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1.
J Wildl Dis ; 54(2): 366-370, 2018 04.
Article in English | MEDLINE | ID: mdl-29286258

ABSTRACT

In the late 20th century, elk ( Cervus canadensis) were reintroduced into southeastern Kentucky, US. This population has since been used as a stock population for additional elk reintroductions in other eastern states. Although reintroduction and translocation practices are effective, they can disseminate vectors and pathogens. Therefore, we surveyed tick species residing on elk hosts a decade after elk reintroduction in Kentucky by examining 263 captured individuals (female=86; male=177) from 2011 to 2013. A total of 1,617 ticks were collected from 255 elk. We found five tick species: American dog ( Dermacentor variabilis), Gulf Coast ( Amblyomma maculatum), winter ( Dermacentor albipictus), deer ( Ixodes scapularis), and Lone Star ( Amblyomma americanum). The most prevalent ticks were winter tick (52.3%) and American dog tick (42.1%). We found no difference between female and male elk in mean intensity of American dog tick (mean=2.6, 95% confidence limits: -2.6, 2.7; P=0.701) or winter tick (mean=3.28, 95% confidence limits: -2.21, 2.07; P=0.274). Our findings demonstrated that the elk population acts as host to a diversity of tick species, suggested a broader distribution of tick species than previously reported in Kentucky, and highlighted the potential for inadvertent spread of ticks through translocation and reintroduction efforts, even on a local scale.


Subject(s)
Arachnid Vectors , Deer/parasitology , Tick Infestations/veterinary , Ticks/classification , Zoonoses/transmission , Animals , Female , Kentucky/epidemiology , Male , Tick Infestations/epidemiology
2.
Br J Pharmacol ; 171(19): 4455-63, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24866741

ABSTRACT

BACKGROUND AND PURPOSE: The non-selective sodium channel inhibitor mexiletine has been found to be effective in several animal models of chronic pain and has become popular in the clinical setting as an orally available alternative to lidocaine. It remains unclear why patients with monogenic pain disorders secondary to gain-of-function SCN9a mutations benefit from a low systemic concentration of mexiletine, which does not usually induce adverse neurological side effects. The aim of this study was, therefore, to investigate the biophysical effects of mexiletine on the L858F primary erythromelalgia NaV 1.7 mutation in vitro. EXPERIMENTAL APPROACH: Human wild-type and L858F-mutated NaV 1.7 channels were expressed in HEK293A cells. Whole-cell currents were recorded by voltage-clamp techniques to characterize the effect of mexiletine on channel gating properties. KEY RESULTS: While the concentration-dependent tonic block of peak currents by mexiletine was similar in wild-type and L858F channels, phasic block was more pronounced in cells transfected with the L858F mutation. Moreover, mexiletine substantially shifted the pathologically-hyperpolarized voltage-dependence of steady-state activation in L858F-mutated channels towards wild-type values and the voltage-dependence of steady-state fast inactivation was shifted to more hyperpolarized potentials, leading to an overall reduction in window currents. CONCLUSION AND IMPLICATIONS: Mexiletine has a normalizing effect on the pathological gating properties of the L858F gain-of-function mutation in NaV 1.7, which, in part, might explain the beneficial effects of systemic treatment with mexiletine in patients with gain-of-function sodium channel disorders.


Subject(s)
Analgesics/pharmacology , Mexiletine/pharmacology , NAV1.7 Voltage-Gated Sodium Channel/physiology , Sodium Channel Blockers/pharmacology , Erythromelalgia , HEK293 Cells , Humans , Ion Channel Gating/drug effects , Mutagenesis, Site-Directed , Mutation , NAV1.7 Voltage-Gated Sodium Channel/genetics , Patch-Clamp Techniques
3.
Nat Commun ; 4: 1682, 2013.
Article in English | MEDLINE | ID: mdl-23575686

ABSTRACT

Aberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1-/- mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1-Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.


Subject(s)
Guanine Nucleotide Exchange Factors/physiology , Hyperalgesia/etiology , Ion Channels/physiology , Animals , Base Sequence , Cells, Cultured , Guanine Nucleotide Exchange Factors/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Oligodeoxyribonucleotides , Signal Transduction
4.
J Parasitol ; 98(3): 674-5, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22150391

ABSTRACT

Toxoplasma gondii is a significant worldwide parasitic protozoan. In the present study, prevalence of antibodies of T. gondii was examined from 29 free-ranging black bears ( Ursus americanus ) from south-central Florida where the host species was listed as state threatened during this project. Overall T. gondii prevalence was found to be 44.8%, specifically 46.2% in male and 43.8% in female U. americanus , using a modified agglutination test (1:25 titer). Seroprevalence differences between sexes were not significant (P > 0.05). Results of the present study add supportive data to the growing body of evidence suggesting that U. americanus has one of the highest T. gondii seroprevalences among all known intermediate hosts. In addition, our data emphasize the importance of understanding parasitic disease dynamics from a conservation perspective.


Subject(s)
Antibodies, Protozoan/blood , Toxoplasma/immunology , Toxoplasmosis, Animal/epidemiology , Ursidae/parasitology , Agglutination Tests/veterinary , Animals , Female , Florida/epidemiology , Male , Seroepidemiologic Studies , Sex Distribution
5.
J Med Genet ; 46(4): 249-53, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19028728

ABSTRACT

BACKGROUND: Autosomal recessive primary microcephaly (MCPH) is a model disease to study human neurogenesis. In affected individuals the brain grows at a reduced rate during fetal life resulting in a small but structurally normal brain and mental retardation. The condition is genetically heterogeneous with mutations in ASPM being most commonly reported. METHODS AND RESULTS: We have examined this further by studying three cohorts of microcephalic children to extend both the phenotype and the mutation spectrum. Firstly, in 99 consecutively ascertained consanguineous families with a strict diagnosis of MCPH, 41 (41%) were homozygous at the MCPH5 locus and all but two families had mutations. Thus, 39% of consanguineous MCPH families had homozygous ASPM mutations. Secondly, in 27 non-consanguineous, predominantly Caucasian families with a strict diagnosis of MCPH, 11 (40%) had ASPM mutations. Thirdly, in 45 families with a less restricted phenotype including microcephaly and mental retardation, but regardless of other neurological features, only 3 (7%) had an ASPM mutation. This report contains 27 novel mutations and almost doubles the number of MCPH associated ASPM mutations known to 57. All but one of the mutations lead to the use of a premature termination codon, 23 were nonsense mutations, 28 deletions or insertions, 5 splicing, and 1 was a translocation. Seventeen of the 57 mutations were recurrent. There were no definitive missense mutations found nor was there any mutation/phenotype correlation. ASPM mutations were found in all ethnic groups studied. CONCLUSION: This study confirms that mutations in ASPM are the most common cause of MCPH, that ASPM mutations are restricted to individuals with an MCPH phenotype, and that ASPM testing in primary microcephaly is clinically useful.


Subject(s)
Mutation , Nerve Tissue Proteins/genetics , Child , Consanguinity , DNA Mutational Analysis , Family Health , Female , Genes, Recessive , Humans , Male
6.
J Med Genet ; 43(9): 750-4, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16679491

ABSTRACT

BACKGROUND: The VACTERL with hydrocephalus (VACTERL-H) phenotype is recognised to be a severe manifestation of autosomal recessive Fanconi anaemia. Several families have been described in which the VACTERL-H phenotype segregates as an X linked syndrome. The mutations which cause X linked VACTERL-H syndrome are not known. OBJECTIVE: To determine if mutations in FANCB, which are known to cause Fanconi anaemia complementation group B, are a cause of X linked VACTERL-H syndrome. METHODS: A three generation pedigree with X linked VACTERL-H syndrome was investigated. X inactivation was tested in carrier females, and fibroblasts from an affected male fetus were analysed for increased sensitivity to diepoxybutane. FANCB coding exons and flanking splice sites were screened for mutations by direct sequencing of polymerase chain reaction (PCR) fragments amplified from genomic DNA. cDNA from affected fetal fibroblasts was analysed by PCR and direct sequencing using specific exonic primers. RESULTS: A FANCB mutation which results in a premature stop codon by causing skipping of exon 7 was identified. Chromosomes from the affected fetus showed increased sensitivity to diepoxybutane, and carrier women were found to have 100% skewed X inactivation in blood. CONCLUSIONS: Mutations in FANCB are a cause of X linked VACTERL-H syndrome. The data presented are of relevance to the genetic counselling of families with isolated male cases of VACTERL-H and Fanconi anaemia.


Subject(s)
Abnormalities, Multiple/genetics , Fanconi Anemia Complementation Group Proteins/genetics , Genes, X-Linked/genetics , Hydrocephalus/genetics , Adult , Case-Control Studies , Chromosome Breakage , DNA Mutational Analysis , Exons/genetics , Female , Fetus/abnormalities , Fetus/diagnostic imaging , Humans , Introns/genetics , Male , Mutation/genetics , Pedigree , RNA Splice Sites/genetics , Radiography , Syndrome
7.
J Med Genet ; 40(3): 169-74, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12624134

ABSTRACT

A female patient with non-syndromic mental retardation was shown by high resolution GTL banding to have inherited an apparently balanced translocation, 46,X,t(X;8)(q28;q12)mat. Replication studies in the mother and daughter showed a skewed X inactivation pattern in lymphocytes, with the normal X chromosome preferentially inactivated. The mother also had significant intellectual disability. To investigate the possibility that a novel candidate gene for XLMR was disrupted at the X chromosome translocation breakpoint, we mapped the breakpoint using fluorescence in situ hybridisation (FISH). This showed that the four known genes involved in non-syndromic mental retardation in Xq28, FMR2, SLC6A8, MECP2, and GDI1, were not involved in the translocation. Intriguingly, we found that the X chromosome breakpoint in the daughter could not be defined by a single breakpoint spanning genomic clone and further analysis showed a 650 kb submicroscopic duplication between DXS7067 and DXS7060 on either side of the X chromosome translocation breakpoint. This duplicated region contains 11 characterised genes, of which nine are expressed in brain. Duplication of one or several of the genes within the 650 kb interval is likely to be responsible for the mental retardation phenotype seen in our patient. Xq28 appears to be an unstable region of the human genome and genomic rearrangements are recognised as major causes of two single gene defects, haemophilia A and incontinentia pigmenti, which map within Xq28. This patient therefore provides further evidence for the instability of this genomic region.


Subject(s)
Chromosomes, Human, Pair 8/genetics , Chromosomes, Human, X/genetics , Intellectual Disability/genetics , Sex Chromosome Aberrations , Translocation, Genetic , Child , Child, Preschool , Chromosome Breakage , Female , Gene Duplication , Genetic Linkage , Humans , In Situ Hybridization, Fluorescence , Intellectual Disability/pathology , Karyotyping , Microsatellite Repeats , Syndrome
8.
Neuroscience ; 114(4): 883-9, 2002.
Article in English | MEDLINE | ID: mdl-12379244

ABSTRACT

Homeobox genes are important regulators of cellular identity. Several homeobox genes are known to be specifically expressed in subsets of neurons in the forebrain, exclusively, or in distinct combinations. In this study, we explored the expression of homeobox genes in the forebrain of the adult rat by a degenerate polymerase chain reaction cloning strategy. We identified the expression of 12 homeobox genes, several of which display a remarkable restricted expression pattern in the adult brain. We demonstrated the expression of goosecoid in a very small set of neurons in the hypothalamus. By using Otp as a marker, these goosecoid-positive cells were found to constitute a small area just beside the paraventricular nucleus. Furthermore, we found expression of Rx in the pineal gland, along with Alx4. Rx was additionally found in the posterior pituitary and in cells aligning the bottom of the third ventricle. These findings form a starting point to reveal functions of the described homeobox genes in the forebrain.


Subject(s)
DNA-Binding Proteins , Eye Proteins , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/genetics , Hypothalamus/embryology , Hypothalamus/physiology , Repressor Proteins , Transcription Factors , Age Factors , Amino Acid Sequence , Animals , Cloning, Molecular , Goosecoid Protein , Mice , Molecular Sequence Data , Proteins/genetics , Rats , Reverse Transcriptase Polymerase Chain Reaction
9.
Neuroscience ; 109(2): 287-98, 2002.
Article in English | MEDLINE | ID: mdl-11801365

ABSTRACT

The specific combination of homeobox genes is proposed to be decisive in the terminal differentiation of neuronal systems. In order to identify combined expression of homeobox genes in the ventral forebrain, a reverse transcriptase-polymerase chain reaction strategy using degenerated primers was employed. We identified, amongst others, Lhx7 and Gbx1, displaying a marked overlapping expression in septal and pallidal areas. Gbx1 and Lhx7 were both expressed in those adult brain nuclei that collectively form the basal forebrain cholinergic system, a prime target of neurodegeneration in Alzheimer's disease. Indeed, we detected Lhx7 within cholinergic neurons, whereas the related Lhx6 gene was found in adjacent neurons. From these data we suggest that combined expression of Lhx7 and Gbx1 plays a role in the development of the cholinergic system of the basal forebrain. It is speculated that both genes remain participating in molecular processes in the adult cholinergic neurons, and can be employed to study regulation and survival of these neurons under normal and pathological conditions.


Subject(s)
Cell Differentiation/genetics , Cholinergic Fibers/metabolism , Genes, Homeobox/genetics , Homeodomain Proteins/genetics , Neurons/metabolism , Telencephalon/embryology , Aging/genetics , Animals , Cell Lineage/genetics , Choline O-Acetyltransferase/metabolism , Cholinergic Fibers/ultrastructure , DNA/genetics , DNA/isolation & purification , Fetus , Gene Expression Regulation, Developmental/physiology , Immunohistochemistry , In Situ Hybridization , LIM-Homeodomain Proteins , Mice , Molecular Sequence Data , Neurons/cytology , RNA, Messenger/metabolism , Rats , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Telencephalon/cytology , Telencephalon/growth & development , Transcription Factors
10.
J Neurochem ; 75(5): 1818-25, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11032870

ABSTRACT

Three different transcripts of the homeodomain gene termed pituitary homeobox (Ptx) 2 (Pitx2/Brx/Rieg/Solurshin/Arp) were cloned from different species encoding proteins belonging to the paired-like family of homeodomain proteins. Ptx2a (324 amino acids), Ptx2b (271 amino acids), and Ptx2c (318 amino acids) share the C terminus, including the homeodomain, and have different N termini. Here we report the comparative analysis of all three different Ptx2 splice variants for their transcriptional activity and their expression pattern in the adult rat brain. Ptx2 is able to trans-activate via different model promoters in different cell lines. A mild difference in trans-activating potential is observed among the splice variants, but the underlying mechanism is at present unknown. It is surprising that all Ptx2 transcripts displayed an identical expression pattern in the brain. This markedly restricted pattern is limited to the following brain areas: the anterior and intermediate lobes of the pituitary gland, the subthalamic nucleus, the posterior hypothalamic nucleus, the mammillary bodies, the red nucleus, and the deep gray layer of the superior colliculus. The data presented suggest that all variants of Ptx2 are involved in the development and regulation of distinct neuronal cell groups and the pituitary gland.


Subject(s)
Alternative Splicing/genetics , Brain/metabolism , Homeodomain Proteins/genetics , Nuclear Proteins , Transcription, Genetic , Animals , Cell Line , Cloning, Molecular , Gene Expression , Homeodomain Proteins/biosynthesis , In Situ Hybridization , Organ Specificity/genetics , Pituitary Gland/metabolism , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Rats , Transcription Factors , Transcriptional Activation , Transfection , Homeobox Protein PITX2
11.
Nat Neurosci ; 3(4): 337-41, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10725922

ABSTRACT

We identified the LIM homeodomain transcription factor Lmx1b in the mesencephalic dopamine (mesDA) systems of embryos and adults. Analysis of spatiotemporal expression in Lmx1b null mutants and wild-type mice implicated a cascade involving Lmx1b in the early development of mesDA neurons. Although disruption of this cascade did not block induction of tyrosine hydroxylase (TH), a key enzyme in DA synthesis, or Nurr1, a nuclear hormone receptor, Lmx1b knockout mice failed to induce the mesDA-specific homeodomain gene Ptx3 in TH-positive neurons. Eventually, this small set of TH-positive neurons was lost during embryonic maturation. The data suggest that at least two molecular cascades operate during the specification of the mesDA system, one specifying neurotransmitter phenotype and another essential for other aspects of mesDA neuron differentiation.


Subject(s)
DNA-Binding Proteins , Dopamine/physiology , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Neurons/cytology , Substantia Nigra/cytology , Age Factors , Amino Acid Sequence , Animals , C-Reactive Protein/analysis , C-Reactive Protein/genetics , Cell Differentiation/physiology , DNA Primers , Homeodomain Proteins/analysis , Humans , LIM-Homeodomain Proteins , Mice , Mice, Knockout , Molecular Sequence Data , Neurons/enzymology , Nuclear Receptor Subfamily 4, Group A, Member 2 , RNA, Messenger/analysis , Rats , Serum Amyloid P-Component/analysis , Serum Amyloid P-Component/genetics , Transcription Factors/genetics , Tyrosine 3-Monooxygenase/analysis , Tyrosine 3-Monooxygenase/metabolism
12.
Proc Natl Acad Sci U S A ; 95(7): 4013-8, 1998 Mar 31.
Article in English | MEDLINE | ID: mdl-9520484

ABSTRACT

Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing and mature dopaminergic neurons. Recent studies have demonstrated that Nurr1 is essential for the induction of phenotypic markers of ventral mid-brain dopaminergic neurons whose generation is specified by the floor plate-derived morphogenic signal sonic hedgehog (SHH), but the precise role of Nurr1 in this differentiative pathway has not been established. To provide further insights into the role of Nurr1 in the final differentiation pathway, we have examined the fate of dopamine cell precursors in Nurr1 null mutant mice. Here we demonstrate that Nurr1 functions at the later stages of dopamine cell development to drive differentiation of ventral mesencephalic late dopaminergic precursor neurons. In the absence of Nurr1, neuroepithelial cells that give rise to dopaminergic neurons adopt a normal ventral localization and neuronal phenotype characterized by expression of the homeodomain transcription factor and mesencephalic marker, Ptx-3, at embryonic day 11.5. However, these late precursors fail to induce a dopaminergic phenotype, indicating that Nurr1 is essential for specifying commitment of mesencephalic precursors to the full dopaminergic phenotype. Further, as development progresses, these mid-brain dopamine precursor cells degenerate in the absence of Nurr1, resulting in loss of Ptx-3 expression and a concomitant increase in apoptosis of ventral midbrain neurons in newborn null mutant mice. Taken together, these data indicate that Nurr1 is essential for both survival and final differentiation of ventral mesencephalic late dopaminergic precursor neurons into a complete dopaminergic phenotype.


Subject(s)
DNA-Binding Proteins , Dopamine/physiology , Mesencephalon/cytology , Mesencephalon/physiology , Neurons/cytology , Neurons/physiology , Stem Cells/cytology , Transcription Factors/physiology , Animals , Cell Differentiation/physiology , Cell Survival , Gene Deletion , Mice , Mice, Mutant Strains , Nerve Tissue Proteins/physiology , Nuclear Receptor Subfamily 4, Group A, Member 2 , Stem Cells/physiology
13.
Proc Natl Acad Sci U S A ; 94(24): 13305-10, 1997 Nov 25.
Article in English | MEDLINE | ID: mdl-9371841

ABSTRACT

The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. We have identified a bicoid-related homeobox gene, Ptx3, a member of the Ptx-subfamily, that is uniquely expressed in these neurons. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of Ptx3-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxydopamine-lesioned rats, an animal model for this disease. Thus, Ptx3 is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage.


Subject(s)
Dopamine/metabolism , Membrane Proteins/genetics , Mesencephalon/metabolism , Neurons/metabolism , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , DNA, Complementary , Ectodysplasins , Membrane Proteins/chemistry , Mesencephalon/cytology , Mice , Molecular Sequence Data , Rats , Sequence Homology, Amino Acid
14.
Brain Res Mol Brain Res ; 30(1): 131-6, 1995 May.
Article in English | MEDLINE | ID: mdl-7609634

ABSTRACT

The chicken ovalbumin upstream promoter transcription factor, COUP-TF I, and the protein ARP-1 (COUP-TF II) are two highly homologous orphan receptors of the nuclear hormone receptor family. In this study we investigated their expression patterns in the adult nervous system of the mouse. In situ hybridizations were performed on brain sections with 35S-labeled cRNA probes derived from the 3'-non-coding regions of the mARP-1 and mCOUP-TF I mRNAs. Both COUP-TF I and ARP-1 were shown to be expressed in the adult brain and they displayed restricted and distinct expression patterns. COUP-TF I transcripts were predominantly found in the rostral and caudal parts of the adult mouse brain, whereas ARP-1 transcripts prevaled in the middle part of the brain. High expression of COUP-TF I was detected in the olfactory nucleus, in neocortex layers I/II and V/VL, in the dentate gyrus and in areas CA1/CA3/CA4 of the hippocampus, and in the granular layer of the cerebellum. Only low amounts of COUP-TF I mRNA were detected in the ventral, the laterodorsal and in the interanteromedial thalamic nuclei. Small amounts of COUP-TF I transcripts were also found in the epithelial layer of the ventricle and in arachnoid membranes. High expression of ARP-I was detected in the reticular, the ventral lateral and the gelatinosus thalamic nuclei. Other hot spots of ARP-1 mRNA expression were the amygdaloid nucleus and the arachnoid membranes. Lower amounts of ARP-1 transcripts were found in the anterior and lateral hypothalamic areas, in the suprachiasmatic nucleus, and in the choroid plexus.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Brain/metabolism , DNA-Binding Proteins/biosynthesis , Receptors, Glucocorticoid/biosynthesis , Receptors, Steroid , Transcription Factors/biosynthesis , Animals , Autoradiography , COUP Transcription Factor I , COUP Transcription Factor II , COUP Transcription Factors , DNA-Binding Proteins/genetics , Hippocampus/metabolism , In Situ Hybridization , Mice , Mice, Inbred Strains , RNA, Messenger/metabolism , Receptors, Glucocorticoid/genetics , Telencephalon/metabolism , Transcription Factors/genetics
15.
Mol Cell Endocrinol ; 101(1-2): 237-45, 1994 May.
Article in English | MEDLINE | ID: mdl-9397958

ABSTRACT

An alternatively spliced mRNA coding for a variant estrogen receptor (ER) missing exon 4 (ERdelta4) was detected in the breast tumor cell line MCF7 and meningioma tissue by using the reversed transcriptase PCR technique. The trans-activational properties of this mutant ER were assessed in embryo carcinoma P19EC and human choriocarcinoma JEG3 cells by co-transfection of the ERdelta4 expression vector with an oxytocin promoter construct containing an estrogen-responsive element. ERdelta4 did not trans-activate the oxytocin promoter in either a hormone-dependent or -independent manner. Co-transfection of ERdelta4 together with the wtER did not show any interference of ERdelta4 on the stimulation of the oxytocin promoter by the wtER. ERdelta4 was translated in vitro. Its capacity to bind estradiol, and the binding of the variant to a synthetic estrogen-responsive element were compared to those of the wild-type receptor. ERdelta4 did not bind to a synthetic estrogen-responsive element, nor did it bind estradiol. Hence, ERdelta4 appears to be a silent variant and we speculate that it is without any role in tumor progression.


Subject(s)
Alternative Splicing , Breast Neoplasms/metabolism , Exons/genetics , Meningioma/metabolism , Receptors, Estrogen/genetics , Estrogens/metabolism , Female , Humans , Receptors, Estrogen/metabolism , Tumor Cells, Cultured
16.
J Biol Chem ; 269(21): 15046-53, 1994 May 27.
Article in English | MEDLINE | ID: mdl-8195142

ABSTRACT

The orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TF I) fully prevented not only the activation of the oxytocin gene by retinoic acid and thyroid hormone but also completely repressed the estrogen-dependent stimulation in transfected P19 EC cells. DNase I footprinting showed that the COUP-TF I protein bound to the 5'-flanking region of the oxytocin gene at the site of the distal composite hormone response element, which mediates the responses to estrogen, retinoic acid, and thyroid hormone. Electrophoretic mobility shift assay using this composite hormone response element as probe showed that COUP-TF I and the estrogen receptor competed for binding but did not form a heterodimer. The binding by COUP-TF I was stronger than the binding of the estrogen receptor. Thus, the mechanism of repression involves occupancy of integrated binding sites. By mutagenesis of the composite hormone response element, the COUP-TF I binding site and the estrogen response element could be separated, resulting in functional dissociation of the repressive action of COUP-TF I and the induction by estrogen. The results show that repression of gene expression by COUP-TF I is not limited to receptors that act through heterodimerization but also extends to the homodimer-forming estrogen receptor in a context-dependent manner. This interaction between COUP-TF I and the estrogen receptor may provide a physiological mechanism of selective antagonism of gene regulation by estrogens.


Subject(s)
DNA-Binding Proteins/metabolism , Estrogens/pharmacology , Ovalbumin/genetics , Oxytocin/genetics , Promoter Regions, Genetic , Transcription Factors/metabolism , Animals , Base Sequence , Binding, Competitive , COUP Transcription Factor I , Chickens , Gene Expression Regulation , Molecular Sequence Data , Mutation , Oligodeoxyribonucleotides , Rats
18.
Mol Endocrinol ; 7(1): 47-57, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8383287

ABSTRACT

Transcription factors of the steroid/thyroid hormone receptor superfamily are mediators of development and regulation of the brain. Previous studies have shown that the hypothalamic oxytocin (OT) gene is a potential target of these receptors, since its promoter is stimulated by estrogens and thyroid hormone. Here it is shown that the rat OT promoter is stimulated (at least 20-fold) by retinoic acid through two distinct regions in the 5'-flanking region. The major retinoic acid response element was located between nucleotides -172 and -148 and a minor one between nucleotides -112 and -77, as concluded from the transactivation of 5'-deletion mutants and binding to promoter elements by the retinoic acid receptor. Since the -172/-148 element also conferred estrogen and thyroid hormone responsiveness, it can be considered a composite hormone response element. This element contains a natural variant of the direct repeat of the half-site AGGTCA with spacing zero (DR-0) as well as a palindrome. Analysis of the core sequences of this element by site-directed mutagenesis showed that each of the three TGACC motifs integral to this element contributes to the multihormone sensitivity, but the contribution of each motif is different for the individual receptors. In neonatal rats, vitamin A deficiency and retinoic acid supplementation did not cause changes in hypothalamic OT mRNA levels and OT peptide levels in the pituitary gland and plasma. Gel-retarded protein-DNA complexes were formed between the composite hormone response element and extracts of the hypothalamic supraoptic and paraventricular nuclei. The composite hormone response element has a unique configuration and integrates responses of multiple members of the steroid/thyroid hormone receptor superfamily.


Subject(s)
Gene Expression Regulation , Hypothalamus/metabolism , Multigene Family , Oxytocin/genetics , Promoter Regions, Genetic , Receptors, Cell Surface/physiology , Regulatory Sequences, Nucleic Acid , Transcription Factors/physiology , Transcriptional Activation , Animals , Base Sequence , Carrier Proteins/genetics , Carrier Proteins/physiology , Male , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxytocin/biosynthesis , Protein Binding , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , Receptors, Cell Surface/genetics , Receptors, Estrogen/genetics , Receptors, Estrogen/physiology , Receptors, Retinoic Acid , Receptors, Thyroid Hormone/genetics , Receptors, Thyroid Hormone/physiology , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Signal Transduction , Transcription Factors/genetics , Tretinoin/pharmacology
19.
Br J Surg ; 79(10): 1004-10, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1422706

ABSTRACT

Medical information is increasingly stored in electronic format, enabling faster and more flexible access to the literature. Online, compact disc and floppy disc databases are widely available. The origins and development of these different database media are described. The strengths and weaknesses of each, and the ways in which they complement each other, are examined. Ease of access to medical information can result in data management problems; the role of bibliographic software in ensuring full exploitation of the electronic information revolution is therefore emphasized.


Subject(s)
Medical Informatics , CD-ROM , Compact Disks , Databases, Bibliographic , MEDLINE/organization & administration , MEDLINE/standards , Medical Informatics/organization & administration , Medical Informatics/standards , Online Systems , Software
20.
J Biol Chem ; 267(6): 3771-7, 1992 Feb 25.
Article in English | MEDLINE | ID: mdl-1371278

ABSTRACT

Endocrine factors involved in the transcriptional regulation of the oxytocin (OT) gene were investigated in heterologous expression systems. Plasmids having a 5'-flanking region of the rat OT gene (-363/+16) or the human OT gene (-382/+41) cloned in front of the firefly luciferase gene were co-transfected with an expression vector for the rat thyroid hormone receptor alpha in P19 embryonal carcinoma (EC) cells. Thyroid hormone (T3) stimulated the activity of the rat and human OT promoters about 10-fold. In MCF-7 breast tumor cells transfected with the human OT promoter-luciferase fusion gene, T3 stimulation through endogenous thyroid hormone receptors was about 5-fold. Co-transfection experiments in P19EC cells using 5' deletion mutants of the rat OT gene showed that thyroid hormone responsiveness was located in two regions, one located between nucleotides -195 and -172, the other between nucleotides -172 and -148. Each region accounted for about 3-fold T3 stimulation. Gel retardation analysis using extracts from HeLa cells over-producing the c-erbA/TR alpha protein showed specific binding to the -172/-148 element, while no binding occurred on the -195/-172 element. The -172/-148 element which contains the imperfect estrogen response element, GGTGACCTTGACC, has inverted as well as direct repeats of the TGACC motif. Mutagenesis of TGACC motifs separately reduced thyroid hormone responsiveness by about 50%. However, simultaneous mutation of two TGACC motifs abolished the responsiveness to T3 completely. There was no cooperativity between the activated thyroid hormone and estrogen receptors in transfected MCF-7 cells nor in thyroid hormone receptor and estrogen receptor co-transfected P19EC cells. Negative interactions between these two receptors were observed and gel retardation assays showed interaction between the two receptors proteins. It was shown in an in vivo experiment that treatment of rats with thyroid hormone increased hypothalamic OT mRNA levels, the pituitary OT content, as well as OT levels in blood. The results reveal thyroid hormone as a physiological regulator of OT gene expression, which stimulates OT promoter activity directly through interaction with a thyroid hormone-response element in the OT gene.


Subject(s)
Gene Expression Regulation , Oxytocin/genetics , Thyroid Hormones/physiology , Animals , Base Sequence , Blotting, Northern , Brain/metabolism , Cell Line , Coleoptera/enzymology , HeLa Cells , Humans , Luciferases/genetics , Male , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , RNA/genetics , Rats , Rats, Inbred Strains , Receptors, Estrogen/metabolism , Receptors, Thyroid Hormone/metabolism , Sequence Homology, Nucleic Acid , Transcription, Genetic , Transfection
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