demonstrated a lack of enzymatic activity for each enzyme. GC–MS experiments showed that a mutant deficient in both enzymes did not produce mannitol. Mutants deficient in mannitol 1-phosphate 5-dehydrogenase or mannitol dehydrogenase alone produced 11.5 and 65.7 %, respectively, of wild type levels. All mutants grew on mannitol as a sole carbon source, however, the double mutant and mutant deficient in mannitol 1-phosphate 5-dehydrogenase grew poorly. Our data demonstrate that mannitol 1-phosphate 5-dehydrogenase and mannitol dehydrogenase are essential enzymes in mannitol metabolism in A. alternata, but do not support mannitol metabolism operating as a cycle.}, number={4}, journal={FUNGAL GENETICS AND BIOLOGY}, author={Velez, Heriberto and Glassbrook, Norman J. and Daub, Margaret E.}, year={2007}, month={Apr}, pages={258–268} }