bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of arginine vasopressin mRNA in rat fetal hypothalamic cell culture. Role of protein kinases and glucocorticoids
Authors: Hu S-B , Tannahill L A , Lightman S L 
Published: December 2008

ABSTRACT
Studies have been performed to investigate the regulation of arginine vasopressin (AVP) mRNA expression in fetal hypothalamic cultures. AVP mRNA-positive neurones were identified by in-situ hybridization histochemistry, and changes in mRNA expression were quantitated by nuclease protection assay. Both protein kinase C and protein kinase A activators increased the expression of AVP mRNA, in contrast to dexamethasone, which inhibited the responses to both protein kinase C and protein kinase A activation.
Developmental expression of the relaxin gene in the porcine corpus luteum
Authors: Bagnell C A , Zhang Q , Ohleth K , Connor M L , Downey B R , Tsang B K , Ainsworth L 
Published: December 2008

ABSTRACT
Northern analysis and in-situ hybridization were used to follow the development of relaxin gene expression in the newly forming corpus luteum (CL) after ovulation and throughout luteal development. Alkaline phosphatase (AP) was used as a marker of theca-derived lutein cells and the relationship between AP-positive and relaxin mRNA-containing cells was assessed. Ovaries from prepubertal pigs treated with pregnant mares serum gonadotrophin (PMSG)/human chorionic gonadotrophin (hCG) were collected during the periovulatory period and at various times during 19 days after ovulation. In addition, CL from cyclic pigs on days 10 and 16 were used to monitor relaxin gene expression in small and large luteal cells. Northern analysis revealed that relaxin gene expression increased with CL development in the PMSG/hCG-treated pig, reaching maximal levels at around day 14 post-ovulation. Thereafter, as the CL regressed, the level of relaxin mRNA declined. In CL from cyclic pigs at day 10 of the cycle, only small luteal cells expressed relaxin mRNA. However, by day 16 of the cycle, large luteal cells were the source of relaxin gene expression. In-situ hybridization studies revealed that in the early CL (up to 30 h post-ovulation), the relaxin gene transcript was observed in cells along the margins of the CL and in the core of the infolding follicle wall corresponding to the AP-positive, luteinized theca cell layer. As luteinization progressed, the theca and granulosa cell layers could no longer be distinguished morphologically (from 54 h after ovulation until day 9). However, the pattern of relaxin hybridization persisted along the periphery in bands of cells penetrating the CL, and coincided with areas of AP staining, indicating that the theca lutein cells were the site of relaxin gene expression. At day 14, relaxin hybridization and AP staining were distributed throughout the luteal tissue. With CL regression both AP staining and relaxin hybridization declined. This pattern of relaxin hybridization in the CL of the gonadotrophin-primed pig was identical to that observed in cyclic pigs on days 10 and 16 of the cycle. These findings indicate that theca interna cells retain their ability to express the relaxin gene following ovulation and luteinization. In the early CL, the small theca-derived lutein cells are the source of relaxin transcript. However, as the CL becomes fully differentiated, the large granulosa-derived lutein cells acquire the capacity to express the relaxin message.
Identification of a functional role for the 3′ region of the human oestrogen receptor gene
Authors: Keaveney M , Parker M G , Gannon F 
Published: December 2008

ABSTRACT
A well-conserved feature of the steroid receptor gene family is the presence of an exceptionally long 3′ untranslated region (UTR). Analysis of this sequence from the human oestrogen receptor (hER) gene showed the presence of a number of AT-rich regions that included thirteen repeats of the ATTTA motif, an element known to have a destabilizing effect in other systems. In the region 3′ of the gene there were a further eight copies of this pentamer. Also located in this sequence were two members of the Alu repetitive family in inverse orientation and in a tandem arrangement. Transfection experiments in which the 3′ UTR and 3′ flanking sequence were included in chloramphenicol acetyltransferase expression vectors revealed a large destabilization effect with several different fragments. This inherent instability appears to be determined by the primary nucleotide sequence but may act in conjunction with other factors. This posttranscriptional regulatory mechanism may contribute to the control of the level of the hER mRNA.
Human chorionic gonadotrophin (CG)-induced down-regulation of the rat luteal LH/CG receptor results in part from the down-regulation of its synthesis, involving increased alternative processing of the primary transcript
Authors: Lakkakorpi J T , Pietilä E M , Aatsinki J T , Rajaniemi H J 
Published: December 2008

ABSTRACT
To elucidate the molecular mechanisms involved in the homologous regulation of LH/chorionic gonadotrophin (CG) receptors, the receptor and its mRNA levels were analysed in the same pseudopregnant rat ovarian samples after human (h)CG-induced down-regulation using a binding assay, ligand blotting, immunoblotting and Northern blotting together with the polymerase chain reaction (PCR). Treatment of the animals with 500 IU hCG resulted in a loss of 125I-labelled hCG binding and the 90 kDa receptor on the ligand and immunoblots within 12 and 24 h respectively, followed by a transient partial recovery on days 4 and 5, while a distinct decline occurred only on day 7 in the controls. Northern blots of total ovarian RNA, as probed with a 293 bp AvaI/HindIII fragment from the extracellular domain of PCR-generated full-length rat LH/CG receptor cDNA, revealed six major mRNAs of 7·0, 4·2, 2·8, 2·0, 1·4 and 1·1 kb. The 4·2 kb mRNA, which was the most abundant, possibly encodes the 90 kDa receptor, while the smaller species probably represent alternatively spliced forms of the LH/CG receptor pre-mRNA, as also supported by the finding that PCR produced three cDNA bands of 2·1, 2·0 and 1·8 kb when oligomers derived from the N and C termini of rat LH/CG receptor cDNA were used as primers and rat ovarian total RNA as a template. Treatment with hCG led to the down-regulation of all six mRNAs in a fashion parallel to the changes in receptor protein. No smaller receptor components capable of binding radiolabelled hCG or receptor antibody appeared on the ligand or immunoblots prior to or during down-regulation or the subsequent transient period of up-regulation, suggesting that the smaller mRNA species are translated in minute amounts in vivo or are not translated at all. Laser densitometric analysis of the Northern blots revealed that the amounts of the four smallest mRNA species increased during the period of down-regulation in relation to the 4·2 kb mRNA, and correspondingly decreased during the subsequent period of up-regulation, indicating changes in the alternative splicing of the primary transcript. The data suggest that hCG-induced transient down-regulation of the LH/CG receptor results in part from down-regulation of its mRNA levels, and that changes in alternative processing of the receptor pre-mRNA may play a regulatory role in the expression of functional LH/CG receptor during down- and up-regulation.
Changes in progesterone and oestrogen receptor mRNA and protein during maternal recognition of pregnancy and luteolysis in ewes
Authors: Ott T L , Zhou Y , Mirando M A , Stevens C , Harney J P , Ogle T F , Bazer F W 
Published: December 2008

ABSTRACT
This study characterized changes in levels of mRNA and protein for endometrial oestrogen receptors (ERs) and progesterone receptors (PRs) during luteolysis and maternal recognition of pregnancy. For cyclic and pregnant ewes, endometrium was collected on days 10, 12, 14, or 16 post-oestrus (4 ewes/day for each status) for the measurement of ER and PR mRNA and protein. The amount of receptor mRNA is expressed in relative units above background, measured from radiographs of dot-blot hybridization of total endometrial RNA with ER and PR cDNAs. At hysterectomy, jugular vein blood samples were collected and assayed for progesterone, total corpus luteum weight was recorded and, in vitro, endometrial oxytocin-stimulated inositol phosphate formation was estimated. In pregnant ewes, plasma progesterone increased gradually between days 10 and 16 (P<0·01), corpus luteum weight was stable at approximately 08 g and oxytocin did not stimulate endometrial formation of inositol phosphates in vitro. In contrast, in cyclic ewes, plasma progesterone decreased from day 10 to day 16 (P<0·01), corpus luteum weight decreased after day 14 to approximately 0·48 g (P=0·05) and oxytocin stimulated an increase of approximately 1300% in the endometrial formation of inositol phosphates on day 16. cDNAs specifically hybridized with 1·6 and 31 kb transcripts for PR mRNA and a 6·5 kb transcript for ER mRNA. In cyclic ewes, the amount of PR mRNA increased from day 10 to maximum levels on days 14–16. The number of PRs decreased from day 10 (225 pmol/mg DNA) to day 12 (0·98 pmol/mg DNA) and then increased from day 14 to day 16 (2·8 pmol/mg DNA). In pregnant ewes, PR mRNA levels were greatest on days 10–12 and decreased by approximately 50% by day 16. In contrast, the number of PRs was relatively unchanged from day 10 to day 16 (1·53 to 103 pmol/mg DNA). In cyclic ewes, the amount of ER mRNA was lowest at day 10 and increased fivefold by day 16. The number of ERs remained relatively unchanged from day 10 to day 14 (607 pmol/mg DNA) and increased by day 16 (1612 pmol/mg DNA). In pregnant ewes, ER mRNA decreased by approximately 80% from day 12 to day 16. Similarly, the number of ERs decreased from day 10 to day 16 (5·41 to 205 pmol/mg DNA). Correlations between ER mRNA and PR mRNA (r=0·68), ERs and PRs (r = 0·50) and ER mRNA and ERs (r=0·50) were high (P<0·01). PR mRNA and PRs, PR mRNA and ERs, and ER mRNA and PRs were not correlated (P>0·1). Pregnancy had the apparent effect of stabilizing the number of endometrial PRs and inhibiting ER production by decreasing both the amount of ER mRNA and ER protein.
Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland
Authors: Sellar R E , Taylor P L , Lamb R F , Zabavnik J , Anderson L , Eidne K A 
Published: December 2008

ABSTRACT
We have isolated the TRH receptor (TRH-R) from a rat anterior pituitary cDNA library, determined its sequence and examined its functional characteristics. Expression studies were carried out in Xenopus oocytes and in COS-7 cells. Microinjection of sense RNA transcripts into Xenopus oocytes showed electrophysiological responses of between 800 and 1000 nA under voltage-clamp conditions. COS-7 cells were transiently transfected with the cDNA clone under the control of a cytomegalovirus promoter and inositol phosphate (IP) measurements carried out. In TRH-R transfected cells, TRH (100 nm) produced an approximately twofold increase in total IP production. In-situ hybridization in the rat anterior pituitary revealed a heterogeneous distribution of label, a characteristic pattern of TRH-R expression. The rat 3·3 kb insert coded for a protein of 411 amino acids compared with 393 for the mouse protein. Over its length, the rat TRH-R protein showed considerable homology with that of the mouse, except for a deletion of 232 bp in the 3′-coding region. This deletion did not appear to affect the functional characteristics of the receptor, as shown by electrophysiological studies with Xenopus oocytes and by transfection of the cDNA into COS-7 cells. The sequence given for the 3′-untranslated region is 1·5 kb longer than that reported for the mouse receptor, and extends to the poly(A) tail.
Localization of inhibin/activin subunit mRNAs during the luteal phase in the primate ovary
Authors: Fraser H M , Lunn S F , Cowen G M , Saunders P T K 
Published: December 2008

ABSTRACT
Localization of inhibin/activin subunit mRNAs within the macaque ovary from the immediate pre-ovulatory period of the menstrual cycle, when serum immunoreactive inhibin begins to rise, to day 9 of the luteal phase, when serum inhibin concentrations are maximal, was investigated using in-situ hybridization. Ovaries were studied on the day of the LH surge (day 0) and on days 2, 5, and 9 of the luteal phase by hybridizing frozen tissue sections with radio-labelled riboprobes specific to the inhibin/activin α-, βA-and βB-subunits. After autoradiographic exposure for 10 and 21 days, grain concentrations were quantified by image analysis. Moderate expression of α-, βA- and βB-subunit mRNA was present within the granulosa cells of the pre-ovulatory follicle (day 0). The granulosa-lutein cells of the corpora lutea expressed high levels of α-subunit at days 2, 5 and 9. mRNAs for βA and βB were detected at low but significant levels in all of the corpora lutea. All healthy antral follicles exhibited a high level of expression of βB-subunit mRNA in the granulosa cells. On day 2 after ovulation these follicles also expressed high α- and moderate βA-subunit mRNA. On day 9 the βB-inhibin mRNA in antral follicles was found in association with low expression of the other subunits. Small follicles in ovaries on day 2 expressed moderate α- and low levels of βB-subunit mRNA, while mRNA for βA was absent. α-subunit mRNA expression was present on day 5 while neither βA- nor βB-subunit mRNA was detected. On day 9 a proportion of small follicles expressed α- and βA-subunit mRNA. These results demonstrate that marked differences are present in the levels of expression of the three inhibin/activin subunit genes between follicles and the corpus luteum. The predominance of the βB-subunit mRNA within antral follicles would be consistent with the synthesis of activin. The predominance of the α-subunit combined with the low expression of the β-subunits in the corpus luteum suggests that both biologically active inhibin and free α-subunit are produced by the primate corpus luteum.
Molecular cloning and characterization of the cyclic AMP-responsive ovine CYP11A1 (cholesterol side-chain cleavage) gene promoter: DNase 1 protection of conserved consensus elements
Authors: Pestell R G , Hammond V E , Crawford R J 
Published: December 2008

ABSTRACT
DNA elements governing transcription of the ovine cytochrome P-450 side-chain cleavage (CYP11A1) gene were investigated. Three overlapping genomic clones for the ovine CYP11A1 gene were isolated and characterized. The transcriptional start site was located 51 nucleotides upstream from the initiating methionine. Gene transfer experiments were conducted in murine adrenocortical Y1 cells and human choriocarcinoma JEG-3 cells using chloramphenicol acetyltransferase reporter gene constructs containing promoter fragments from −2700 to −177 bp.
The results demonstrate that DNA elements sufficient to convey a basal level of expression and cyclic AMP (cAMP) responsiveness lie within 177 bp of the transcriptional start, although the possibility that additional regulatory elements reside outside this 177 bp has not been excluded. The ovine 5′ flanking sequence demonstrated 92% homology with the bovine sequence, extending over the entire fragment. In contrast, only four significant regions of conservation between the ovine, murine, rat and human CYP11A1 promoters were found. These regions are positioned within 200 bp upstream of the transcriptional start site.
DNase 1 footprinting was performed to identify DNA elements able to bind nuclear proteins. Primary adrenocortical and placental tissues from sheep were used as the source of nuclear extracts to detect DNA-protein interactions relevant to CYP11A1 gene expression in vivo. Five regions of protection were detected in the first −634 bp of the ovine CYP11A1 promoter. Three of these elements corresponded to the regions which are well-conserved between species. The other two elements resembled activating protein-1 (AP-1) and AP-4 sites and overlapping AP-2/Sp1 sites, and are conserved in the bovine gene but not in other species.
Nuclear protein extracts from adrenals of sheep with different serum ACTH levels (i.e. ACTH-treated, dexamethasone-treated and untreated sheep) protected similar regions of the ovine CYP11A1 promoter fragment. Similarly, the regions protected did not differ when nuclear protein from JEG-3 cells treated with cAMP was compared with that of untreated JEG-3 cells. These results suggest that induction of CYP11A1 gene transcription by ACTH in the ovine adrenal and by cAMP in JEG-3 cells in culture is not mediated by changes in binding of the proteins that interact directly with these footprinted elements.
The elements footprinted by extracts from primary ovine tissue lie within the 177 bp sufficient for cAMP-regulated expression. The correspondence of these elements either to regions conserved between species or to known consensus binding sites suggests that these sequences are cis elements involved in regulating transcription of the ovine CYP11A1 gene in vivo.
Endocrine and cardiac paracrine actions of insulin-like growth factor-I (IGF-I) during thyroid dysfunction in the rat: is IGF-I implicated in the mechanism of heart weight/body weight change during abnormal thyroid function?
Authors: Thomas M R , Miell J P , Taylor A M , Ross R J M , Arnao J R , Jewitt D E , McGregor A M 
Published: December 2008

ABSTRACT
Thyroid hormones are essential for the normal growth and development of many tissues. In the rat, hypothyroidism is associated with growth impairment, and hyperthyroidism with the development of a hypercatabolic state and skeletal muscle wasting but, paradoxically, cardiac hypertrophy. The mechanism by which thyroid hormone produces cardiac hypertrophy and myosin isoenzyme changes remains unclear. The role of IGF-I, an anabolic hormone with both paracrine and endocrine actions, in producing cardiac hypertrophy was investigated during this study in hyperthyroid, hypothyroid and control rats. A treated hypothyroid group was also included in order to assess the effect of acute normalization of thyroid function.
Body weight was significantly lower in the hyperthyroid (mean±s.e.m.; 535·5±24·9 g, P<0·05), hypothyroid (245·3±9·8 g, P<0·001) and treated hypothyroid (265·3±9·8 g, P<0·001) animals when compared with controls (618·5±28·6 g). Heart weight/body weight ratios were, however, significantly increased in the hyperthyroid (2·74 ± 0·11×10−3, P<0·01) and treated hypothyroid (2·87±0·07 ×10−3, P<0·001) animals when compared with controls (2·26±0·03 × 10−3). Serum IGF-I concentrations were similar in the control and hyperthyroid rats (0·91±0·07 vs 0·78±0·04 U/ml, P=0·26), but bioactivity was reduced by 70% in hyperthyroid serum, suggesting a circulating inhibitor of IGF. Serum IGF-I levels (0·12±0·03 U/ml, P<0·001) and bioactivity (0·12±0·04 U/ml, P<0·001) were significantly lower in the hypothyroid group. Liver IGF-I mRNA levels were not statistically different in the control and hyperthyroid animals, but were significantly reduced in the hypothyroid animals (P<0·05 vs control). Heart IGF-I mRNA levels were similar in the control and hypothyroid rats, but were significantly increased in the hyperthyroid and treated hypothyroid animals (increased by 32% in hyperthyroidism, P<0·05; increased by 57% in treated hypothyroidism, P<0·01). Cardiac IGF-I was significantly elevated in hyperthyroidism (0·16±0·01 U/mg heart tissue, P<0·01), was low in hypothyroidism (0·08±0·01 U/mg, P<0·01) and was normalized in the treated hypothyroid group (0·11 ± 0·01 U/mg vs control, 0·13±0·01 U/mg).
Low body mass during both hypothyroidism and hyperthyroidism is therefore associated with reduced systemic IGF bioactivity. In hypothyroidism there is a primary defect in the endocrine function of IGF-I, while in hyperthyroidism serum IGF bioactivity is reduced in the presence of normal endocrine production of this anabolic hormone. In contrast, the paracrine actions of IGF-I are increased in the heart during hyperthyroidism, and this hormone appears to play a part in the development of hyperthyroid cardiac hypertrophy.
Expression of human thyroid peroxidase in the yeasts Saccharomyces cerevisiae and Hansenula polymorpha
Authors: Wedlock N , Furmaniak J , Fowler S , Kiso Y , Bednarek J , Baumann-Antczak A , Morteo C , Sudbery P , Hinchcliff A , Smith B Rees 
Published: December 2008

ABSTRACT
Saccharomyces cerevisiae and the methylotrophic yeast Hansenula polymorpha have been used to express both full-length and a large hydrophilic domain of human thyroid peroxidase (TPO). Expression of TPO in S. cerevisiae, using the natural signal sequence or the yeast α-mating factor (MFα) signal sequence, resulted in undetectable or very low levels of recombinant TPO production. However, TPO was expressed when the natural TPO leader sequence was replaced by the yeast STE2 signal sequence. This recombinant TPO reacted with both rabbit anti-human TPO polyclonal and mouse anti-human TPO monoclonal antibodies on Western blots. In the case of H. polymorpha, TPO expression was achieved when the natural TPO leader sequence was replaced by the MFα leader and the construct placed under the control of the methanol-regulated promoter from the methanol oxidase gene. The recombinant TPO produced in H. polymorpha reacted with both TPO polyclonal and TPO monoclonal antibodies. No TPO was produced when the signal sequence of SUC2 (invertase) or the TPO natural signal sequence was used to direct expression.
Lysine residues 2 and 104 of the human chorionic gonadotrophin β subunit influence receptor binding
Authors: Xia H , Huang J , Chen T-M , Puett D 
Published: December 2008

ABSTRACT
Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common α subunit and a hormone-specific β subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian β subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-β. Using site-directed mutagenesis, each of these two lysines in hCG-β was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine α subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine α subunit bound to hCG-β (Glu2) and to hCG-β (Glu104), as well as with the control, i.e. the bovine α subunit bound to the hCG-β wild-type subunit. In two in-vitro assays, one a competitive binding assay with 125I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant β subunit exhibited bioactivity, but their potencies were lower than that of the bovine α subunit bound to the hCG-β wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-β participate, either directly or indirectly, in receptor binding.
Increased tachykinin receptor gene expression in asthmatic lung and its modulation by steroids
Authors: Adcock I M , Peters M , Gelder C , Shirasaki H , Brown C R , Barnes P J 
Published: December 2008

ABSTRACT
Substance P has several inflammatory effects on the airways mediated via neurokinin 1 receptors (NK1Rs) and, if released from sensory nerves, may amplify the chronic inflammation seen in asthma. Northern blot analysis of NK1R mRNA in lung showed a 52 ± 10% (s.e.m.; P<0·01) increase in mRNA in the asthmatic lung compared with non-asthmatic control tissue. NK1R mRNA was reduced by 84·5 ± 1·9% after incubation with dexamethasone (1 μm) for 3 h (P<0·01). In contrast, NK2R mRNA was unaltered in asthmatic lungs and dexamethasone treatment had no effect on the level of NK2R mRNA. These results suggest that chronic inflammation in asthma may result in increased NK1R gene expression and that this effect is reversed by glucocorticosteroids.