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E A Linton

Publications and source records attributed to E A Linton.

At least 37 records · Page 2Linked to original sources
PubMed1998

Levels of corticotrophin-releasing hormone receptor subtype 1 mRNA in pregnancy and during labour in human myometrium measured by quantitative competitive PCR.

It is suggested that corticotrophin-releasing hormone (CRH) is involved in parturition. We have previously reported the presence of the CRH receptor subtype 1 (CRH R1) in human uterine myocytes. The aim of the present study was to investigate whether expression of the CRH R1 in myometrial tissue changes in pregnancy and labour. We used a quantitative competitive PCR method to measure the mRNA levels of this receptor in non-pregnant and in term pregnant myometrium before and at different stages of labour. The levels of mRNA for the housekeeping gene for glucocerebrosidase (GCB) were also determined. The results were expressed as a ratio of CRH R1 and GCB mRNA levels. We have found that in pregnancy the CRH R1 is down-regulated from a ratio of 0.093+/-0.011 in non-pregnant myometrium to 0.012+/-0.005 (P<0.001) in term non-labouring myometrium. No significant changes were observed in the CRH R1:GCB ratio in tissues sampled within 13 h (0.013+/-0.004) from the start of labour. In summary, normalised levels of CRH R1 are down-regulated in pregnancy and do not change during labour. We speculate that our results do not support a direct role for the CRH R1 receptor in myometrial stimulation.

Adult↗
PubMed1998

Expression of corticotrophin-releasing hormone receptor mRNA and protein in the human myometrium.

The reported effects of corticotrophin-releasing hormone (CRH) on human myometrium support the existence of specific receptors for the hormone in this tissue. We have used the reverse transcriptase-polymerase chain reaction (RT-PCR) technique to study the expression of mRNA coding for the CRH R1 and R2 receptors. RT-PCR of total RNA from both nonpregnant and pregnant myometrium using specific primers resulted in amplification products of the expected sizes for the R1 alpha and R2 alpha CRH receptors. The identity of these amplification products was confirmed by specific restriction digests and sequencing. Immunohistochemistry using a rabbit antibody raised against a specific domain of the CRH R1 receptor demonstrated that the R1 mRNA is translated into protein and confirmed that it is the uterine smooth muscle cells from both nonpregnant and pregnant women that bear this receptor. Our results suggest that CRH may play a role in human pregnancy at the myometrium.

Adult↗
PubMed1996

Corticotrophin-releasing hormone receptor type 1: generation and characterization of polyclonal antipeptide antibodies and their localization in pituitary cells and cortical neurones in vitro.

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stress. CRH acts by first binding to specific receptors on the plasma membrane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH-R1). In this paper, we have chosen three different peptide sequences within the CRH-R1 molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on the surface of the native protein, for antibody production. Some of these fragments produced antipeptide antibodies of good titre which cross-reacted with the CRH-R1 receptor expressed in transiently transfected COS-7 cells and in tissue extracts from rat cerebellum, cortex, pituitary gland and human myometrium, both in Western blots and in liquid-phase radioimmunoassay. We used immunofluorescence techniques to localize the CRH receptor in transiently transfected COS-7 cells, primary cultures of rat anterior pituitary (AP) cells, the corticotroph-tumour cells AtT20 D16-16 and cortical neurons in primary culture. Our results indicate IR-CRH-R1 receptors have a punctate distribution on the plasma membrane of AP cells and AtT20 D16-16 cells. Whilst in AP cells their appearance is a fine punctate pattern, in AtT20 cells, they appear as large patches which could account for receptor clusters. Within primary cortical neurons, their distribution does not appear to be polarized. Our results suggest that distribution of CRH-R1 receptors within the different cell-types investigated depends not only on the amino acid sequence but also on cellular factors.

Animals↗
PubMed1995

Co-localisation of autoimmune antibodies specific for double stranded DNA with procorticotrophin-releasing hormone within the nucleus of stably transfected CHO-K1 cells.

Human autoantibodies and corticotrophin-releasing hormone (CRH)-specific antibodies have been used in a double-labelling immunofluorescence technique to demonstrate that immunoreactive CRH structures are co-localised with immunostaining produced by double stranded DNA-specific human autoantibodies within the nucleus of cultured ovarian cells of Chinese hamsters (CHO-K1). This co-localisation was confirmed using confocal microscopy. A metabolic labelling technique was used to investigate the role of the cytoskeleton in mediating nuclear translocation of proCRH within stably transfected CHO-K1 cells and showed that microtubule and actin disrupting agents had no effect upon the nuclear translocation of proCRH. These results, therefore, suggest that nuclear translocation of proCRH is not affected by drugs which disrupt the cytoskeleton and, consequently, modify the diameter of the nuclear pores.

Actins↗
PubMed1995

Identification and isolation of corticotrophin-releasing hormone-positive cells from the human placenta.

During human pregnancy, plasma corticotrophin-releasing hormone (CRH) levels rise from undetectable amounts prior to 20 weeks gestation to reach a peak near term, with an exponential rise during the final 5 weeks of gestation. Within hours of parturition plasma levels fall and rapidly return to undetectable baseline measurements. The appearance of CRH in maternal plasma has been attributed to the placental production and subsequent release into the maternal circulation of this hormone. Previous studies have shown that human placental extracts contain a CRH-like peptide and this has been reinforced by the observation of CRH mRNA in placental tissue. Initial attempts to identify the site of production using immunocytochemistry have led to conflicting results. This study attempts to clarify this situation by using a variety of highly specific anti-CRH antibodies to show the cellular expression of placental CRH. Intense CRH staining was observed in the syncytial trophoblast layer in first trimester and term chorionic villi, whilst the underlying cytotrophoblast appeared to be negative. The fetal membranes also contained CRH immunoreactivity with the cytotrophoblast cells in the chorionic membrane displaying the most intense staining. CRH immunoactivity was also observed in the amnion and in some cells in the decidua. As a model of cellular CRH expression, cytotrophoblast cells from term chorionic membrane were isolated and shown to be positive for CRH.

Chorionic Villi↗
PubMed1995

Generation and characterization of an antiserum reactive with a proteolytic processing site within rat procorticotrophin-releasing hormone.

In this paper we report the generation of an antibody specific for the cleavage site within procorticotrophin-releasing hormone (proCRH) at the N-terminus proCRH/CRH (1-41) junction. Using radioimmunoassay techniques were show that the antibody generated (781) cross-reacts specifically with the proCRH (137-150) Tyr fragment, corresponding to the cleavage site within the full length precursor molecule. The anti-cleavage site antibody does not crossreact with the endoproteolytic products originated from the CRH precursor molecule, i.e. CRH (1-41) or proCRH (125-151) or with any of the CRH-immunoreactive fragments tested i.e. CRH (36-41), CRH (1-20) and CRH (30-41). It also shows no cross-reactivity with CRH-related substances from other species, i.e. urotensin I (fish) and sauvagine (frog). The cleavage site antibody (781), recognizes the full length proCRH molecule in Western blotting and in liquid phase radioimmunoassay from transfected CHO-K1 cells expressing the full length pre-proCRH cDNA. Using immunofluorescence and immunoprecipitation techniques followed by SDS-PAGE and autoradiography, we confirm the presence of the intact CRH precursor molecule within the nucleus and the cytoplasm of stably transfected CHO-K1 cells expressing immunoreactive proCRH. The immunofluorescence studies using primary cultures of hypothalamic neurons, show that immunoreactive (IR) proCRH is localized within the perinuclear region and was also seen along the neuronal processes where it accumulates at their tips. Our results, therefore, show that this antibody will be an invaluable tool in the study of intracellular trafficking in relation to the endoproteolytic processing of the CRH precursor molecule.

Amino Acid Sequence↗
PubMed1995

Expression of biologically active procorticotrophin-releasing hormone (proCRH) in stably transfected CHO-K1 cells: characterization of nuclear proCRH.

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre-proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within the trans-Golgi network, where they are finally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellular compartment. We have recently established stably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR-CRH displayed an apparent molecular weight approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule. In this paper, we further characterized the bitopological, i.e. nuclear and cytoplasmic localization of proCRH within transfected CHO-K1 cells. Immunoreactive nuclear CRH was not extractable using detergents (Triton X-100 and CHAPS), 10 mM salt washes or RNase digestion but could be abolished by digestion with DNase I. These results therefore suggest that nuclear proCRH is in close association with DNA/chromatin. Treatment of transfected cells with inhibitors of protein and RNA synthesis for up to 24 h had no effect upon immunoreactive nuclear CRH, indicating that it is very stable with a long half life. Brefeldin A treatment had no effect upon the nuclear translocation of newly synthesized proCRH, suggesting that late stages of the secretory pathway (i.e. post rough endoplasmic reticulum compartments) of the transfected cells do not play a role in proCRH nuclear transport. We also demonstrate that proCRH synthesized within stably transfected CHO-K1 cells is capable of stimulating ACTH release from primary cultures of anterior pituitary cells, therefore showing for the first time that the intact precursor is also biologically active and could act as an ACTH secretagogue in-vivo.

Adrenocorticotropic Hormone↗
PubMed1995

Corticotrophin-releasing hormone and corticotrophin-releasing hormone binding protein in normal and pre-eclamptic human pregnancies.

OBJECTIVE: To measure the plasma levels of corticotrophin-releasing hormone and corticotrophin-releasing hormone binding protein in normal pregnancy and in pregnancies complicated by pre-eclampsia. SETTING: John Radcliffe Hospital, Oxford and St Thomas's Hospital, London. SUBJECTS: One hundred and twenty pregnant women sampled prospectively throughout gestation, of whom 91 experienced a normal pregnancy and eight developed pre-eclampsia; in a second study, 10 women with severe pre-eclampsia, presenting at a range of gestational ages, were sampled once and compared with appropriately matched normal pregnant women. MAIN OUTCOME MEASURE: Plasma levels of corticotrophin-releasing hormone determined by immunoradiometric assay. Plasma levels of corticotrophin-releasing hormone binding protein measured by direct radioimmunoassay. RESULTS: In the prospective study, plasma samples from women with pre-eclampsia exhibited higher (390.2 versus 292.7 pmol/l at 36 weeks) levels of corticotrophin-releasing hormone and significantly lower (5.24 versus 8.14 nmol/l at 36 weeks, P < 0.002) levels of corticotrophin-releasing hormone binding protein than normal controls. In the second, single time point study a significant elevation in CRH (P < 0.002) and reduction in CRH-BP (P < 0.001) was found in pre-eclamptic pregnancies compared with controls. CONCLUSIONS: In human pregnancies complicated by pre-eclampsia there is an elevated level of corticotrophin releasing hormone whilst there is less corticotrophin-releasing hormone binding protein; therefore there is a net increase in free potentially bioactive hormone which may play a role in the pathology of the disease.

Adolescent↗
PubMed1995

Corticotrophin-releasing hormone (CRH)-binding protein interference with CRH antibody binding: implications for direct CRH immunoassay.

Direct immunoassay of plasma corticotrophin-releasing hormone (CRH) is potentially subject to interference from high levels of CRH-binding protein (CRH-BP) that exist in the human circulation. In this study, we tested the effect of CRH-free, native CRH-BP (6.4 nmol/l) purified from human plasma, CRH-BP diluent alone, normal human plasma (containing 5.8 nmol endogenous CRH-BP/l) and normal sheep plasma (containing no CRH-BP) on the binding of 125I-labelled CRH tracer to five N-terminal and four C-terminal CRH antibodies. All anti-(1-20)CRH N-terminal antibody dilution curves displayed marked inhibition of binding in the presence of purified CRH-BP and human plasma in comparison with the curves with the control diluent or sheep plasma. Almost no inhibition of binding was obtained with any of the C-terminal antibodies (all directed against epitopes within the last six amino acids of CRH) and the four dilution curves were nearly superimposable. Liquid-phase CRH IRMAs were then developed with different combinations of two of each of the N- and C-terminal antibodies, using radiolabelled IgG prepared from purified C-terminal antisera as tracer and raw N-terminal antisera as the link antibodies to the separating system. The addition of dilutions of purified CRH-BP over the range 1.25-20 nmol/l to the IRMA standard curve in assay buffer resulted in a dose-dependent reduction in the signal; with 5 nmol CRH-BP/l, a level commonly found in human plasma, the reduction in binding was 67% and 81% in two different IRMAs at a CRH concentration of 631 pmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Corticotrophin-releasing hormone-binding protein in human fetal plasma.

During pregnancy maternal plasma corticotrophin-releasing hormone (CRH) levels rise 1000-fold whilst fetal plasma levels are often 100-fold higher than the concentrations seen in normal non-pregnant human plasma. Despite these high CRH levels neither the maternal nor fetal pituitary releases excessive amounts of ACTH. A specific CRH-binding protein (CRHBP) exists in the maternal circulation which is able to bind and inactivate the ACTH releasing activity of CRH. In this study we have used a specific CRHBP radioimmunoassay to determine the level of CRHBP in fetal and maternal plasma samples. Fetal samples were collected by cordocentesis between 20 and 33 weeks gestation and matched maternal samples were taken by venepuncture at the same time. In a second study, plasma samples were collected from 8 women at fortnightly intervals from week 20 to term, at labour and post-partum. A fetal sample, taken from the umbilical vein, was collected immediately post-delivery. The mean maternal CRHBP concentration for the samples collected between 20 and 33 weeks (n = 23) was 8.12 nmol/l and the fetal level was 8.62 nmol/l. Data from the second study showed that at term the maternal CRHBP concentration decreased significantly (P < 0.025) to 6.32 nmol/l. The fetal CRHBP level also decreased significantly (P < 0.001) at term to a level of 5.84 nmol/l. The CRHBP in both fetal and maternal plasma was shown to be functional by 125I-CRH binding and gel permeation chromatography. The capacity of maternal and fetal plasma to bind 125I-CRH decreased at term in agreement with the quantitation of plasma CRHBP by radioimmunoassay.

Carrier Proteins↗
PubMed1993

Prediction of protein antigenic sites in human corticotrophin-releasing hormone precursor.

1. The primary structure of human corticotrophin-releasing hormone precursor (h pre-proCRH) has been analysed using a number of computer algorithms to identify the areas of highest predicted antigenicity. 2. These results were correlated with crossreactivity data obtained from studies of antibodies produced in rabbits by immunizing with h pre-proCRH, and a number of related peptides. 3. Six areas of high predicted antigenicity were identified in h pre-proCRH by the prediction routines utilized. Two of these corresponded almost exactly to the two putative cleavage sites of the prohormone, and a third lay within the C-terminal region of one of the products of post-translational processing of the prohormone, i.e. CRH(1-41). 4. Experimental crossreactivity data also indicated that a number of structural factors (e.g. Omega loops, peptide conformation) may also be involved in recognition of peptide fragments by antibodies.

Algorithms↗
PubMed1993

Alterations in hypothalamic NPY and CRF in anorexic tumor-bearing rats.

Regional hypothalamic concentrations of neuropeptide Y (NPY) and corticotropin-releasing factor (CRF), respectively a stimulant and an inhibitor of feeding behavior, were investigated in hypothalamic nuclei in rats carrying the Yoshida sarcoma. Tumor-bearing rats (n = 10), non-tumor-bearing controls (n = 10), and food-restricted rats (n = 10), which did not carry tumors but were pair-fed to match the reduced food intake of the tumor-bearing group, were studied after 10 days. NPY concentrations in the arcuate nucleus (ARC, the main site of NPY synthesis) were significantly increased above controls (P < 0.01) in both tumor-bearing and food-restricted groups. However, NPY concentrations in the paraventricular nucleus (PVN, an NPY-sensitive site of NPY release) showed opposing changes, with a 25% decrease (P = 0.052) in the tumor-bearing but a 48% increase (P < 0.01) in the food-restricted group. CRF concentrations in both the PVN and the ARC were significantly reduced (P < 0.01) in the food-restricted group, but remained close to control values in the tumor-bearing group (P not significant). Changes in hypothalamic appetite-regulating neuropeptides in cancer anorexia, which may result from the action of cytokines produced by a host defense response or the tumor itself, may account for reduced feeding. Such changes may include impaired activity of NPY or failure of CRF activity to be suppressed after underfeeding and weight loss.

Adipose Tissue↗
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

Plasma measurements of corticotrophin-releasing hormone-binding protein in normal and abnormal human pregnancy.

Using corticotrophin-releasing hormone-binding protein (CRHBP) purified from human plasma and a 25 amino acid peptide corresponding to the C-terminus of CRHBP we have been able to produce rabbit polyclonal antisera specific for CRHBP. This has allowed the development of a radioimmunoassay which is able to detect CRHBP specifically in human plasma regardless of the presence of endogenous CRH. We have used this assay to estimate the level of CRHBP in non-pregnant human plasma to be approximately 20 nmol/l with a range of 9.1-40.6 nmol/l. We have also examined sequential plasma samples taken from 84 normal pregnant women at fortnightly intervals from 16 weeks gestation through to term. Four women were also sampled during labour and the first week postpartum. The median plasma level of CRHBP at week 16 of normal pregnancy was 21.59 nmol/l, levels rose slightly during the early part of the third trimester (26.76 nmol/l at week 30, (P < 0.01) and fell markedly towards term (19.72 nmol/l, P < 0.01) with only 8.70 nmol/l at labour. CRHBP levels returned to normal non-pregnant levels within 48 h of parturition suggesting a role for the fetoplacental unit in CRHBP production. In eight pregnancies complicated by diabetes, CRHBP levels at each gestational age were similar to those recorded for normal pregnancy. However, in pregnancies complicated by pre-term labour (n = 9) and pre-eclampsia (n = 7), plasma CRHBP levels were significantly reduced (P < 0.01).

Carrier Proteins↗