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Biomedical subjects

D P Behan

Publications and source records attributed to D P Behan.

At least 19 recordsLinked to original sources
PubMed1998

Displacement of insulin-like growth factors from their binding proteins as a potential treatment for stroke.

Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role in normal growth and brain development and protect brain cells from several forms of injury. The effects of IGFs are mediated by type-I and type-II receptors and modulated by potentially six specific binding proteins that form high-affinity complexes with IGFs in blood and cerebrospinal fluid (CSF) and under most circumstances inactivate them. Because brain injury is commonly associated with increases in IGFs and their associated binding proteins, we hypothesized that displacement of this large "pool" of endogenous IGF from the binding proteins would elevate "free" IGF levels to elicit neuroprotective effects comparable to those produced by administration of exogenous IGF. A human IGF-I analog [(Leu24, 59, 60, Ala31)hIGF-I] with high affinity to IGF-binding proteins (Ki = 0.3-3.9 nM) and no biological activity at the IGF receptors (Ki = >10,000 nM) increased the levels of "free, bioavailable" IGF-I in the CSF. Intracerebroventricular administration of this analog up to 1h after an ischemic insult to the rat brain had a potent neuroprotective action comparable to IGF-I. This novel strategy for increasing "free" IGF levels in the brain may be useful for the treatment of stroke and other neurodegenerative diseases.

Animals↗
PubMed1997

Enhancement of performance in multiple learning tasks by corticotropin-releasing factor-binding protein ligand inhibitors.

Evidence favors a role for corticotropin-releasing factor (CRF) in learning and memory processes. A binding protein (CRF-BP) with the ability to inactivate CRF provides a novel target to modulate endogenous levels of CRF. The present studies employed three measures of information processing in rats in order to examine the impact of CRF system activation resulting from administration of CRF-BP ligand inhibitors, which increase levels of "free CRF." Acquisition of a visual discrimination paradigm and retention of a inhibitory avoidance task were dose dependently facilitated by central administration of a CRF-BP ligand inhibitor. CRF-BP ligand inhibitor treatment also improved performance in an active avoidance paradigm in aged animals. No nonspecific anorexic effects of the active dose of CRF-BP ligand inhibitor were detected in a food intake test. Moreover, the magnitude of in vivo efficacy of the CRF-BP ligand inhibitor peptide in producing a mild increase in motor activity was dissociated from that of a postsynaptic CRF receptor agonist that exerted robust and long-lasting activity increases. Thus, CRF-BP ligand inhibitors appear to elicit generalized learning enhancement effects without mimicking the robust nonspecific behavioral actions of a CRF receptor agonist.

Animals↗
PubMed1997

Corticotropin-releasing factor (CRF), CRF-binding protein (CRF-BP), and CRF/CRF-BP complex in Alzheimer's disease and control postmortem human brain.

In Alzheimer's disease (AD) there are dramatic reductions in human corticotropin-releasing factor (hCRF) concentration and reciprocal increases in CRF receptor density in the cortex. hCRF-binding protein (hCRF-BP), hCRF/hCRF-BP complex, and "free" hCRF were measured in 10 brain regions from control and AD postmortem human tissue. In the control brains hCRF-BP was heterogenously distributed and levels were at least 10-fold higher on a molar basis than total hCRF levels, suggesting that one major role of the binding protein is to limit the actions of hCRF at the hCRF receptors. Concordant with this hypothesis, the percentage of total hCRF that was in the bound inactive form ranged from 65 to 90% in most areas examined, with the exception of the caudate and globus pallidus where only 15 and 40% were complexed, respectively. hCRF-BP concentrations were similar in the control and AD groups except for Brodmann area (BA) 39 where there was a small but significant decrease in the AD group. Complexed hCRF levels were significantly decreased in BA 8/BA 9, BA 22, BA 39, nucleus basalis, and globus pallidus in the Alzheimer's group and free hCRF levels were significantly decreased only in three brain areas, BA 4, BA 39, and caudate; substantial (40%) but nonsignificant decreases were also noted in BA 8/BA 9 and BA 22. These data demonstrate that (1) a large proportion of the total hCRF in human brain is complexed to hCRF-BP and thus unavailable for hCRF receptor activation, (2) reductions in total hCRF alone do not necessarily predict reductions in bioactive free hCRF, and (3) total hCRF levels and hCRF-BP levels appear to be the main factors determining the quantity of bound and free hCRF in human brain.

Aged↗
PubMed1996

Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal.

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of "free" CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

Animals↗
PubMed1996

Urocortin interaction with corticotropin-releasing factor (CRF) binding protein (CRF-BP): a novel mechanism for elevating "free' CRF levels in human brain.

Here we demonstrate that urocortin, a new mammalian member of the corticotropin-releasing factor (CRF) neuropeptide family has high affinity for both the recombinant human CRF binding protein (CRF-BP) and for a membrane-associated form of the protein solubilized from postmortem human cerebrocortical brain tissue. The rank order of binding potency for both the human recombinant CRF-BP and for the solubilized human brain CRF-BP is: urotensin > hCRF > urocortin > sauvagine. The bound hCRF/hCRF-BP complex was detected in the postmortem human brain tissue using an ELISA assay specific for the hCRF/hCRF-BP complex. A large proportion (65%) of the endogenous hCRF was found to be complexed to the CRF-BP and thus unavailable for CRF receptor activation. Incubation of human brain postmortem tissue extracts with urocortin and urotensin resulted in a dramatic decrease in hCRF/hCRF-BP levels and a concomitant increase in "free' hCRF levels. Thus, urocortin and other putative CRF-related peptides may elevate endogenous levels of "free' hCRF in brain by displacing hCRF from the binding protein. These data define an indirect endogenous mechanism for activation of CRF receptors by new mammalian members of the CRF family of neuropeptides.

Binding Sites↗
PubMed1996

Characterization of a sheep brain corticotropin releasing factor binding protein.

We report here the identification, purification and cDNA cloning of a corticotropin releasing factor (CRF) binding protein(s) (CRF-BP) from sheep brain. Native sheep and rat brain CRF-BP and recombinant rat CRF-BP were shown to be N-glycosylated. Two membrane associated forms of brain CRF-BPs of 33 and 35 kDa were purified from sheep brain homogenates after solubilization in the presence of detergent. N-Terminal sequence analysis revealed that the 35 kDa protein is proteolytically cleaved near the N-terminus giving rise to an 18 amino acid peptide and a 33 kDa CRF-BP. Both the purified 33 and 35 kDa ovine CRF-BPs could be specifically cross linked to ovine [125I]CRF and human [125I]CRF. In contrast, recombinant rat CRF-BP can only be cross-linked to human [125I]CRF. A 1.7 kb cDNA clone (Basil 7) encoding an open reading frame for a 324 amino acid CRF-BP precursor was cloned from a sheep brain lambda gtlO cDNA library and was shown to have 85% and 87% amino acid homology to the rat and human proteins, respectively. Competitive binding analysis of the recombinant sheep CRF-BP (Basil 7) expressed in CHO cells revealed that it binds human and ovine CRF with high affinity. However, the recombinant sheep CRF-BP (Basil 7) had approximately 50-fold higher affinity for human CRF than for the ovine peptide. These data present the first biochemical proof that CRF-BP is in the brain and provides evidence for the existence of different forms of CRF-BP which have evolved across species to regulate CRF.

Amino Acid Sequence↗
PubMed1996

Corticotrophin-releasing factor receptors: from molecular biology to drug design.

Corticotrophin-releasing factor (CRF) acts within both the brain and the periphery to coordinate the overall response of the body to stress. The involvement of the CRF systems in a variety of both CNS and peripheral disease states has stimulated great interest in this peptide as a potential site of therapeutic intervention. The recent cloning of multiple CRF receptor subtypes has precipitated a new era in CRF research that has allowed precise molecular, pharmacological and anatomical examination of mammalian CRF receptors. In this article, Derek Chalmers and colleagues highlight the major differences between the two classes of CRF receptors, CRF1 and CRF2, and a functionally related CRF-binding protein, and discuss the relevance of these sites to the ongoing development of CRF-based therapeutics.

Amino Acid Sequence↗
PubMed1996

Measurement of corticotropin-releasing factor (CRF), CRF-binding protein (CRF-BP), and CRF/CRF-BP complex in human plasma by two-site enzyme-linked immunoabsorbant assay.

The actions of human corticotropin-releasing factor (hCRF) in brain, pituitary, and plasma are modulated by a 37-kDa protein [CRF-binding protein (CRF-BP)] that binds to hCRF and neutralizes the peptide's biological activity, suggesting that only the free unbound peptide is biologically active. To accurately predict the biological consequences resulting from changes in total hCRF levels, we have developed two-site enzyme-linked immunosorbent assays (ELISAs) for hCRF-BP, free hCRF, and the hCRF-BP/hCRF complex. The assays were validated by measuring each factor in 1) maternal plasma at times when CRF and hCRF-BP levels are altered, and 2) plasma from normal elderly human subjects who have undergone a hCRF stimulation test. The hCRF-BP ELISA has a sensitivity of 2.7 fmol and a range of detection from 2.7-8000 fmol. Both the hCRF and hCRF-BP/ hCRF assays have a sensitivity of 0.4 fmol, with a useful range of detection from 0.4-40 fmol. Maternal plasma hCRF-BP levels remained unaltered between the 16-21 and 34-39 month gestational age groups. However, levels rose from 0.88 +/- 0.069 nmol/L in the 16-21 month gestational age group to 1.01 +/- 0.09 nmol/L in the 28-33 month gestational age group. Bound hCRF levels dramatically rose from undetectable at 16-21 months of gestation to 200 +/- 69 and 442 +/- 106 pmol/L in the 28-33 and 34-39 month gestational age groups, respectively. In comparison, free hCRF levels remained low throughout gestation, but dramatically rose to 318 +/- 120 pmol/L from 34-39 months of gestation. Binding site occupancy on the hCRF-BP decreased when bound and free hCRF levels were elevated. After treating the third trimester plasma sample with the high affinity hCRF-BP ligand, alpha-helical CRF-(9-41), all of the bound hCRF was displaced from the binding protein, and free hCRF levels rose from 87 to 284 pmol/L. The plasma hCRF-BP level was 0.9 +/- 0.08 nmol/L in normal human volunteers (10 men and 9 women; mean +/- SD age, 74.2 +/- 7.7 yr), decreased to 60% of basal levels 15 min after a bolus injection of 1 microgram/kg synthetic hCRF, and gradually returned to preinjection levels after 120 min. Conversely, bound and free hCRF levels increased from undetectable levels before hCRF injection to 0.58 +/- 0.03 nmol/L at 15 min and then rapidly decreased to undetectable levels at 120 min. These data validate the ELISAs in combination with high affinity hCRF-BP ligands for measuring bound and free hCRF in human plasma and suggest the utility of these assays for further determining alterations in peripheral CRF in conditions such as pregnancy.

Aged↗
PubMed1996

Nature of ligand affinity and dimerization of corticotrophin-releasing factor-binding protein may be detected by circular dichroism.

As the association of corticotrophin-releasing factor (CRF) with its binding protein (BP) to form a dimer complex (CRF2/BP2) appears to be dependent on the nature of the ligand we have compared the circular dichroism difference spectra after association of the BP with ovine (o) CRF, human (h) CRF and the alpha-helical CRF (9-41) antagonist. All three ligands caused a negative change in molar ellipticity above 210 nm, with oCRF having the least and hCRF the greatest effect. Below 210 nm there was a marked divergence of difference spectra, with the reaction with the natural peptides, hCRF and oCRF, resulting in a positive change in ellipticity, whilst that with the antagonist produced a negative change. In view of the BP spectrum indicating predominantly beta-sheet and the peptides showing mainly alpha-helix these results were interpreted as the changes above 210 nm being due to dimerization and below 210 nm to a change in the conformation of ligand on binding. The opposite change in alpha-helicity of the antagonist observed on binding compared with the two natural CRF peptides could have fundamental pharmacological implications.

Animals↗
PubMed1996

Regulation of corticotropin-releasing factor-binding protein expression in cultured rat astrocytes.

The regulation of brain corticotropin-releasing factor (CRF)-binding protein (BP), an endogenous modulator of the CRF family of neuropeptides, has been difficult to pursue due to a lack of basal expression in a known cell line or primary cells in vitro. In light of the ability of intracellular factors to modulate neuronal and glial function, we examined the effects of a variety of signal transduction modulators on CRF-BP expression in cultured astrocytes. In particular, the effect of agents that stimulate protein kinase A and protein kinase C pathways was evaluated. CRF-BP was measured using a ligand immunoradiometric assay. Forskolin, dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine treatment resulted in a dose-dependent increase in CRF-BP levels detected in the medium from astrocytes and neurons. The increase in CRF-BP expression was not due to increased cell proliferation as measured by [3H]thymidine incorporation. In addition, treatment of the astrocytes with phorbol myristate acetate, a protein kinase C activator, caused a robust increase in CRF-BP levels in the medium. Steroids such as dexamethasone, corticosterone, hydrocortisone and, to a lesser extent, dehydroepiandosterone inhibited the stimulated release of CRF-BP from astrocytes. These data define a primary role for intracellular messengers in regulating CRF-BP expression in neurons and astrocytes.

Animals↗
PubMed1996

Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding protein: implications for the treatment of CNS disorders.

The actions of CRF in the brain and in the periphery are mediated through multiple binding sites. There are three receptors, CRF1, CRF2 alpha and CRF2 beta, which encode 411, 415 and 431 amino acid proteins and transduce signals via the stimulation of intracellular cAMP production. The recent identification of high-affinity non-peptide CRF receptor antagonists should allow for rapid progress in drug development of CRF receptor antagonists. In addition to the receptors, the actions of CRF in brain and in the periphery can also be modulated by a binding protein of 322 amino acids. Ligands of CRF-BP, such as CRF (6-33) can elevate brain levels of 'free' CRF and improve learning and memory without stress-like side effects of CRF receptor agonists. Urocortin, a mammalian CRF-related peptide with close sequence homology to fish urotensin, interacts with CRF1, CRF2 receptors and with CRF-BP. These data indicate that CRF receptor antagonists may be useful for the treatment of the disease states where CRF is elevated such as anxiety and depression, anorexia nervosa and stroke and that ligand inhibitors of CRF-BP may be used to elevate brain levels of 'free' urocortin and other CRF-related peptides.

Animals↗
PubMed1995

Displacement of corticotropin releasing factor from its binding protein as a possible treatment for Alzheimer's disease.

In Alzheimer's disease (AD) there are dramatic reductions in the content of corticotropin releasing factor (CRF), reciprocal increases in CRF receptors, and morphological abnormalities in CRF neurons in affected brain areas. Cognitive impairment in AD patients is associated with a lower cerebrospinal fluid concentration of CRF, which is known to induce increases in learning and memory in rodents. This suggests that CRF deficits contribute to cognitive impairment. The identification in post-mortem brain of CRF-binding protein (CRF-BP), a high-affinity binding protein that inactivates CRF, and the differential distribution of CRF-BP and CRF receptors, provides the potential for improving learning and memory without stress effects of CRF receptor agonists. Here we show that ligands that dissociate CRF from CRF-BP increase brain levels of 'free CRF' in AD to control levels and show cognition-enhancing properties in models of learning and memory in animals without the characteristic stress effects of CRF receptor agonists.

Adult↗
PubMed1995

Corticotropin releasing factor binding protein (CRF-BP) is expressed in neuronal and astrocytic cells.

Corticotropin releasing factor (CRF) binding protein (CRF-BP) was measured in media and cell lysates of primary rat astrocytes, microglia and neurons with the use of a ligand immunoradiometric assay (LIRMA). A low basal level of CRF-BP was detected in the media and cell lysates from primary neuronal and astrocyte cells after 48 h in culture. No basal expression of CRF-BP was detected in cell lysates or media from primary microglial cultures. The CRF-BP expressed in cultured astrocytes and neurons had the same pharmacological characteristics as the human recombinant molecule. After forskolin, IBMX or forskolin/IBMX treatment, a robust increase in secreted CRF-BP levels in the media from astrocytes and neurons, but not microglia, was observed. An increase in CRF-BP-like immunoreactivity in cell lysates was also observed after IBMX/forskolin treatment. In situ hybridization analysis revealed that CRF-BP mRNA was increased in primary cultured astrocytes after IBMX/forskolin stimulation suggesting that regulation was at the level of gene transcription. 'Axon sparing' lesions produced with 0.12 M quinolinic acid in PBS injected intracerebrally (unilaterally into dorsal hippocampus) resulted in loss of CRF-BP expression in neurons. These data provide evidence for the differential localization and regulation of CRF-BP in different cell types in brain and suggest that CRF-BP expression may be locally increased in disease states associated with astrocytosis and gliosis.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Corticotropin releasing factor (CRF) binding protein: a novel regulator of CRF and related peptides.

A 37-kDa corticotropin releasing factor (CRF) binding protein (CRF-BP) was purified from human plasma by repeated affinity purification and subsequently sequenced and cloned. The human and rat CRF-BP cDNAs encode proteins of 322 amino acids with one putative signal sequence, one N-glycosylation site, and 10 conserved cysteines. Human CRF-BP binds human CRF with high affinity but has low affinity for the ovine peptide. In contrast, sheep CRF-BP binds human and ovine CRF with high affinity. The CRF-BP gene consists of seven exons and six introns and is located on chromosome 13 and loci 5q of the mouse and human genomes, respectively. CRF-BP inhibits the adrenocorticotrophic hormone (ACTH) releasing properties of CRF in vitro. CRF-BP dimerizes after binding CRF and clears the peptide from blood. This clearance mechanism protects the maternal pituitary gland from elevated plasma CRF levels found during the third trimester of human pregnancy. CRF-BP is expressed in the brains of all species so far tested but is uniquely expressed in human liver and placenta. In brain, CRF-BP is membrane associated and is predominantly expressed in the cerebral cortex and subcortical limbic structures. In some brain areas CRF-BP colocalizes with CRF and CRF receptors. The protein is also present in pituitary corticotropes, where it is under positive glucocorticoid control, and is likely to locally modulate CRF-induced ACTH secretion. The ligand requirements of the CRF receptor and the CRF-BP can be distinguished in that central human CRF fragments, such as CRF (6-33) and CRF (9-33), have high affinity for CRF-BP but low affinity for the CRF receptor. The binding protein's ability to inhibit CRF-induced ACTH secretion can be reversed by CRF (6-33) and CRF (9-33), suggesting that ligand inhibitors may have utility in elevating free CRF levels in disease states associated with decreased CRF. Thus, by controlling the amount of free CRF which activates CRF receptors, it is likely that the CRF-BP is an important modulator of CRF both in the CNS and in the periphery.

Amino Acid Sequence↗
PubMed1995

Ligand requirements of the human corticotropin-releasing factor-binding protein.

CRF-binding protein (CRF-BP), identified as a 37-kilodalton human serum protein, binds human (h) CRF (Kd = 0.17 +/- 0.01 nM) and blocks hCRF's ability to stimulate ACTH release by pituitary cells in vitro. The present study examines ligand requirements of CRF-BP by testing the affinity of recombinant CRF-BP for synthetic analogs of CRF and peptides in the CRF family. The relative affinities of various fragments of hCRF or related peptides for CRF-BP indicate that residues 9-28 are crucial for ligand binding. CRF-BP binds human/rat CRF and urotensin-I with high affinity, sauvagine with moderate affinity, and ovine (o) CRF with low affinity. The marked difference in the affinity of CRF-BP for oCRF (Ki = 1100 +/- 97 nM) compared to hCRF (Ki = 0.17 +/- 0.01 nM), when considered with the importance of the central domain, suggests that amino acids 22, 23, and/or 25 are critical for binding. Altering oCRF residues 22, 23, or 25 individually or collectively to match those of hCRF increases the affinity of CRF-BP for these ligands; [Ala22, Arg23, Glu25]oCRF, in which all three of these central amino acids are substituted by their hCRF counterparts, binds CRF-BP with an affinity equal to that of hCRF. CRF-BP has differential affinities for CRF receptor antagonists, binding alpha-helical CRF-(9-41) with high affinity and [D-Phe12, Nle21,38]hCRF-(12-41) with low affinity. Thus, the structural requirements for binding to CRF-BP can clearly be distinguished from those for CRF receptor recognition of both agonists and antagonists. Peptides such as hCRF-(9-33), with low biological activity but which retain high affinity for the binding protein, can competitively override the effects of CRF-BP to block CRF-induced ACTH secretion, raising the possibility that whereas endogenous CRF-BP serves to limit the distribution or duration of action of CRF, specific pharmacological inhibitors of the ligand-binding protein interaction might be used to therapeutically elevate free CRF levels.

Amino Acid Sequence↗
PubMed1994

Assignment of disulfide bonds in corticotropin-releasing factor-binding protein.

We have previously isolated, cloned, and characterized a protein that specifically binds and inactivates the peptide corticotropin-releasing factor. The integrity of the disulfide bonds in the binding protein is essential for this activity as reduction abolishes the protein's ability to bind corticotropin-releasing factor. The disulfide arrangement of the 10 cysteines present in the mature protein was established by analysis of proteolytically cleaved protein and sequence analysis of cystine containing fragments. A pattern is observed where each cysteine is connected to the next one in a sequential manner. Inspection of the genomic DNA encoding for this protein reveals that four of the domains defined by disulfide linkage coincide with four different exons.

Amino Acid Sequence↗