bluuebunny/crossref_metadata_2025 huggingface dataset:
Amplification or rearrangement of the β-human chorionic gonadotrophin (β-hCG)—human LH gene cluster is not responsible for the ectopic production of β-hCG by bladder tumour cells
Authors: Iles R. K. , Czepulkowski B. H. , Young B. D. , Chard T.
Published: December 2008

The β-subunit of human chorionic gonadotrophin (hCG) is coded on chromosome 19 by the β-hCG-hLH gene cluster. Genomic DNA has been isolated from bladder tumour cell lines which ectopically express β-hCG. The β-hCG—hLH gene cluster was probed for possible rearrangement or amplification and cells karyotyped for chromosome 19 abnormalities. No rearrangement or amplification of the gene cluster and no consistent abnormalities of chromosome 19 were found. The expression of β-hCG by bladder tumours is therefore likely to be the result of altered gene regulation and not a rearrangement or amplification of this gene cluster.
The cytostatic effects of α-interferon may be mediated by transforming growth factor-β
Authors: Kerr D. J. , Pragnell I. B. , Sproul A. , Cowan S. , Murray T. , George D. , Leake R.
Published: December 2008

There is some evidence to suggest that transforming growth factor-β (TGF-β) mediates the cytostatic effects of the anti-oestrogen tamoxifen. In this study we have demonstrated that α-interferon has a significant anti-proliferative effect on the oestrogen receptor-positive human breast cancer cell line ZR-75. There is decreased phenotypic expression of the oestrogen receptors (to about 30% of control values) and increased TGF-β mRNA. Under the growth conditions used here, ZR-75 cells had approximately 5800 TGF-β binding sites per cell, with an apparent dissociation constant of 70 pm, and we have shown that the anti-proliferative effects of α-interferon can be reduced by 60% by co-treating the cells with a TGF-β polyclonal antibody. The cytostatic effects of α-interferon may therefore be mediated by TGF-β in this human breast cancer cell line.
Gastric inhibitory polypeptide: a gut hormone with anabolic functions
Authors: Beck B.
Published: December 2008

The gastrointestinal hormone, gastric inhibitory polypeptide (GIP), has been isolated and characterized because of its enterogastrone-type effects. It is also named glucose-dependent insulinotropic polypeptide and is actually considered to be the main incretin factor of the entero-insular axis. Besides these well-described effects on gastric secretion and pancreatic β cells, it also has direct metabolic effects on other tissues and organs, such as adipose tissue, liver, muscle, gastrointestinal tract and brain. In adipose tissue it is involved in the activation and regulation of lipoprotein lipase (LPL); it also inhibits glucagon-induced lipolysis and potentiates the effect of insulin on incorporation of fatty acids into triglycerides. It may play a role in the development of obesity because of the hypersensitivity of adipose tissue of obese animals to some of these actions. In the liver it does not modify insulin extraction, and its incretin effects are due only to the stimulation of insulin secretion and synthesis. It reduces hepatic glucose output and inhibits glucagon-stimulated glycogenolysis. It might increase glucose utilization in peripheral tissues such as muscle. GIP also has an effect on the volume and/or electrolyte composition of intestinal secretion and saliva. The functional importance of its effect on the hormones of the anterior pituitary lobe remains to be established, as it has never been detected in the brain.

Its links with insulin are very close and the presence of insulin is sometimes necessary for the greater efficiency of both hormones. GIP can be considered as a true metabolic hormone, with most of its functions tending to increase anabolism.
Evidence that the bovine ovary secretes large amounts of monomeric inhibin α subunit and its isolation from bovine follicular fluid
Authors: Knight P. G. , Beard A. J. , Wrathall J. H. M. , Castillo R. J.
Published: December 2008

Analysis of bovine follicular fluid (FF) using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with a sensitive immunoblotting procedure resolved several components that were immunoreactive with an antiserum directed against the n-terminus of the α subunit of human inhibin (hIα(1–32)). Under non-reducing conditions, three intensely stained bands having apparent Mr values of 116 000, 44 000 and 25 000 were present, whilst under reducing conditions only two intensely stained bands (Mr 43 000 and 21 000) were detected. The Mr 44 000 and 25 000 immunoreactive forms (non-reducing conditions) were also demonstrated in bovine utero-ovarian vein and peripheral venous plasma after subjecting samples (40 ml) to immunoaffinity concentration using Sepharose beads coupled to anti-hIα(1–32), SDS-PAGE and immunoblotting. The same approach revealed the presence of the smaller (Mr 25 000) form in bovine granulosa cell-conditioned culture medium (GCCM). Gel-permeation chromatography (Sephacryl S-200), immunoaffinity chromatography (Sepharose-anti-hIα(1–32)) and reversed-phase high-performance liquid chromatography (RP-HPLC; C18 and C8 columns) were employed to isolate from bFF (30 ml, 195 g protein) 750 μg protein which appeared essentially homogeneous by RP-HPLC and SDS-PAGE and had an Mr of 25 000 (non-reducing conditions)/21 000 (reducing conditions), identical to that of the immunoreactive component of lowest Mr found in bovine FF, utero-ovarian vein plasma, peripheral plasma and GCCM. The isolated material was highly immunoreactive with antisera against both hIα(1–32) and purified Mr 32 000 bovine inhibin but was devoid of biological activity when tested in a rat pituitary cell inhibin bioassay. Amino-terminal analysis revealed an amino acid sequence (residues 1–14) identical to that reported elsewhere for the α subunit (Mr 20 000/21 000) of bovine inhibin.

In conclusion, the present study has revealed that the bovine ovary secretes considerable quantities of monomeric inhibin α subunit. The unexpected presence of this material in peripheral blood is likely to hinder attempts to obtain physiologically relevant data on circulating levels of inhibin in cattle using conventional radioimmunoassays.
A monoclonal antibody to human insulin-like growth factor-I: characterization, use in radioimmunoassay and effect on the biological activities of the growth factor
Authors: Morrell D. J. , Dadi H. , More J. , Taylor A. M. , Dabestani A. , Buchanan C. R. , Holder A. T. , Preece M. A
Published: December 2008

A monoclonal antibody (BPL-M23) to insulin-like growth factor-I (IGF-I) was obtained following immunization of BALB/c mice with human IGF-I conjugated to ovalbumin. The affinity constant of BPL-M23 for IGF-I was 10·5 litres/nmol and the cross-reactivities of IGF-II, multiplication-stimulating activity III-2 and insulin were 08, 003 and less than 0·0001 % respectively. Porcine, bovine, ovine and rabbit sera, but not rat or mouse sera, showed substantial reactivity with the antibody.

Comparison of radioimmunoassay analyses of 54 human serum samples from normal subjects and acromegalic and GH-deficient patients using BPL-M23 and a polyclonal rabbit antiserum (R557A) to human IGF-I showed a high correlation, indicating the usefulness of the monoclonal antibody in radioimmunoassay.

Monoclonal antibody BPL-M23 was capable of abolishing the sulphation, mitogenic and insulin-like activities of IGF-I in in-vitro bioassays, suggesting that these activities may rely upon the same receptor-binding site which is near to the antibody-binding site.
Thyrotrophin-releasing hormone (TRH)-induced growth hormone secretion in fowl: binding of TRH to pituitary membranes
Authors: Harvey S. , Baidwan J. S.
Published: December 2008

Heat-labile specific binding sites for [3H]3-methylhistidine2-TRH ([3H]Me-TRH) were demonstrated on chicken adenohypophysial membranes. These binding sites were of both high affinity (dissociation constant, Kd = 15·53 nm) and low capacity (maximum binding capacity, Bmax = 8·73 pmol/g tissue) and of low affinity (Kd = 104·5 nm) and high capacity (Bmax = 32·41 pmol/g). Binding of [3H]Me-TRH to the pituitary membranes was greater at 4 °C than at 39 °C and occurred with rate constants (k1) of 1·6× 106 and 3·39×106m-1 min-1 respectively. Dissociation of [3H]Me-TRH binding at 4 °C occurred with a rate constant (k-1) of 0·125 min-1. Binding sites for [3H]Me-TRH were found in the cephalic lobe of the pituitary gland (the location of most lactotrophs and thyrotrophs) and in the caudal lobe (the location of most somatotrophs). The number of binding sites was greater in the caudal lobe than in the cephalic lobe, although the affinity of [3H]Me-TRH binding did not differ. The binding of [3H]Me-TRH to caudal lobe membranes was displaced by Me-TRH, TRH, pGlu-His-Pro-Gly-NH2 and [Glu1]-TRH, with half-maximal effective doses of 33 nm, 70·7 nm, 1·23 μm and 22 μm respectively, but not by [Phe2]-TRH, TRH free acid or His-Pro-diketopiperazine. The number of caudal lobe binding sites for [3H]Me-TRH in old birds was less than that in young ones, and the number of binding sites was increased in birds deprived of food for 48 h. TRH-induced GH secretion in birds would thus appear to be mediated by specific receptors on caudal lobe somatotrophs, and these results suggest that physiological changes in GH secretion (during growth and periods of fasting) are causally related to the abundance of TRH-binding sites.
Differential binding of thyroid hormone receptors to mouse glandular kallikrein gene promoters: evidence for multiple binding regions in the mGK-6 gene
Authors: Barlow J. W. , Raggatt L. E. , Drinkwater C. C. , Lyons I. G. , Richards R. I.
Published: December 2008

We have used a DNA-cellulose competition assay to investigate the binding of thyroid hormone receptors to fragments of the mouse glandular kallikrein genes and the human and rat GH genes. Nuclear extracts from human lymphoblastoid IM-9 cells were incubated with [125I]tri-iodothyronine ([125I]T3) and DNA-cellulose. The ability of cloned gene fragments to compete for radiolabelled receptors bound to DNA-cellulose was compared with that of DNA from pBR322. As previously observed, a 900 bp fragment from the human GH gene showed preferential binding to the thyroid hormone receptor. High-affinity binding was observed with a synthetic fragment of the rat GH gene encompassing positions − 163 to − 192 but not with a similar fragment from positions −224 to −192. Preferential binding was also observed with fragments of the mouse glandular kallikrein gene, mGK-6. Binding to the entire gene and fragments containing 2300 and 776 bp of the promoter region was identical. Detectable but reduced binding was seen with a shorter fragment. These results suggest that the T3 receptor binds to multiple sites within the first 776 bp of the mGK-6 gene promoter. Potential thyroid hormone response elements can be identified within this region of the gene. In contrast, the kallikrein gene mGK-3, which shows a different response to thyroid hormone from that of mGK-6, showed no significant binding in the comparable promoter region.
Treatment of ovariectomized heifers with bovine follicular fluid specifically suppresses pituitary levels of FSH-β mRNA
Authors: Beard A. J. , Savva D. , Glencross R. G. , McLeod B. J. , Knight P. G.
Published: December 2008

To investigate the inhibin-induced suppression of FSH secretion by the anterior pituitary, chronically ovariectomized heifers (three per group) were treated for 56–58 h with either steroid-free bovine follicular fluid (bFF; 8 ml i.v. every 8 h) or 0·9% (w/v) NaCl (8 ml i.v. every 8 h). Blood was withdrawn at 8-h intervals for analysis of plasma concentrations of FSH and LH by radioimmunoassay. At the end of the treatment period, heifers were slaughtered and pituitary glands recovered for determination of gonadotrophin contents and levels of mRNA encoding FSH-β, LH-β, TSH-β and common α glycoprotein hormone subunits using [32P]cDNA probes in total RNA dot and Northern blot assays. Treatment with bFF markedly suppressed plasma FSH by 85% (P<0·001 compared with pretreatment period), but did not affect plasma LH concentrations. Plasma FSH and LH concentrations did not vary significantly in the saline-injected control heifers. The level of FSH-β subunit mRNA was reduced by 60% (P<0·001) in heifers treated with bFF, whereas no significant differences between control and bFF-treated heifers were observed in the levels of mRNA encoding LH-β, TSH-β or common α subunits. Treatments with bFF, however, did not affect pituitary content of either FSH or LH.

These results support the conclusion that inhibin exerts its selective suppressive effect on the secretion of FSH by the bovine pituitary, at least in part, by directly inhibiting expression of the gene encoding the FSH-β subunit.
Lactate alters plasma membrane potential, increases the concentration of cytosolic Ca2+ and stimulates the secretion of insulin by the hamster β-cell line HIT-T15
Authors: Meats J. E. , Tuersley M. D. , Best L. , Lynch A. M. , Tomlinson S.
Published: December 2008

We have studied the effect of lactate on a number of intracellular events which may be important in controlling the secretion of insulin by the hamster β-cell line HIT-T15. Using the fluorescent dye Oxonol V, as well as intracellular recording techniques to measure changes in membrane potential, we found that lactate, glucose, K+ and tolbutamide caused depolarization of HIT cells, while valinomycin resulted in hyperpolarization. Consistent with these findings was the observation that 10 mm lactate caused an increase of 69·0±18·4% (s.e.m., n=6) in the level of free cytosolic Ca2+ within HIT cells (assessed by fluorescence of quin 2). This was probably due to influx of Ca2+ through voltage-sensitive calcium channels, since it was dependent upon the concentration of extracellular Ca2+ and inhibited by verapamil. Lactate also caused cytosolic acidification in HIT cells and increased the secretion of insulin. These findings are consistent with the view that the electrogenic efflux of lactate could be a determinant in the activation of HIT cells by lactate and possibly by glucose.
C4A gene deletion: association with Graves' disease
Authors: Ratanachaiyavong S. , Lloyd L. , McGregor A. M.
Published: December 2008

The association of HLA class I and class II antigens, particularly HLA-B8,DR3, with a variety of autoimmune diseases has been well documented. The C4A*Q0 (non-expressed C4A) allele which is in linkage disequilibrium with HLA-B8,DR3 has also been reported to be associated with systemic lupus erythematosus, insulin-dependent diabetes mellitus and Graves' disease. However, the number of studies has been limited by the requirement of family data for the assignment of the C4A*Q0 allele based on C4 protein typing. Recently, with the availability of a C4 cDNA probe, a C4A gene deletion associated with HLA-B8,DR3 has been reported in normal individuals. We have tried to resolve the problem of assigning the C4A*Q0 allele by using both phenotypic and genotypic approaches and have determined the significance of the C4A*Q0 allele in 80 unrelated patients with Graves' disease and in 50 normal control subjects. Our results demonstrate a strong association of the C4A*Q0 allele with Graves' disease (56 versus 26%; P< 0·002, relative risk=3·7) and in particular in association with HLA-B8 and/or DR3 (92 versus 70·6%; P<0·04) when compared with normal controls. All the C4A*Q0 alleles that were associated with HLA-B8 and/or DR3 were due to a C4A gene deletion. Of the C4A*Q0 alleles in Graves' disease, 94% (compared with 82% in the control group) could be detected by C4 DNA analysis using either TaqI or EcoRI restriction endonucleases. It is suggested that a combination of C4 protein typing with C4 DNA analysis is the best approach for the determination of the C4A*Q0 allele in unrelated individuals without access to family data.
Structure of rhesus monkey relaxin predicted by analysis of the single-copy rhesus monkey relaxin gene
Authors: Crawford R. J. , Hammond V. E. , Roche P. J. , Johnston P. D. , Tregear G. W.
Published: December 2008

The gene encoding rhesus monkey relaxin has been investigated. A cDNA library was prepared using corpus luteal RNA from a pregnant rhesus monkey, cDNA clones encoding relaxin were isolated and the nucleotide sequence was determined. The amino acid sequence of rhesus monkey preprorelaxin, predicted from the cDNA, demonstrates that the sequence has not been strongly conserved when compared with that of man, although features characteristic of the relaxin molecule have been maintained. This structural information will allow production of rhesus monkey relaxin, leading to studies investigating the bioactivity of relaxin in a homologous primate system. Southern blot analysis indicated that there is only one relaxin gene in the rhesus monkey and baboon genomes. In this respect these primate genomes are different from the human genome which contains two relaxin genes.
Subunit assembly and secretion of transthyretin: studies in a cell-free translation system and in microinjected Xenopus oocytes
Authors: Docherty K. , Shennan K. I. J. , Marsden R. F. , Ramsden D. B.
Published: December 2008

Transthyretin (TTR), or thyroid-binding prealbumin, is a protein of 55 kDa, composed of four identical subunits, which is synthesized by the liver and choroid plexus epithelium. In order to study the subunit assembly and secretion of TTR, cRNA encoding TTR was translated in a rabbit reticulocyte lysate or microinjected into Xenopus oocytes, and radiolabelled biosynthetic products were immunoprecipitated with an antibody against TTR and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. In the cell-free translation system in the absence of dog pancreatic microsomes, a single protein of Mr 17 000 was synthesized. In the presence of dog pancreatic microsomes, two proteins of Mr 15 000 and 37 000 were observed. The Mr 17 000 protein was identified as pre-TTR and the Mr 15 000 and 37 000 proteins as monomeric and dimeric forms of TTR.

When the mRNA was microinjected into Xenopus oocytes both Mr 15 000 and 37 000 proteins were secreted into the media. It was shown that, under the SDS-PAGE conditions used in this study, the TTR tetramer dissociated to the dimeric form (Mr 37 000), but that there was no, or at least very little, further breakdown to the monomer. Therefore, to determine whether tetrameric or dimeric forms of TTR were secreted from the oocytes, the media from microinjected ooctyes were subjected to gel permeation chromatography under non-dissociating conditions, and the eluted fractions analysed by SDS-PAGE. TTR eluted from the column as a dimer; there was no tetramer or monomer. The dimer, however, was completely dissociated to the monomer when analysed by SDS-PAGE, which suggested that incomplete or incorrect subunit assembly had occurred. These results lead to the conclusion that liver- and choroid plexus-specific factors may be required for correct subunit assembly of TTR.
Desensitization of the β-adrenergic receptor of the S49 murine lymphoma cell: mechanism of receptor loss and recovery
Authors: Rich K. A. , Iyengar R.
Published: December 2008

The effect of prolonged exposure of S49 murine lymphoma cells to (−) isoprenaline on the levels of β-adrenergic receptors and the stimulatory regulatory component of adenylyl cyclase (Gs) was investigated. Exposure of wild-type S49 cells (WT cells) to isoprenaline for 16 h resulted in an 84% decrease in β-adrenergic receptors and a 78% decrease in catecholamine-stimulated adenylyl cyclase activity. The desensitization of adenylyl cyclase was homologous since no change in stimulation by any other effectors was observed. Similar treatment of cyc- S49 cells, a mutant that lacks Gs, resulted in only a 10–15% receptor loss despite a 50% decrease in catecholamine stimulation of reconstituted adenylyl cyclase activity. By quantitative extraction of Gs from control and isoprenaline-treated WT cellmembranes and reconstitution into untreated cyc- cell membranes, it was demonstrated that the levels and behaviour of Gs in WT cell membranes were unchanged after isoprenaline treatment, despite a substantial loss of receptors from the cell surface. The β-adrenergic receptors lost from the cell surface could not be identified in any intracellular membrane fraction. Addition of cycloheximide to the culture medium of WT cells previously treated with isoprenaline resulted in a complete blockade of the restoration of β-adrenergic receptors which was otherwise observed within 16 h after removal of the agonist.

It is concluded that, in the S49 cell line, prolonged exposure to agonists results in a loss of β-adrenergic receptors by a process dependent on the presence of Gs. However, the loss of receptors is selective since under conditions where the majority of receptors are lost, there is no measurable change in the level of cell surface membrane-bound Gs. The lost receptors appear to be degraded, since the restoration of receptor levels after removal of the agonist is dependent on protein synthesis.
Procholecystokinin and proenkephalin A mRNA expression is modulated by cyclic AMP and noradrenaline
Authors: Monstein H. J. , Folkesson R. , Geijer T.
Published: December 2008

Regulation of the expression of procholecystokinin (proCCK) and proenkephalin A mRNA was studied in the human neuroblastoma cell line SK-N-MC. Cells were treated with dibutyryl-3′,5′-cyclic AMP (dbcAMP), noradrenaline or isoproterenol, a β-adrenoceptor agonist. Levels of proCCK and proenkephalin A mRNA were determined by Northern blot analysis with proCCK- and proenkephalin A-specific cRNA hybridization probes 9 h after drug treatments. ProCCK and proenkephalin A mRNA were co-expressed in SK-N-MC cells. ProCCK mRNA levels were increased 1·5–2·5 times by dbcAMP, noradrenaline and isoproterenol when compared with controls. The level of proenkephalin A mRNA increased approximately two to three times under the same drug conditions, whereas the level of N-myc mRNA did not change significantly. These results suggest that expression of proCCK and proenkephalin A mRNA may be regulated by a similar cAMP-dependent mechanism in the SK-N-MC cell line.
Developmental and hormonal regulation of a mouse mammary tumour virus glycoprotein in normal mouse mammary epithelium
Authors: Bolander F. F. , Blackstone M. E.
Published: December 2008

A radioimmunoassay was developed and validated for the major glycoprotein (gp58) of the mouse mammary tumour virus (MMTV). Using this assay, the expression of gp58 during pregnancy and lactation was found to parallel that for MMTV RNA. In particular, there was a very rapid induction in late pregnancy and a decline in late lactation, although some residual expression persisted well into involution. In cultures of normal mouse mammary tissue, induction of gp58 occurred after a 24-h lag period and began to reach a plateau after 3 days. Both the insulin and prolactin dose—response curves for gp58 resembled those for MMTV RNA; in contrast, the effects of steroid hormones on gp58 and MMTV RNA were disparate. Although progesterone stimulated the RNA, it only slightly increased gp58 levels; however, the presence of cortisol greatly augmented this stimulation, despite the inability of cortisol to induce RNA at physiological concentrations. These results suggest that insulin, prolactin and progesterone act primarily at the level of RNA accumulation in normal mammary epithelium, while cortisol affects some more distal event.
Thyroidal inhibition of growth hormone secretion in fowl: tri-iodothyronine-induced down-regulation of thyrotrophin-releasing hormone-binding sites on pituitary membranes
Authors: Harvey S. , Baidwan J. S.
Published: December 2008

The number, but not affinity, of binding sites for [3H]3-methyl-histidine2-TRH ([3H]Me-TRH) on chicken adenohypophysial plasma membranes was increased in chickens made hypothyroid by goitrogen (methimazole) treatment (50 mg/kg per day for 7 days), which also increased circulating GH concentrations. Daily i.p. injection of thyroxine (T4; 100 μg/kg for 7 days) had no effect on [3H]Me-TRH binding to pituitary membranes, although it suppressed endogenous GH secretion. Binding of [3H]Me-TRH to pituitary caudal lobe membranes was, however, suppressed by tri-iodothyronine (T3) injected chronically (100 μg/kg per day, i.p., for 7 days) or acutely (100 μg/kg, 2 h before being killed). The suppression of [3H]Me-TRH binding and inhibition of GH secretion following T3 administration was dose related. Binding of [3H]Me-TRH to caudal lobe membranes was also suppressed following the incubation of pituitary glands with T3 in vitro, and the response was both dose and time related.

These results suggest that T3 inhibits GH secretion in fowl by a down-regulation of pituitary TRH receptors. However, other mechanisms are involved in thyroidal inhibition of GH release in birds, since T4 had no effects on [3H]Me-TRH binding yet suppressed GH secretion in vivo.
Expression of the human pro-opiomelanocortin gene introduced into a rat glial cell line
Authors: Usui T. , Nakai Y. , Tsukada T. , Takahashi H. , Fukata J. , Naito Y. , Nakaishi S. , Tominaga T. , Murakami N. , Imura H.
Published: December 2008

A fragment of human genomic DNA containing the entire pro-opiomelanocortin (POMC) gene was introduced by transfection into the rat glial cell line C6. Blot analysis using poly(A)-rich RNA from the transformed C6 cells showed several hybridization bands. One band was similar in size (1·2 kb) to the POMC mRNA of human pituitary, while two were larger (2·6 and 2·2 kb) and the fourth smaller (800 bp). S1 nuclease mapping revealed that the POMC transcripts in transformed C6 cells were similar to those in non-pituitary tissues. Immunoreactive ACTH (ir-ACTH) was measurable in both the culture medium and cells. Gel chromatography showed that ir-ACTH in the medium eluted at a position identical to that of so-called big ACTH (approximately 40 kDa) which is found in the plasma of patients with ectopic ACTH syndrome. The human POMC gene could thus be expressed in the non-pituitary rat glial cell line C6, although the transcripts and translation products in C6 cells differ from those in the human pituitary. These results suggest that the transformed C6 cell may be a useful tool for studying the regulation of human POMC gene expression in non-pituitary cells.
MULTIPLE DNA ELEMENTS DETERMINE BASAL AND THYROID HORMONE REGULATED EXPRESSION OF THE HUMAN GLYCOPROTEIN α SUBUNIT GENE IN PITUITARY CELLS
Authors: Balfour N.J. , Franklyn J.A. , Gurr J.A. , Sheppard M.C.
Published: December 2008

Basal and thyroid hormone-regulated expression of constructs containing varying lengths of 5′ flanking sequence of the human glycoprotein hormone α subunit gene ligated to the reporter gene encoding chloramphenicol acetyl transferase (CAT) were examined in transfected pituitary GH3 cells. CAT gene expression was quantified by measurement of CAT enzyme activity. In addition, an RNase protection assay was used to confirm changes in CAT mRNA levels and to verify regulation of transcription from the authentic α gene start site. The pattern of basal α promoter activity indicated the presence of inhibitory DNA elements between −98 and −172 and upstream of −846 relative to the transcriptional start site, as well as a stimulatory element between −346 and −846. Each of the transfected constructs exhibited tri-iodothyronine inhibition of α promoter activity, suggesting the presence of an inhibitory thyroid hormone response element within 98 bp of the α gene transcriptional start site.
Desensitization of thyrotrophin-releasing hormone (TRH)-induced growth hormone secretion in chickens: coincident down-regulation of TRH binding to pituitary membranes
Authors: Harvey S. , Baidwan J. S.
Published: December 2008

Release of GH is stimulated by TRH in chickens. However, for 60 min following a priming injection of TRH, a second injection of TRH is unable to provoke further GH release. TRH binds to the plasma membranes of the pituitary caudal lobe, in which somatotrophs predominate, although the magnitude of [3H]3-methyl-histidine2-TRH ([3H]Me-TRH) binding was reduced (by 25–50%) 30 min after the i.v. administration of a dose of TRH (5 μg/kg maximally effective in provoking GH release. A significant reduction in [3H]Me-TRH binding to caudal lobe membranes was also observed within 15 min of TRH administration and was maintained for at least 60 min. Control levels of [3H]Me-TRH binding were restored 2 h after TRH injection, coincident with the restoration of GH responsiveness to TRH challenge. The suppression of [3H]Me-TRH binding to pituitary membranes 30 min after in-vivo TRH administration was dose related, whereas the maximal (10 min) GH response to TRH was biphasic. The suppression of [3H]Me-TRH binding to chicken pituitary membranes was due to direct pituitary actions of TRH and could be induced by a 30-min exposure to 100 nm TRH in vitro.

These results demonstrate that avian pituitary TRH-binding sites differ greatly from mammalian ones in the timing of the onset and duration of down-regulation. Pituitary TRH-binding sites in birds are rapidly and transiently down-regulated following TRH administration in vivo, coincident with the period of GH refractoriness to TRH challenge.
Molecular cloning and sequence analysis of the cDNA for the putative β subunit of the type-II gonadotrophin from the European eel
Authors: Quérat B. , Moumni M. , Jutisz M. , Fontaine Y.A. , Counis R.
Published: December 2008

Oestradiol treatment enhances type-II gonadotrophin (GTH-II) synthesis in the European eel (Anguilla anguilla) at the silver stage. As a first step in studying the molecular mechanisms involved in this stimulation, we cloned and characterized the cDNA encoding the β subunit of eel GTH-II.

A cDNA library was constituted in λgt10 from oestradiol-treated eels. It was screened using an oligodeoxyribonucleotide mixed probe designed to be complementary to a highly conserved region of cDNAs from several LH-related β subunits. Several clones were obtained and four were subcloned in pUC13 and sequenced. The longest clones comprised a 420 bp coding sequence, plus 5′ and 3′ untranslated regions of 36 and 172 bp respectively. Comparison with GTH-II from other teleost fish permitted the localization of the putative cleavage site of a 24 amino acid signal peptide. The resulting 116 amino acid apopeptide had well-conserved cysteine positions and a putative N-linked glycosylation site; homology was 70–80% with GTH–II from other fish, 45% with LH from mammals and birds, 38% with mammalian FSH and only 35% with fish GTH-I. Preliminary results indicated a strong positive effect of oestradiol treatment on the level of the putative GTH-II β-subunit mRNA. This supports our proposal that the European eel provides a suitable model for studying the positive regulation of gonadotrophin synthesis by gonadal steroids.
Absence of specific cell-surface binding of tissue plasminogen activator in uterine cells
Authors: Ayub M. , Jenkins N. , White J. O.
Published: December 2008

Tissue plasminogen activator (tPA), an arginine-specific serine protease, is an oestrogen-regulated protein in uterine and breast cancer tissue. It contains a domain which shares homology with epidermal growth factor (EGF).

The aim of the present study was to determine whether specific tPA receptors or EGF receptors mediate the binding of tPA to cells and whether tPA possesses intrinsic mitogenic activity. The binding of 125I-labelled tPA to rat uterine and liver membranes was shown to be non-specific and could not be displaced by unlabelled tPA or EGF. Furthermore, acid washing of cell membranes did not unmask specific tPA-binding sites. In contrast, 125I-labelled EGF binding to both rat uterine and liver membranes was displaced in a dose-dependent manner by unlabelled EGF, and Scatchard analysis of the binding data revealed dissociation constant (Kd) values of 2·4 and 0·71 nm respectively. Unlabelled tPA (up to 20 000-fold excess) did not displace 125I-labelled EGF binding to these membranes.

A study of the binding of 125I-labelled tPA and 125I-labelled EGF to endometrial carcinoma cells (Ishikawa), cervical carcinoma cells (HOG-1) and vulval carcinoma cells (A431) showed that up to a 100-fold excess of EGF or a 1000-fold excess of tPA did not displace 125I-labelled tPA binding to these cells. In contrast, 125I-labelled EGF binding was displaced by unlabelled EGF (Kd values for Ishikawa and HOG-1 cells were 2·72 and 1·92 nm respectively) but not by unlabelled tPA (1000-fold excess).

Treatment of Ishikawa and HOG-1 cells with EGF (20 ng/ml) for up to 6 days stimulated [3H]thymidine incorporation 1·3- to 2-fold and 3·5- to 4·7-fold respectively. Human recombinant tPA (10 IU/ml) was without effect on both cell lines over 6 days of treatment.

It was concluded that although tPA contains an EGF-like domain it does not bind specifically to membrane preparations from rat uterine or liver cells or to cultured uterine cancer cells, and does not displace bound 125I-labelled EGF. Furthermore, tPA does not have mitogenic properties similar to those of EGF.
Molecular cloning and DNA sequence analysis of preproinsulin genes in the NON mouse, an animal model of human non-obese, non-insulin-dependent diabetes mellitus
Authors: Sawa T. , Ohgaku S. , Morioka H. , Yano S.
Published: December 2008

Two insulin genes of the NON mouse, an animal model of human non-obese, non-insulin-dependent diabetes mellitus, were isolated and characterized to examine the hypothesis that these genes are structurally different from those of normal mice. The NON mouse was found to have two non-allelic insulin genes, as does the normal mouse, and no structural differences were found between the normal and NON mouse in the nucleotide sequence of the insulin gene, including that of the 5′-transcriptional regulatory region, and in the deduced amino acid sequence. There was, however, an additional 113 bp sequence and seven point mutations in a further 5′-flanking region, and three point mutations in the 3′-flanking region of the insulin II gene. We conclude that reduced expression of insulin genes in the NON mouse is not due to the structural change in the known transcriptional regulatory region, although the effect on insulin II gene expression of an additional sequence upstream of the 5′-flanking region, as the negative regulatory factor, remains to be elucidated.
Characterization and cloning of a bovine insulin-like growth factor-binding protein
Authors: Upton F. Z. , Szabo L. , Wallace J. C. , Ballard F. J.
Published: December 2008

The primary structure of the insulin-like growth factor-binding protein (IGFBP) produced by the bovine kidney cell line, MDBK, has been deduced from the cDNA clone. The MDBK binding protein precursor consists of a hydrophobic pre-peptide of at least 26 amino acids and a mature protein of 284 amino acids. The predicted protein sequence shares extensive sequence similarity with both the rat (82%) and human (89%) IGFBP-2s, so that the MDBK binding protein is clearly the bovine counterpart of IGFBP-2. The protein has limited similarity with classes 1 (31%) and 3 (31%) human IGFBPs, except that all 18 cysteine residues are conserved. Other features deduced from the bovine IGFBP-2 cDNA include: an abundance of leucine in the pre-peptide, an Arg-Gly-Asp sequence, absence of N-linked glycosylation sites, and an imperfect polyadenylation signal as well as an ATTTA motif in the 3′ non-coding DNA. Western blotting indicated that this binding protein is widely distributed in bovine fluids as well as in media conditioned by bovine cell lines. Proteins immunologically related to bovine IGFBP-2 were detected not only in sheep, but also in chickens, indicating that this IGFBP is not exclusively mammalian.
Association of glucocorticoid receptors with prostate nuclear sites for androgen receptors and with androgen response elements
Authors: Davies P. , Rushmere N. K.
Published: December 2008

Ventral prostate glands of intact normal rats contained low levels (2500 molecules/cell) of high-affinity (dissociation constant (Kd) 0·57 nmol/l) glucocorticoid receptors (GR). Levels of GR increased 2·8-fold 1 day after castration, and 4·3-fold 3 days after castration. Nuclear GR increased from a normal value of 1150 molecules/nucleus to 5200 molecules/nucleus 3 days after castration. The greater increase in intranuclear GR was in that associated with oligomeric chromatin. Although nuclear GR never approached the normal population of nuclear androgen receptors (AR; approximately 16000 molecules/nucleus), the selective rise in chromatin-associated receptors ensured that almost 60% of chromatin sites remained occupied. GR associated with prostate nuclear structures in a similar manner to AR, and exogenous GR bound saturably and with high affinity (Kd 100 pmol/1) to a similar number of sites as did AR. Both steroid receptors apparently competed for the same sites. In DNA—cellulose competition analyses, synthetic oligonucleotides containing glucocorticoid response elements or putative androgen response elements competed similarly against immobilized non-specific DNA for both AR and GR. In view of these data and information from other sources, it is probable that the role of GR in the prostate should be assessed with a view to understanding its action under conditions of androgen deprivation.
The gene for the β subunit of porcine LH: clusters of GC boxes and CACCC elements
Authors: Ezashi T. , Hirai T. , Kato T. , Wakabayashi K. , Kato Y.
Published: December 2008

The gene for the β subunit of porcine LH (LH-β) was cloned from a genomic library constructed in EMBL3. The nucleotide sequence was determined for the entire gene transcriptional unit of porcine LH-β in addition to 1277 and 372 bp of the 5′- and 3′-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the LH-β gene is present as a single copy. The transcriptional unit of porcine LH-β spanned 1107 bp and contained three exons interrupted by two introns of 326 and 289 bp. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues −16/−15 and +41/+42 were highly conserved in the rat, human and bovine LH-β genes. In the 5′-flanking region, one TATA box and two CCAAT boxes were present. The steroid-responsive element was not found up to 1277 bases upstream of the transcription start site. The potential AP-2 factor-responsive elements appeared nine times within the sequence that was determined, and four of them were located in the 5′-flanking region. Two distal AP-2 elements were arranged in an inverted repeat forming a 16 bp palindromic sequence. This feature suggested that hypothalamic gonadotrophin-releasing hormone stimulates expression of the LH-β gene, predominantly by a signal-transduction system with the protein kinase C cascade and a mediator, the AP-2 factor. A further characteristic feature of the porcine LH-β gene was the presence of clusters of GC boxes and CACCC elements in the 5′-flanking region and the downstream sequence. Co-existence of these regulatory elements with other elements, such as the AP-2 element or CCAAT box, was also found. The porcine LH-β gene shows a structure distinct from the porcine FSH-β and common α genes, which are counterparts of the LH-β gene, reflecting differential control of their synthesis during gametogenesis.
Differential association of endogenous proenkephalin-derived peptides with membranes of microsomes from rat striatum, adrenal medulla and heart ventricle
Authors: Vindrola O. , Ase A. , Aloyz R. , Saravia F. , Finkielman S. , Nahmod V. E.
Published: December 2008

Proenkephalin-derived peptides, in common with other prohormones, are associated with membranes of microsomes and secretory granules in the bovine adrenal medulla. Post-translational processing of the precursor molecule varies depending upon the tissue. The relationship between post-translational events in different tissues was examined by studying the membrane association of endogenous proenkephalin-derived peptides in the crude microsomal fraction of rat adrenal medulla, brain striatum and heart ventricle. [Met]-Enkephalin and synenkephalin (proenkephalin(1–70)) immunoreactivities were quantified by radioimmunoassay after sequential enzymatic digestion with trypsin and carboxypeptidase B. Between 60 and 75% of total immunoreactive peptides present in intact microsomes of the three tissues were associated with membranes and specifically released with 2 m KSCN (pH 7·4). Analysis of the chromatographic profile of materials present in the soluble and associated fractions produced the following results. In the three tissues the materials associated with microsomal membranes corresponded to peptides larger than 3–5 kDa and displayed synenkephalin and [Met]-enkephalin immunoreactivity. Adrenal and heart microsomes showed a continuous pattern of membrane-associated proenkephalin-derived peptides of high, intermediate and low molecular weights containing the synenkephalin and [Met]-enkephalin sequences. These tissues, however, presented quantitative differences, as the highest concentrations belonged to materials larger and smaller than 12·5 kDa in adrenal and heart microsomes respectively. On the other hand, brain striatal microsomes displayed a discontinuous pattern of associated materials, with the absence of some products of high and intermediate molecular weight. Only in the soluble fraction of striatal microsomes were peptides detected of high and intermediate molecular weight containing the [Met]-enkephalin but not the synenkephalin sequence.

In conclusion, this study demonstrates that the association of proenkephalin-derived peptides with microsomal membranes is a common event in the adrenal medulla, heart ventricle and rat striatum, involving, in all cases, some portion of the C-terminal sequence of the synenkephalin molecule. These observations provide further evidence suggesting that the differential profiles of proenkephalin-derived peptides in the adrenal medulla and striatum may be related to differential post-translational processing instead of changes in processing rate.
Effect of testosterone on regulation of the level of sex steroid-binding protein mRNA in monkey (Macaca fascicularis) liver
Authors: Kottler M. L. , Dang C. D. , Salmon R. , Counis R. , Degrelle H.
Published: December 2008

Two specific oligonucleotide probes complementary to different regions of human sex steroid-binding protein (SBP) cDNA were used to study the levels of hepatic monkey SBP mRNA during different hormonal states. In females the SBP mRNA level was higher than in males and paralleled the serum SBP level. After castration, the SBP concentration increased in the serum but was reduced after testosterone treatment. In contrast, the hepatic SBP mRNA level decreased after castration and was restored by testosterone treatment. These results suggest a high homology of the nucleotide sequence between human and monkey SBP mRNAs. The changes in liver SBP mRNA levels may explain the sex difference in plasma SBP concentrations, but mechanisms other than the regulation of transcription may regulate the plasma concentration in monkeys.
Parathyroid hormone-related peptide in normal human fetal development
Authors: Moniz C. , Burton P. B. J. , Malik A. N. , Dixit M. , Banga J. P. , Nicolaides K. , Quirke P. , Knight D. E. , McGregor A. M.
Published: December 2008

Parathyroid hormone-related peptide (PTHrP) has been detected in fetal serum and amniotic fluid. Using a combination of immunocytochemistry and molecular biology we have detected the peptide and its mRNA in a variety of fetal tissues throughout gestation. Tissue-specific mRNA isoforms were observed, the pattern of hybridization of which changed throughout gestation. In addition, the intensity and pattern of immunocytochemical localization of the peptide was found to vary over the time-period studied (8∓30 weeks). PTHrP is expressed by a variety of tumours associated with the syndrome of humoral hypercalcaemia of malignancy and probably accounts for the hypercalcaemia by virtue of its limited amino acid homology with parathyroid hormone. These data demonstrate for the first time that PTHrP, a tumour-related peptide, is expressed during normal human fetal development, and suggest the possibility that it may function to regulate fetal calcium balance and growth in utero.
Proinsulin processing in electrically permeabilized rat islets of Langerhans
Authors: Slee D. J. , Jones P. M. , Howell S. L.
Published: December 2008

Proinsulin conversion to insulin was studied using electrically permeabilized rat islets of Langerhans. Using high-performance liquid chromotography separation of radiolabelled islet proteins, we have demonstrated that conversion was dependent upon temperature, sensitive to pH and regulated by MgATP. Ammonium acetate was used to collapse the granular pH gradient, over a pH range of 3·5–7·4. Conversion was optimum at pH 4·5–5·5 and was reduced, but not abolished, at pH 7·4. Ca2+ (10 μm) and 4β-phorbol 12-myristate 13-acetate (500 nm), which are insulin secretagogues in permeabilized islets, caused no significant stimulation of conversion.
β-Adrenergic regulation of β-actin mRNA abundance in mouse parotid glands by a post-transcriptional mechanism
Authors: Roberts S. G. E. , Cope G. H. , McDonald C. J.
Published: December 2008

In the first 24 h after a single injection of the β-adrenergic agonist isoprenaline to mice, the level of β-actin mRNA in the parotid glands increased significantly above that observed in untreated mice. The increase was transient, reaching 11 times the normal level 18 h after treatment and declining thereafter. Repeated daily doses of isoprenaline did not result in any further increase in β-actin mRNA. Nuclear transcription experiments showed that there was no increase in the transcription rate of the β-actin gene 8 h after an injection of isoprenaline, although β-actin mRNA levels were increasing at this time. Immunoblotting revealed an increase in β-actin protein in parotid gland samples after isoprenaline treatment, although the increase was not to the same extent as the mRNA, perhaps indicating that degradation of β-actin had also increased. Using immunocytochemistry it was found that β-actin was located mainly in the apical cortex of the normal acinar cell. There was a significant decrease in cortical β-actin 24 h after isoprenaline treatment, suggesting that the β-actin was under the process of redistribution.

From these data we propose that isoprenaline caused an increase in β-actin synthesis by a post-transcriptional mechanism and a redistribution of β-actin in preparation for the well-known subsequent change in morphology and function of the parotid glands.
Cloning of the inhibin/activin βB subunit gene from the Booroola Merino sheep
Authors: Rodgers R. J.
Published: December 2008

The gene encoding the inhibin/activin βB subunit was isolated from a Booroola Merino ewe which had two copies of the unidentified fertility gene. Both the DNA sequence (>87%) and amino acid sequence (>93%) were highly homologous to those of the human and rat genes. The upstream region of the gene was similar to that of the human and rat genes, having SP1 binding sites and no TATA or CAAT boxes and, like that of the rat, but unlike that of the human gene, no cyclic AMP response elements, suggesting that the βB gene of sheep and rats is regulated differently from that of man.