bluuebunny/crossref_metadata_2025_split huggingface dataset:
The nature and significance of multiple isoforms of the oestrogen receptor in breast tumours
Authors: Puddefoot J R , Baker V A , Bakkers B , Marsigliante S , Barker S , Panahy C , Goode A W , Carpenter R , Vinson G P 
Published: December 2008

ABSTRACT
Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6·1, 6·3, 6·6 and 6·8. Of these the first (pI 6·1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6·3 and pI 6·8 were significantly correlated with the presence of progesterone receptors.
To characterize the isoforms more fully, ER isoforms labelled by [3H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-3H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6·3, 6·8 and 6·6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6·3, 6·8 and 6·6 isoforms, could form dimers.
We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome.
Localization of ovine follistatin and α and βA inhibin mRNA in the sheep ovary during the oestrous cycle
Authors: Tisdall D J , Hudson N , Smith P , McNatty K P 
Published: December 2008

ABSTRACT
The sites of follistatin and α and βA inhibin gene expression were examined by in situ hybridization in sheep ovaries during the early and mid-luteal phases (days 3 and 10) of the oestrous cycle and a prostaglandin F2α (PGF2α)-induced follicular phase.
Follistatin mRNA was detected in the granulosa cells of preantral, antral and early atretic follicles at all stages of the oestrous cycle, and in the corpora lutea at the early and mid-luteal stages of the cycle. However, only low levels of expression of follistatin were observed in the presumptive preovulatory follicle at 56 h after treatment with PGF2α.
Both α and βA inhibin were shown to be expressed in ovaries at all stages of the oestrous cycle. In situ hybridization localized α subunit mRNA to the granulosa cells of most, but not all, healthy antral follicles, and to no other ovarian cell type. In contrast, expression of the βA subunit was confined to a few medium-to-large healthy antral follicles. In antral follicles expressing βA inhibin, mRNAs for α inhibin and follistatin were always detected, but the converse was not true. Unlike follistatin, no α and βA inhibin expression was seen in preantral follicles, developing corpora lutea, or follicles undergoing atresia.
These results show that, in the adult sheep ovary, follistatin gene expression is a constitutive event in all growing follicles from the early preantral stage, and also provide indirect evidence of the involvement of follistatin, but not inhibin or activin, in the early stages of ovarian follicle development in sheep.
Translocation of protein kinase C isozymes in lactotroph-enriched rat anterior pituitary cell cultures: differential effects of substance P and phorbol ester
Authors: Mau S E , Særmark T , Vilhardt H 
Published: December 2008

ABSTRACT
Translocation of protein kinase C (PKC) from the cytosol to the plasma membranes is believed to reflect activation of the enzyme. We have studied translocation of PKC in lactotroph-enriched anterior pituitary cell cultures by measuring the incorporation of γ-32P from [γ-32P]ATP into a synthetic peptide substrate, MBP4–14, and by immunoblotting of PKC isozymes. Using cells permeabilized with digitonin the effects of PKC cofactors on the distribution of the enzyme were studied. Ca+ (50 nm) and dioctanoyl-sn-glycerol had no effect when tested alone, but in combination they caused a redistribution of PKC from the soluble to the particulate fraction. Arachidonic acid needed Ca+ to induce translocation of PKC, while being ineffective under Ca2+-free conditions. Western blot analysis of partly purified PKC from lactotroph-enriched pituitary cells revealed the presence of the α, β, δ, and ζ isozymes. 12-O-Tetradecanoylphorbol 13-acetate (TPA) and substance P displayed different patterns of redistribution of PKC isozyme immunoreactivity from soluble to membrane-attached forms. Thus, TPA induced time- and dose-dependent (mean effective concentration (EC50)=1 nm) translocation of the α, β and δ species, while substance P stimulated time- and dose-dependent (EC50=1 nm) redistribution of the α and β isozymes. ζ subtype immunoreactivity could not be translocated by either agonist; neither could the immunoreactivity of ζ be down-regulated by long-term treatment (24 h) with TPA.
The results indicate that simultaneous activation of phospholipases C and A2 induces a synergistic activation of PKC. Finally it is suggested that substance P may exert some of its effects in lactotrophs by translocation of PKC isozymes α and β.
Effect of in vivo oestradiol treatment on cell-free transcription in trout liver nuclear extracts
Authors: Delaunay F , Pakdel F , Valotaire Y 
Published: December 2008

ABSTRACT
In order to perform later studies on the transcriptional regulation of hormone-dependent genes in fish liver, we firstly examined the potential of trout liver nuclear extracts in a cell-free transcription system. As reporter genes, we used DNA sequences without G (G-free cassettes) under the control of three promoters derived from the 5′ flanking sequence of the Xenopus vitellogenin B1 gene; two of them were responsive to the oestrogen receptor (ER) through oestrogen responsive elements (ERE). Maximal transcriptional activity was obtained within a range of 40–130 μg protein per extract depending on the extract preparation. Transcription was maximal in reactions carried out at 25 °C.
Similar transcriptional activities for the three promoters were observed when transcription was performed in extracts from untreated male trout. In contrast, we observed a 4·5- to 6-fold increase in the transcription with ERE-containing promoters in comparison with that with the minimal promoter bearing only a TATA box when extracts from oestradiol-treated male trout were used. This effect was correlated with the increase in the nuclear ER concentration induced by in vivo hormonal treatment. This enhanced transcription was specifically inhibited by the addition of a 25- to 100-fold excess of ERE oligonucleotide competitor.
These data demonstrated, therefore, that transcription was ERE-dependent in this system and suggest strongly that it was mediated by the trout ER. Addition of oestradiol or the anti-oestrogens hydroxytamoxifen or ICI 164384 had no effect on the transcriptional activity of the two ERE-containing promoters, indicating that transcription was hormone-independent in trout liver nuclear extracts.
Dual activity of human pituitary thyrotrophin isoforms on thyroid cell growth
Authors: Medri G , Sergi I , Papandréou M-J , Beck-Peccoz P , Verrier B , Ronin C 
Published: December 2008

ABSTRACT
Alkaline (pI 8·6–7·5) and neutral (pI 7·0–6·0) isoforms of human TSH have been isolated from a highly purified intrapituitary preparation by isoelectric focusing and compared for their respective actions on thyroid cell proliferation. Both TSH isoforms displayed the same ability to bind to porcine thyroid membranes as the original hormone preparation, indicating a similar recognition at the receptor sites. Alkaline forms showed a higher potency in inducing either cyclic AMP (cAMP) production or [3H]thymidine incorporation in FRTL-5 cells (half-maximal effective doses (ED50 values)=0·25 and 0·29 nm respectively) compared with their neutral counterparts (ED50 values=0·66 and 0·70 nm respectively). Increasing the concentration of alkaline forms in the presence of a half-maximal concentration of neutral TSH resulted in a profound inhibition of cell growth without a significant change in cAMP. Conversely, increasing the amount of neutral forms in the presence of a half-maximal dose of alkaline TSH resulted in an additive response for cAMP production but not in cell proliferation.
To assess whether glycosylation might be responsible for the variation in hormone action, both alkaline and neutral TSH isoforms were tested for recognition of their carbohydrate chains by concanavalin A (Con A) and ricin. No major difference was found in binding to Con A, indicating that the contribution of carbohydrates to changes in hormone pI was not related to core branching. Very few galactose residues were accessible in either hormone fraction since little binding to ricin was observed. Isoelectric focusing of TSH forms before and after neuraminidase treatment revealed that neutral forms had a higher sialic acid content than alkaline TSH.
In conclusion, the current findings show that TSH isoforms differentially affect cAMP production and cell growth. TSH fractions with a high sialic acid content and a low mitogenic activity behave as antagonists to the more active forms for cell proliferation. It is suggested that physiological control of TSH action at the thyroid gland may reside in the respective amounts of various TSH forms which, once bound to their receptor, can induce variable activation of post-receptor events while controlling cell proliferation.
Isolation and characterization of ovine IGFBP-4: protein purification and cDNA sequence
Authors: Carr J M , Grant P A , Francis G L , Owens J A , Wallace J C , Walton P E 
Published: December 2008

ABSTRACT
Three different molecular mass forms of IGF-binding proteins (IGFBPs) were purified from ovine plasma by IGF-I affinity chromatography and reverse-phase HPLC: a 46 kDa doublet and 29 kDa and 24 kDa forms. Amino-terminal sequence analysis confirmed that these proteins were ovine (o)IGFBP-3 (46 kDa) and two molecular size variants of oIGFBP-4. oIGFBP-3 and the 29 kDa form of oIGFBP-4 were shown to be N-glycosylated. Isoelectric points were determined to be at approximately pH 6 for oIGFBP-3 and at pH 7 and pH 7·5 for the 29 and 24 kDa forms of oIGFBP-4 respectively. The two different molecular mass variants of oIGFBP-4 had similar IGF-binding properties. Compared with human IGFBP-3 and oIGFBP-3, the two variants of oIGFBP-4 exhibited lower relative binding to amino-terminally modified IGF-I analogues in a competitive IGF-binding assay. The full protein sequence of oIGFBP-4, as deduced from the cDNA sequence, showed a high degree of identity with rat (90%), human (96%) and bovine (98%) IGFBP-4. The cDNA sequence also showed homology over regions of the 3′ non-coding sequence, particularly in comparison with bovine IGFBP-4 (96%). Northern analysis of mRNA for oIGFBP-4 indicated a 26 kb major transcript and two minor transcripts of approximately 21 and 1·8 kb. oIGFBP-4 mRNA transcripts were detected in adult ewe liver>kidney>lung>>heart and also in several fetal tissues, thus suggesting tissue-specific and developmental regulation. The availability of purified oIGFBP-4 and oIGFBP-3 as well as DNA probes for oIGFBP-4 will enable further study of the properties and functions of these proteins, as well as the establishment of specific assays for these IGFBPs.
Production and characterization of recombinant chicken insulin-like growth factor-II from Escherichia coli
Authors: Upton Z , Francis G L , Kita K , Wallace J C , Ballard F J 
Published: December 2008

ABSTRACT
Recombinant chicken (c)IGF-II has been produced in Escherichia coli after first modifying a plasmid that coded for a human (h)IGF-II fusion protein. The cIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography and refolded. Recombinant cIGF-II was then released from the fusion protein using a genetically engineered serine protease and purified to homogeneity by reverse-phase HPLC. In vitro analysis of recombinant cIGF-II revealed differences between cIGF-II and its human counterpart. Recombinant cIGF-II was less potent than hIGF-II in stimulating protein synthesis in rat myoblasts. This appeared to be due to a decreased affinity for the type-1 IGF receptor. The human and chicken peptides were similar, however, in studies assessing binding to the type-2 IGF receptor and to IGF-binding proteins. Moreover, recombinant cIGF-II and hIGF-II were equipotent in both biological and receptor binding studies in chick embryo fibroblasts, suggesting that there may be a difference between mammalian and avian type-1 IGF receptors.
Developmental and glucocorticoid regulation of pituitary 11 β-hydroxysteroid dehydrogenase 1 gene expression in the ovine fetus and lamb
Authors: Yang K , Matthews S G , Challis J R G 
Published: December 2008

ABSTRACT
To examine the role of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in the control of glucocorticoid actions in the ovine pituitary during development, we have sought developmental changes in the distribution and the level of 11β-HSD1 mRNA by in situ hybridization. In the pars distalis, 11β-HSD1 mRNA was present by day 60; its amount did not change significantly until term (days 145–147) when it increased dramatically. The level of 11β-HSD1 mRNA increased further during the postnatal period. In contrast, 11β-HSD1 mRNA in the pars intermedia was not detectable until day 135; it increased in amount at days 140–143, but did not change significantly thereafter through to adulthood.
We have also measured levels of both dehydrogenase and reductase activities of 11β-HSD1 in the pars distalis of fetal sheep at day 140 and term, and of postnatal sheep at 1–2 months of age, to determine whether changes in 11β-HSD1 mRNA are reflected in the levels of enzyme activities. There were progressive increases in both dehydrogenase and reductase activities from day 140 to 1–2 months postnatally, although dehydrogenase activity was consistently higher than reductase activity.
Finally, we have determined the effect of short-term intrafetal cortisol infusion (5 μg/min for 12 h) on levels of pituitary 11β-HSD1 mRNA by in situ hybridization. There was no effect of cortisol infusion on 11β-HSD1 mRNA expression.
The present results demonstrate that 11β-HSD mRNA and enzyme activity in the pars distalis of fetal sheep increase dramatically at term when plasma levels of both ACTH and cortisol are elevated. This suggests that 11β-HSD1 may contribute to the proposed resetting of cortisol negative feedback within the fetal pituitary at that time.
Growth hormone releasing peptide (GHRP-6) stimulates phosphatidylinositol (PI) turnover in human pituitary somatotroph cells
Authors: Lei T. , Buchfelder M. , Fahlbusch R. , Adams E. F. 
Published: December 2008

ABSTRACT
Growth hormone releasing peptide (GHRP-6) is a synthetic hexapeptide which specifically stimulates secretion of growth hormone (GH) by pituitary somatotrophs. The precise intracellular mechanism by which this is achieved has not been deciphered although it is known to involve protein kinase C (PKC) and Ca2+ but to be cAMP-independent. We have used cell cultures of human pituitary somatotrophinomas to demonstrate powerful effects of GHRP-6 on membrane phosphatidylinositol (PI) turnover, a second messenger system which leads to activation of PKC and mobilisation of intracellular Ca2+ reserves. Incubation of somatotrophinoma cells with GHRP-6 led to a dose-dependent stimulation of rate of PI turnover. GH secretion was increased in parallel. Effects were discernable after only 15 minutes incubation and rose to a maximum at 2 hours. PI turnover was stimulated by GHRP-6 in 8 of 8 tumours examined, effects ranging from 2.1–7.9 fold increases. Stimulation of GH secretion by GHRP-6 was independent of presence of gsp oncogenes, emphasising the cAMP-independent nature of its effects. These results provide evidence that the GH-stimulatory effects of GHRP-6 are achieved through activation of the PI second messenger system and thus support earlier findings that PKC and Ca2+ play central roles in mediating the effects of GHRP-6.
(PI) into diacylglycerol (DAG) and inositol phosphates (Nishizuka, 1988; Berridge and Irvine, 1989), raising the possibility that GHRP-6 acts by stimulating this intracellular second messenger system. To test this hypothesis, we have examined whether GHRP-6 is able to promote increased PI turnover in human pituitary somatotrophinoma cells in culture.
Expression of tilapia prepro-melanin-concentrating hormone mRNA in hypothalamic and neurohypophysial cells
Authors: Gröneveld D , Eckhardt E R M , Coenen A J M , Martens G J M , Balm P H M , Bonga S E Wendelaar 
Published: December 2008

ABSTRACT
Melanin-concentrating hormone (MCH) is a neuropeptide involved in background adaptation in teleost fish, and in multiple regulatory functions in mammals and fish. To study the expression of the MCH preprohormone (ppMCH) in teleosts, we first cloned a hypothalamic cDNA encoding the complete ppMCH of tilapia (Oreochromis mossambicus), and a cRNA probe derived from a 270 bp ppMCH cDNA fragment was used for the expression studies. The level of ppMCH mRNA expression in tilapia hypothalamus, measured by dot blot analysis, was significantly higher in fish adapted to a white background than in black-adapted animals, which is in accordance with the reported MCH plasma and tissue concentrations in fish. Northern blot analysis not only revealed a strong ppMCH mRNA signal in the hypothalamus, but also the presence of ppMCH mRNA in the neurointermediate lobe (NIL) of the pituitary. In situ hybridization and immunocytochemistry showed that ppMCH mRNA as well as MCH immunoreactivity are located in perikarya of two hypothalamic regions, namely in the nucleus lateralis tuberis (NLT) and the nucleus recessus lateralis (NRL). Quantitative analysis by dot blot hybridization revealed about eight times more ppMCH mRNA in the NLT than in the NRL and NIL of mature tilapias. ppMCH mRNA in the NIL could be localized to cell bodies of the neurohypophysis, which were also MCH immunoreactive.
Modulation of glucose transport by parathyroid hormone and insulin in UMR 106–01, a clonal rat osteogenic sarcoma cell line
Authors: Thomas D M , Rogers S D , Sleeman M W , Pasquini G M , Bringhurst F R , Ng K W , Zajac J D , Best J D 
Published: December 2008

ABSTRACT
This study characterizes the actions of insulin and parathyroid hormone (PTH) on the glucose transport system in the rat osteogenic sarcoma cell line UMR 106–01, which expresses a number of features of the osteoblast phenotype. Using [1,2-3H]2-deoxyglucose (2-DOG) as a label, UMR 106–01 cells were shown to possess a glucose transport system which was enhanced by insulin. In contrast, PTH influenced glucose transport in a biphasic manner with a stimulatory effect at 1 h and a more potent inhibitory effect at 16 h on basal and insulin-stimulated 2-DOG transport. To explore the mechanism of PTH action, a direct agonist of cAMP-dependent protein kinase (PKA) was tested. 8-Bromo-cAMP had no acute stimulatory effect but inhibited basal and insulin-stimulated 2-DOG transport at 16 h. This result suggested that the prolonged, but not the acute, effect of PTH was mediated by the generation of cAMP. Further studies with the cell line UMR 4–7, a UMR 106–01 clone stably transfected with an inducible mutant inactive regulatory subunit of PKA, confirmed that the inhibitory but not the stimulatory effect of PTH was mediated by the PKA pathway. Northern blot data indicated that the prolonged inhibitory effects of PTH and 8-bromo-cAMP on glucose transport were likely to be mediated in part by reduction in the levels of GLUT1 (HepG2/brain glucose transporter) mRNA.
Consequences of antisense human chorionic gonadotrophin-α subunit cDNA expression in human choriocarcinoma JAR cells
Authors: Cao H , Lei Z M , Rao Ch V 
Published: December 2008

ABSTRACT
The biosynthesis of human chorionic gonadotrophin (hCG) is a hallmark endocrine function of human choriocarcinoma cells. The present study investigated the consequences of greatly diminishing this synthesis in JAR cells by stably transfecting them with pRSV-antisense hCG-α cDNA expression vector. The vector directs the synthesis of antisense hCG-α subunit mRNA which would then bind to sense hCG-α subunit mRNA, thus blocking its translation and consequently dimer hCG protein synthesis.
The transfection with pRSV-antisense hCG-α cDNA resulted in a dramatic decrease in hCG secretion as compared with untransfected parental cells or those transfected with an empty vector used for the selection of clones. The decreased secretion was due to a decreased synthesis which in turn was due to a fall in steady-state hCG-α and -β subunit mRNA levels. The decrease of hCG-β subunit transcripts was unexpected and it was not due to contamination of antisense hCG-α cDNA construct with hCG-β sequence. The transcription of hCG-α and -β subunit genes was not altered in transfected cells suggesting that increased degradation was responsible for decreased steadystate hCG subunit mRNA levels. Despite the decreased hCG levels, the transfected cells maintained normal hCG receptor levels, responded to epidermal growth factor stimulation of hCG synthesis and secretion and grew at the same rate as the control parental cells and those transfected with an empty vector.
Absence of D2S dopamine receptor in the prolactin-secreting MMQ pituitary clone: characterization of a wild D2L receptor coupled to native transduction mechanisms
Authors: Ventra C , Meucci O , Grimaldi M , Scorziello A , Porcellini A , Schettini G 
Published: December 2008

ABSTRACT
We used the PCR amplification technique in an attempt to characterize further the dopamine D2L receptor expressed in the prolactin-secreting pituitary MMQ cell clone, derived from the prolactinand ACTH-secreting Buffalo rat 7315a pituitary tumour. By semiquantitative PCR amplification we were unable to detect the mRNA encoding the D2S receptor isoform, which derives from the wellknown process of alternative splicing, producing two D2 receptor subtypes (D2L and D2S) in such tissues as the anterior pituitary and the corpus striatum. Although the pharmacology of the D2 receptor has been established in many studies on both native receptors and transfected receptor isoforms, because of the lack of tissues naturally expressing only one receptor isoform, MMQ cells represent the first example of cells uniquely or prevalently expressing only the D2L receptor, conceivably coupled to its native transduction mechanisms. These considerations prompted us to evaluate the pharmacology and the second messenger systems known to be modulated by dopamine. Scatchard analysis of [3H]spiperone binding re-suited in a linear plot, consistent with the existence of a single class of binding sites, with a Kd of 0·055±0·002 nm and a Bmax of 27±3·5 fmol/mg protein. Competition experiments confirmed the GTP-dependence and the order of potency for agonist and antagonist ligands consistent with binding to a D2 receptor. The inhibitory effects of dopamine on adenylyl cyclase activity, inositol phosphate production and intracellular free calcium concentrations, the latter presumably via the opening of K+ channels, and prolactin secretion, as well as the reversal of the effect by the D2-selective antagonist (−)sulpiride and pretreatment with pertussis toxin, are consistent with the known biological actions of dopamine at D2 receptors. Based on our observations, the MMQ cell line can be considered a useful tool for investigating ligand-receptor interactions to develop new selective dopaminergic D2L ligands for the therapy of dopamine-related disorders such as schizophrenia, depression, Parkinson's disease and drug addiction.