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Biomedical subjects

P Barrett

Publications and source records attributed to P Barrett.

At least 91 records · Page 5Linked to original sources
PubMed1994

Melatonin receptors: localization, molecular pharmacology and physiological significance.

A pre-requisite to understanding the physiological mechanisms of action of melatonin is the identification of the target sites where the hormone acts. The radioligand 2-[125I]iodo-melatonin has been used extensively to localize binding sites in both the brain and peripheral tissues. In general these binding sites have been found to be high affinity, with Kd in the low picomolar range, and selective for structural analogues of melatonin. Also the affinity of these sites can generally be modulated by guanine nucleotides, consistent with the notion that they are putative G-protein coupled receptors. However, only a few studies have demonstrated that these putative receptors mediate biochemical and cellular responses. In the pars tuberalis (PT) and pars distalis (PD) of the pituitary, the amphibian melanophore and vertebrate retina, evidence indicates that melatonin acts to inhibit intracellular cyclic AMP through a G-protein coupled mechanism, demonstrating that this is a common signal transduction pathway for many melatonin receptors. However in the pars distalis the inhibition of calcium influx and membrane potential are also important mediators of melatonin effects. How many different forms or states of the melatonin receptor exist is unknown, but clearly the identification of the structure of the melatonin receptor(s) and its ability to interact with different G-proteins and signal transduction pathways are quintessential to our understanding of the physiological mechanisms of action of melatonin. In parallel the recent development of new melatonin analogues will greatly aid our understanding of the pharmacology of the melatonin receptor both in terms of the development of potent melatonin receptor antagonists and for the definition of receptor sub-types. The wide species and phylogenic diversity of melatonin binding sites in the brain has probably generated more questions than answers. Nevertheless the localization of melatonin receptors to the suprachiasmatic nucleus of the hypothalamus is at least consistent with circadian effects within the foetus and the adult. In contrast the PT of the pituitary presents an enigma in relation to the seasonal effects of melatonin. A model of how melatonin might mediate the timing of the circannual events through the PT is proposed.

Animals↗
PubMed1994

Autoradiographic anomaly in 125I-melatonin binding revealed in ovine adrenal.

Conventional in vitro autoradiographical techniques have been used to screen a number of ovine peripheral tissues for the presence of specific 2-[125I]iodomelatonin binding sites. Intense specific labelling (defined as that displaced by 1 microM melatonin) was observed in the adrenal cortex, and less intense specific binding in the spleen. Subsequent attempts to characterize these adrenal binding sites using in vitro binding assays were unsuccessful. The level of specific binding to both crude membranes and to dispersed whole cell preparations was negligible, and was not correlated with increasing protein or cell concentration. Using quantitative in vitro autoradiography, melatonin was found to competitively inhibit 2-[125I]iodomelatonin binding to sections of the adrenal cortex, but with a considerably lower affinity (IC50 of 1 microM) than that obtained for the ovine pars tuberalis (IC50 of 150 pM) under identical conditions. These results suggest that under the conditions of in vitro autoradiography, 2-[125I]iodomelatonin binds to a finite number of non-receptor sites which are restricted to the ovine adrenal gland. The ability to visualize these sites in the present study may arise through the use of an inappropriately high excess of unlabelled melatonin (1 microM). Consequently to avoid this potential problem, non-specific labelling should perhaps more appropriately be defined as binding in the presence of a concentration of melatonin at approximately 100-fold the value of the Kd (i.e. 10 nM).

Adrenal Glands↗
PubMed1994

Evidence for multiple forms of melatonin receptor-G-protein complexes by solubilization and gel electrophoresis.

The daily production of melatonin from the pineal gland influences circadian and seasonal behaviour and physiology. To further understand how melatonin may function, it is important to characterize the receptor and signal transduction systems. Using the detergent digitonin, we were able to solubilize the receptor from the ovine pars tuberalis (PT) membrane. The receptor was isolated as a complex associated with its heterotrimeric G-protein. In the solubilized state, pre-bound 125I-2-iodomelatonin was stable at 4 degrees C, but was displaceable by GTP gamma S. The receptor-G-protein complex could be separated by molecular mass using native polyacrylamide gel electrophoresis. We demonstrate that the receptor-complex has a molecular mass of 525 kDa and differs from solubilized receptor-complexes isolated from either the lizard brain, chicken brain or the ovine hippocampus. Furthermore the receptor complex isolated from the hippocampus had the lowest molecular mass of these tissues (365 kDa) and was found not to be sensitive to GTP gamma S. This may indicate the existence of a distinct non-G-protein coupled form of the receptor.

Animals↗
PubMed1994

Melatonin regulates the phosphorylation of CREB in ovine pars tuberalis.

This study investigated whether melatonin could modulate the phosphorylation of the calcium/cyclic AMP response-element binding-protein (CREB) within primary cell cultures of ovine pars tuberalis (oPT) and pars distalis (oPD). Gel shift assays confirmed the presence of nuclear factors able to alter the electrophoretic mobility of a 32P-labelled CRE oligonucleotide. Two shifted bands were observed probably due to monomer and dimer binding to the CRE. Each band was supershifted by antisera directed against both CREB and the phosphorylated form of CREB (P-CREB), consistent with a specific role of CREB proteins in transcriptional regulation. To study the physiological role of CREB, the nuclear immunoreactivity for P-CREB was followed in primary cultures of oPT given different pharmacological treatments. Cells stimulated with forskolin responded with a robust time- and dose-dependent increase in nuclear phospho-CREB immunoreactivity (P-CREB-ir), confirming that activation of this transcription factor occurred through the cyclic AMP-PKA pathway. Maximal stimulation was achieved within 15 min and persisted for up to 1 h. Treatment with melatonin alone did not alter basal P-CREB-ir levels, yet melatonin inhibited the forskolin-induced increase in P-CREB-ir in a dose-dependent manner (IC50 of between 10(-10) M and 10(-8) M melatonin when tested against 1 microM forskolin). In contrast, in primary cultures of oPD, melatonin failed to block forskolin-stimulated increases in either the content of cyclic AMP or the intensity of nuclear P-CREB-ir, confirming that the action of melatonin upon P-CREB-ir is tissue specific. These results demonstrate that, consistent with its inhibitory effect on the activation of PKA within oPT, melatonin prevents or reverses the phosphorylation of CREB induced by activation of the cyclic AMP signal transduction pathway. Therefore melatonin has the potential to regulate gene expression in the oPT by acting upon the CREB transcription factor. However, this paper also shows that 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates PKC also leads to the phosphorylation of CREB in oPT cells, suggesting the potential involvement of other signal transduction pathways in the transcriptional regulation of these cells.

Animals↗
PubMed1994

Pneumopericardium: a rare cause of cardiac tamponade in an infant on a positive pressure ventilation.

Cardiac tamponade most commonly results from accumulation of blood, or other fluids, within the pericardial sac. However, rarely air tamponade can lead to tension pneumopericardium and death. Positive pressure ventilation in infants carries a risk of barotrauma and presents as pneumothorax, pneumomediastinum or rarely pneumopericardium. We present an infant with cardiac tamponade due to pneumopericardium to illustrate the classical signs and symptoms, and the management of this rare condition.

Cardiac Tamponade↗
PubMed1994

p72, a marker protein for melatonin action in ovine pars tuberalis cells: its regulation by protein kinase A and protein kinase C and differential secretion relative to prolactin.

The function of the pars tuberalis as a mediator of the action of melatonin remains elusive. As a direct method of assessing the potential role of secretory proteins, ovine pars tuberalis cells have been cultured and radiolabelled with 35S-methionine, and the accumulation of specific radioactive products in the medium, measured after separation by SDS-PAGE and fluorography. The synthesis and secretion of a number of labelled proteins are increased by forskolin (1 microM) and inhibited dose dependently by melatonin (IC50, 300 pM), although consistently a 72-kD protein (p72), is the most intensely labelled of these. Thus, 72 acts as a useful marker of cellular activity for melatonin, whereas prolactin (p23) provides a melatonin non-responsive marker in ovine pars tuberalis cell cultures. The synthesis and secretion of p72 and other melatonin-sensitive proteins is regulated through the cyclic AMP/protein kinase A second-messenger pathway, as analogues of cyclic AMP mimic the action of forskolin, yet 1,9-dideoxyforskolin, a forskolin analogue that is not active on adenylate cyclase, has no effect. However, the phorbol ester, phorbol-12,13-myristate acetate, also regulates the synthesis and secretion of the same profile of proteins as forskolin indicating a potential role for protein kinase C, which occurs through an independent rather than a synergistic pathway. The differential effects of nocadazole (1 microM) and extracellular calcium depletion upon p72 and prolactin secretion indicates that p72 is secreted by a calcium and microtubule independent pathway, in contrast to prolactin. These observations in conjunction with the absence of dense-core storage vesicles in melatonin-responsive cells of the ovine PT are consistent with constitutive secretion of p72 from the latter and regulated secretion of prolactin from melatonin non-responsive cells. Using immunoprecipitation de novo synthesis and secretion of either LH or LH-like proteins from ovine pars tuberalis cells could not be detected under the conditions used. The absence of 125I-(Des-Gly10[D-Ala6]-LHRH-ethylamide) binding over most, but not all, of the ovine pars tuberalis supports the contention that the majority of the cells of the ovine pars tuberalis are not gonadotrophs. These results provide further support for the unique function for the pars tuberalis.

Animals↗
PubMed1994

Cloning and expression of a new member of the melanocyte-stimulating hormone receptor family.

A new member of the G protein-coupled receptor superfamily has been isolated from an ovine genomic library with a probe generated by the application of the PCR technique, using cDNA synthesized on a mRNA template isolated from the ovine pars tuberalis. This genomic clone encodes a novel receptor of 325 amino acids with seven transmembrane domains. These domains share homology with other members of this family, but the best homology is with the recently cloned human MC-1 (50% in the transmembrane domains) and MC-3 (69% in the transmembrane domains) MSH receptors and the human ACTH (42% in the transmembrane domains) receptor. When this receptor was expressed in Cos7 cells, it was able to bind a potent analogue of alpha-MSH, [Nle4,D-Phe7]-alpha-MSH (NDP-MSH), with high affinity. This binding could be displaced by pro-opiomelanocortin-derived and related peptides, with the order of potency NDP-MSH > alpha-MSH = ACTH > beta-MSH and with no effect of gamma-MSH, delta-MSH or beta-endorphin. The expressed receptor was demonstrated to be functionally coupled to the adenylate cyclase second messenger pathway, with alpha-MSH, beta-MSH and ACTH stimulating cyclic AMP production. The amount of the mRNA for this receptor was found to be very low. The tissue distribution of this receptor could only be observed using the reverse transcription-PCR technique and the receptor was found to be present in a number of somatic tissues. These data indicate that this is a new and distinct member of the melanocortin receptor family.

Amino Acid Sequence↗
PubMed1993

The nature of schizotypy.

The matrix of intercorrelations between scales of schizotypy presented by Kendler and Hewitt in 1992 was reanalyzed, and results rather different from those reported by the original authors were found. The new structure shows good agreement with the theory of personality disorder published by Eysenck in 1987. In all, the different scales seem to fall into three groups or factors, identified as Neuroticism (N), Extraversion (E), and Psychoticism (P). It is doubtful if there is a common element left over once these three groups have been eliminated.

Affective Symptoms↗
PubMed1993

Prediction of cancer and coronary heart disease as a function of method of questionnaire administration.

We have tested the hypothesis in this study that different methods of administering a questionnaire produce differential approximations to truthful admission of undesirable personality traits and behaviours. Four different methods of administration produced different levels of trust and understanding, using the current prediction among healthy subjects of death by cancer or coronary heart disease 13 years later as the criterion. There were significant differences in the accuracy of the predictions, depending crucially on the method of administration of the questionnaires. Best predictions were achieved for subjects when both trust and understanding had been increased by interviewers' suitable participation; worst results were achieved for subjects when no special effort was made to increase either. Intermediate results were found for procedures which increased either trust or understanding. It is argued that the success or failure of studies investigating the influence of personality and stress on diseases like cancer and coronary heart disease may depend crucially on the adopted method of interrogation.

Cardiovascular Diseases↗
PubMed1992

Anaesthesia and the patient with latex allergy.

Reports of severe life-threatening anaphylaxis to latex are increasing. A case of latex anaphylaxis occurring during surgery is reported. Sudden cardiorespiratory collapse 25 min after the start of surgery was treated with oxygen, fluid, epinephrine, hydrocortisone, and benadryl. Two months later, skin testing to latex was positive but intradermal testing to the drugs used during anaesthesia was negative. Anaesthetists should be aware of this clinical entity. Latex allergy should be considered in the differential diagnosis of intraoperative anaphylaxis. Fortunately, it is usually preventable by obtaining a positive history, recognising that it occurs in particular subsets of patients and by avoiding latex products. Skin testing to latex is available and may assist in the recognition of latex sensitivity.

Anaphylaxis↗
PubMed1991

Melatonin receptor mRNA expression in Xenopus oocytes: inhibition of G-protein-activated response.

Melatonin is the major endocrine product of the pineal gland in the mammalian brain and plays a variety of roles in photoperiodic functions. In order to investigate melatonin receptors, poly(A)+ RNA was extracted from pars tuberalis of the ovine pituitary and injected into oocytes of Xenopus laevis. After 3-5 days of incubation, functional melatonin receptors were expressed. Receptors were revealed by their inhibitory effect upon oscillatory currents resulting from AlF4-induced activation of G-proteins in the oocyte membrane under voltage clamp conditions. The effect of melatonin was dose-dependent, non-desensitizing and was not observed in uninjected oocytes.

Aluminum↗
PubMed1991

Intracellular signalling in the ovine pars tuberalis: an investigation using aluminium fluoride and melatonin.

The effect of aluminium fluoride (AlF4-) has been studied on inositol phosphate accumulation, calcium mobilization, cyclic AMP production and [2-125I]iodomelatonin binding in ovine pars tuberalis cells. These cells have high-affinity receptors for, and respond to, melatonin through inhibition of forskolin-stimulated adenylate cyclase. In the presence of 10 mM LiCl, AlF4- stimulated the net accumulation of inositol monophosphate and inositol bisphosphate. Consistent with these findings, AlF4- increased intracellular calcium; although this response was attenuated in calcium-depleted medium, indicating that the calcium response comprises both intracellular and extracellular components. Melatonin was ineffective on either basal or AlF4(-)-stimulated turnover of inositol phosphates. In concordance with the inositol phosphate response, melatonin had no effect on either the AlF4(-)-stimulated or the basal calcium levels. AlF4- blocked the increase in cyclic AMP stimulation by 1 microM forskolin, being as effective as melatonin, achieving approximately 90% inhibition. AlF4- also attenuated the binding of [2-125I]iodomelatonin to ovine pars tuberalis membranes by 15%. At the concentration used, these results are consistent with the interpretation that AlF4- activates many G protein-mediated responses, and thus imply that the inhibitory pathway for cyclic AMP predominates over the stimulatory arm, whereas there can only be a stimulatory pathway linked to phosphoinositide metabolism in ovine pars tuberalis cells.

Aluminum↗
PubMed1990

Late onset adrenal hyperplasia in a group of Irish females who presented with hirsutism, irregular menses and/or cystic acne.

The aims of this study were to determine the frequency of late-onset adrenal hyperplasia due specifically to 21-hydroxylase deficiency in a group of Irish women who presented at a Dublin Clinic with symptoms of hyperandrogenism, including hirsutism, menstrual irregularities and/or cystic acne, and to determine if those with 21-hydroxylase deficiency showed particular HLA associations. 119 women had blood samples taken basally and 1 h after an injection of 0.25 mg synacthen with the following hormones profiled: 17-hydroxyprogesterone, 11-deoxycortisol, androstenedione, testosterone, DHEAS and cortisol. Blood sampling was carried out between 0900 and 1000 h during the early follicular phase of the menstrual cycle (when applicable). Ninety-six subjects were new referrals to the Clinic for investigation of hyperandrogenism and 23 were acting as controls. In this study, 6% of patients showed evidence of partial 21-hydroxylase deficiency. In addition, 3 of the 6 with partial 21-hydroxylase deficiency had normal baseline levels of 17-hydroxyprogesterone, with the biochemical abnormality becoming manifest only on synacthen stimulation. Late-onset adrenal hyperplasia due to partial deficiency of this enzyme should always be considered as a possible diagnosis in women who present with symptoms of hyperandrogenism. Synacthen stimulation is an important diagnostic tool in elucidating partial enzyme deficiency as baseline 17-hydroxyprogesterone may be normal in such patients.

Acne Vulgaris↗
PubMed1990

Calcium ion as intracellular messenger and cellular toxin.

Ca2+ serves a nearly universal intracellular messenger function in cell activation, but excess Ca2+ is also a cellular toxin. The possibility of Ca2+ intoxication is minimized by an elaborate autoregulatory system in which changes in Ca2+ influx rate across the plasma membrane are rapidly compensated for by parallel changes in Ca2+ efflux rate. By this mean, cellular Ca2+ homestasis is maintained so that minimal changes in total cell calcium and cytosolic Ca2+ concentration occur during sustained Ca2(+)-mediated responses. Rather than a sustained increase in cytosolic Ca2+ concentration, it is the localized cycling of Ca2+ across the plasma membrane that is the critically important Ca2+ messenger during the sustained phase of cellular responses mediated via surface receptors linked to the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 hydrolysis gives rise to inositol(1,4,5)trisphosphate (IP3) and diacylglycerol (DAG). The IP3 acts to release Ca2+ from an intracellular pool, thereby causing a transient rise in cytosolic Ca2+ concentration. This transient Ca2+ signal activates calmodulin-dependent protein kinases transiently, and hence, causes the transient phosphorylation of a subset of cellular proteins that mediate the initial phase of the response. The DAG brings about the association of protein kinase C (PKC) with the plasma membrane where a receptor-mediated increase in Ca2+ cycling across the membrane regulates PKC activity. The sustained phosphorylation of a second subset of proteins by PKC mediates the sustained phase of the response. Hence, Ca2+ serves as a messenger during both phases of the cellular response, but its cellular sites of action, its mechanisms of generation, and its molecular targets differ during the initial and sustained phases of the response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Multiple modifications in the phosphoproteins bound to stored messenger RNA in Xenopus oocytes.

Messenger RNA molecules accumulated in amphibian oocytes are stabilized and blocked from translation through association with a defined set of phosphoproteins. Phosphoproteins of 60 kDa and 56 kDa (pp60 and pp56) isolated from messenger ribonucleoprotein particles of Xenopus laevis oocytes can be bound in vitro to mRNA sequences. After phospholabelling in vitro, both pp60 and pp56 show a range of ionic forms, which resolve on two-dimensional gel electrophoresis as a series of pairs with identical charge. The similarities between pp60 and pp56 in their ionic properties suggest a common protein primary structure. This suggestion gains further support from proteinase digestion analysis of pp60 and pp56: practically identical size patterns of phospholabelled fragments are generated using a range of different proteinases. However, in spite of their structural similarities, pp60 and pp56 are recognised as antigenically distinct from each other by using polyclonal antibodies. It is concluded from these, and other, observations that pp60 and pp56 are members of a family of structurally similar polypeptides which are subjected to multiple secondary modifications. Of these modifications, phosphorylation appears to be instrumental in establishing tight binding to mRNA, while antigenicity appears to be determined by some other modification. The role of microheterogeneity in the structure of RNA-binding proteins is discussed in relation to the differential activation of mRNA sequences for translation during early development.

Animals↗