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PubMed2000

Modulation of the far-upstream enhancer of the rat alpha-fetoprotein gene by members of the ROR alpha, Rev-erb alpha, and Rev-erb beta groups of monomeric orphan nuclear receptors.

Expression of the oncodevelopmental alpha-fetoprotein (AFP) gene is tightly regulated and occurs in the yolk sac, fetal liver and intestine, and cancerous liver cells. Transcription of the AFP gene is under the control of three enhancers that are very tissue specific. We have shown that the most upstream of these enhancers, located at -6 kb, works through the combined action of liver-enriched factors and nuclear receptors that bind to three regions of this DNA regulatory element. This study showed that orphan nuclear receptors of the ROR alpha, Re-verb alpha, and Rev-erb beta groups can bind as monomers with high affinity and specificity to an evolutionarily conserved AGGTCA motif in the functionally important region 1 of this AFP enhancer. Transient transfection experiments performed with human HepG2 hepatoma cells showed that overproduction of ROR alpha 4 stimulated the activity of the AFP enhancer in a dose-dependent manner, while that of Rev-erb alpha and Rev-erb beta had the opposite effect. These effects were highly specific and required the integrity of the AGGTCA motif. The action of these nuclear receptors also occurred in the context of the entire 7-kb regulatory region of the rat AFP gene. These results suggest that altering the amounts or activities of these orphan receptors in cells of hepatic or endodermal origin could modulate AFP gene expression in response to a variety of developmental or carcinogenic stimuli.

Animals↗
PubMed2001

X-ray structure of the orphan nuclear receptor RORbeta ligand-binding domain in the active conformation.

The retinoic acid-related orphan receptor beta (RORbeta) exhibits a highly restricted neuronal-specific expression pattern in brain, retina and pineal gland. So far, neither a natural RORbeta target gene nor a functional ligand have been identified, and the physiological role of the receptor is not well understood. We present the crystal structure of the ligand-binding domain (LBD) of RORbeta containing a bound stearate ligand and complexed with a coactivator peptide. In the crystal, the monomeric LBD adopts the canonical agonist-bound form. The fatty acid ligand-coactivator peptide combined action stabilizes the transcriptionally active conformation. The large ligand-binding pocket is strictly hydrophobic on the AF-2 side and more polar on the beta-sheet side where the carboxylate group of the ligand binds. Site-directed mutagenesis experiments validate the significance of the present structure. Homology modeling of the other isotypes will help to design isotype-selective agonists and antagonists that can be used to characterize the physiological functions of RORs. In addition, our crystallization strategy can be extended to other orphan nuclear receptors, providing a powerful tool to delineate their functions.

Amino Acid Sequence↗
PubMed1995

Oncogenic conversion of a novel orphan nuclear receptor by chromosome translocation.

A recurrent t(9;22) (q22;q12) chromosome translocation has been described in extraskeletal myxoid chondrosarcoma (EMC). Fluorescent in situ hybridization experiments performed on one EMC tumour indicated that the chromosome 22 breakpoint occurred in the EWS gene. Northern blot analysis revealed an aberrant EWS transcript which is cloned by a modified RT-PCR procedure. This transcript consists of an in-frame fusion of the 5' end of EWS to a previously unidentified gene, which was named TEC. This fusion transcript was detected in six of eight EMC studied, and three different junction types between the two genes were found. In all junction types, the putative translation product contained the amino-terminal transactivation domain of EWS linked to the entire TEC protein. Homology analysis showed that the predicted TEC protein contains a DNA-binding domain characteristic of nuclear receptors. The highest identity scores were observed with the NURR1 family of orphan nuclear receptors. These receptors are involved in the control of cell proliferation and differentiation by modulating the response to growth factors and retinoic acid. This work provides, after the PML/RAR alpha gene fusion, the second example of the oncogenic conversion of a nuclear receptor and the first example involving the orphan subfamily. Analysis of the disturbance induced by the EWS/TEc protein in the nuclear receptor network and their target genes may lead to new approaches for EMC treatment.

Amino Acid Sequence↗
PubMed2006

Genomic analyses facilitate identification of receptors and signalling pathways for growth differentiation factor 9 and related orphan bone morphogenetic protein/growth differentiation factor ligands.

Recent advances in genomic sequencing allow a new paradigm in hormonal research, and a comparative genomic approach facilitates the identification of receptors and signalling mechanisms for orphan ligands of the transforming growth factor beta (TGFbeta) superfamily. Instead of purifying growth differentiation factor 9 (GDF9) receptor proteins for identification, we hypothesized that GDF9, like other ligands in the TGFbeta family, activates type II and type I serine/threonine kinase receptors. Because searches of the human genome for genes with sequence homology to known serine/threonine kinase receptors failed to reveal uncharacterized receptor genes, GDF9 likely interacts with the known type II and type I activin receptor-like kinase (ALK) receptors in granulosa cells. We found that co-treatment with the bone morphogenetic protein (BMP) type II receptor (BMPRII) ectodomain blocks GDF9 activity. Likewise, in a GDF9-non-responsive cell line, overexpression of ALK5, but none of the other six type I receptors, conferred GDF9 responsiveness. The roles of BMPRII and ALK5 as receptors for GDF9 were validated in granulosa cells using gene "knock-down" approaches. Furthermore, we demonstrated the roles of BMPRII, ALK3 and ALK6 as the receptors for the orphan ligands GDF6, GDF7 and BMP10. Thus, evolutionary tracing of polypeptide ligands, receptors and downstream signalling molecules in their respective 'subgenomes' facilitates a new approach for hormonal research.

Animals↗
PubMed2004

Neotenic formation in laboratory colonies of the termite Coptotermes gestroi after orphaning.

The termite Coptotermes gestroi (Wasmann 1896) (Rhinotermitidae: Coptotermitinae) is an exotic species in Brazil and information concerning its reproductive developmental biology is scarce. We induced the formation of neotenics in laboratory colonies through orphaning experiments. Orphaning experiments were conducted in three-year old colonies of C. gestroi kept under laboratory conditions. After three months, eight nymphoid neotenics were observed in one colony after queen removal. Histological analysis showed that these neotenics were non-functional. The results suggest that these individuals may have arisen from the first nymphal instar (N1) or from an early N1 instar after one or two larval moults. Neotenics also were recorded on two incipient colonies of C. gestroi that lost the queen naturally.

Animals↗
PubMed1995

Transcriptional activation by the orphan nuclear receptor ARP-1.

ARP-1 is a ubiquitous orphan nuclear receptor that binds to a site (site A) in the apolipoprotein AI (apoAI) liver-specific enhancer and represses its transcriptional activity in hepatoblastoma HepG2 cells. Electrophoretic mobility shift analysis of HepG2 cell nuclear extracts showed that in addition to ARP-1, site A also binds the orphan nuclear receptors Ear-2 and HNF-4. In in vitro transcription assays, Hela cell nuclear extracts which contain ARP-1 had no effect on transcription from a basal promoter linked to multiple copies of site A. However, supplementation of these extracts with excess amounts of recombinant ARP-1 resulted in significant stimulation. Supplementation of the extracts with purified polypeptides representing fusions between the ARP-1 N- or C-terminal domains and the yeast activator GAL4 DNA binding domain also stimulated transcription from a basal promoter linked to multiple GAL4 DNA binding sites. Co-immunoprecipitation assays using ARP-1-selective antibodies revealed specific physical interactions between ARP-1 and the basal transcription factor TFIIB. We conclude that ARP-1 possesses intrinsic transcription activation potential which is modulated, at least in part, by the intracellular balance of other nuclear receptors that also bind to its cognate DNA binding site.

Apolipoproteins A↗
PubMed1996

Transcriptional repression by the orphan steroid receptor RVR/Rev-erb beta is dependent on the signature motif and helix 5 in the E region: functional evidence for a biological role of RVR in myogenesis.

RVR/Rev-erb beta/BD73 is an orphan steroid receptor that has no known ligand in the "classical' sense. RVR binds as a monomer to an element which consists of an A/T-rich sequence upstream of the consensus hexameric half-site. However, RVR does not activate transcription and blocks transactivation of this element by ROR/RZR. The mechanism of RVR action remains obscure, hence we used the GAL4 hybrid system to identify and characterize an active transcriptional silencer in the ligand binding domain (LBD) of RVR. Rigorous deletion and mutational analysis demonstrated that this repressor domain is encoded by amino acids 416-449 of RVR. Furthermore, we demonstrated that efficient repression is dependent on the so-called LBD-specific signature motif, (F/W)AKxxxxFxxLxxxDQxxLL (which spans loop3-4 and helix 4) and helix 5 (H5; identified in the crystal structures of the steroid receptor LBDs). Although RVR is expressed in many adult tissues, including skeletal muscle, and during embryogenesis, its physiological function in differentiation and mammalian development remains unknown. Since other 'orphans', e.g. COUP-TF II and Rev-erbA alpha, have been demonstrated to regulate muscle and adipocyte differentiation, we investigated the expression and functional role of RVR during mouse myogenesis. In C2C12 myogenic cells, RVR mRNA was detected in proliferating myoblasts and was suppressed when the cells were induced to differentiate into post-mitotic, multinucleated myotubes by serum withdrawal. This decrease in RVR mRNA correlated with the appearance of muscle-specific markers (e.g. myogenin mRNA). RVR 'loss of function' studies by constitutive over-expression of a dominant negative RVR delta E resulted in increased levels of p21Cip1/Waf1 and myogenin mRNAs after serum withdrawal. Time course studies indicated that expression of RVR delta E mRNA results in the precocious induction and accumulation of myogenin and p21 mRNAs after serum withdrawal. In addition, we demonstrated that over-expression of the COUP-TF II and Rev-erbA alpha receptors in C2C12 cells completely blocked induction of p21 mRNA after serum withdrawal. In conclusion, our studies identified a potent transcriptional repression domain in RVR, characterized critical amino acids within the silencing region and provide evidence for the physiological role of RVR during myogenesis.

Amino Acid Sequence↗
PubMed1997

Orphan neuropeptide NocII, a putative pronociceptin maturation product, stimulates locomotion in mice.

NocII is a heptadecapeptide whose sequence lies immediately downstream of nociceptin, the newly discovered natural agonist of the ORL1 receptor, in pronociceptin, nociceptin's precursor polypeptide. Since the sequence of NocII is framed by putative convertase excision sites and it totally conserved across murine and human species, we have sought to determine whether this orphan neuropeptide might by physiologically significant, i.e. endowed with central biological activity in vivo. Intracerebroventricular administration of 10 and 100 ng of NocII increased locomotion in mice. However, unlike nociceptin, which stimulates both the horizontal and vertical (rearing) components of locomotion, NocII affected only the horizontal component. The motor stimulant action of NocII appears to depend largely on dopamine transmission since it is totally reversed by the D1 or the D2 dopamine receptor antagonists SCH 23390 and haloperidol. NocII does not modify the number of explored holes in the hole board test, indicating that, unlike nociceptin, the orphan peptide does not affect exploratory behavior in mice.

Amino Acid Sequence↗
PubMed2003

A comparison between gross and net count methods when searching for orphan radioactive sources.

Different numerical strategies in searching for orphan radioactive sources in the environment by means of a mobile detector system have been evaluated. A carborne 3- by 3-inch NaI(Tl) spectrometric system was used with an unshielded 2 GBq 137Cs source as a test source. In this paper, a previous method (MB method), based on a moving average algorithm applied on the gross count rate, was extended and compared with three moving average algorithm methods involving different natural background subtraction strategies. For each method the distance from the road that an orphan source can be detected with a probability of 50% (the critical distance, CD) when driving by is determined. The CD for a 2 GBq 137Cs source improved from 105 m to 130 m when the interference from 40K was subtracted from the 137Cs spectral window. However, when the contribution of other natural gamma-emitting nuclides also was subtracted, the ability to find the 137Cs source was reduced.

Algorithms↗
PubMed2005

Orphans of the AIDS epidemic: an examination of clinical level problems of children.

OBJECTIVE: The percentage of children with clinical levels of internalizing and externalizing problems before and after maternal death from acquired immunodeficiency syndrome was contrasted with two comparison groups: children whose mothers were human immunodeficiency virus infected but continued to live and children whose mothers were not infected. METHOD: One hundred five 6- to 11-year-old African-American children from New Orleans and their mothers or caregivers participated. Mothers and caregivers completed the Child Behavior Checklist. RESULTS: The findings indicated that, relative to those in one of the two control groups, more children in the orphans group had clinical levels of internalizing and externalizing problems before their mother's death and clinical levels of internalizing problems 2 years after her death. No differences emerged at 6 months after the mother's death. CONCLUSIONS: The differences that emerged and the high percentage of children in the orphans group in the borderline clinical or clinical range (52%-73% across the three assessments) indicate the importance of intervention efforts for these children beginning before the mother's death and continuing after her death.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Orphan comparisons and indirect meta-analysis: a case study on antidepressant efficacy in dysthymia comparing tricyclic antidepressants, selective serotonin reuptake inhibitors, and monoamine oxidase inhibitors by using general linear models.

Direct comparisons of the efficacy of competing interventions are not always available in the literature. This situation leads to the presence of clinically relevant "orphan comparisons" of therapeutic interventions which have never been compared head-to-head. To overcome this limitation, simple methods for indirect meta-analysis have been suggested. Nevertheless, their results are prone to bias when more than 1 indirect comparison is tested because of the likely duplication of data for some comparisons. In contrast, general linear models can be used to extend simple indirect meta-analysis beyond 1 indirect comparison by fitting to incomplete data using maximum likelihood within the framework of multitreatment comparisons. This study presents a tutorial application of general linear models to the comparative efficacy of several antidepressants in dysthymia (tricyclic antidepressants, selective serotonin reuptake inhibitors, and monoamine oxidase inhibitors. Working with previously published data comparing the efficacy of antidepressants with placebo, it is shown that tricyclic antidepressants and selective serotonin reuptake inhibitors present similar efficacy (odds ratio = 1.19, P = 0.37; relative risk = 1.10, P = 0.24; risk difference = 0.03, P = 0.53), whereas monoamine oxidase inhibitors outperform both tricyclic antidepressants and selective serotonin reuptake inhibitors, at least for some effect scales (odds ratio = 1.57, P = 0.05; relative risk = 1.25, P = 0.05; risk difference = 0.09, P = 0.08). This finding, which is an instance of a relevant orphan comparison and could not be obtained otherwise, could motivate the conduct of clinical trials or focused systematic reviews to support or refute its importance through appropriate head-to-head comparisons.

Antidepressive Agents, Tricyclic↗
PubMed2004

Recipient death during a live donor liver transplantation: who gets the "orphan" graft?

The limited availability of deceased organ donors, prolongation of waiting time, and increasing number of patients dying awaiting transplantation have contributed to the increased use of adult-to-adult living-donor liver transplant. In the event that the intended recipient dies after the donor graft has been procured but before it has been transplanted, what should be done with the graft? A structured, nine-item oral survey of 26 experts in liver transplantation was conducted in June and July 2003. Respondents were selected primarily because of their extensive experience with liver transplantation, especially adult-to-adult living-donor transplant. All respondents said the surgical team should try to use the available graft for another recipient. Twenty-one respondents believed consent from the donor or the donor's family was required for allocation, whereas 19% believed consent desirable but not required. Nine respondents recommended an allocation organization place the graft, whereas 17 respondents recommended placement within the donor hospital. Two of the respondents had previously encountered this situation, whereas four had experienced an intraoperative recipient death before procurement of a live donor graft. On the basis of the responses, we offer the following recommendations for handling orphan liver grafts: (1) obtain predonation informed consent from all donors that indicates what the donor would want to have done with the "orphan graft" in all cases of living-donor liver transplantation; (2) avoid the premature removal of the donor graft until the recipient hepatectomy and survival are likely; (3) if a live donor graft has been procured and cannot be transplanted into the intended recipient, and if informed consent has been obtained before the donor operation, the organ should be reallocated without delay to minimize cold ischemia time and maximize the utility of the graft.

Female↗
PubMed1998

BXR, an embryonic orphan nuclear receptor activated by a novel class of endogenous benzoate metabolites.

Nuclear receptors are ligand-modulated transcription factors that respond to steroids, retinoids, and thyroid hormones to control development and body physiology. Orphan nuclear receptors, which lack identified ligands, provide a unique, and largely untapped, resource to discover new principles of physiologic homeostasis. We describe the isolation and characterization of the vertebrate orphan receptor, BXR, which heterodimerizes with RXR and binds high-affinity DNA sites composed of a variant thyroid hormone response element. A bioactivity-guided screen of embryonic extracts revealed that BXR is activatable by low-molecular-weight molecules with spectral patterns distinct from known nuclear receptor ligands. Mass spectrometry and 1H NMR analysis identified alkyl esters of amino and hydroxy benzoic acids as potent, stereoselective activators. In vitro cofactor association studies, along with competable binding of radiolabeled compounds, establish these molecules as bona fide ligands. Benzoates comprise a new molecular class of nuclear receptor ligand and their activity suggests that BXR may control a previously unsuspected vertebrate signaling pathway.

Amino Acid Sequence↗
PubMed2004

Cloning of a novel neuronally expressed orphan G-protein-coupled receptor which is up-regulated by erythropoietin, interacts with microtubule-associated protein 1b and colocalizes with the 5-hydroxytryptamine 2a receptor.

G-protein-coupled receptors (GPCRs) are the largest group of cell surface molecules involved in signal transduction and are receptors for a wide variety of stimuli ranging from light, calcium and odourants to biogenic amines and peptides. It is assumed that systematic genomic data-mining has identified the overwhelming majority of all remaining GPCRs in the genome. Here we report the cloning of a novel orphan GPCR which was identified in a search for erythropoietin-induced genes in the brain as a strongly up-regulated gene. This unknown gene coded for a protein which had a seven-transmembrane topology and key features typical of GPCRs of the A family but a low overall identity to all known GPCRs. The protein, coded ee3, has an unusually high evolutionary conservation and is expressed in neurons in diverse areas of the CNS with relation to integrative functions or motor tasks. A yeast two-hybrid screen for interacting proteins revealed binding to the microtubule-associated protein (MAP) 1b. Coupling to MAP1a has been described for another cognate GPCR, the 5-hydroxytryptamine (5HT) 2a receptor. Surprisingly, we found complete colocalization of ee3 and the 5HT2a receptor. The interaction with MAP1b proved to be critical for the stability or folding of ee3 as in mice lacking MAP1b the ee3 protein was undetectable by immunohistochemistry, although messenger RNA levels remained unchanged. We propose that ee3 is a highly interesting new orphan GPCR with potential connections to erythropoietin and 5HT2a receptor signalling.

Amino Acid Sequence↗
PubMed2005

Cloning and characterization of a family C orphan G-protein coupled receptor.

Members of the family C receptors within the G-protein coupled receptor superfamily include the metabotropic glutamate receptors, GABA(B) receptors, the calcium-sensing receptor (CaSR), the V2R pheromone receptors, the T1R taste receptors, and a small group of uncharacterized orphan receptors. We have cloned and studied the mouse GPRC6A family C orphan receptor. The open reading frame codes for a protein with highest sequence identity to the fish 5.24 odorant receptor and the mammalian CaSR. The gene structure shows a striking resemblance to that of the CaSR. Results from RT-PCR analyses showed that mouse GPRC6A mRNA is expressed in mouse brain, skeletal muscle, heart, lung, spleen, kidney, liver, and in the early stage mouse embryo. Immunocytochemical analysis of the cloned mouse GPRC6A cDNA expressed in human embryonic kidney 293 cells demonstrated that GPRC6A was present on the plasma membrane, as well as in the endoplasmic reticulum and nuclear envelope membranes of transfected cells. A chimeric cDNA construct in which the extracellular ligand binding domain of the fish 5.24 amino acid-activated odorant receptor was ligated to the complementary downstream sequence of the mouse GPRC6A receptor indicated that GPRC6A is coupled to phosphoinositol turnover and release of intracellular calcium. Further studies with mouse GPRC6A expressed in Xenopus laevis oocytes demonstrated that this receptor possesses a pharmacological profile resembling that of the fish 5.24 odorant receptor. These findings suggest that GPRC6A may function as the receptor component of a novel cellular transmitter system in mammals.

Amino Acid Sequence↗
PubMed2003

Progress in methodology. Improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors.

Seven-transmembrane G-protein-coupled receptors play a central role in physiology by facilitating cell communication through recognition of a wide range of ligands. Even more important, they represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and hence their functions, remain to be identified. These receptors are referred to as "orphan" receptors. A pre-requisite for the identification of ligands activating orphan receptors is powerful assay systems. Until now, reporter gene assays have not been in common use in this process. Here, we summarize our development of improved reporter gene assays. We optimized reporter gene assays in respect of (i) the promoter region of the construct, (ii) the reporter enzyme used, (iii) and the assay procedure. Furthermore, an unique fluorescence-based clone selection step was introduced, allowing rapid selection of the most sensitive reporter cell clones when establishing stable reporter cell lines. Mathematical formulae are provided to enable a simple and reliable comparison between different cell lines, when tested with a compound of interest. The resulting reporter cell lines responded in a very sensitive way to the stimulation of various test receptors. The reporter system was termed HighTRACE (high-throughput reporter assay with clone election). Its high assay quality makes it suitable as a primary screening tool. Ligands for two recently unknown 7TM receptors were identified using the HighTRACE system i.e., two cell surface free fatty acid receptors, GPR40 (FFA1R) and GPR43 (FFA2R). The identification was accomplished using a reverse pharmacology approach.

Cell Line↗
PubMed1996

Survival of orphans in 19th century Sweden--the importance of remarriages.

The study aims at elucidating the fate of orphans in 19th century Sweden. A cohort of all children born in the region of Sundsvall was followed. The Cox regression model was used. The population increased 12-fold in the sawmill parish, and doubled in the other parishes. Every 13th child lost either his or her mother or father. The death risk represented by being a motherless infant exceeded every other death risk for the child, and was further enhanced if parents were crofters or coming from the sawmill parish. For children beyond the age of 1 year, factors other than parental deprivation became decisive. Step-parents had an immense positive influence on the survival of the children. The social network worked relatively well for those children who were orphans beyond the age of 1 year. The motherless infants had the worst outcome. The best chance for survival was found for children with step-parents.

Age Factors↗
PubMed1992

Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors.

Steroid-thyroid hormone receptors typically bind as dimers to DNA sequences that contain repeated elements termed half-sites. NGFI-B, an early response protein and orphan member of this receptor superfamily, binds to a DNA sequence that contains only one half-site (5'-AAAGGTCA-3'). A domain separate from the NGFI-B zinc fingers, termed the A box, was identified and is required for recognition of the two adenine-thymidine (A-T) base pairs at the 5' end of the NGFI-B DNA binding element. In addition, a domain downstream of the zinc fingers of the orphan receptor H-2 region II binding protein, termed the T box, determined binding to tandem repeats of the estrogen receptor half-site (5'-AGGTCA-3').

Amino Acid Sequence↗