bluuebunny/crossref_metadata_2025 huggingface dataset:
Gestational- and labour-associated changes in the relative abundance of prostaglandin G/H synthase-1 and -2 mRNA in ovine placenta
Authors: Rice G E , Freed K A , Aitken M A , Jacobs R A
Published: December 2008

The aim of this study was to establish the gestational- and labour-associated variation in the relative abundance of prostaglandin synthase-1 (PGHS-1) and prostaglandin synthase-2 (PGHS-2) mRNA in ovine placenta (cotyledons). Cotyledons were collected from non-labouring ewes at 40–145 days of gestation (n=25) and from ewes in active labour (145–147 days, n=5). The relative abundance of PGHS-1 and PGHS-2 mRNA transcripts was determined by Northern blot analysis and laser densitometry, using a 2·3 kb sheep and a 1·2kb mouse cDNA probe respectively. Data were expressed as a ratio of PGHS transcript hybridization/18S rRNA hybridization. During pregnancy, the relative abundance of PGHS-2 mRNA increased sevenfold, from 0·19±0·04 at 40–85 days (n=5) to 1·39±0·05 at 140–145 days (n=4) (P<0·01). PGHS-1 mRNA relative abundance did not change significantly (P>0·05) during gestation. Neither PGHS-1 nor PGHS-2 mRNA relative abundance changed significantly in association with labour onset at term (n=5) when compared with the relative abundance observed at 140–145 days (n=4) (P>0·05). The data obtained in this study are consistent with the hypothesis that PGHS-1 is constitutively expressed in ovine placenta during pregnancy and at the time of labour, and that PGHS-2 is induced during the second half of pregnancy. It remains to be established to what extent these two isozymes contribute to the net prostaglandin-forming capacity of the ovine placenta, particularly at the time of labour.
The relationship between decidualization and prolactin mRNA and production at different stages of human pregnancy
Authors: Wu W-X , Brooks J , Glasier A F , McNeilly A S
Published: December 2008

Within the human utero-placental unit only decidualized stromal cells express mRNA for prolactin. However, it is not clear if the level of prolactin production is related to the number of decidualized cells or the capacity of individual decidual cells to synthesize prolactin, either or both of which parameters may change during pregnancy. In the present study, prolactin production at different stages of human pregnancy was examined using quantitative in situ hybridization to assess decidual prolactin mRNA abundance, immunocytochemistry to examine the prolactin content inside decidual cells and RIA to measure decidual prolactin output into amniotic fluid. Throughout pregnancy the proportion of stromal cells showing positive immunostaining and mRNA for prolactin increased. There was a parallel increase in decidual cell size which was correlated with an increase in prolactin gene expression and intensity of immuno-staining for prolactin in individual decidual cells. These changes in decidual cells were consistent with the changes in the concentration of prolactin in amniotic fluid. These results suggest that there is a close link between the level of prolactin gene expression and production of prolactin by individual decidual cells, which in turn is directly related to the process of decidualization that continues throughout human pregnancy.
Influence of promoter and signal peptide on the expression and secretion of recombinant porcine LH extracellular domain in baculovirus/lepidopteran cells or the caterpillar system
Authors: Bozon V , Remy J-J , Pajot-Augy E , Couture L , Biache G , Severini M , Salesse R
Published: December 2008

Overexpression of the porcine LH receptor (pLHR) ectodomain has been achieved using the baculovirusinsect cell system but mostly in an aggregated form with no secretion. In order to carry this out, new baculoviruses were selected to produce the pLHR ectodomain in insect Sf9 cells and caterpillars. In pLHR-P10–297 and pLHR-mel-319 baculoviruses, pLHR cDNA was under the control of the P10 promoter and the polyhedrin gene promoter respectively.

The constructs contained either the porcine signal peptide (pLHR-P10–297) or the insect signal peptide of melittin (pLHR-mol-319). Infected cells produced 1 × 105−3 × 105 receptors/cell 3 days after infection. The recombinant LH receptor ectodomains produced were secreted in a biologically active form and bound the hormone with high affinity. Infected caterpillars produced a larger amount of active pLHR ectodomain than insect cells. The products were not secreted into the haemolymph however.

Promoter and/or signal peptide modifications therefore enabled pLHR recombinant ectodomain secretion in a biologically active form, using the baculovirus—lepidopteran cell system. Moreover, moderate levels of expression seem to allow the production of biologically active ectodomain.
Transcriptional induction of rat mineralocorticoid receptor gene in neurones by corticosteroids
Authors: Castrén M , Trapp T , Berninger B , Castrén E , Holsboer F
Published: December 2008

We investigated the mechanisms by which corticosteroids regulate the expression of the mineralocorticoid receptor (MR) in neurones. Aldosterone and dexamethasone produced a dose-dependent increase of MR mRNA levels in cultured primary hippocampal neurones. Transient transfection of neuroblastoma cells showed that corticosteroids directly activate the rat MR promoter, indicating that the steroid-induced increase in the MR mRNA concentration is at least partially transcriptional. Progressive 5′ deletions of the MR promoter sequence revealed that the promoter induction cannot be assigned to a single element. An oligonucleotide comprising a consensus half-glucocorticoid responsive element located at – 319 bp in the MR promoter stimulated the corticosteroid-induced activation of the heterologous promoter. Cloning three of these enhancers in tandem greatly potentiated the responses to glucocorticoids and mineralocorticoids, suggesting that although this element is a weak enhancer it can, in combination with other enhancer elements, induce MR gene expression by both types of corticosteroid receptors.
Development of cytochrome P450 aromatase mRNA levels and enzyme activity in ovaries of normal and hypogonadal (hpg) mice
Authors: Gray S A , Mannan M A , O'Shaughnessy P J
Published: December 2008

The cytochrome P450 aromatase (P450arom) enzyme is required for bioconversion of androgen to oestrogen. In this study ovarian P450arom mRNA and enzyme activity have been measured during development in normal mice and hypogonadal (hpg) mice which lack circulating gonadotrophins. A semi-quantitative reverse transcription-PCR (RTPCR) technique was used to measure cytochrome P450arom mRNA levels and aromatase enzyme activity was measured directly. Using RT-PCR, P450arom mRNA was detectable in the adult mouse ovary and also in the uterus, kidney, brain and skeletal muscle but not in cardiac smooth muscle. In the normal mouse, P450arom mRNA was detectable in the ovary on the day of birth (day 1) and levels increased significantly up to day 15 with the most marked changes seen between days 1 and 5. Aromatase activity was also detectable at all ages in the ovary and increased significantly between days 1 and 7. In ovaries from [ill] mice, normal levels of P450arom mRNA were present on day 1 but there was no significant change in P450arom mRNA at later ages up to day 15. These results show that in the newborn mouse ovary, which contains only primordial follicles, there is a basal expression of P450arom mRNA which is not gonadotrophindependent. After 1 day, however, gonadotrophins are required for normal expression of ovarian P450arom and this coincides with development of primary and secondary follicles.
A 33 kDa Pit-1-like protein binds to the distal region of the human thyrotrophin α-subunit gene
Authors: Kim D S , Yoon J H , Ahn S K , Kim K E , Seong R H , Hong S H , Kim K , Ryu K , Park S D
Published: December 2008

Our previous studies demonstrated that at least two DNA regions with upstream limits between positions −223 to −190 and positions −151 to −135 of the human TSH gene are important for transcriptional regulation by TRH in GH3 rat pituitary cells. The proximal region (−151 to −135 bp) including the cAMP-responsive element (CRE) was required for the induction of the TSH gene by TRH, while the distal region (−223 to −190 bp) containing an element similar to the binding site for the pituitary-specific transcription factor, Pit-1, was necessary to amplify the effects of TRH. To determine whether a pituitary-specific nuclear protein, in addition to the CRE-binding protein, is involved in the molecular mechanism of TRH regulation, a gel retardation assay and Southwestern blot analysis were performed on the distal region with GH3 cell nuclear extracts. GH3 extracts generated a distinct DNA—protein complex that was effectively eliminated in the presence of excess unlabelled DNA fragment, and TRH treatment increased the affinity of protein binding remarkably. Excess Pit-1 DNA-binding sequence from the rat prolactin gene inhibited formation of the complex, but mutation of the Pit-1 consensus sequence in the distal region did not eliminate the complex. In addition, Southwestern experiments showed that a 33 kDa nuclear protein present in GH3 cells bound to this region and its binding affinity was increased slightly 2 h after TRH treatment, with the maximal increase (fivefold) at 3 h, which was similar to the results when using gel retardation. Phosphatase treatment of nuclear protein also resulted in a loss of binding affinity. Taken together, these data indicate that the interaction of a pituitary-specific nuclear protein, identical or closely related to Pit-1, with the distal region may be involved in the TRH stimulation of human TSH gene expression.
IGF, type I IGF receptor and IGF-binding protein mRNA expression in the developing mouse lung
Authors: Schuller A G P , van Neck J W , Beukenholdt R W , Zwarthoff E C , Drop S L S
Published: December 2008

The IGFs are important mitogens involved in lung growth and development. The regulation of IGF action depends not only on the expression of IGFs and IGF receptors, but also on the modulation of IGF activity by IGF-binding proteins (IGFBPs). In this study, we describe the mRNA expression of IGF-I, IGF-II, type I IGF receptor, IGFBP-2, IGFBP-4 and IGFBP-5 during mouse lung development as studied by in situ hybridization techniques. The IGF, type I IGF receptor and IGFBP-2, -4 and -5 genes were expressed in developing lung as early as embryonal day 12·5. Expression of IGFBPs-1, -3 and -6 was below detection. IGF and IGFBP-2 mRNAs were expressed both in mesenchymal and epithelial cells. Type I IGF receptor transcripts were also observed throughout the developing lung, with the exception of the epithelial cells of the bronchi after embryonal day 15. Furthermore, mRNA expression of IGFBPs-4 and -5 was noted in neighbouring cell types, and after embryonal day 15, co-expression of the type I IGF receptor and IGFBP-4 transcripts was detected. The observed expression patterns imply that the IGFBP-2, -4 and -5 genes are differentially regulated during embryonic development and suggest that each may have a discrete function. A possible role for IGFBPs-2, -4 and -5 is to participate in the regulation of cell-specific IGF responses during mouse lung development.
Two differing precursor genes for the salmon-type gonadotropin-releasing hormone exist in salmonids
Authors: Ashihara M , Suzuki M , Kubokawa K , Yoshiura Y , Kobayashi M , Urano A , Aida K
Published: December 2008

Salmon gonadotropin-releasing hormone (sGnRH) is considered to have an important role in the control of reproduction in salmonid fish. As a basis for understanding the physiological functioning of sGnRH at the molecular level, we characterized the nucleotide sequences of two types of cDNAs encoding the precursors of sGnRH in sockeye salmon (ss), Oncorhynchus nerka, by a cloning strategy based on reverse transcription-PCR. The two types of cDNAs are referred to as ss-pro-sGnRH-I and -II, and consisted of 435 and 481 bases respectively. Both precursors are predicted to contain a signal peptide, the hormone and a GnRH-associated peptide that is attached to the hormone via a Gly-Lys-Arg sequence. The presence of two types of mRNAs hybridizing with either cDNA was confirmed by Northern blot analysis of brain RNA from sockeye salmon, masu salmon, O. masou, and rainbow trout, O. mykiss. The ss-pro-sGnRH-I cDNA had 97·2% and 82·8% overall identity with sGnRH cDNA from masu salmon and putative sGnRH cDNA deduced from the gene of the Atlantic salmon, Salmo salar respectively, whereas the ss-pro-sGnRH-II cDNA had 80·0% and 91·2% overall identity with the former and the latter respectively. The nucleotide sequences of ss-sGnRH-I and -II cDNAs showed less similarity (79·3%). These results indicated that each salmonid species possesses two differing sGnRH genes. The results of Southern blot analysis using genomic DNA extracted from individuals support this evidence in sockeye salmon, masu salmon and rainbow trout.
Semi-quantitative analysis of interleukin-1α, interleukin-6 and interleukin-8 mRNA expression by human thyrocytes
Authors: Watson P F , Pickerill A P , Davies R , Weetman A P
Published: December 2008

It has been suggested that the thyroid itself may contribute to the inflammatory process observed in autoimmune thyroiditis by releasing the cytokines interleukin-1α (IL-1α), interleukin-6 (IL-6) and interleukin-8 (IL-8), but studies of cytokine gene expression in thyrocytes have been limited and conflicting. A semi-quantitative reverse transcription-PCR technique has been used to investigate the expression of IL-1α, IL-6 and IL-8 mRNA in the human thyroid cell line HTori3 and in cultures of primary human thyroid follicular cells (TFCs). Cytokine mRNA levels were examined over a 24-h period, and the modulatory effects of exogenous IL-1α, interferon-γ (IFN-γ) and TSH investigated. Basal expression of IL-1α, IL-6 and IL-8 mRNA was detected in HTori3 and primary TFC cultures. Stimulation with IL-1 (10 U/ml) for 12 h produced an increase in the level of IL-1α mRNA in both primary TFC and HTori3 cultures. IL-6 and IL-8 mRNA levels were increased by the addition of IL-1 in both cell types, and this effect was detected throughout the 24-h time-course. IFN-γ (100 U/ml) had no significant effect on cytokine gene expression. A higher concentration of IFN-γ (500 U/ml) had no significant effect on the expression of IL-1α or IL-8 but produced an increase in the level of IL-6 mRNA in primary cultures and in HTori3 cells. Addition of TSH (1 mU/ml) produced an increase in the level of IL-1α mRNA in primary TFC and HTori3 cells, at 12 and 24 h. TSH had no significant effect on the expression of IL-6 or IL-8 mRNA. These results demonstrate that human TFCs constitutively express IL-1α, IL-6 and IL-8 mRNA and that this expression can be modulated by IL-1, IFN-γ and TSH.
Characterization of an estrogen-responsive element implicated in regulation of the rainbow trout estrogen receptor gene
Authors: Le Dréan Y , Lazennec G , Kern L , Saligaut D , Pakdel F , Valotaire Y
Published: December 2008

We previously reported that the expression of the rainbow trout estrogen receptor (rtER) gene is markedly increased by estradiol (E2). In this paper, we have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyl transferase (CAT), linked to 5′ flanking regions of the rtER gene promoter, to identify cis-elements responsible for E2 inducibility. Deletion analysis localized an estrogen-responsive element (ERE), at position +242, with one mutation on the first base compared with the consensus sequence. This element confers estrogen responsiveness to CAT reporter linked to both the herpes simplex virus thymidine kinase promoter and the homologous rtER promoter. Moreover, using a 0·2 kb fragment of the rtER promoter encompassing the ERE and the rtER DNA binding domain obtained from a bacterial expression system, DNase I footprinting experiments demonstrated a specific protection covering 20 bp (+240/+260) containing the ERE sequence. Based on these studies, we believe that this ERE sequence, identified in the rtER gene promoter, may be a major cis-acting element involved in the regulation of the gene by estrogen.
Characterization of human relaxin gene regulation in the relaxin-expressing human prostate adenocarcinoma cell line LNCaP.FGC
Authors: Gunnersen J M , Roche P J , Tregear G W , Crawford R J
Published: December 2008

Relaxin is a peptide hormone which is produced in human reproductive tissues including the ovary and prostate gland. Little is known of the molecular events regulating relaxin gene transcription. We have studied this question using gene transfer of relaxin promoter/reporter gene constructs into a relaxin-expressing cell line. A number of human cell lines expressed relaxin as detected by reverse transcription-PCR. In one of these lines, the prostate adenocarcinoma cell line LNCaP.FGC, relaxin mRNA was also detected by Northern blot analysis. The DNA sequences of the proximal 5′-flanking regions (∼900 nucleotides) of the two human relaxin genes, HI and H2, were determined. Deletion constructs containing portions of the 5′-flanking regions of HI and H2 linked to the bacterial chloramphenicol acetyl transferase reporter gene were prepared. The expression of the reporter gene constructs was analysed in the LNCaP.FGC cell line and the results of these transient transfection assays have led to the identification of positive and negative regulatory regions within the 5′-flanking DNA. A difference in activity of the H1 and H2 gene promoters in this prostate cell line was observed, with the H2 promoter being more active. This situation may mimic that occurring in vivo since the relaxin secreted from the prostate gland into seminal fluid is the product of the H2 gene.
Functional analysis of the human pro-opiomelanocortin promoter in the small cell lung carcinoma cell line DMS-79
Authors: Picon A , Leblond-Francillard M , Raffin-Sanson M-L , Lenne F , Bertagna X , de Keyzer Y
Published: December 2008

DMS-79 is a human cell line derived from a small cell lung carcinoma (SCLC), which expresses the pro-opiomelanocortin (POMC) gene. We took it as a model in which to study the mechanism of POMC gene expression in non-pituitary tumours. DMS-79 reproduces the usual characteristics of POMC expression in these tumours: precursor processing is altered and gene expression is essentially unresponsive to glucocorticoids. POMC gene structure appeared normal by Southern blot analysis, indicating that gene rearrangement was not responsible for its expression in DMS-79. Indeed, using transient expression of human POMC—luciferase fusion genes in DMS-79, we showed that (1) the normal human POMC promoter was functional in DMS-79, and (2) the same proximal promoter region (−417;+21) produced the full transcriptional activity in DMS-79 and in the mouse pituitary cell line AtT-20. Progressive 5′ deletion analysis revealed differences between AtT-20 and DMS-79: region (−161;−376) was active in AtT-20 and not in DMS-79, whereas region (−95;−161) was active in both cell lines and (−376;−417) was only active in DMS-79. The latter partially overlaps a motif homologous to the DE-2 rat element which confers the tissue-specific expression of POMC in AtT-20 cells; however, this motif had no effect in DMS-79. These data suggest that POMC gene transcription is achieved through a different set of transacting factors in DMS-79 and AtT-20. Altogether, our results provide evidence that DMS-79 is a valid model of tumours responsible for the ectopic ACTH syndrome and that the mechanism of POMC gene expression in these SCLC cells is different from that in pituitary cells.
A novel action of epidermal growth factor in rat granulosa cells: its potentiation of gonadotrophin action
Authors: Hattori M-A , Yoshino E , Shinohara Y , Horiuchi R , Kojima I
Published: December 2008

It is well known that epidermal growth factor (EGF) induces down-regulation of LH receptors and desensitization to gonadotrophin stimulation in gonadal cells, including granulosa cells. In a previous study we showed that EGF receptor levels in rat granulosa cells were increased up to fourfold after 96 h of culture with human GH in the presence of FSH, and the present study has evaluated the action of EGF on these cells. The induced EGF receptors were identical in size to the pre-existing receptors as assessed by affinity labelling with 125I-EGF. After 48 h in culture, various amounts of EGF (0·5–10 ng) were added and the cells were cultured for a further 48 h. The addition of EGF caused down-regulation of LH receptors in cells expressing high levels of EGF receptors. However, this down-regulation was less than that in control cells. After the cells were washed, cAMP synthesis in response to human chorionic gonadotrophin (hCG) increased by two to three times the control value and this increase was closely correlated with an increase in EGF receptor content. However, stimulation with cholera toxin or forskolin showed no such augmentation, indicating that it may not be due to quantitative alterations in G proteins and their effector systems. Induction of EGF potentiation required long-term exposure to EGF, for at least more than 24 h. In addition, progesterone synthesis was sensitive to stimulation with lower doses of hCG. These findings indicate that the activation of hGH-induced EGF receptors may potentiate gonadotrophin action in granulosa cells.
Pancreastatin: further evidence for its consideration as a regulatory peptide
Authors: Sánchez-Margalet V , Lucas M , Goberna R
Published: December 2008

Pancreastatin is a 49 amino acid peptide first isolated, purified and characterized from the porcine pancreas, and whose biological activity in different tissues can be assigned to the C-terminal part of the molecule. Pancreastatin has a prohormonal precursor, chromogranin A (CGA), which is a glycoprotein present in neuroendocrine cells, including the endocrine pancreas. Both intracellular and extracellular processing of CGA can yield pancreastatin. This processing is tissue-specific, with the pancreatic islet and antral gastric endocrine cells being the major source of fully processed pancreastatin. Most of the circulating CGA is secreted by chromaffin tissue. Therefore, peripheral processing of CGA is probably the major indirect source of pancreastatin. Pancreastatin seems to have a general modulatory control on endocrine (insulin, glucagon, parathormone) and exocrine (pancreatic, gastric) secretion from tissues close to the source of production. This has led to the assumption that pancreastatin may be a peptide with an autocrine and paracrine function. It has recently been revealed to be a peptide with a metabolic function counter-regulatory to insulin action. This effect, in conjunction with the inhibitory effect on insulin and pancreatic exocrine secretion, points to a role in the physiology of stress. The molecular mechanism of the glycogenolytic effect of pancreastatin is better known, although further work is still needed. In general, more studies should be carried out at the molecular level to investigate the mechanism of action of pancreastatin and thus to clarify its physiological role in the neuroendocrine system.
Developmental expression of the cyclo-oxygenase-1 and cyclo-oxygenase-2 genes in the peri-implantation mouse uterus and their differential regulation by the blastocyst and ovarian steroids
Authors: Chakraborty I , Das S K , Wang J , Dey S K
Published: December 2008

Cyclo-oxygenase (COX) is a rate-limiting enzyme that converts arachidonic acid to prostaglandins (PGs) and exists in two isoforms, COX-1 and COX-2. In the rodent, increased uterine vascular permeability at sites of blastocyst apposition is one of the earliest prerequisite events in the implantation process. This event is preceded by generalized uterine edema and luminal closure, and coincides with the initial attachment reaction between the trophectoderm and luminal epithelium. Vasoactive PGs are implicated in these processes. Here we demonstrate that COX genes are differentially regulated in the peri-implantation mouse uterus. During the preimplantation period (days 1–4), the COX-1 gene was expressed in the uterine epithelium mainly on day 4 until the initiation of attachment reaction in the evening after which the expression was downregulated. This COX-1 expression coincides with the generalized uterine edema required for luminal closure. In contrast, the COX-2 gene was expressed in the luminal epithelium and subepithelial stromal cells at the anti-mesometrial pole exclusively surrounding the blastocyst at the time of attachment reaction on day 4 and persisted through the morning of day 5. This uterine gene was not expressed at the sites of blastocyst apposition during progesterone (P4) treated delayed implantation, but was readily induced in the uterus surrounding the activated blastocysts after termination of the delay by estradiol-17β (E2). The results suggest that PG synthesis catalyzed by COX-2 is important for localized increased uterine vascular permeability and attachment reaction. The COX-1 gene that was downregulated from the time of attachment reaction on day 4 was again expressed in the mesometrial and anti-mesometrial secondary decidual beds on days 7 and 8. These results suggest that PGs generated by COX-1 are involved in decidualization and/or continued localized endometrial vascular permeability observed during this period. In contrast, the COX-2 gene, expressed at the anti-mesometrial pole on days 4 and 5, switched its expression to the mesometrial pole from day 6 onward. These results suggest that PGs produced at this site by COX-2 are involved in angiogenesis for the establishment of placenta. In the ovariectomized mice, the COX-1 gene was induced in the epithelium by a combined treatment with P4 and E2. However, P4 and/or E2 treatments failed to influence the uterine COX-2 gene. Overall, the results suggest that the uterine COX-1 gene is influenced by ovarian steroids, while the COX-2 gene is regulated by the implanting blastocyst during early pregnancy.
Developmental and tissue-regulated expression of IGF-I and IGF-II mRNAs in Sparus aurata
Authors: Duguay S J , Lai-Zhang J , Steiner D F , Funkenstein B , Chan S J
Published: December 2008

Recent studies have shown that homologues of the mammalian IGF-I and -II genes are also found in teleosts. We report here the cDNAs coding for IGF-I and IGF-II cloned from the gilthead seabream, Sparus aurata. Sequence comparisons revealed that both IGFs have been well conserved among teleosts, although Sparus IGF-I is shorter by three amino acid residues due to truncated B-and C-domains. Using the cloned cDNAs as probes, the relative expression of IGF-I and IGF-II mRNAs were assayed in different Sparus tissues. Sparus liver clearly contained the highest level of IGF-I mRNA while relatively high levels of IGF-II mRNA were found in liver, heart and gill using the ribonuclease protection assay. After GH administration the amount of IGF-I mRNA was increased by 220% in liver but no changes in IGF-II mRNA levels were detected in any tissue. We also assayed the expression of IGF-I and IGF-II in Sparus during early development. The IGF-II mRNA level was highest in larva 1 day after hatching and decreased thereafter. In contrast, IGF-I mRNA was detected in 1-day-old larva but there was an increase in expression in 12- and 16-day-old larva. These results demonstrated that the expression of IGF-I and IGF-II is highly regulated in teleosts and suggest that they play distinct roles during growth and development.
Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells
Authors: Nicholson L B , Vlase H , Graves P , Nilsson M , Molne J , Huang G C , Morgenthaler N G , Davies T F , McGregor A M , Banga J P
Published: December 2008

We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2b and H-2d) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22–35 (A10 and All, both IgG2b from H-2b animals), amino acids 402–415 (A7, IgG2b from H-2b animals) and amino acids 147–228 (A9, IgG1 from H-2d animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and All was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and All, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.
The human placenta expresses transcription enhancer factor-1 but there is no correlation with the expression of placental lactogen
Authors: Quinn G , Boam D S W , Davis J R E , Glazier J D , Mylona P , Sides K , Sibley C P
Published: December 2008

A transcriptional enhancer which has a consensus binding sequence for transcription enhancer factor-1 (TEF-1) has been found 3′ of the hPL3 gene. We examined whether TEF-1 is expressed by the human placenta and whether such expression is co-ordinated with that of human placental lactogen (hPL). Probing Northern blots of total RNA from first trimester and term placenta, the choriocarcinoma-derived cell line JAr and primary cultured cytotrophoblast cells with a cDNA for TEF-1 revealed transcripts of 12–13 kb and 3–4 kb. The level of TEF-1 expression was the same in first trimester as compared with term placenta and in undifferentiated JAr as compared with differentiated cytotrophoblast cells. hPL expression was tenfold higher in term compared with first trimester placenta and, whilst detectable in cytotrophoblast cells, was undetectable in JAr cells. These data show that TEF-1 is expressed by the placenta but is not co-ordinated with hPL expression.
Anterior pituitary vasoactive intestinal peptide mRNA is colocalised with prolactin mRNA in hyperoestrogenised rats
Authors: Chew L-J , Seah V , Murphy D , Carter D
Published: December 2008

It is well established that oestrogens can stimulate prolactin (PRL) secretion as well as the expression of the vasoactive intestinal peptide (VIP) gene whose product is also a potent PRL secretagogue. Previous evidence has supported both an autocrine and a paracrine role for pituitary VIP in PRL release in vitro; however, the cellular origin of VIP in pituitary tissue still remains poorly defined. In these studies, we have demonstrated by in situ hybridisation that VIP RNA is detected in the anterior pituitaries of chronically hyperoestrogenised rats, but not in those of untreated animals. Using a double-probe labelling procedure, VIP RNA has been shown to be present in a subpopulation of PRL-producing cells, while colocalisation of VIP and GH RNA was not observed. VIP gene expression in the rat anterior pituitary gland was characterised by the presence of two alternatively polyadenylated transcripts, 1·7 kb and 1·0 kb in size. We have generated a probe specific for the 1·7 kb transcript and double-labelling studies also showed definitive colocalisation with PRL mRNA. Our results demonstrating the presence of VIP RNA in PRL-producing cells thus suggest that VIP may play an autocrine role in PRL hypersecretion under conditions of oestrogen-induced hyperplasia.
The ARP-1 orphan receptor represses steroid-mediated stimulation of human placental lactogen gene expression
Authors: Stephanou A , Shah M , Richardson B , Handwerger S
Published: December 2008

Studies were performed to determine whether ARP-1, which is an orphan receptor of the steroid receptor superfamily, inhibits basal activity of the human placental lactogen (hPL) promoter and the increase in hPL promoter activity in response to the receptors for thyroid hormone (TR) and retinoic acid (RAR). Co-transfection of an ARP-1 expression vector into BeWo choriocarcinoma cells, along with an expression vector containing 1·2 kb of the hPL promoter coupled to a CAT reporter gene, resulted in a dose-dependent inhibition of basal CAT activity. In addition, ARP-1 inhibited the stimulation of CAT activity by RARα and TRβ expression vectors. Mobility shift assays demonstrated that ARP-1 binds specifically to a composite steroid response element on the hPL promoter that confers retinoic acid and T3 responsiveness. The results implicate an inhibitory role for ARP-1 in the regulation of hPL gene expression and strongly suggest that hPL gene expression is regulated, at least in part, by the interaction of stimulatory and inhibitory members of the steroid receptor superfamily.
Mutational analysis of the estrogen receptor ligand-binding domain: influence of ligand structure and stereochemistry on transactivation
Authors: Kohno H , Bocchinfuso W P , Gandini O , Curtis S W , Korach K S
Published: December 2008

The mouse estrogen receptor (mER) exhibits ligand stereochemical specificity for indenestrol A (IA), a stilbestrol estrogen. IA has a chiral C3 methyl group, and the mER preferentially binds the S-enantiomer (IA-S), resulting in elevated biological activity when compared with the IA-R enantiomer. To elucidate the mechanisms for this stereochemical recognition, we have constructed a series of mERs with individual amino acid substitutions at Met521, His528, Met532, and Val537. The abilities of yeast-expressed wild-type and mutant mERs to transactivate an estrogenresponsive reporter gene construct were measured in the presence of diethylstilbestrol (DES) and IA enantiomers. The concentration of IA-S required to induce half-maximal transactivation by wild-type mER was 10-fold lower than IA-R, which is attributed to the 15-fold greater binding affinity for IA-S. Wild-type mER displayed similar dose—response curves for IA-R and demethyl IA, which lacks a C3 methyl group, demonstrating that the presence and correct orientation of the C3 methyl group on the IA compound is required for high-affinity ligand binding and transcriptional activity. Each mutant exhibited a reduced preference for IA-S enantiomer with respect to transactivation, suggesting that this region of the mER functions in ligand stereochemical recognition and activation. A mutation at Met532 diminished DES-and IA-S-induced transactivation by 7·5-fold and 40-fold respectively, with minimal change on their binding affinity. These data suggest that Met532 is required for transactivation induced by the potent agonist, IA-S, and the M532G mutation effectively uncouples IA-S ligand binding from transactivation. Use of these stereochemically different ligands in combination with mutagenesis of the mER demonstrates that ligand structure could influence transactivation by specifically altering the conformation of the mER AF-2 region.
Design and construction of a submarine outfall in Murcia, Spain
Authors: Hernández T. , Gonzalez J. M. , Palazón A. V. , Conradi C.
Published: December 2008

This paper describes the design and construction of a submarine outfall in the province of Murcia, Spain. The construction design was produced by Iberinsa in March 2004 and construction took place in 2004–2005. The factors which influenced the design are identified and the construction programme is described in detail. The actions taken to safeguard the environment during the construction and to monitor the environmental impacts, particularly with respect to a marine meadow that lies off the coast, are also reported.
Assessing breaching risk in coastal gravel barriers
Authors: Hartley L. , Pontee N.
Published: December 2008

This paper presents a method to assess the breaching risk to coastal barriers under a no-active-intervention (NAI) scenario. The approach categorises breaches as small high-level breaches, which operate periodically; to larger permanent tidal inlets with water flowing in/out on each tide. Assessing the risk of breaching involves four stages that consider the most probable location of a breach; the possible breach dimensions; the permanence of the breach; and the timeline of breach formation. The most probable location for a breach to form is assessed by considering the past and future shoreline behaviour, the susceptibility of beach profiles to overwashing, the current defence standard and probability of defence failure. The potential permanence and size of the breach is evaluated by considering a stability rating based on the ratio of the tidal prism to total annual littoral drift. The potential cross-sectional area of a breach is calculated by considering the relationship between the cross-sectional area and tidal prism. The results from this analysis are brought together in a written description of breach formation over time. The gravel barrier between Aldeburgh and Orford Ness on the Suffolk coast in the UK is used as a case study. The approach described within this paper is generic and can be used to assess the breaching risk for other vulnerable coastal barrier features under an NAI scenario.
Extension to the port of Gijón, Spain
Authors: Díaz Rato J. L. , Moyano Retamero J. , Miguel Riestra M. de
Published: December 2008

The port of Gijón is Spain's leading dry bulk-cargo port with annual traffic in excess of 21·5 Mt. Its main unloading terminal - designed for an annual traffic of 12 Mt - is at present unloading more than 16 Mt, with rates of occupation that are greater than advisable and limits on vessel draughts. This paper reports on the current port extension project, which involves building a new breakwater with a total length of 3834 m and creating a basin of 145 ha sheltered water. This will enable three 230 000 t deadweight bulk carriers with a draught of 20 m to berth simultaneously. It will also provide the port with a new solid bulk terminal that has an unloading capacity of over 25 Mt and a storage area of 60 ha, which will enable up to 2 Mt of iron ore and coal to be stocked. The €580 million, part-EU-funded project is due for completion by 2010.
Large breakwaters in deep water in northern Spain
Authors: Molinero Guillén P.
Published: December 2008

The north-eastern coast of Spain is rugged and densely populated. To build new ports, land must therefore be recovered from the sea in areas that are very deep and subject to strong wave action. Designing, testing, managing and performing port construction projects is thus a great technological challenge. It has resulted in world-record-setting infrastructures, including large main breakwaters and enormous volumes of material in zones with unusual depths and exposed to extreme climate conditions. In the 1970s the ports of Abra at Bilbao, Gijón and San Ciprian (Lugo) were enlarged. At the end of the century the growth of port activity in northern Spain required the construction of the following breakwaters: Ferrol’s outer port, with a 1515 m long breakwater armoured with 90 t blocks, an 858 m long, 20 m deep quay and a 90 ha platform; Gijon port enlargement project, with a main breakwater 3834 m long, a 1380 m long, 23 m deep quay and a 145 ha platform; and a new port installations at Langosteira Point in La Coruña, with a 3354 m long, 40 m deep breakwater, a 921 m long, 16 m deep quay and a 145 ha platform.
Auditor Specialization, Auditor Dominance, and Audit Fees: The Role of Investment Opportunities
Authors: Cahan Steven F. , Godfrey Jayne M. , Hamilton Jane , Jeter Debra C.
Published: December 2008

ABSTRACT: A report issued by the U.S. General Accounting Office (GAO) in 2003 identified auditors’ industry expertise as a critical factor for firms choosing an auditor, and highlighted the extreme levels of auditor concentration in some industries. We posit that the investment opportunity set (IOS) plays a fundamental role in determining whether an industry is an attractive target for auditor specialization. When industry-specific IOS is high, specialist auditors make costly investments in industry-specific knowledge, allowing them to offer a differentiated product and to create entry barriers for other audit firms. When the IOS of firms within an industry is relatively homogeneous, auditors can transfer such knowledge across clients in the industry more easily, resulting in cost savings and scale economies. However, greater homogeneity of IOS in an industry can also increase a client’s aversion to sharing an auditor with its competitors because of concerns about transfers of proprietary information, suggesting that industries with relatively homogeneous IOS are less likely to be dominated by a single auditor. We show that auditor concentration in an industry relates positively to both the level and homogeneity of IOS in the industry, while auditor dominance relates negatively to industry IOS homogeneity. Further, we find that audit fees are positively associated with both levels and homogeneity of industry IOS.
Matching and the Changing Properties of Accounting Earnings over the Last 40 Years
Authors: Dichev Ilia D. , Tang Vicki Wei
Published: December 2008

ABSTRACT: We present a theory that poor matching manifests as noise in the economic relation of advancing expenses to earn revenues. As a result, poor matching decreases the correlation between contemporaneous revenues and expenses, increases earnings volatility, decreases earnings persistence, and induces a negative autocorrelation in earnings changes. The empirical tests document these effects in a sample of the 1,000 largest U.S. firms over the last 40 years. We find a clear and economically substantial trend of declining contemporaneous correlation between revenues and expenses, increased volatility of earnings, declining persistence of earnings, and increased negative autocorrelation in earnings changes. The combined evidence suggests that accounting matching has become worse over time and that this trend has a pronounced effect on the properties of the resulting earnings. This evidence also suggests that the standard-setters’ stated goal of moving away from matching and toward more fair-value accounting is likely to continue and deepen the identified trends in the properties of earnings.
Do Individual Investors Cause Post-Earnings Announcement Drift? Direct Evidence from Personal Trades
Authors: Hirshleifer David A. , Myers James N. , Myers Linda A. , Teoh Siew Hong
Published: December 2008

ABSTRACT: This study tests whether nai¨ve trading by individual investors, or some class of individual investors, causes post-earnings announcement drift (PEAD). Inconsistent with the individual trading hypothesis, individual investor trading fails to subsume any of the power of extreme earnings surprises to predict future abnormal returns. Moreover, individuals are significant net buyers after both negative and positive extreme earnings surprises, consistent with an attention effect, but not with their trades causing PEAD. Finally, we find no indication that trading by individuals explains the concentration of drift at subsequent earnings announcement dates.
Farm-level economic analysis of the US National Johne's Disease Demonstration Herd Project
Authors: Groenendaal Huybert , Wolf Christopher A.
Published: December 2008

Objective—To evaluate farm-level economic costs and benefits related to control of paratuberculosis (Johne's disease) in dairy herds in the National Johne's Disease Demonstration Herd Project (NJDDHP).

Design—Economic analysis.

Sample Population—40 dairy herds enrolled in the NJDDHP.

Procedures—A farm-level economic analysis of the US NJDDHP was performed. Costs and benefits of management-related practices to control Johne's disease were estimated on the basis of results for 40 dairy operations enrolled in the project. From these costs and benefits, the net present value (NPV) for control of Johne's disease was estimated.

Results—Analysis revealed a mean NPV of $34/animal (equivalent to approx $3/animal/y) when there were no testing costs for producers and a mean NPV of −$14/animal when testing costs were borne by the producers.

Conclusions and Clinical Relevance—Management-related practices to control Johne's disease were typically found to be of marginal economic benefit when the costs of testing were not borne by producers. The continuation of the NJDDHP for another 2 to 4 years would allow more precise estimation of the economic benefits of a control program for Johne's disease.
Prevalence of anthelmintic resistance on sheep and goat farms in the southeastern United States
Authors: Howell Sue B. , Burke Joan M. , Miller James E. , Terrill Thomas H. , Valencia Elide , Williams Mimi J. , Williamson Lisa H. , Zajac Anne M. , Kaplan Ray M.
Published: December 2008

Objective—To determine prevalence of anthelmintic resistance on sheep and goat farms in the southeastern United States.

Design—Cross-sectional study.

Animals—Sheep and goats from 46 farms in 8 southern states, Puerto Rico, and St Croix in the US Virgin Islands.

Procedures—Parasite eggs were isolated from fecal samples, and susceptibility to benzimidazole, imidathiazole, and avermectin-milbemycin anthelmintics was evaluated with a commercial larval development assay.

Results—Haemonchus contortus was the most common parasite on 44 of 46 farms; Trichostrongylus colubriformis was the second most commonly identified parasite. Haemonchus contortus from 45 (98%), 25 (54%), 35 (76%), and 11 (24%) farms were resistant to benzimidazole, levamisole, ivermectin, and moxidectin, respectively. Resistance to all 3 classes of anthelmintics was detected on 22 (48%) farms, and resistance to all 3 classes plus moxidectin was detected on 8 farms (17%).

Conclusions and Clinical Relevance—Findings provided strong evidence that anthelmintic resistance is a serious problem on small ruminant farms throughout the southeastern United States. Owing to the frequent movement of animals among regions, the prevalence of resistance in other regions of the United States is likely to also be high. Consequently, testing of parasite eggs for anthelmintic resistance should be a routine part of parasite management on small ruminant farms.