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P Barrett

Publications and source records attributed to P Barrett.

At least 55 records · Page 3Linked to original sources
PubMed1999

Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins.

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

Adenylate Cyclase Toxin↗
PubMed1999

Comparison of the immunogenicity and safety of two 17D yellow fever vaccines.

As part of the clinical validation process of a new working seed of a licensed yellow fever vaccine (new working seed PV26, Stamaril; Pasteur Mérieux Connaught, Lyon, France), the immunogenicity and safety of two batches of this vaccine (PM-YF) were compared with those of another commercially available vaccine (Arilvax; Evans Medical-Wellcome, Liverpool, United Kingdom) in 211 healthy adults. While the geometric mean titer values at days 10-14 and day 28 after vaccination were higher in the PM-YF group, the vaccines provided equivalent seroprotection (titers > or = 1/10) one month after a single vaccine dose (100% PM-YF versus 99% W-YF; P = 0.001, by one-sided equivalence test). Both vaccines were safe. There were no serious local or systemic reactions reported, nor any clinically significant hepatic function abnormalities associated with the use of either vaccine. These two 17D yellow fever vaccines from different European vaccine manufacturers were highly immunogenic and safe, and provided equivalent seroprotection.

Adolescent↗
PubMed1998

Leptin (ob) mRNA and hypothalamic NPY in food-deprived/refed Syrian hamsters.

Food deprivation in the laboratory rat decreases plasma leptin and insulin, elevates glucocorticoid concentration, and increases the activity of the neuropeptide Y (NPY) system and feeding drive. In contrast, Syrian hamsters fail to modify feeding behaviour in response to various food scarcity paradigms. Two components of the neuroendocrine-hormonal response to food deprivation, adipose tissue-derived leptin and hypothalamic NPY, are investigated in the Syrian hamster. ob (leptin) mRNA was less abundant in subcutaneous than abdominal adipose tissue, but not to the extent observed in other rodents. Food deprivation for 48 h reduced ob mRNA in inguinal and retroperitoneal white adipose tissue; gene expression was partially restored by refeeding. In contrast, in epididymal fat there was no effect on ob mRNA. NPY concentrations in hypothalamic nuclei were also unaffected by feeding state. The predicted amino acid sequence of leptin from the Syrian hamster was over 90% homologous with Djungarian hamster and mouse sequences, and the leptin receptor gene (OB-R), and specifically the long intracellular splice variant, OB-Rb, was expressed in the same forebrain and hypothalamic regions that have been described in laboratory mice and rats, including hypothalamic arcuate, dorsomedial, and ventromedial nuclei. The failure of food deprivation to affect NPY and feeding behaviour in Syrian hamsters is unlikely to be due to defects in the leptin system, although there may be region-specific differences in the regulation of leptin signaling in laboratory rats and Syrian hamsters.

Adipose Tissue↗
PubMed1998

The ovine melatonin-related receptor: cloning and preliminary distribution and binding studies.

A melatonin-related receptor was cloned from an ovine genomic library. The sequenced gene has a similar structure to that of the melatonin receptor gene family and consists of two exons separated by an intron of approximately 3 kb. Exon 1 and exon 2 of the ovine melatonin-related receptor encode a protein of 575 amino acids which is 73.8% homologous to the human melatonin-related receptor and shows 40.9% homology with the ovine Mel1a melatonin receptor. COS-7 cells transiently expressing ovine melatonin-related receptors did not bind 2-[125]iodomelatonin or 3H-melatonin. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization studies revealed expression of the ovine melatonin-related receptor in the hypothalamus, pituitary, retina and retinal pigment epithelium. Furthermore, expression of the ovine melatonin-related receptor is shown to be coincident with Mel1a and 2-[125I]iodomelatonin binding in the pituitary and serotonin N-acetyl transferase (arylalkylamine N-acetyl transferase, AANAT) expression in the retina. Expression patterns and similarity with the melatonin receptor gene family suggest a role for this novel G protein-coupled receptor in control and regulation of endocrine function and retinal physiology.

Amino Acid Sequence↗
PubMed1998

oPer1 is an early response gene under photoperiodic regulation in the ovine pars tuberalis.

Mammalian Per1 (or RIGUI) is a recently described putative clock gene that is expressed in the suprachiasmatic nucleus. It is also expressed in the pars tuberalis (PT) of the pituitary, where melatonin appears to drive its expression. This study examines the regulation of Per1 expression. In ovine PT cells, oPer1 is an early response gene transiently expressed after stimulation with forskolin, but melatonin has no independent effect on its expression. In sheep, PT tissue photoperiodic background influences the magnitude or timing of expression of oPer1 2 h after lights-on. These data demonstrate that oPer1 mRNA is elevated in the PT following the decline in night-time melatonin, and that the amplitude or timing of this elevation is dependent upon the duration of the nocturnal melatonin signal.

Animals↗
PubMed1998

The use of DNA fingerprinting to assess monozygotic twinning in Meishan and Landrace x large white pigs.

The extent of embryo mortality is usually estimated by the difference between the numbers of corpora lutea and embryos in the same animal, which assumes that each corpora lutea represents one potential embryo. Recent observations from the authors' laboratory reveal situations in which the number of embryos exceeds the number of corpora lutea, implicating the presence of identical twins. The objective of the present study was to establish DNA fingerprinting techniques to investigate the prevalence of monozygotic twinning in two breeds of pig. DNA fingerprints of every fetus carried by 6 Meishan (MS) and 6 Landrace x Large White (LxLW) gilts on Day 29 +/- 2 of pregnancy were obtained. Five LxLW and 3 MS litters carried no identical fetuses. The remaining gilts carried a pair of fetuses with indistinguishable DNA profiles. No pairs of fetuses were monochorionic. These results suggest that monozygotic twinning in the pig occurs during embryo cleavage or blastocyst development and may be more prevalent in MS compared with indigenous breeds.

Animals↗
PubMed1998

Mel 1a melatonin receptor expression is regulated by protein kinase C and an additional pathway addressed by the protein kinase C inhibitor Ro 31-8220 in ovine pars tuberalis cells.

The expression of the melatonin receptor is positively regulated by cAMP and negatively regulated by melatonin in the ovine pars tuberalis (PT). Furthermore, when PT cells are dispersed in primary culture, both messenger RNA (mRNA) and protein levels spontaneously increase through a process that can be blocked by melatonin, but does not involve cAMP. This suggests that other second messengers may be regulated by melatonin, which, in turn, regulates melatonin receptor mRNA and protein levels. In this study using ribonuclease protection assays, ligand binding, protein kinase C (PKC), and cAMP analysis, we demonstrate that the levels of Mel 1a mRNA and protein expression in ovine PT are reduced by phorbol 12-myristate 13-acetate in a cAMP-independent process. This is indicative of an inhibitory role for PKC in receptor regulation. Melatonin, however, does not act through PKC activation to reduce Mel 1a mRNA or protein levels. Basal PKC activity in PT cells can be inhibited by the PKC inhibitor Ro 31-8220, and this suggests that basal PKC activity may suppress Mel 1a receptor expression. Paradoxically, however, Ro 31-8220 also inhibits melatonin receptor mRNA and protein levels in PT cells by a cAMP-independent mechanism. This suggests that other undefined pathways must play an important role in the physiological self-regulation of Mel 1a receptor expression by melatonin.

Animals↗
PubMed1998

A novel interaction between inhibitory melatonin receptors and protein kinase C-dependent signal transduction in ovine pars tuberalis cells.

This study revealed an important and unexpected finding: namely, that inhibitory melatonin receptors can inhibit a phorbol 12,13 myristate acetate (PMA)-induced, protein kinase C (PKC)-dependent increase in c-fos messenger RNA expression in ovine pars tuberalis (PT) cells. PMA induces dose-dependent stimulation of c-fos expression that is attenuated by melatonin in a dose-dependent and pertussis toxin-sensitive manner. The effect of 100 nM PMA is blocked by Ro31-8220 (1 microM), yet is not mimicked by 4alpha-PMA (100 nM). PMA (100 nM) induces PKC activity in PT cells (P < 0.05) within 5 min, but melatonin has no effect on this response. PMA (100 nM) stimulates both phospholipase D and mitogen-activated protein kinase (MAPK) (p42/44) activities in PT cells, but melatonin has no effect on these responses. The results indicate that neither of these second-messenger activities contribute to the melatonin-sensitive pathway of c-fos activation. The MEK (MAPK kinase) inhibitor, PD98059 (50 microM), does not block the induction of c-fos by PMA, although at the same dose it inhibits PMA-mediated activation of p42/44 MAPK by 50-70%, and activation by forskolin or insulin-like growth factor-I by 100%. These data suggest that p42/44 MAPK may not be the primary mediator of PKC-dependent c-fos induction. In contrast to the effect of melatonin on PMA-mediated c-fos induction in PT cells, in L cells stably transfected with the sheep Mel1 alphabeta receptor, melatonin potentiates the c-fos response in a pertussis toxin-sensitive manner. These data indicate the tissue-specific nature of melatonin receptor signaling, and reveal that a pertussis toxin-sensitive pathway can block PKC-mediated c-fos induction in PT cells.

Animals↗
PubMed1998

Melatonin receptors in the human fetal kidney: 2-[125I]iodomelatonin binding sites correlated with expression of Mel1a and Mel1b receptor genes.

Melatonin receptors in the human fetal kidney were identified and characterized by quantitative in vitro autoradiography using the melatonin agonist, 2-[125I]iodomelatonin. Specific binding was localized to cells in the nephrogenic region at the outer perimeter of the developing kidney and was time-dependent, saturable and inhibited in the presence of guanosine 5'-0-(3-thiotriphosphate) indicative of a G protein-coupled receptor. Expression of the Mel1a and Mel1b melatonin receptors in human fetal kidney was determined using RT-PCR. In situ hybridization confirmed the localization of the Mel1a mRNA transcripts. A role for melatonin in development of the human fetal kidney is postulated.

Autoradiography↗
PubMed1997

The roles of valine 208 and histidine 211 in ligand binding and receptor function of the ovine Mel1a beta melatonin receptor.

Site-directed mutagenesis was used to study two residues, valine 208 and histidine 211, in transmembrane domain 5 of the ovine Mel1a beta melatonin receptor. A series of 4 mutants were constructed (V208A, V208L, H211F, H211L), and each engineered to contain a FLAG-epitope. Immunocytochemistry demonstrated that all the mutants were expressed in COS-7 cells at levels comparable to the FLAG-epitope tagged wild-type Mel1a beta receptor (approximately 120 fmol/mg protein). Ligand binding revealed however that all mutants had reduced affinities for 2-[125I]-iodomelatonin (Kd wild-type 139 pM, Kd mutants 320 to 989 pM). Competition studies, with a series of melatonin analogues, identified a probable interaction between histidine 211 and the 5-methoxy group of melatonin. The wild-type receptor and both valine 208 mutants displayed a dose-dependent melatonin mediated inhibition of cyclic AMP levels in HEK293 cells, with IC50 values in the same rank-order as their melatonin binding affinities. Both H211F and H211L, however, did not display any melatonin mediated effects and may suggest that histidine 211 is critical for melatonin mediated receptor activation.

Amino Acid Sequence↗
PubMed1997

Two orphan seven-transmembrane segment receptors which are expressed in CD4-positive cells support simian immunodeficiency virus infection.

Clinical isolates of primate immunodeficiency viruses, including human immunodeficiency virus type 1 (HIV-1), enter target cells by sequential binding to CD4 and the chemokine receptor CCR5, a member of the seven-transmembrane receptor family. HIV-1 variants which use additional chemokine receptors are present in the central nervous system or emerge during the course of infection. Simian immunodeficiency viruses (SIV) have been shown to use CCR5 as a coreceptor, but no other receptors for these viruses have been identified. Here we show that two orphan seven-transmembrane segment receptors, gpr1 and gpr15, serve as coreceptors for SIV, and are expressed in human alveolar macrophages. The more efficient of these, gpr15, is also expressed in human CD4(+) T lymphocytes and activated rhesus macaque peripheral blood mononuclear cells. The gpr15 and gpr1 proteins lack several hallmarks of chemokine receptors, but share with CCR5 an amino-terminal motif rich in tyrosine residues. These results underscore the potential diversity of seven-transmembrane segment receptors used as entry cofactors by primate immunodeficiency viruses, and may contribute to an understanding of viral variation and pathogenesis.

Amino Acid Sequence↗
PubMed1997

Identification and characterisation of 2-[125I]iodomelatonin binding and Mel1a melatonin receptor expression in the human fetal leptomeninges.

Melatonin binding sites were identified over the leptomeninges surrounding the human fetal brain using quantitative in vitro autoradiography and the melatonin agonist, 2-[125I]iodomelatonin. Binding was found to be saturable and of high affinity (dissociation constant (Kd) = 54 pM and maximal theoretical binding (Bmax) = 13 fmol/mg protein), and inhibited by guanosine-5'-o-(3-thiotriphosphate) (GTPgammaS) suggesting that these binding sites represent G protein-coupled melatonin receptors. RT-PCR performed on mRNA isolated from the human fetal leptomeninges detected expression of the G protein-coupled melatonin receptor Mel1a, but not Mel1b. In situ hybridisation confirmed the localisation of Mel1a mRNA transcripts over the leptomeninges of the fetal brain. The identification of 2-[125I]iodomelatonin and Mel1a melatonin receptor expression in the fetal leptomeninges implies that melatonin may play a role in the early growth and development of the human brain.

Fetus↗
PubMed1997

Cloning and functional analysis of a polymorphic variant of the ovine Mel 1a melatonin receptor.

We have isolated a novel variant of the Mel 1a melatonin receptor from an ovine PT cDNA library. Relative to the reported sequence for the Mel 1a melatonin receptor there are 8 changes in the DNA sequence. Only 3 of these result in amino acid substitutions, one in extracellular loop 3 and two in the carboxy-terminal tail. We have designated the novel variant of the sheep Mel 1a receptor Mel 1a(beta), and correspondingly the previously reported variant Mel 1a(alpha). As minor changes in the primary amino acid sequence of G-protein-coupled receptors can influence their functional characteristics we have accordingly characterized this novel variant of the Mel 1a melatonin receptor. This melatonin receptor displays high affinity binding and inhibits the cAMP second messenger pathway in transfected L-cells demonstrating that this receptor is fully functional. PCR analysis shows Mel 1a(beta) is present in several breeds of sheep and suggests that the Mel 1a(beta) receptor was established early in the evolution of the sheep species.

Animals↗
PubMed1997

Identification of Mel1a melatonin receptors in the human embryonic kidney cell line HEK293: evidence of G protein-coupled melatonin receptors which do not mediate the inhibition of stimulated cyclic AMP levels.

Binding assays using 2-[125I]iodomelatonin revealed high-affinity, guanosine 5'-O-(3-thiotriphosphate) sensitive, melatonin binding sites (B(max) 1.1 fmol/mg protein) in the human embryonic kidney cell line HEK293. Competition studies using the selective melatonin receptor antagonist luzindole and RT-PCR techniques identified these sites as human Mel1a melatonin receptors. Challenge of HEK293 cells with 1 microM melatonin had no effect on forskolin stimulated cyclic AMP levels, whereas in HEK293 cells engineered to stably over-express the human Mel1a melatonin receptor (B(max) > 400 fmol/mg protein) melatonin dose-dependently inhibited stimulated cyclic AMP levels (IC50 7.7 pM). These data may indicate that certain tissues, expressing low levels of G protein-coupled melatonin receptors, do not display melatonin mediated inhibition of cAMP.

Binding, Competitive↗
PubMed1997

Incompleteness of statutory notification of bacterial gastro-intestinal infection.

A study was made of all potentially statutorily notifiable bacterial infections diagnosed in faecal samples submitted from symptomatic patients to a single microbiology laboratory during a six-month period. Salmonella spp, Campylobacter spp or Shigella spp were isolated from 167 patients and 51% of these were formally notified (54% of general practice patients and 47% of hospital patients). Forty-seven percent of cases of food-poisoning (Salmonella spp. and Campylobacter spp) were notified as were 70% of cases of shigella infections. Notification was made on average 9.4d after sending a specimen to the laboratory. A questionnaire used to ascertain the reasons for non-notification in 80 of 85 cases elicited replies in respect of 78 patients. Four patients infected with Salmonella spp or Campylobacter spp were said not to have been suffering from food-poisoning. A variety of reasons was given for failing to notify the others, the most common were forgetfulness, not receiving the result of the specimen, or believing someone else had made the notification.

Campylobacter Infections↗
PubMed1997

Melatonin receptors in the brain and pituitary gland of hypothalamo-pituitary disconnected Soay rams.

In Soay rams in which the pituitary gland has been surgically separated from the hypothalamus, blood prolactin concentrations vary in response to changes in photoperiod and the administration of melatonin, as in intact animals, providing evidence that melatonin acts within the pituitary gland to control prolactin secretion. In this study the presence of potentially functional melatonin receptors in the pars tuberalis and zona tuberalis (PT/ZT) of hypothalamo-pituitary disconnected (HPD) Soay rams is confirmed using both in vitro autoradiography with the ligand 2-(125I)-iodomelatonin and in situ hybridization for the melatonin receptor. There was no effect of the HPD operation on the pattern and quantity of 2-(125I)iodomelatonin binding in the brain demonstrating that this binding is independent of hypothalamic regulation. The possibility that melatonin may control prolactin secretion directly via specific receptors on lactotrophs was investigated using dual in situ hybridization with a (35S) labelled probe for the ovine melatonin receptor (Mel 1a(b)) and a Digoxigenin labelled probe for ovine prolactin. Melatonin receptor gene expression was observed in the PT/ZT in both intact and HPD rams, however, there was no colocalization with prolactin gene expression; only in the ZT was there a close association between cells expressing the melatonin receptor and lactotrophs. The results provide strong support for the view that melatonin acts via the PT/ZT to mediate the effects of photoperiod on the seasonal cycle in prolactin secretion.

Animals↗
PubMed1997

Corticosteroid therapy in Riedel's thyroiditis.

We report a case of Riedel's thyroiditis presenting with a systemic illness, life-threatening stridor and a stony hard goitre. Diagnosis was confirmed by open thyroid biopsy. Treatment with corticosteroid resulted in a dramatic improvement. A possible autoimmune mechanism in the pathogenesis of Riedel's thyroiditis is discussed.

Autoimmune Diseases↗