Search PubMed⌕ Search

Biomedical subjects

D R Weaver

Publications and source records attributed to D R Weaver.

At least 55 records · Page 3Linked to original sources
PubMed1995

Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor.

A G protein-coupled receptor for the pineal hormone melatonin was recently cloned from mammals and designated the Mel1a melatonin receptor. We now report the cloning of a second G protein-coupled melatonin receptor from humans and designate it the Mel1b melatonin receptor. The Mel1b receptor cDNA encodes a protein of 362 amino acids that is 60% identical at the amino acid level to the human Mel1a receptor. Transient expression of the Mel1b receptor in COS-1 cells results in high-affinity 2-[125I]iodomelatonin binding (Kd = 160 +/- 30 pM). In addition, the rank order of inhibition of specific 2-[125I]iodomelatonin binding by eight ligands is similar to that exhibited by the Mel1a melatonin receptor. Functional studies of NIH 3T3 cells stably expressing the Mel1b melatonin receptor indicate that it is coupled to inhibition of adenylyl cyclase. Comparative reverse transcription PCR shows that the Mel1b melatonin receptor is expressed in retina and, to a lesser extent, brain. PCR analysis of human-rodent somatic cell hybrids maps the Mel1b receptor gene (MTNR1B) to human chromosome 11q21-22. The Mel1b melatonin receptor may mediate the reported actions of melatonin in retina and participate in some of the neurobiological effects of melatonin in mammals.

Amino Acid Sequence↗
PubMed1995

c-fos and jun-B mRNAs are transiently expressed in fetal rodent suprachiasmatic nucleus following dopaminergic stimulation.

We examined the time-course of expression of c-fos and jun-B mRNAs in the fetal rat suprachiasmatic nuclei (SCN) following maternal cocaine injection on gestational day 20. Both c-fos and jun-B mRNA levels increased within 20 min, peaked at 40 min and declined to baseline by 120 min after cocaine treatment (30 mg/kg). In mice, the D1-dopamine agonist, SKF 38393, induced c-fos and jun-B mRNAs in the fetal SCN and striatum. Regulated expression of immediate early genes in the fetal SCN may play a role in entrainment of the fetal clock.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1995

Localization of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor mRNAs in rat brain.

Parathyroid hormone (PTH)-related peptide (PTHrP) has been identified in human tumors associated with the syndrome of humoral hypercalcemia of malignancy. PTHrP mRNA is also expressed in a variety of non-malignant tissues, suggesting that PTHrP is an endogenous peptide with as-yet unidentified autocrine or paracrine functions in normal tissues, including brain (Weir et al., Proc. Natl. Acad. Sci., 87 (1990) 108-112). In the present study, we used in situ hybridization to examine the expression of PTHrP and the common receptor for PTH and PTHrP in adult rat brain. Widespread yet anatomically discrete patterns of hybridization were observed using 35S-labeled antisense cRNA probes. PTHrP gene expression was highest in the supramamillary nucleus of the hypothalamus, medial superior olivary nucleus, and in subpopulations of cells in the neostriatum, hippocampus, and cerebral cortex. Other major sites of PTHrP gene expression included the amygdala, midline thalamic nuclei, pontine nuclei, choroid plexus, and the anterior pituitary gland. Highest levels of PTH/PTHrP receptor mRNA were in the mesencephalic portion of the trigeminal nucleus and the trigeminal ganglion, the lateral reticular, pontine and reticulotegmental nuclei, the hypoglossal nucleus and area postrema. Other major sites of PTH/PTHrP receptor expression included the anterodorsal nucleus of the thalamus, basolateral amygdala, entorhinal cortex, parasubiculum, cells in the Purkinje cell layer of the cerebellum, vestibular nuclei, ventral cochlear nucleus, the motor nucleus of the trigeminal, and the facial and external cuneate nuclei. The expression of genes encoding PTHrP and its receptor in discrete areas of the brain suggests that PTHrP may function as a neurotransmitter in the central nervous system.

Animals↗
PubMed1995

Definition of the developmental transition from dopaminergic to photic regulation of c-fos gene expression in the rat suprachiasmatic nucleus.

D1-dopamine receptor stimulation induces c-fos gene expression in the fetal suprachiasmatic nucleus (SCN), but not in the adult rat SCN. Light exposure at night induces c-fos gene expression in the adult SCN. The present experiments were designed to define the developmental transition from dopaminergic to photic regulation of c-fos gene expression in the SCN. Treatment with the D1-dopamine receptor agonist SKF 38393 (10 mg/kg) increased SCN c-fos gene expression during both day and night on postnatal day (PD) 0, 1, and at night on PD 2, but the c-fos response disappeared by PD 4. Photic induction of c-fos gene expression was apparent during both day and night at each age examined, from PD 0 through PD 6. The magnitude and distribution of c-fos expression following light during the daytime was distinguishable from the response to light at night beginning on PD 2, indicating that the circadian clock regulates (gates) the c-fos response by PD 2. Orbital enucleation prevents the induction of c-fos by light at night on PD 2, indicating retinal mediation. Developmental loss of the c-fos response to SKF is not precipitated by the arrival of the retinohypothalamic tract; animals enucleated on PD 0 were insensitive to SKF on PD 6, as were visually intact controls. The results demonstrate that both dopaminergic and photic inputs can regulate SCN c-fos gene expression early in the neonatal period, and that the developmental loss of sensitivity to SKF is not due to the arrival of photic input to the SCN. The developmental transition from dopaminergic to photic regulation of c-fos gene expression roughly parallels the developmental transition from maternal to photic entrainment of the developing biological clock.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1995

Melatonin receptors are for the birds: molecular analysis of two receptor subtypes differentially expressed in chick brain.

Two receptors (CKA and CKB) of the G protein-coupled melatonin receptor family were cloned from chick brain. CKA encodes a protein that is 80% identical at the amino acid level to the human Mel1a melatonin receptor and is thus designated the chick Mel1a melatonin receptor. CKB encodes a protein that is 80% identical to the Xenopus melatonin receptor and defines a new receptor subtype, the Mel1c melatonin receptor, which is distinct from the Mel1a and Mel1b melatonin receptor subtypes. A melatonin receptor family consisting of three subtypes is supported by PCR cloning of distinct melatonin receptor fragments from Xenopus and zebrafish. Expression of CKA and CKB results in similar ligand binding and functional characteristics. The widespread distribution of CKA and CKB mRNA in brain provides a molecular substrate for the profound actions of melatonin in birds.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses.

The pineal hormone melatonin regulates seasonal reproductive function and modulates circadian rhythms in mammals. We now report the cloning and characterization of a high affinity receptor for melatonin from the sheep and human. The receptor cDNAs encode proteins that are members of a newly discovered group within the G protein-coupled receptor family. Expression of the sheep and human receptors in COS-7 cells results in high affinity 2-[125I]iodomelatonin binding and pharmacological characteristics similar to endogenous high affinity receptors. Functional studies of NIH 3T3 cells stably expressing the sheep receptor show that the mammalian melatonin receptor is coupled to inhibition of adenylyl cyclase through a pertussis toxin-sensitive mechanism. In situ hybridization studies of melatonin receptor mRNA in several mammals reveal hybridization signals in the hypophyseal pars tuberalis and hypothalamic suprachiasmatic nucleus. The cloned high affinity receptor likely mediates the reproductive and circadian actions of melatonin in mammals.

Animals↗
PubMed1994

Entrainment of the fetal hamster circadian pacemaker by prenatal injections of the dopamine agonist SKF 38393.

Prenatal treatment with the D1-dopamine receptor agonist SKF 38393 or cocaine induces expression of the immediate-early gene c-fos in the fetal rat suprachiasmatic nucleus (SCN) (Weaver et al., 1992). Because the induction of c-fos gene expression in the SCN has been implicated in the entrainment of circadian rhythms by light in mature animals, the present study investigated whether prenatal dopaminergic activation entrains the fetal circadian pacemaker. Injections of SKF 38393 (8 mg/kg) were given to pregnant, SCN-lesioned hamsters during the last 5 d of gestation and the phases of the offspring's wheel-running activity rhythms were measured on postnatal day 20. Pregnant hamsters were each given two injections/day 12 hr apart, but only one of the injections each day contained SKF 38393. One group of hamsters received the drug at 0800 hr while another group received the drug at 2000 hr. The offspring from these treatment groups showed average phases that differed by 11.3 hr, demonstrating that prenatal SKF 38393 set the phase of the offspring's circadian rhythms. These results suggest that the fetal circadian pacemaker can be entrained by dopaminergic activation. In situ hybridization using cRNA probes demonstrated that a single injection of SKF 38393 on the last day of gestation induced c-fos gene expression in the fetal hamster SCN and that mRNA for the D1-dopamine receptor was present in the SCN at that time. It is possible that maternal entrainment of the fetal circadian pacemaker, which normally occurs during development, is mediated by dopaminergic activation within the fetal hypothalamus.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1993

Serotonin receptor gene expression in the rat suprachiasmatic nuclei.

Serotonin (5HT) is thought to reset the biological clock in the suprachiasmatic nuclei (SCN) in vitro through a postsynaptic 5HT-1a receptor. Thus we examined 5HT receptor gene expression in the SCN by in situ hybridization. On film autoradiograms, 5HT-1c receptor mRNA showed intense SCN hybridization, while 5HT-1b receptor mRNA displayed a weaker signal. Emulsion autoradiograms additionally revealed expression of 5HT-1a and 5HT-2 receptor mRNAs by a few scattered SCN cells. 5HT-3 receptor mRNA was not detected in the SCN, although the transcript was detected elsewhere in the brain. 5HT-1d and -1e receptor mRNAs were not detected in the SCN or elsewhere in brain within the sections examined. The results do not support a major role for postsynaptic 5HT-1a receptors in resetting SCN rhythms.

Animals↗
PubMed1993

A2a adenosine receptor gene expression in developing rat brain.

Adenosine is a neuromodulator in the adult central nervous system. Membrane-bound receptors for adenosine have been identified and cDNAs for A1, A2a, A2b, and A3 adenosine receptor subtypes have been cloned recently. The present study documents the developmental appearance of A2a adenosine receptor gene expression in the rat brain. In situ hybridization using 35S-labeled RNA probes generated from the rat A2a adenosine receptor cDNA revealed receptor gene expression in the striatum on gestational day (GD) 14. Developmental alterations in the pattern of receptor gene expression within the striatum suggest that this receptor mRNA is expressed by striatal neurons soon after they complete neurogenesis. Transient expression of the A2a adenosine receptor mRNA was observed in cerebral cortex, subiculum, parafascicularis nucleus of the thalamus, facial nucleus, trigeminal nucleus, locus coeruleus, area postrema, anterior pituitary gland and in the fetal cerebral vasculature. The ganglia of cranial nerves V, VII, VIII, IX and X expressed A2a adenosine receptor mRNA in fetuses; adults have not been examined. A2a adenosine receptor mRNA was expressed in the carotid body and intermediate lobe of the pituitary during development and also in adult rats. Northern blot analysis revealed that the A2a adenosine receptor transcript is consistent in size (ca 2.5 kb) across the developmental period examined (GD 14 through adult). Previous studies in adult rats have reported that A2a adenosine receptor gene expression is limited to a population of striatal medium spiny neurons. This study documents early developmental expression of the A2a adenosine receptor gene in the striatum and its transient expression elsewhere in the brain and cerebral vasculature. If the A2a adenosine receptor mRNA is translated into receptor protein shortly after the mRNA is expressed, adenosine could influence neuronal differentiation, migration, synaptogenesis, and angiogenesis. Expression of A2a adenosine receptor mRNA in cranial ganglia, carotid body, and intermediate lobe of the pituitary gland similarly suggests novel sites of adenosine action during development and in the adult.

Animals↗
PubMed1993

Fibronectin adsorpton kinetics on phase segregated polyurethaneureas.

The kinetics of fibronectin (FN) adsorption upon three polyurethaneureas (PUU) were measured by Fourier transform infrared spectroscopy. The three polymers had soft blocks composed of polydimethylsiloxane (PDMS), polytetramethyleneoxide (PTMO), and polyethyleneoxide (PEO). On each polymer, the amount of adsorbate increased proportional to the square root of time, but the rates were less than that predicted for purely diffusion controlled adsorption. Adsorption rate constants and sticking coefficients (the fraction of encounters with the surface which result in adsorption) were calculated from the data. The adsorption rate constants are 4.8 x 10(-4), 1.3 x 10(-4), and 2.5 x 10(-5) cm/s on PDMS-PUU, PTMO-PUU and PEO-PUU respectively. The rate constants and sticking coefficients fall within the range of previously reported values for other protein/polymer systems. The sticking coefficients correlate in general with the amount of FN adsorbed at 120 min, and with the extent of conformational change of the adsorbed protein.

Adsorption↗
PubMed1993

Melatonin receptors in human hypothalamus and pituitary: implications for circadian and reproductive responses to melatonin.

Two major physiological roles for the pineal hormone melatonin (MEL) have been identified in vertebrates: the hormone influences circadian rhythmicity and regulates seasonal responses to changes in day length. These effects of MEL are thought to be due to interaction with specific, high affinity MEL receptors in the suprachiasmatic nucleus (SCN) and hypophysial pars tuberalis (PT), respectively. Using the ligand 2-[125I]iodo-MEL ([125I]MEL), we examined putative MEL receptors in these regions in human and monkey tissue specimens by in vitro autoradiography. Specific, high affinity [125I]MEL-binding sites (Kd, 53.3 +/- 13.0 pM) were consistently observed in the human SCN. In contrast, specific [125I]MEL binding was detectable in the PT of only one of the eight human specimens examined. Specific [125I]MEL binding was also detected in the pars distalis of several subjects, but with an inconsistent distribution. In rhesus monkey tissue, MEL receptors were readily detected in the SCN and, as in all other seasonally breeding species examined to date, in the PT. The relative absence of MEL receptors from the human PT suggests that neuroendocrine responses to MEL in humans may occur by fundamentally different mechanisms than those that underlie the photoperiodic regulation of reproduction in seasonally breeding species.

Animals↗
PubMed1992

Circadian and developmental regulation of Oct-2 gene expression in the suprachiasmatic nuclei.

Oct-2 is a transcriptional activating factor that is expressed in the suprachiasmatic nuclei (SCN), the site of a biological clock. We examined in rats whether Oct-2 gene expression is regulated by the circadian pacemaker or by light using quantitative in situ hybridization. The ontogeny of Oct-2 gene expression in the SCN was also studied. Oct-2 mRNA levels remained constant throughout the circadian cycle. In contrast to c-fos mRNA levels which are acutely induced by acute light exposure at night, Oct-2 mRNA levels were not increased by light exposure at night. At gestational day 18, the first age the SCN are anatomically distinct, a prominent Oct-2 hybridization signal was present in the SCN. Our results suggest that Oct-2 is constitutively expressed in the SCN and is present from the time the SCN are discernible as discrete nuclei in fetal brain.

Animals↗
PubMed1992

D1-dopamine receptors activate c-fos expression in the fetal suprachiasmatic nuclei.

The existence of an activatable dopamine system within the hypothalamic suprachiasmatic nuclei (SCN), the site of a biological clock, was investigated in rats during fetal life. In situ hybridization studies revealed that D1-dopamine receptor mRNA was highly expressed in the fetal SCN and not expressed in other hypothalamic regions. Cocaine injected into pregnant rats or directly into rat fetuses on day 20 of gestation selectively activated c-fos gene expression in the fetal SCN; cocaine did not induce c-fos expression elsewhere in the fetal brain or in the maternal SCN. This cocaine-induced activation of c-fos expression in fetal SCN was mediated in part through D1-dopamine receptors, as the cocaine-induced activation was partially blocked by the D1-dopamine receptor antagonist SCH 23390. In addition, the selective D1-dopamine receptor agonist SKF 38393 induced high levels of c-fos expression in the fetal SCN. The presence of an activatable dopamine system within the fetal SCN provides a mechanism through which maternal signals could entrain the fetal biological clock and through which maternally administered psychotropic drugs could alter normal development of the circadian timing system.

Animals↗
PubMed1992

Sticking coefficients of adsorbing proteins.

The protein sticking coefficient, phi, the fraction of collisions that result in adsorption, is a function of the molecular interactions between the protein and the surface. A random walk and diffusion-to-capture model was used to describe the kinetics of protein adsorption. The assumption of a constant sticking coefficient leads to a first-order model of the kinetics. A solution of the problem of adsorption from a semi-infinite medium with first-order kinetics at the boundary was obtained by numerical simulation on the computer. The results of the computer simulations match the time dependence observed experimentally. A correlation was developed to estimate phi from experimental data. phi has been found to be in the range 10(-5)-10(-8) for several protein adsorption kinetic studies reported in the literature.

Adsorption↗
PubMed1992

Molecular cloning of the rat A2 adenosine receptor: selective co-expression with D2 dopamine receptors in rat striatum.

A cDNA fragment homologous to other G protein-coupled receptors was isolated from rat brain using the PCR method and demonstrated to be abundantly expressed in striatum. Using this fragment as a probe, a 2.1 kb full-length cDNA was isolated from a rat striatal cDNA library. This cDNA encodes a protein of 410 amino acids and is highly homologous to previously isolated adenosine receptor cDNAs. Expression of this cDNA in COS cells revealed high affinity (Kd = 38.6 nM) and saturable binding of the A2 adenosine receptor-selective ligand [3H]CGS 21680. Agonist displacement profile of [3H]CGS 21680 binding was consistent with an adenosine receptor of the A2 subtype (NECA greater than (R)-PIA greater than CPA greater than (S)-PIA). In situ hybridization demonstrated that rat A2 adenosine receptor mRNA was co-expressed in the same striatal neurons as D2 dopamine receptor mRNA, and never co-expressed with striatal D1 dopamine receptor mRNA. Several lines of evidence have previously suggested that dopamine-induced changes in motor behavior can be modulated by adenosine analogs acting at the A2 subtype of adenosine receptor in the forebrain. The co-expression of D2 dopamine and A2 adenosine receptors in a subset of striatal cells provides an anatomical basis for dopaminergic-adenosinergic interactions on motor behavior.

Adenosine↗
PubMed1992

Adenosine receptor gene expression in rat kidney.

Adenosine is an important modulator of renal function. Adenosine produced and released within the kidney is thought to participate in the metabolic regulation of glomerular filtration (tubuloglomerular feedback), as well as in regulating renal excretory function and renin secretion. The recent cloning of cDNAs encoding the A1 and A2a adenosine receptors from rat brain allows direct examination of potential sites of adenosine action within the rat kidney. Northern blot analysis of rat kidney poly(A)+ RNA revealed that A1 adenosine receptor mRNA was more abundant in kidney than the A2a adenosine receptor transcript. In situ hybridization with 35S-labeled cRNA probes was used to localize A1 and A2a adenosine receptor mRNAs within the kidney. A1 adenosine receptor mRNA was most abundant in the collecting ducts of the papilla and inner medulla. Collecting ducts in the outermost portion of the inner stripe of the outer medulla and cells of the juxtaglomerular apparatus also expressed A1 adenosine receptor mRNA. A2a adenosine receptor mRNA was localized to the renal papilla. The distribution of A1 and A2a adenosine receptor mRNAs within the rat kidney supports previously postulated roles for adenosine in the regulation of renal hemodynamics, excretory function, and renin secretion.

Animals↗
PubMed1992

Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype.

A novel adenosine receptor subtype has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA, designated RFL9, encodes a protein of 332 amino acids. The structure of RFL9 is most similar to that of the recently cloned rat A2-adenosine receptor, with a sequence identity of 73% within the presumed seven transmembrane domains. Expression of RFL9 in COS-6M cells resulted in ligand binding and functional activity characteristics of an adenosine receptor that is coupled positively to adenylyl cyclase. Examination of the tissue distribution of RFL9 mRNA by Northern blot analysis showed a restricted distribution with highest levels expressed in large intestine, cecum, and urinary bladder; this pattern was distinct from that of either the A1- or A2-adenosine receptor mRNAs. In situ hybridization studies of RFL9 mRNA showed no specific hybridization pattern in brain, but a hybridization signal was readily observed in the hypophyseal pars tuberalis. Thus, RFL9 encodes a novel A2-adenosine receptor subtype.

Amino Acid Sequence↗