bluuebunny/crossref_metadata_2025_split huggingface dataset:
Agonist-induced internalisation of the angiotensin II-binding sites from plasma membranes of isolated rat hepatocytes
Authors: Montiel M , Caro M C , Jiménez E 
Published: December 2004

Angiotensin II (Ang II) provokes rapid internalisation of its receptor from plasma membranes in isolated rat hepatocytes. After 10 min stimulation with Ang II, plasma membrane lost about 60% of its 125I-Ang II-binding capacity. Internalisation was blocked by phenylarsine oxide (PhAsO), whereas okadaic acid, which markedly reduced the sustained phase of calcium mobilization, did not have a preventive effect on Ang II–receptor complex sequestration. These data suggest that Ang II receptor internalisation is probably independent of a phosphorylation/dephosphorylation cycle of critical serine/threonine residues in the receptor molecule. To establish a relationship between sequestration of the Ang II receptor and the physical properties of the Ang II-binding sites, 125I-Ang II–receptor complex profiles were analysed by isoelectric focusing. In plasma membrane preparations two predominant Ang II-binding sites, migrating to pI 6·8 and 6·5 were found. After exposure to Ang II, cells lost 125I-Ang II-binding capacity to the Ang II–receptor complex migrating at pI 6·8 which was prevented in PhAsO-treated cells. Pretreatment of hepatocytes with okadaic acid did not modify Ang II–receptor complex profiles, indicating that the binding sites corresponding to pI 6·5 and pI 6·8 do not represent a phosphorylated and/or non-phosphorylated form of the Ang II receptor. The results show that the Ang II–receptor complex isoform at pI 6·8 represents a functional form of the type-1 Ang II receptor. Further studies are necessary to identify the Ang II-related nature of the binding sites corresponding to pI 6·5.
Journal of Endocrinology (1997) 152, 407–412
IGF-II, IGF-binding proteins and IGF receptors in pancreatic β-cell lines
Authors: Katz L E L , Bhala A , Camron E , Nunn S E , Hintz R L , Cohen P 
Published: December 2004

The IGFs are mitogenic agents which are closely linked to regulatory processes in carbohydrate metabolism. Because limited information is available on the occurrence of the IGF system in the pancreatic β-cell milieu, we evaluated the presence of IGFs, IGF receptors, and IGF-binding proteins (IGFBPs) in the β-cell lines βTC3 and HIT T-15. Serum-free conditioned media (SFCM) from βTC3 cells contained IGF-II at concentrations greater than 100 ng/ml. High (15 kDa) and low (7·5 kDa) molecular weight IGF-II were detected both by column chromatography followed by RIA and by immunoblotting. GH (10–1000 ng/ml) conditioning of βTC3 cells stimulated IGF-II secretion in a dose-dependent manner. IGF-II mRNA was detected in βTC3 cells using Northern blots, and also showed a GH-dependent relationship. IGF-II peptide was detected in SFCM from HIT cells, albeit at lower concentrations. To evaluate the presence of IGF receptors in β-cell lines, affinity cross-linking studies were performed on βTC3 cells, demonstrating type I IGF receptors which bound iodinated IGF-II with high affinity, iodinated IGF-I with lesser affinity, and had minimal appreciable binding to iodinated insulin. Type II IGF receptors were not detected. SFCM from βTC3 and HIT cells was subjected to Western ligand blotting, which disclosed the presence of two major IGFBPs of 29 kDa and 24 kDa, characteristic of IGFBP-2 and IGFBP-4. The identity of the specific IGFBPs was confirmed by immunoprecipitation and Northern blotting. Varying the glucose concentration had no significant effect on the levels of IGFBPs, nor did preconditioning with GH, IGF-I, IGF-II, insulin, or glucagon. Levels of both IGFBPs in βTC3 cell-conditioned media increased in the presence of dexamethasone at concentrations of 10−6 m or greater. In summary, we present evidence that β-cell lines comprise an environment for GH and IGF action. We speculate that IGFs, their receptors and binding proteins function as a complex interactive system which regulates β-cell growth and function.
Journal of Endocrinology (1997) 152, 455–464
Protein kinase C dependent induction of pp125FAK in monocytes by colony stimulating factor-GM: evidence for a synergistic effect by the cytokine and 1,25 dihydroxyvitamin D3
Authors: Kanan Raed M , Rathod Hersha , Datta Harish K 
Published: December 2004

Abstract
The differentiation of monocytes into osteoclasts has been recently achieved in vitro in a suitable milieu containing morphogens that includes 1,25 dihydroxyvitamin D3, colony stimulating factors, interleukins and the presence of cells of osteoblastic lineage. However, the precise role of these factors in the osteoclastic differentiation process has not yet been examined. Since our previous studies have shown that osteoclasts express a much higher level of focal adhesion kinase (pp125FAK) than cells of macrophage/monocytic lineage, the present study was carried out to ascertain which morphogens are involved in increasing the expression of the kinase during the differentiation of monocytes to osteoclasts. We demonstrate that a marked increase in the expression of pp125FAK occurs only after prolonged exposure to hCSF-GM and combination of hCSF-GM and 1,25 (OH)2 D3. The hCSF-GM was found to be a more potent stimulator of pp125FAK induction than 1,25 (OH)2 D3; interestingly, the presence of both hCSF-GM and 1,25 (OH)2 D3 showed co-operative effect. Furthermore, the presence of a protein kinase C inhibitor, bisindolylmaleimide (GF 109203X), blocked hCSF-GM-mediated induction of focal adhesion kinase, implicating an important role for protein kinase C in the induction of pp125FAK.
IGF-I has a dual effect on insulin release from isolated, perifused adult rat islets of Langerhans
Authors: Hill D J , Sedran R J , Brenner S L , McDonald T J 
Published: December 2004

Abstract
We examined and compared the actions of IGF-I and -II on the release of insulin from isolated, intact rat islets of Langerhans within a perifusion system. Islets were isolated from adult male rats by collagenase digestion and Ficoll gradient separation, and were maintained in tissue culture for 48 h before perifusion. Following an equilibration period, islets were perifused with medium containing 2·7 mm glucose from 0 to 30 min, and 2·7 11·1 or 16·7 mm glucose from 30 to 90 min. All chambers then received medium with 2·7 mm glucose from 90 to 120 min. Various doses (6·7–53 nm) of IGF-I, des(1–3) IGF-I or IGF-II were given either as a pulse between 30 and 35 min, or continuously from 30 to 90 min. Insulin was measured in effluent medium by RIA. When 11·1 mm glucose was administered after 30 min an immediate increase in insulin release occurred, from a baseline of 1–3 pmol/fraction to approximately 7 pmol/fraction. The elevated rate of release was maintained until 90 min, and fell when the glucose concentration was lowered. Glucose at 16·7 mm was a less effective insulin secretogogue than was 11·1 mm. When islets received a pulse infusion of IGF-I (13·3 nm) at 30 min in the presence of 11·1 mm glucose, a statistically significant increase (P<0·005) in insulin release occurred, of approximately 10 pmol/fraction in excess of that seen with glucose alone. The IGF-I-stimulated insulin release was still higher than controls at 115 min. When the concentration of IGF-I was altered between 6·7 nm and 53 nm, maximum insulin release was achieved with 13·3 nm IGF I, both lower and higher concentrations being less effective. A significant inhibition of insulin release occurred with 53 nm IGF-I compared with glucose alone. IGF-II (13·3 nm) did not significantly increase insulin release, while 53 nm IGF-II significantly inhibited release of insulin relative to controls. Des(1–3) IGF-I (13·3 nm), which has a reduced binding affinity for IGF-binding proteins (IGFBPs), administered with 11·1 mm glucose caused an immediate increase in insulin release, which fell to control values within 30 min. Western ligand blot analysis identified four IGFBP species in perifused islets, of 46 kDa, 35 kDa, 28 kDa and 19 kDa respectively, of which the 28 kDa species was identified immunologically as IGFBP-1. When IGF-I was administered continuously from 30 to 90 min it inhibited glucose-stimulated insulin release at all concentrations used. The results suggest that under perifusion conditions, IGF-I can act both as a potent insulin secretogogue, augmenting the actions of glucose, and as an inhibitor of insulin release, depending on concentration and kinetics of administration.
Journal of Endocrinology (1997) 153, 15–25
A micromethod for quantitative determination of iodoamino acids in thyroglobulin
Authors: Baudry N , Mallet B , Lejeune P J , Vinet L , Franc J L 
Published: December 2004

Abstract
We describe a new method for quantification of iodoamino acids after enzymatic hydrolysis of thyroglobulin. The procedure involves separation of monoiodotyrosine (MIT), diiodotyrosine, tri-iodothyronine and thyroxine by reverse phase HPLC with a Vydac C18 stationary phase and a mobile phase of water–acetonitrile–acetic acid. The separation is monitored by sensitive spectrophotometric detection through a 96-well microplate system based on the catalytic Sandell–Kolthoff reaction of iodide on the oxidation of arsenic(III) by cerium(IV).
This new microassay is particularly convenient because of its high sensitivity and its rapidity (less than 2 h). It can detect 1 pmol MIT and 0·5 pmol of the other three iodoamino acids with a recovery higher than 96%. Moreover, the 96-well microplate system allows many samples to be tested simultaneously and avoids the use of radiolabeled iodine.
Journal of Endocrinology (1997) 153, 99–104
Analysis of a protein kinase C-α mutation in human pituitary tumours
Authors: Schiemann U , Assert R , Moskopp D , Gellner R , Hengst K , Gullotta F , Domschke W , Pfeiffer A 
Published: December 2004

Abstract
It is generally accepted that protein kinase C-α (PKC-α) is an important enzyme in the cellular regulation of growth and differentiation by phosphorylating proteins. Recent studies have described a point mutation of PKC-α (position 908 of the genetic sequence, codon GAC becoming GGC) in invasive human pituitary tumours which leads to an exchange of amino acids in the protein. We investigated 11 human pituitary tumours to evaluate the data obtained previously. cDNA was subcloned and up to ten individual clones were sequenced from each tumour, resulting in 85 clones analyzed in total. All of the pituitary adenomas showed a normal wild-type sequence of PKC-α DNA. Even if the tumour was 'invasive' (infiltration of the dura mater) no mutation at position 908 of the sequence was found. Moreover, using Western blot analyses we did not observe any differences in PKC-α protein expression in invasive as compared with non-invasive pituitary adenomas. Until now we have been unable to confirm the data of other investigators, suggesting that mutated PKC-α is an inconsistent feature of invasive pituitary tumours.
Journal of Endocrinology (1997) 153, 131–137
Differential regulation of hypothalamic pituitary corticotropin releasing hormone receptors during development of adjuvant-induced arthritis in the rat
Authors: Aguilera G , Jessop D S , Harbuz M S , Kiss A , Lightman S L 
Published: December 2004

Abstract
The expression of corticotropin releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN) and CRH receptor mRNA in the PVN and anterior pituitary was studied during development of adjuvant-induced arthritis in Piebald–Viral–Glaxo rats, using in situ hybridization techniques. As previously shown with i.p. hypertonic saline injection, basal and immobilization stress-stimulated CRH mRNA levels in the PVN were significantly lower than in controls 14 days after adjuvant injection. However, 7 days after injection, preceding the onset of inflammation, the increase of CRH mRNA following immobilization was significantly higher than in control rats. In contrast to other chronic stress paradigms, inflammation stress failed to induce type-1 CRH receptor (CRH-R1) mRNA in the PVN, either at 7 days, or at 14 days after adjuvant injection, when inflammation is present. The ability of acute immobilization to induce CRH-R1 mRNA in the PVN was not affected 14 days after adjuvant injection but parallel to the CRH peptide mRNA response it was markedly potentiated at 7 days. Pro-opiomelanocorpin (POMC) mRNA levels in the anterior pituitary increased significantly 14 days after adjuvant injection, and they were unaffected by 1 h immobilization. While CRH binding in the pituitary decreased significantly 14 days after adjuvant injection, CRH-Rl mRNA was unchanged. This study shows biphasic hypothalamic responses to acute stress during development of adjuvant-induced arthritis, with enhanced CRH peptide and CRH-Rl mRNAs responses at 7 days, preceding the onset of inflammation, and blunted CRH mRNA responses at 14 days at the height of the inflammatory response. The lack of CRH receptor expression in the PVN in this model of chronic inflammation stress associated to low hypothalamic CRH peptide levels supports the view that positive feedback regulation by CRH is necessary to maintain enhanced CRH expression during chronic stress.
Journal of Endocrinology (1997) 153, 185–191
Magnetic resonance imaging as a monitor of kidney enlargement and regression in experimental diabetes in rats
Authors: Christiansen T , Stødkilde-Jørgensen H , Flyvbjerg A 
Published: December 2004

Abstract
The aim of the present study was to use magnetic resonance imaging as a non-invasive measure of renal enlargement and regression in experimental diabetes in rats. Rats were randomized into three groups: (1) non-diabetic control animals, (2) diabetic animals, untreated for 30 days after streptozotozin treatment and (3) diabetic animals treated with insulin from day 21 to day 30 after streptozotocin administration. Magnetic resonance images were obtained from anaesthetized rats and kidney, cortex, medulla and pelvis volumes were calculated throughout the experiment until day 30. In untreated diabetic animals, a marked increase in kidney volume was seen 4 days after the induction of diabetes (P<0·05) and after 30 days this increase amounted to 73%, with similar increases in the cortex (63%) and medulla (69%). No change was seen in the pelvis volume. Insulin administration on day 21 to untreated diabetic rats abruptly reduced kidney volume by 21%, cortex volume by 27% and medulla volume by 18%, and after 4 days all three parameters were similar to those seen in non-diabetic controls. In conclusion, the present study has demonstrated the applicability of magnetic resonance imaging as a non-invasive measure of renal enlargement and regression in experimental diabetes in rats. In untreated hyperglycaemic diabetic rats, with manifest diabetic renal enlargement, acute regression in renal volume was observed concomitant with insulin-induced normoglycaemia.
Journal of Endocrinology (1997) 153, 193–198
Rapidly reversible binding of rabbit prolactin to the rabbit prolactin receptor accounts for the differences between homologous and heterologous binding
Authors: Petridou B , Cahoreau C , Djiane J 
Published: December 2004

Abstract
The binding of radioiodinated rabbit (rb) prolactin (PRL) to rabbit mammary membranes is low and its affinity constant, 0·02 nm−1, calculated from heterologous inhibition assays, is about 300 times lower than that of ovine (o) PRL. Although the differences between homologous and heterologous binding are well documented in different species, the reasons for such differences are still unknown.
Here we show that the low affinity of rbPRL for the native receptor does not affect its in vitro bioactivity compared with that of oPRL. We also show that rbPRL displays high specific binding to the baculovirus-expressed recombinant receptor and further establish that its lower affinity for binding to the homologous receptor is due to its faster and more complete dissociation compared with that of oPRL. Hormone binding affinity for full-length and carboxy-terminal truncated rbPRL receptor mutants expressed in mammalian or in baculovirus-infected cells was not affected by partial truncation of the cytoplasmic domain of the receptor, whereas the affinity for oPRL increased and that for rbPRL decreased upon truncation of both the cytoplasmic and membrane domains. The affinity of rbPRL for the native receptor is two orders of magnitude lower than that for the recombinant receptor. Affinity cross-linking and binding experiments showed that this difference in affinities is not related to selective cleavage of the native microsomal receptor during the binding reaction; however, this difference may be related to cell context-dependent differences in the oligomerization state of the receptor.
Thus, obviously, the cloned receptor is alone sufficient for binding to rbPRL without requiring any receptor-associated protein. The lower affinity for rbPRL binding to its homologous receptor in comparison with higher affinity binding of oPRL to the same receptor is attributable to differences in their dissociation kinetics and in the conformational requirements of the receptor–hormone interaction site for binding to the two hormones.
Journal of Endocrinology (1997) 153, 207–219
Development, validation and application of a two-site enzyme-linked immunosorbent assay for activin-AB
Authors: Evans L W , Muttukrishna S , Knight P G , Groome N P 
Published: December 2004

Abstract
Monoclonal antibodies, specific for the βa and βb subunits of activin, were used to develop a new two-site ELISA for activin-AB. The assay had a detection limit of 0·19 ng/ml. High concentrations of activin-AB were found in bovine, ovine and porcine follicular fluids (FF), with less in human FF (1310, 1730, 688 and 7 ng/ml respectively). Recovery of spiked activin-AB standard from human, bovine and ovine FFs and from homogenized human placental extracts averaged 91%, 115%, 115% and 94% respectively. Within-plate coefficients of variation for different concentrations of activin-AB were between 1·3% and 2·67%. The between-plate coefficient of variation was 5·5%. Crossreactivity experiments showed the high specificity of the assay for activin-AB, with inhibin-A, inhibin-B, follistatin, activin-A and activin-B all cross-reacting <0·2%. Incubation with high concentrations of follistatin (500 ng/ml) prior to assay did not affect the recovery of activin-AB. Samples of bovine, porcine, ovine and human FF gave dose responses parallel to that of the standard, as did bovine granulosa cell-conditioned media. In human and porcine FF, levels of activin-A and activin-AB were similar whereas, in bovine and ovine FF, activin-A levels were approximately threefold higher than activin-AB levels. As we have reported previously for activin-A, nearly all of the endogenous activin-AB in bovine FF was detected in the eluate from gel permeation chromatography with an Mr of >700 000 indicating its association with higher molecular weight binding protein(s). By contrast, after denaturation, immunoreactive activin-AB was detected with an Mr of ∼25 000 consistent with the complete dissociation from binding proteins.
Activin-A was detected in relatively high concentrations in human FF (∼5 ng/ml), homogenized placental extracts (4·35–95·5 ng/g), sera from pregnant women (>4 ng/ml) and amniotic fluid (3–13 ng/ml), and in much lower concentrations in postmenopausal serum (500 pg/ml), normal cycle serum (100–200 pg/ml), serum from gonadotrophin-treated women (200 pg/ml) and normal adult male serum (225 pg/ml). Activin-A was also found in the culture media from explants of human amnion, chorion, maternal decidua and placenta. In marked contrast, activin-AB was undetectable (<0·19 ng/ml) in all of these samples with the exception of human FF (∼7 ng/ml).
In conclusion, we have developed a sensitive and specific ELISA to measure total (bound+free) activin-AB. Preliminary results show a more restricted distribution of this isoform compared with activin-A. The presence of high levels of both activin-A and activin-AB in FF suggests a function for both isoforms in the developing ovarian follicle.
Journal of Endocrinology (1997) 153, 221–230
Insulin-like growth factor-I (IGF-I) production by bovine granulosa cells in vitro and peripheral IGF-I measurement in cattle serum: an evaluation of IGF-binding protein extraction protocols
Authors: Gutiérrez C G , Campbell B K , Armstrong D G , Webb R 
Published: December 2004

Abstract
Insulin-like growth factor-binding protein (IGFBP) extraction protocols were tested for their efficacy in removing IGFBPs from bovine plasma and bovine granulosa cell culture medium compared with standard acid exclusion chromatography. Traditional extraction methods, acidification, Sep–Pak, ethanol:acetone:acetic acid (EAA) and EAA-cryoprecipitation (EAA-C), failed to remove all the IGFBPs from both granulosa cell culture medium and plasma. However, EAA and EAA-C treatment of plasma samples did give values similar to those obtained by acid exclusion HPLC, when corrected for extraction efficiency. There was an inverse relationship between insulin-like growth factor-I (IGF-I) concentration in plasma samples, as measured using HPLC chromatography, and IGF-I concentration after EAA extraction. Furthermore, the interference caused by residual IGFBPs differed between samples taken from animals given various treatments that altered peripheral IGF-I concentrations.
As for plasma samples, EAA was the most effective extraction method for culture media, but residual IGFBPs caused an overestimation of IGF-I concentrations. In culture media, but not plasma, it was possible to block the interference of IGFBPs in the IGF-I assay, in both extracted and non-extracted culture samples, by the addition of excess IGF-II. Using this assay procedure, no IGF-I production by bovine granulosa cells was detected. This was confirmed by HPLC acid chromatography.
It is concluded that HPLC extraction is needed for the accurate measurement of peripheral IGF-I concentrations. For granulosa cell culture media it is possible to measure IGF-I concentrations in non-extracted samples if the IGFBPs are blocked by adding IGF-II. Using either this assay, or after HPLC acid chromatography, no IGF-I was detected in culture media, suggesting that IGF-I is not produced by non-luteinised bovine granulosa cells.
Journal of Endocrinology (1997) 153, 231–240