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Biomedical subjects

P Barrett

Publications and source records attributed to P Barrett.

At least 37 records · Page 2Linked to original sources
PubMed2001

Characterization of an antibody to the human melatonin mt1 receptor.

Melatonin acts via high affinity, G-protein coupled, seven transmembrane domain receptors. To precisely localize these receptors, antibodies were raised in chickens against a 15 amino acid fragment at the intracellular C-terminal region of the human melatonin receptor subtype mt1 (DSSNDVADRVKWKPS, mt(1338-352)). A chimeric form of the receptor with a hydrophilic Flag peptide (DYKDDDDK) in sequence with the extracellular N-terminus (Flag-mt1) was generated by polymerase chain reaction and expressed in mammalian cell lines. An IgY antibody (Y31), which gave high antibody titres by enzyme-linked immunosorbent assay, was used to localize Flag-mt1 in stably transfected cells by immunofluoresence. Flag-mt1 localization with Y31 was identical to that obtained with the M5 antibody directed against the Flag epitope and was mainly localized to the Golgi apparatus with some staining at the cell surface. No staining was seen in untransfected cells with either antibody. Y31 staining was abolished using antibody preabsorbed with peptide antigen. Y31 immunofluorescence in fetal human kidney sections was restricted to nephrogenic regions and matched that of 2-((125)I)iodomelatonin binding and mt1 gene expression by in situ hybridization. Y31 was used to immunoprecipitate biotinylated membrane proteins from Flag-mt1 stably transfected and untransfected CHO cells. Western blotting of immunoprecipitated proteins revealed two major bands specific to stably transfected cells, one at 63 kDa and one at 86 kDa. The first band almost certainly corresponds to the glycosylated form of Flag-mt1 and the second band to receptor dimers. Thus, Y31 antibody is suitable for use in detecting the human mt1 receptor subtype in tissues and in transfected cells.

Amino Acid Sequence↗
PubMed2001

The differential regulation of CART gene expression in a pituitary cell line and primary cell cultures of ovine pars tuberalis cells.

The cocaine-amphetamine regulated transcript (CART) encodes for a protein which has an important role in the regulation of appetite and body weight. To date, no details of the molecular events and signal transduction pathways which regulate this gene are available. We report the identification of CART gene expression in the GH3 pituitary cell line. We have used activators of the cAMP or protein kinase C (PKC) signal transduction pathways to show that, in GH3 cells, CART is transcriptionally up-regulated by activators of the cAMP signal transduction pathway. We also identify CART gene expression in ovine pars tuberalis (PT) tissue and primary cell cultures. In PT cells in contrast to GH3 cells, CART gene expression is upregulated by activators of the PKC signal transduction pathway. Cultured cells have provided a valuable resource for the detailed analysis of specific regulatory mechanisms underlying transcriptional or translational regulation of genes, signal transduction events and many other cellular processes. GH3 and PT cells may therefore provide a resource for the further detailed molecular analysis of the events regulating CART gene expression and processing.

Animals↗
PubMed2001

Localization of the melatonin-related receptor in the rodent brain and peripheral tissues.

Previous studies have provided a limited examination of the expression of the orphan melatonin-related receptor in the pituitary and hypothalamus of human and sheep and retinal tissue in the sheep. The present study reports evidence of conservation of expression in regions of the hypothalamus (dorsal medial hypothalamus, lateral hypothalamus, arcuate nucleus), the epithelial layer lining the third ventricle and the paraventricular thalamic nucleus of the mouse, rat and hamster. An extensive and detailed analysis of melatonin-related receptor mRNA expression in the mouse central nervous system and peripheral tissues is presented. Mapping the distribution throughout the entire mouse brain has revealed new sites of expression in a number of brain nuclei, including preoptic areas, parabrachial nuclei and widespread distribution in the olfactory bulb. Reverse transcriptase-polymerase chain reaction was performed with RNA isolated from peripheral tissues revealing expression of the melatonin-related receptor mRNA in the mouse kidney, adrenal gland, intestine, stomach, heart, lung, skin, testis and ovary. These results suggest a conserved function in neuroendocrine regulation and a potential role in coordinating physiological responses in the central nervous system and peripheral tissues.

Animals↗
PubMed2001

ADCON-L and hypotension during lumbar microdiscectomy.

ADCON-L anti-adhesion gel is used as an antifibrotic agent in surgery for herniated lumbar disc. We report seven patients undergoing lumbar microdiscectomy, in whom ADCON-L gel was applied to the nerve root before closure of the surgical site. After the administration of ADCON-L, tachycardia and hypotension were noted, and were quickly and effectively reversed by i.v. fluids and ephedrine. Since its routine introduction into our practice, we have given ADCON-L on 212 occasions, with an incidence of adverse haemodynamic reactions of 3.3%. Adverse cardiovascular reactions do not appear to have been reported before.

Adult↗
PubMed2001

Prevention of anxiety symptoms in primary school children: preliminary results from a universal school-based trial.

Preliminary data are presented on the effectiveness of a universal school-based intervention for the prevention of anxiety symptoms in primary school children. A sample of 489 children (aged 10-12 years) were assigned to one of three intervention conditions: a psychologist-led preventive intervention, a teacher-led preventive intervention, or a usual care (standard curriculum) with monitoring condition. The intervention offered was the Friends for Children programme, a 12-session cognitive-behavioural intervention, originally based upon Kendall's (1994) Coping Cat programme. Participants in both intervention conditions reported fewer symptoms of anxiety at post-intervention than participants in the usual care condition. These preliminary results suggest that universal programmes for childhood anxiety are promising intervention strategies that can be successfully delivered to a school-based population and integrated into the classroom curriculum.

Analysis of Variance↗
PubMed2000

Trends in the frequency and predictive value of reporting high grade abnormalities in cervical smears.

BACKGROUND: The "organized approach" to cervical screening in Australia includes standardized quality assurance measures for laboratories. This study examines changes in the frequency and the positive predictive value of reporting severe abnormalities in cervical smears over a 3-year period as a guide to the effects of implementing these measures. METHODS: The results of screening in 6-month periods from January 1995 to December 1997 were determined. Biopsy follow-up for results in the high grade epithelial abnormality ("HGEA") and "inconclusive: possible HGEA" categories was obtained from the Western Australian Cervical Cytology Registry (CCR). RESULTS: Approximately 40,000 smears were examined in each 6-month period. The frequencies of reporting HGEA were 0.47%, 0.59%, 0.79%, 0.85%, and 0.84%, and 0.91% for the study periods (P < 0.001). For the inconclusive category, they were 0.24%, 0.18%, 0.24%, 0.31%, 0.38%, and 0.35% (P < 0.001). Biopsy follow-up was available for 83. 9%, 80.5%, 89.9%, 92.4%, 93.1%, and 90.3% of the HGEA results and for 78.6%, 71.7%, 80.5%, 75.0%, 87.1%, and 85.9% of the inconclusive results over the study periods. The yield of high grade lesions for the biopsied cases was 82.6%, 82.3%, 83.1%, 79.5%, 80.9%, and 79% for HGEA cases and 58.2%, 41.9%, 60.6%, 52.8%, 47.5%, and 54.1% for inconclusive cases. CONCLUSIONS: There was a doubling in the reporting of HGEA results, whereas the positive predictive value for biopsied cases remained at about 80%. Reporting rates for inconclusive: possible HGEA cases also doubled, but the yield of biopsy-proven, high grade lesions remained at about 50%. These changes occurred in the absence of ancillary testing and with targeted rescreening methods. A high rate of reporting HGEA, in combination with a high positive predictive value, is among the most important indicators of cervical cytology laboratory performance. Large improvements in results may occur using conventional methods of quality assurance. Cancer (Cancer Cytopathol)

Adenocarcinoma↗
PubMed2000

Chimeric melatonin mt1 and melatonin-related receptors. Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt1 receptor.

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt(1) receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[(125)I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[(125)I]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt(1) receptor sequences identified specific 2-[(125)I]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt(1) receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.

Animals↗
PubMed2000

Characterisation of human melatonin mt(1) and MT(2) receptors by CRE-luciferase reporter assay.

A cyclic AMP response element (CRE)-luciferase reporter gene assay was used to characterise the functional responses of human melatonin mt(1) and human melatonin MT(2) receptors, stably expressed in the human embryonic kidney cell line HEK293, to a series of six naphthalenic analogues of melatonin. By comparison to the observed melatonin-mediated inhibition of stimulated luciferase levels the naphthalenic series was identified as comprising agonists, partial agonists and one antagonist of melatonin mt(1) and melatonin MT(2) receptor function. Three of the agonist/partial agonist members of this series were also identified as displaying a functional selectivity for the melatonin MT(2) receptor. Competitive displacement of 2-[125I]iodomelatonin binding to the ovine pars tuberalis melatonin ML(1) receptor demonstrated a close correlation to the observed functional luciferase responses of the human melatonin mt(1) receptor. We conclude that the CRE-luciferase reporter gene assay provides an effective functional screening method for the pharmacological characterisation of human melatonin receptor subtypes.

Acetamides↗
PubMed2000

Angiotensin-converting enzyme inhibition by quinapril blocks the albuminuric effect of atrial natriuretic peptide in Type 1 diabetes and microalbuminuria.

AIMS: This study examined the effect of angiotensin-converting enzyme inhibition, administered at doses with no effect on systemic blood pressure, on the albuminuric action of atrial natriuretic peptide (ANP). METHODS: Seven Type 1 diabetic patients with established microalbuminuria participated in a two limb, single-blind, placebo controlled study. Subjects were administered quinapril 10 mg daily or placebo for 7 days prior to study. On the study day, subjects were euglycaemic clamped and subsequently fluid loaded (20 ml/kg tap water orally plus urinary losses). At steady state diuresis, a 1 h intravenous infusion of ANP 0.05 mg.kg(-1) x min(-1) was administered. Urine was collected at 15-min intervals for estimation of albumin-creatinine ratio (ACR). Results were analysed by ANOVA. RESULTS: Baseline mean arterial pressure was similar after pre-treatment with quinapril and placebo (98.7 +/- 3.8 vs. 100 +/- 4.5 mmHg, mean +/- SD, P > 0.5), and was unaltered by ANP infusion on either study day. Baseline ACR was similar on quinapril and placebo (P = 0.13). ANP infusion induced a rise in urine ACR with placebo (58.4 +/- 40.2 to 393.6 +/- 262.9 mg/mmol, P = 0.006), but not with quinapril (29.3 +/- 10.7 to 81.5 +/- 43 mg/mmol, P = 0.15). The urine ACR response to ANP infusion was higher with placebo than with quinapril (P = 0.02). CONCLUSIONS: Quinapril blocks the albuminuric effect of intravenous infusion of ANP in subjects with Type 1 diabetes mellitus and established microalbuminuria. This action is independent of changes in mean arterial pressure and creatinine clearance.

Albuminuria↗
PubMed2000

A role for tyrosine phosphorylation in the regulation and sensitization of adenylate cyclase by melatonin.

Mimicking short photoperiod melatonin signals (16 h exposure) on primary cell cultures of melatonin target cells of the ovine pars tuberalis (PT) results in an enhanced cAMP response to forskolin stimulation relative to untreated cells, a phenomenon termed sensitization. The sensitized response of PT cells may be an important aspect of the interpretation of the melatonin signal to initiate appropriate seasonal physiological responses. The aim of this study is to add to our understanding of the molecular mechanisms involved in the sensitization of PT cells by melatonin. We demonstrate that sensitization of PT cells by melatonin is mediated via a G(i)-coupled melatonin receptor. The sensitized cAMP response is not only obtained with the pharmacological tool forskolin, but also with cholera toxin, an activator of G(salpha). Changes in the level of G(salpha) or G(ialpha) G-protein subunits are ruled out as part of the sensitization mechanism. However, changes in tyrosine phosphorylation may be involved as tyrosine kinase inhibitors sensitize ovine PT cells and tyrosine phosphatase inhibitors significantly blunt adenylate cyclase activity, including the sensitized response to melatonin. The adenylate cyclase isoforms mediating the sensitized response may be broad as 7 of the 9 isoforms of adenylate cyclase are expressed in the PT.

Adenylate Cyclase Toxin↗
PubMed2000

Photoperiod regulates growth, puberty and hypothalamic neuropeptide and receptor gene expression in female Siberian hamsters.

In seasonal mammals, both the growth and reproductive axes are regulated by photoperiod. Female Siberian hamsters were kept, for up to 12 weeks, in long-day (LD) or short-day (SD) photoperiod, from weaning at 3 weeks of age (Exp 1). LD hamsters had characteristically faster growth and higher asymptotic body weight, adiposity, and leptin gene expression in adipose tissue. Only LD females attained puberty. Gene expression in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), POMC, and melanocortin 3-receptor (MC3-R) was higher in LD but did not change from weaning levels in SD. In contrast, gene expression in the arcuate nucleus for cocaine and amphetamine-regulated transcript (CART) was higher in SD than LD, a difference that was apparent at 2 weeks post weaning. Transfer of SD females to LD at 15 weeks post weaning (Exp 2) increased body weight, leptin signal, and gene expression for POMC but failed to induce normal puberty onset or to increase gene expression for OB-Rb and MC3-R. Therefore, photoperiodic regulation of puberty may be modulated by age, by photoperiodic history, and by changes in leptin signaling and the activity of the leptin-sensitive hypothalamic melanocortin system (POMC, MC3-R). A role for CART in photoperiodic regulation of growth is suggested, because the changes in CART gene expression preceded significant divergence of growth trajectories in the opposite photoperiods.

Agouti Signaling Protein↗
PubMed1999

Decoding photoperiodic time through Per1 and ICER gene amplitude.

The mammalian Per1 gene is expressed in the suprachiasmatic nucleus of the hypothalamus, where it is thought to play a critical role in the generation of circadian rhythms. Per1 mRNA also is expressed in other tissues. Its expression in the pars tuberalis (PT) of the pituitary is noteworthy because, like the suprachiasmatic nucleus, it is a known site of action of melatonin. The duration of the nocturnal melatonin signal encodes photoperiodic time, and many species use this to coordinate physiological adaptations with the yearly climatic cycle. This study reveals how the duration of photoperiodic time, conveyed through melatonin, is decoded as amplitude of Per1 and ICER (inducible cAMP early repressor) gene expression in the PT. Syrian hamsters display a robust and transient peak of Per1 and ICER gene expression 3 h after lights-on (Zeitgeber time 3) in the PT, under both long (16 h light/8 h dark) and short (8 h light/16 h dark) photoperiods. However, the amplitude of these peaks is greatly attenuated under a short photoperiod. The data show how amplitude of these genes may be important to the long-term measurement of photoperiodic time intervals.

Animals↗
PubMed1999

Practical methods to increase use of advance medical directives.

OBJECTIVE: To compare the effectiveness of two means for increasing the use of advance medical directives: written materials only versus written materials and an educational videotape. DESIGN: Population-based, randomized controlled trial with 3-month follow up. SETTING: Kaiser Permanente Colorado Region, a not-for-profit group-model health maintenance organization. PARTICIPANTS: A population-based sample of all 1,302 members aged 75 years and older who used the Franklin Medical Office, excluding 55 persons who died or disenrolled during the study period or were identified by their physicians as blind or cognitively impaired. INTERVENTIONS: All subjects were mailed a 10-page cartoon-illustrated educational pamphlet on patient choices, a selection of Colorado advance medical directive forms, and a guide to their completion; 619 subjects also were mailed a 20-minute videotape on advance directives. Both groups had access to a study nurse for assistance in completing and placing advance medical directives. MEASUREMENTS AND MAIN RESULTS: The main outcome measure is the proportion of subjects who placed a directive in their medical record for the first time. Placement rates increased almost identically, from 21.2% to 35.0% in the written materials-only group and from 18.9% to 32.6% in the group receiving the videotape (95% confidence interval for difference -0.04, 0.04, p =.952). CONCLUSIONS: In an elderly population with a substantial baseline placement rate, mailing of written materials substantially increased placement of an advance directive in the medical record, but the addition of a videotape did not. Mailing the video did increase the use of treatment trials and made patients more aware of reasons not to use advance directives.

Advance Directives↗
PubMed1999

The thermal effects of Kirschner wire fixation on small bones.

The aim of this study was to assess bone heating caused by the passage of fine (<2 mm) K-wires of different types. Stainless steel K-wires of trocar and diamond point configurations (0.8-2.0 mm) were drilled into the metatarsal bones from a freshly amputated lower limb at a constant force. Temperature measurements were made by miniature thermocouples inserted into the bones, at 3 second intervals over a period of 3 minutes while each K-wire was drilled three times. The temperature reached varied with the tip configurations and the diameter of the K-wires. Regardless of point configuration, thinner wires generated more heat than thicker ones.

Body Temperature↗
PubMed1999

Ovine arylalkylamine N-acetyltransferase in the pineal and pituitary glands: differences in function and regulation.

The enzyme arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) has been conventionally linked with the biosynthesis of melatonin within the pineal gland and retina. This study establishes that AANAT messenger RNA (mRNA) and functional enzyme occurs within the pars tuberalis (PT) and to a lesser degree within the pars distalis (PD) of the sheep pituitary gland; expression in these tissues is approximately 1/15th (PT) and 1/300th (PD) of that in the ovine pineal gland. AANAT mRNA in the PT appears to be expressed in the same cells as the Mel1a receptor. No evidence was obtained to indicate that either PT or PD cells have the ability to synthesize melatonin, suggesting that this enzyme plays a different functional role in the pituitary. We also found that cAMP regulation of the abundance of AANAT mRNA differs between the PT and pineal gland. Forskolin (10 microM) has no effect on pineal AANAT mRNA levels, yet represses expression in the PT. This suppressive influence could be mediated by ICER (inducible cAMP response early repressor), which is induced by forskolin in both tissues. Although it appears that the specific function and regulation of AANAT in the pituitary gland differ from that in the pineal gland, it seems likely that AANAT may play a role in the broader area of signal transduction through the biotransformation of amines.

Animals↗