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Biomedical subjects

E Potter

Publications and source records attributed to E Potter.

At least 19 recordsLinked to original sources
PubMed1998

Ectopic expression of the CRF-binding protein: minor impact on HPA axis regulation but induction of sexually dimorphic weight gain.

Corticotrophin-releasing factor (CRF) and urocortin possess a high-affinity binding protein. Although the CRF binding protein (BP) can sequester these ligands and inhibit their activity, the endogenous activity of this protein is not understood. Therefore, transgenic mouse lines that over-express the CRF-BP were created. The transgene was constructed by ligating rat CRF-BP cDNA (1.1 kb) between a mouse metallothionein-I promoter (1.8 kb) and a nonfunctional human growth hormone gene sequence (2.1 kb) in a modified pBR322 plasmid and microinjecting the transgene into C57BL/6 x SJL hybrid ova. The transgene was expressed in 50% in both male and female progeny. All transgenic lines were maintained by crossing transgenic animals with wild-type C57BL/6 mates. Reverse-transcriptase (RT) PCR of the CRF-BP transgene showed that it is widely expressed not only in the brain and pituitary, but also peripheral tissues including the liver, kidney and spleen. Transgenic animals of both sexes showed significant increases in weight gain as established by analysis of variance; however, the weight gain profiles for each sex were distinct. High levels of circulating CRF-BP were detected in the transgenic animals, but the basal ACTH and corticosterone levels were not significantly decreased compared to wild-type littermates. The hypothalamopituitary-adrenal (HPA) axis was stimulated by systemic inflammation induced with lipopolysaccharide (LPS). An expected increase in transgene expression was observed and was accompanied by a significant attenuation of ACTH secretion at 3 h after LPS injection in the transgenic males but not the females. These data suggest that HPA axis regulation is significantly affected only with very high circulating levels of CRF-BP. Moreover, this work supports previous studies that implicate CRF and urocortin in the regulation of appetite and the binding protein expression may play a sexually dimorphic role in regulating this and other responses.

Adrenal Glands↗
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

Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary.

Corticotropin-releasing factor (CRF) is a major hypophysiotropic peptide regulating pituitary-adrenal response to stress, and it is also widely expressed in the central nervous system. The recent cloning of cDNAs encoding the human and rat CRF receptors has enabled us to map the distribution of cells expressing CRF receptor mRNA in rat brain and pituitary by in situ hybridization. Receptor expression in the forebrain is dominated by widespread signal throughout all areas of the neo-, olfactory, and hippocampal cortices. Other prominent sites of CRF receptor mRNA expression include subcortical limbic structures in the septal region and amygdala. In the diencephalon, low levels of expression are seen in a few discrete ventral thalamic and medial hypothalamic nuclei. CRF receptor expression in hypothalamic neurosecretory structures, including the paraventricular nucleus and median eminence, is generally low. In the brainstem, certain relay nuclei associated with the somatic (including trigeminal), auditory, vestibular, and visceral sensory systems, constituted prominent sites of CRF receptor mRNA expression. In addition, high levels of this transcript are present in the cerebellar cortex and deep nuclei, along with many precerebellar nuclei. In the pituitary, moderate levels of CRF receptor mRNA expression were detected throughout the intermediate lobe and in a subset of cells in the anterior lobe identified as corticotropes by concurrent immunolabeling. Overall, the central distribution of CRF receptor mRNA expression is similar to, though more expansive than, that of regions reported to bind CRF, and it shows limited overlap with loci expressing CRF-binding protein. Interestingly, CRF receptor mRNA is low or undetectable in several cell groups implicated as central sites of CRF action.

Adrenocorticotropic Hormone↗
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↗
PubMed1994

Association of human corticotropin-releasing hormone to its binding protein in blood may trigger clearance of the complex.

Late in the last trimester of human pregnancy, as plasma CRH levels rise, the concentration of circulating CRH-binding protein (CRH-BP) falls. We have investigated, using nonpregnant subjects, the hypothesis that CRH has a negative effect on plasma levels of CRH-BP. A specific RIA developed with the aid of recombinant binding protein has been used to measure CRH-BP. Subjects given iv infusions of human CRH for 10 h showed a sustained fall in plasma CRH-BP for the duration of the infusion. Intravenous bolus injection of human CRH produced a rapid reduction in CRH-BP levels to 54% of the basal value, whereas ovine CRH was without effect, even though both peptides are cleared from the plasma at similar rates and have similar effects on the pituitary-adrenal axis. The rapid clearance was concluded to be related to ligand affinity, as ovine CRH has a 200-fold lower affinity than human CRH for CRH-BP. We suggest that the rising levels of CRH are responsible for the reduction in CRH-BP concentrations observed in late pregnancy, and that this reduction is triggered by the binding of CRH-BP to its ligand.

Animals↗
PubMed1993

The functional neuroanatomy of corticotropin-releasing factor.

Descriptions of the central distribution of corticotropin-releasing factor (CRF) have been taken as generally supporting the proposition that this neuropeptide is involved in the mediation of complementary neuroendocrine, autonomic and behavioural responses to stress. The hypothalamic paraventricular nucleus (PVN) is recognized as the principal source of CRF in hypophysial portal plasma; CRF mRNA and peptide expression in parvocellular neurosecretory neurons are regulated negatively by adrenal steroids and positively by many stressors. Consistent with the latter, the hypophysiotropic zone of the PVN receives a rich, and biochemically differentiated, afferent supply that provides visceral, somatic and special sensory systems with access to the 'CRF neuron'. Within the PVN, CRF is also expressed, and differentially regulated, in oxytocinergic magnocellular neurosecretory neurons and in autonomic-related projection neurons. CRF expression in at least some extrahypothalamic cell groups (olfactory bulb, Barrington's nucleus) is responsive to certain stressful stimuli, but not to perturbations of the steroid environment. Refinement of our understanding of the central distribution of CRF has been provided by the recognition that most CRF antisera cross-react with an amidated dipeptide encoded by the melanin-concentrating hormone precursor, and by the likelihood that some central sites of CRF peptide expression may be muted or masked by the presence of a CRF-binding protein (CRF-BP). The CRF-BP is expressed prominently in the telencephalon, where it is co-localized with CRF in some neurons, and in anterior pituitary corticotrophs.

Afferent Pathways↗
PubMed1993

Cloning and structure of the human corticotrophin releasing factor-binding protein gene (CRHBP)

The human CRF-binding protein gene has been cloned and mapped to the distal region of chromosome 13 and loci 5q in the mouse and human genomes, respectively. The gene consists of 7 exons and 6 introns. The mature protein has 10 cysteines and 5 tandem disulfide bridges 4 of which are contained within exons 3, 5, 6, and 7. One bridge is shared by exons 3 and 4. The signal peptide and the first 3 amino acids of the mature protein were coded for by an extreme 5' exon. Primer extension analyses revealed the transcriptional initiation site to be located 32 bp downstream from a consensus TATA box. The promoter sequence contained a number of putative promoter elements including an AP-1 site, three ER-half sites, the immunoglobulin enhancer elements NF-kappa B and INF-1, and the liver-specific enhancers LFA1 and LFB1.

Animals↗
PubMed1993

Corticotropin releasing hormone-binding protein (CRH-BP): plasma levels decrease during the third trimester of normal human pregnancy.

In pregnancy, maternal plasma corticotropin releasing hormone (CRH) concentrations rise substantially in the third trimester and fall rapidly post-partum. A binding protein (BP) specific for CRH exists in the human circulation which inactivates CRH, thus possibly explaining why maternal ACTH does not rise outside normal limits throughout gestation. We here describe the measurement of CRH-BP directly in plasma during human pregnancy using a radioimmunoassay that is not affected by the presence of the high plasma levels of CRH that occur at this time. In 119 healthy non-pregnant individuals, mean CRH-BP levels were 4.46 nmol/L +/- 1.0 (SD), with a wide range of 1.81-7.24 nmol/L. Plasma CRH-BP in 34 pregnant women randomly sampled during the first and second trimesters also averaged 4.46 nmol/L +/- 1.54, with individual values ranging from 1.59-7.51 nmol/L and there was no correlation of CRH-BP levels with gestational age. In a group of 14 women sampled sequentially throughout the third trimester, plasma CRH-BP averaged 4.56 nmol/L +/- 1.70 at 30-35 weeks gestation and fell dramatically to 1.84 nmol/L +/- 0.43 at weeks 38-40 (P < 0.001). The post partum recovery in CRH-BP levels occurred within 48 hours of delivery. These results indicate that there is an increase in the availability of free, potentially bioactive CRH at term to stimulate the release of ACTH from the maternal pituitary and/or to act at a peripheral, non-pituitary CRH receptor(s).

Adult↗
PubMed1993

Corticotropin-releasing factor-binding protein is produced by human placenta and intrauterine tissues.

CRF circulates in high concentration in pregnant woman. It is produced by the placenta and the other intrauterine tissues (maternal decidua, amnion, and chorion). Recently, a CRF-binding protein (CRF-BP) has been identified and cloned. It binds the circulating CRF, reducing its biological action during pregnancy. Liver is the major source of CRF-BP. The aim of the present study was to evaluate whether human placenta and intrauterine tissues produce CRF-BP. The localization of mRNA and immune CRF-BP by in situ hybridization and immunohistochemistry, respectively, was performed. Antisense and sense riboprobes synthesized from a fragment of human CRF-BP cRNA and a specific rabbit anti-hCRF-BP serum was used. The syncytial layer of placental villi at term intensely expressed CRF-BP mRNA and immunoreactivity, whereas rare positively hybridized cells were observed within the cytotrophoblasts and mesenchymal cells. Large decidual cells, amniotic epithelial cells, and chorionic cytotrophoblast stained positively for CRF-BP mRNA and protein. Control sections collected from the same tissues failed to show any positive localization of sense strand cRNA probe and antiserum preadsorbed with immunogen. Finally, the addition of recombinant CRF-BP to human cultured placental cells significantly decreased CRF-induced ACTH release, with a dose-dependent effect. The present data show that local production of CRF-BP occurs in human trophoblast and intrauterine tissues and may represent one of the major mechanisms used by targets tissues to control CRF activity during pregnancy.

Adrenocorticotropic Hormone↗
PubMed1992

The central distribution of a corticotropin-releasing factor (CRF)-binding protein predicts multiple sites and modes of interaction with CRF.

In recent studies to clone and characterize genes coding for the corticotropin-releasing factor-binding protein (CRF-BP), analysis of the tissue distribution of the CRF-BP gene indicated a high level of expression in the rat brain. We have now characterized by immunohistochemical and hybridization histochemical means the cellular localization of CRF-BP protein and mRNA expression, respectively. Results from both approaches converged to indicate that CRF-BP is expressed predominantly in the cerebral cortex, including all major archi-, paleo-, and neocortical fields. Other prominent sites of mRNA and protein expression include subcortical limbic system structures (amygdala, bed nucleus of the stria terminalis), sensory relays associated with the auditory, olfactory, vestibular, and trigeminal systems, severe raphe nuclei, and a number of cell groups in the brainstem reticular core. Expression in the hypothalamus appears largely limited to the ventral premammillary and dorsomedial nuclei; only isolated CRF-BP-stained cells are apparent in neurosecretory cell groups. Dual immunostaining for CRF and CRF-BP revealed a partial colocalization in some of these regions. In addition, prominent CRF-BP-stained terminal fields have been identified in association with CRF-expressing cell groups in circumscribed hypothalamic and limbic structures. In the anterior pituitary, CRF-BP mRNA and immunoreactivity were colocalized with corticotropin-immunoreactivity in a majority of corticotropes. Thus, CRF-BP could serve to modify the actions of CRF by intra- and intercellular mechanisms, in CRF-related pathways in the central nervous system and pituitary.

Adrenocorticotropic Hormone↗
PubMed1991

Cloning and characterization of the cDNAs for human and rat corticotropin releasing factor-binding proteins.

Corticotropin-releasing factor (CRF), is a potent stimulator of synthesis and secretion of preopiomelanocortin-derived peptides. Although CRF concentrations in the human peripheral circulation are normally low, they increase throughout pregnancy and fall rapidly after parturition. Maternal plasma CRF probably originates from the placenta, which responds to the bioactive peptide and produces the peptide and its messenger RNA. Even though CRF concentrations in late gestational maternal plasma are similar to those in rat hypothalamic portal blood and to those that can stimulate release of adrenocorticotropic hormone (ACTH) in vitro, maternal plasma ACTH concentrations increase only slightly with advancing gestation and remain within the normal range. Several groups have now reported the existence of a CRF-binding protein in human plasma which inactivates CRF and which has been proposed to prevent inappropriate pituitary-adrenal stimulation in pregnancy. The binding protein was recently purified from human plasma. We have now isolated and partially sequenced the binding protein, allowing us to clone and characterize its complementary DNA from human liver and rat brain. Expression of the cDNAs for human and rat binding protein in COS7 cells showed that these proteins bind CRF with the same affinity as the native human protein. Both rat and human recombinant binding proteins inhibit CRF binding to a CRF antibody and inhibit CRF-induced ACTH release by pituitary cells in vitro.

Adrenocorticotropic Hormone↗
PubMed1990

Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro.

Activin-A, a homodimeric protein composed of two inhibin beta A-subunits, was first isolated from gonadal fluids based upon its ability to stimulate FSH secretion and biosynthesis, but was also observed to suppress GH secretion. The present report describes the effects of activin on the biosynthesis of GH and the proliferation of pituitary somatotrophs. In pituitary cells cultured in the presence of 0.7 nM activin for 3 days, GH secretion was decreased by 50% compared to the control value. Inhibition of GH biosynthesis, measured by quantitative immunoprecipitation of [35S]methionine-labeled cells, could be observed after 24 h of activin treatment, and maximal (70%) inhibition of GH biosynthesis was observed after 3 days. Activin inhibited basal as well as GH-releasing factor (GRF)-, glucocorticoid-, and thyroid hormone-stimulated GH biosynthesis. Inhibin, which is known to reverse the effect of activin on FSH secretion, did not reverse the effect of activin on GH biosynthesis. Treatment of somatotrophs with activin for 3 days completely inhibited the growth-promoting effect of GRF on somatotrophs. However, no effect of activin on GRF-stimulated expression of the c-fos protooncogene was observed. These data demonstrate that activin, in addition to its stimulatory effect on FSH secretion, is able to inhibit both expression of GH and growth of somatotropic cells.

Activins↗
PubMed1989

Effects of growth and insulin treatment on the levels of insulin receptors and their mRNA in Hep G2 cells.

We have studied the variations in the number of insulin receptor and insulin receptor mRNA levels in (Hep G2) cells in response to growth and insulin treatment. The levels of insulin receptors are relatively low in growing cells. After approximately 5 days in culture, if cells are not refed they cease to divide and the number of receptors/cell increases, reaching 4 times the initial values by the 9th day. Refeeding the cells completely prevented both growth arrest and the increase in insulin receptor number. Insulin added daily to cells at 0.33 microM caused receptor down-regulation but did not prevent a 3-fold increase in binding with growth arrest. Pulse-chase studies of metabolically labeled ([35S]methionine) cells showed that the receptor degradation rate (apparent t 1/2, 18-20 h) was comparable in rapidly growing versus growth-arrested cells. The increased receptor level in non-refed cells is not due to generation of a soluble factor by confluent cells, nor is it caused by depletion of insulin, glucose, or insulin-like growth factor I from the culture medium. The levels of insulin receptor mRNA measured on Northern blots increased in growth-arrested cells in parallel to the increase in receptor number. The mRNA value begins to increase from the 3rd day in culture and by the 9th day reaches a level 6.0 times that on the 3rd day. Chronic insulin-induced receptor down-regulation did not alter insulin receptor mRNA levels at any time point studied. These data demonstrate that the increase in insulin receptor number/cell in growth-arrested cells is paralleled by an increase in insulin receptor mRNA content with no change in the receptor degradation rates. This suggests that the increase in the number of insulin receptors is due to enhanced receptor synthesis due to increased receptor mRNA content. Conversely, down-regulation of the insulin receptor does not affect the level of insulin receptor mRNA and thus must be due to increased receptor degradation.

Blotting, Northern↗
PubMed1988

The John Henryism and Framingham type A scales. Measurement properties in elderly blacks and whites.

In 1984-1985, the authors interviewed 1,017 participants in the Charleston Heart Study in Charleston County, South Carolina to investigate the measurement properties of the Framingham Type A Scale in elderly blacks and whites and those of the John Henryism Scale for Active Coping, which was originally designed for use in black populations. They conclude that the Framingham Type A and John Henryism scales do in fact measure two quite different behavior patterns, and do have different correlates in elderly blacks and whites. The results are of particular interest since the Charleston Heart Study sample includes an oversampling of high socioeconomic status black males (n = 69). In this respect, it is unique among long-term cardiovascular studies and provides the opportunity to estimate separate race and socioeconomic status effects, as well as to examine their interaction.

Adaptation, Psychological↗
PubMed1988

Severe Raynaud's phenomenon in a patient with antinuclear antibody-negative systemic lupus erythematosus.

There is a well-recognized subset of patients with clinical findings consistent with systemic lupus erythematosus (SLE) but with negative antinuclear antibodies (ANA). Most of these patients have significant cutaneous involvement with little central nervous system or renal pathology. The following case report describes such a patient whose presentation was suggestive of SLE but who was ANA negative despite repeated testing. Additionally, the patient was found to have severe Raynaud's phenomenon and cutaneous vasculitis. This case is noteworthy because of the prominence of vascular insufficiency as a presenting feature of ANA-negative SLE.

Adult↗