bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression, distribution, regulation and function of IGFs in the ovine fetal pituitary
Authors: Lü F , Yang K , Han V K M , Challis J R G 
Published: December 2008

ABSTRACT
Activation of the fetal pituitary-adrenal axis is crucial for fetal organ maturation and the onset of parturition in sheep. Many factors including corticotrophin-releasing hormone (CRH) and arginine vasopressin secreted from the hypothalamus, and growth factors produced within the pituitary may be involved in the regulation of maturation of the fetal pituitary gland. IGFs have mitogenic and differentiation-promoting capacities in a variety of organs and are synthesized as paracrine factors within developing tissues. However, there is little information concerning the synthesis, distribution, regulation and function of IGFs in the fetal pituitary gland at different times during pregnancy. Therefore, we have localized IGF-I and IGF-II mRNAs and peptides, and determined the effect of cortisol on the level of IGF-II mRNAs in the pituitary glands of developing sheep fetuses. We examined the possible effects of IGFs on corticotroph function in cultures of adenohypophysial cells from term fetuses.
Seven species of IGF-II transcripts of 1·2–6·0 kb were identified by Northern blot analysis in the pituitary gland of fetuses between day 60 of gestation and term (day 145). The levels of IGF-II mRNAs did not change significantly during pregnancy, although there was a trend for the presence of higher levels of IGF-II mRNAs at day 60 of gestation. IGF-I mRNA was not detectable. By in situ hybridization, IGF-II mRNA was localized to non-endocrine cells and to cells lining the blood vessels of the pars distalis, to some presumed endocrine cells in the pars distalis and pars intermedia, and to clusters of cells in the pars nervosa. In contrast, IGF-I and IGF-II peptides were detected in the presumed endocrine cells in the pars distalis and pars intermedia but not in the pars nervosa. Incubation of adenohypophysial cells from term fetuses with IGF-I, but not IGF-II, for 48 h increased specific 125I-Tyr-ovine CRH binding. However, neither IGF-I nor IGF-II had any significant effects on the basal or CRH-stimulated immunoreactive (ir)-ACTH output, the level of POMC mRNA or the number of ir-ACTH positive cells. Infusion of cortisol to fetuses starting at day 96 of gestation for 100 h or at days 120–125 of gestation for 84 h did not affect the level of IGF-II mRNAs in the pars distalis but decreased the levels of POMC mRNA.
These results are consistent with IGFs having the potential to influence fetal pituitary function, although probably on cell types other than the corticotrophs. The likely sources of IGFs may be predominantly local (IGF-II) or from extrapituitary sources (IGF-I).
Multiple isoforms of the oestrogen receptor in endometrial cancer
Authors: Marsigliante S , Muscella A , Ciardo V , Puddefoot J R , Leo G , Vinson G P , Storelli C 
Published: December 2008

ABSTRACT
We evaluated the presence and variability of oestrogen receptor (ER) isoforms in endometrial cancer by using [3H]oestradiol-labelled ERs and the H222 monoclonal antibody obtained from the Abbott enzyme immunoassay kit.
Using isoelectric focusing (IEF), endometrial ER was shown to be composed of four different species, with pI values of 6·1, 6·3, 6·6 and 6·8, indistinguishable from the isoforms found in normal rat uterus, and human breast and larynx carcinomas. The isoforms at pI 6·3, 6·6 and 6·8, all sedimenting at 4S by sucrose gradient fractionation, showed, on two-dimensional SDS electrophoresis, relative masses of 50, 70 and 65 kDa respectively, equal to the masses previously found in breast cancer. These isoforms did not alter their pI values during IEF fractionation performed in a linear gradient of urea, while the pI 6·1, sedimenting at 8S, generated a new isoform at about 9 mol/l urea with pI 7·2 and a relative mass of 65 kDa. The urea-dissociated isoform (pI 7·2) was able approximately to double the antibody binding with respect to the non-dissociated oligomer, which suggested that some epitopes are 'masked', i.e. not accessible to the antibodies when ER is present in its complexed form. The evidence thus suggested that the oligomer at pI 6·1 contained a single 65 kDa ER form which, as a monomer, focused at pI 7·2.
The variability in the ER isoform profile found in endometrial cancer was similar to the variability previously reported in breast and larynx carcinomas. The balance between these isoforms could be a dynamic parameter involved in the functionality of this receptor and consequently in cell transformation.
Calcium-activated proteases in the bovine parathyroid gland: potential role in degradation of parathyroid hormone to peptide fragments
Authors: Watson P H , Mortimer S T , Wang K K W , Croall D E , Hanley D A 
Published: December 2008

ABSTRACT
Our studies suggest that protein kinase C is involved in low calcium (Ca2+)-stimulated secretion of parathyroid hormone (PTH) but not directly in high Ca2+-stimulated intracellular degradation of PTH to secreted carboxyl-terminal fragments (C-PTH), an important component of Ca2+-regulated PTH secretion. The present study was undertaken to determine the presence of calciumactivated proteases, 84 kDa (micro)-calpain and 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bovine parathyroid tissue using antibodies raised against bovine heart micro- and milli-calpain detected both isoforms of calpain. Western blotting of total bovine parathyroid cell protein prepared from primary cell cultures confirmed the presence of both isoforms of calpain, demonstrated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain and the cleavage products were separated by reverse-phase HPLC. Eluant fractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH peak from bPTH which co-eluted with the major C-PTH secreted by parathyroid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of either micro-calpain or Ca2+, to 71·5% when a 5:1 molar ratio of bPTH to calpain was used. Amino acid sequencing and analysis of the immunoreactive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH47–84 and bPTH69–84. In summary, both isoforms of calpain are present in the bovine parathyroid and calpains may play a role in the Ca2+-dependent degradation of PTH to secreted C-terminal fragments.
Evidence for alternative splicing of the chicken vasoactive intestinal polypeptide gene transcript
Authors: Talbot R T , Dunn I C , Wilson P W , Sang H M , Sharp P J 
Published: December 2008

ABSTRACT
Two forms of chicken vasoactive intestinal polypeptide (VIP) mRNA have been identified by reverse transcription (RT)-PCR and RNase protection assay. The shorter form of chicken VIP mRNA encodes a protein that does not contain an analogue of rat peptide histidine isoleucine (PHI) 1–27 or human peptide histidine methionine 1–27. The larger form encodes both VIP and a chicken analogue of PHI 1–27 in the same protein product.
Three VIP cDNAs isolated from a chicken hypothalamic cDNA library were derived from the shorter mRNA. Sequence analysis of the longest clone identified an open reading frame that codes for a 165 amino acid preproVIP protein and contains two polyadenylation signals. In situ hybridisation with an oligonucleotide probe from the VIP cDNA sequence showed that VIP-encoding mRNA occurs in cells in the basal hypothalamus, an area of the brain known to contain VIP neurosecretory neurones. RT-PCR of total RNA from liver, kidney, gut, pancreas, pituitary, cerebellum, forebrain and hypothalamus, using primers derived from the VIP cDNA sequence, showed that the shorter form of VIP mRNA is present in all of these tissues. The sequence of the longer form of VIP mRNA was obtained by sequencing a portion of the VIP gene from genomic DNA. This revealed a potential exon that was not represented in the VIP cDNA clones analysed. RT-PCR with primers from this sequence showed that it was expressed in the gut and hypothalamus. RNase protection assays confirmed the presence of the two forms of mRNA in gut and hypothalamus. The relative proportions of the two mRNA forms were: 97·8% VIP only, 2·2% PHI/VIP in the hypothalamus and 98·5% VIP only, 1·5% PHI/VIP in the gut.
In conclusion, chicken VIP mRNA is alternatively spliced. The shortest form, which encodes a preproprotein containing only the VIP peptide, is the most abundant. The longer form of chicken VIP mRNA encodes a preproprotein containing sequences for both VIP and a chicken form of PHI.
Influence of xenobiotics on rainbow trout liver estrogen receptor and vitellogenin gene expression
Authors: Flouriot G , Pakdel F , Ducouret B , Valotaire Y 
Published: December 2008

ABSTRACT
Rainbow trout hepatocyte primary culture was used to test the influence of some xenobiotics on the expression of two genes implicated in reproduction, those for the estrogen receptor (ER) and vitellogenin (Vg). We showed that chlordecone, nonylphenol, a polychlorobiphenol (PCB) mixture (Aroclor 1245) and lindane were able to induce ER and Vg mRNA accumulation. Antiestrogens, 4-hydroxytamoxifen and ICI 164,384, prevented the effects of the xenobiotics, indicating that the induction of gene expression is mediated by the ER. Among these four xenobiotics, only chlordecone and nonylphenol were able to displace the binding of [3H]estradiol to ER-enriched COS-1 extracts, and to activate an estrogen-dependent reporter gene (ERE-TK-CAT) cotransfected with an expression vector containing ER cDNA. The results suggest that chlordecone and nonylphenol are direct inducers of rainbow trout ER and Vg gene expression, whereas PCBs and lindane act through their hepatic metabolites. Moreover, pentachlorophenol acts as an antagonist of the induction by estradiol of rainbow trout ER and Vg gene expression.
Secretagogue-induced [Ca2+]i changes in single rat pancreatic islets and correlation with simultaneously measured insulin release
Authors: Martin F , Reig J A , Soria B 
Published: December 2008

ABSTRACT
The effects of secretagogues (glucose, tolbutamide and phorbol esters) on simultaneously measured intracellular free calcium concentration ([Ca2+]i) and insulin release were studied in rat pancreatic islets of Langerhans. Stimulatory concentrations (11 mm) of glucose caused a transient [Ca2+]i increase followed by an almost flat second phase. Increasing glucose concentrations to 16·7 mm in steps caused a further increase in [Ca2+]i. In contrast with mouse islets, rat islets scarcely showed glucose-induced [Ca2+]i oscillations. Digital image analysis showed that [Ca2+]i changes occurred synchronously across the whole islet. As expected, simultaneously measured insulin release was biphasic with a clear second phase. This clearly indicated that in rat islets there is a lack of correlation between [Ca2+]i and insulin release. This was further explored using agents which separately promoted the first (tolbutamide, 200 μm) and second (phorbol-12-myristate-13-acetate; PMA; 5 nm) phases of insulin release. Tolbutamide induced a transient increase in [Ca2+]i paralleled by a transient increase in insulin release, whereas PMA induced a slow increase in insulin release without a clear change in [Ca2+]i. These results suggest that in rat islets the first phase of insulin release is calcium dependent, whereas the second phase is related to the activation of protein kinase C (PKC). However, the glucose-induced second phase of insulin release did not coincide with an increase in membrane-associated PKC activity. Other messengers may contribute to this late phase of insulin release.
Intrauterine injection of ovine interferon-τ alters oestrogen receptor and oxytocin receptor expression in the endometrium of cyclic ewes
Authors: Spencer T E , Ing N H , Ott T L , Mayes J S , Becker W C , Watson G H , Mirando M A , Bazer F W 
Published: December 2008

ABSTRACT
This study determined the effects of intrauterine injections of recombinant ovine interferon-τ (roIFN-τ; 2 × 107 antiviral units/day) or control proteins (6 mg/day) from day 11 to day 14 post-oestrus (oestrus=day 0) on endometrial expression of receptors for oestrogen, progesterone and oxytocin in cyclic ewes. Plasma concentrations of progesterone were greater on day 15 in ewes receiving roIFN-τ compared with control proteins (P<0·02, treatment × day). Ewes injected with roIFN-τ had lower endometrial levels of oestrogen receptor mRNA (P<0·10) and protein (P<0·01) on day 15 compared with ewes receiving control proteins. In situ hybridization analysis indicated that oestrogen receptor mRNA was more abundant in the luminal and glandular epithelium of control ewes compared with roIFN-τ-treated ewes. Immunoreactive oestrogen receptor was also present in the luminal and glandular epithelium of control, but not roIFN-τ-treated ewes. Endometrial levels of progesterone receptor mRNA and protein were not different (P>0·10) between control and roIFN-τ-treated ewes. In situ hybridization analyses indicated that progesterone receptor mRNA abundance was low in endometrial epithelium and stroma of both control and roIFN-τ-injected ewes. Immunoreactive progesterone receptors were present in the endometrial stroma and epithelium of control ewes, but confined to the stroma of roIFN-τ-treated ewes. Oxytocin receptor density was lower (P<0·10) in the endometrium of ewes injected with roIFN-τ than control proteins; however, oxytocin receptor affinity was not affected (P>0·10) by treatment. Concentrations of 13,14-dihydro-15-keto-prostaglandin F2α (PGFM) were not increased by exogenous oxytocin administration in control and roIFN-τ-treated ewes on days 10 or 12 post-oestrus. However, on day 14, control ewes responded to oxytocin with increased plasma concentrations of PGFM, whereas ewes receiving roIFN-τ remained unresponsive to oxytocin. These results indicate that the antiluteolytic effects of IFN-τ are to prevent increases in endometrial oestrogen receptor mRNA and protein and oxytocin receptor density which abrogates uterine release of prostaglandin F2α during maternal recognition of pregnancy. IFN-τ may inhibit the synthesis of oestrogen receptor mRNA by a transcriptional or post-transcriptional regulatory mechanism to suppress oxytocin receptor formation during early pregnancy in ewes.
Prolactin and growth hormone mRNA and protein characterization in SMtTW rat pituitary tumours
Authors: Delhase M , Rajas F , Verdood P , Remy C , Chevallier P , Velkeniers B , Trouillas J , Hooghe-Peters E L 
Published: December 2008

ABSTRACT
We have combined different techniques to analyse passages of five different rat spontaneous pituitary tumours (SMtTW) that were transplanted under the kidney capsule. These tumours were secreting prolactin (PRL), GH or both hormones. RIA, immunocytochemistry (ICC) and Western blot analysis were applied to characterize the hormone(s) stored (ICC and Western blot) and secreted (RIA). mRNA content was analysed by PCR, Northern blot analysis and in situ hybridization.
The data point not only to the reliability of the techniques used at both protein and RNA levels for each tumour studied but also to the complementarity of some techniques. For example, whereas Northern blot analysis demonstrates the presence and size of hormone mRNA, in situ hybridization indicates the percentage of cells expressing a given hormone mRNA and allows the presence of one population (or more) of cells in a given tumour to be identified.
Moreover, the tumours were compared with normal rat pituitary. Although the PRL and GH mRNAs were identical in size, the amount of mRNA was lower in the tumours. At the protein level, the PRL and GH variants exhibited a different pattern of expression in tumours compared with the normal rat pituitary.
The biological significance of these differences is discussed.
Multiple alternative splice isoforms of parathyroid hormone-related peptide mRNA in human cell lines
Authors: Nakamura M , Hashimoto T , Furuyama J-i , Kakudo K 
Published: December 2008

ABSTRACT
Parathyroid hormone-related peptide (PTHrP) is thought to be responsible for hypercalcemia in some patients with malignant tumors. The PTHrP gene has seven exons, giving rise to three types of PTHrP isoform through alternative splicing. We studied the expression of mRNAs in 14 human cell lines using the reverse transcription-PCR method, to examine tissue-specific expression. All the cell lines expressed at least two types of PTHrP transcript. Most cell lines expressed all four types of PTHrP mRNA isoform. However, a rhabdomyosarcoma cell line, RD, and a bladder carcinoma cell line, T24, expressed only two types. These results may suggest that PTHrP mRNA is expressed in the majority of tumor and normal tissues and that it shows less tissue- or tumor-specificity.
Expression of human insulin-like growth factor-binding protein-3 in transgenic mice
Authors: Murphy L J , Molnar P , Lu X , Huang H 
Published: December 2008

ABSTRACT
Transgenic mice which expressed human IGF-binding protein-3 (hIGFBP-3) were generated by pronuclear injection of an hIGFBP-3 cDNA driven by the mouse metallothionein 1 promoter. Two of the seven founder mice had measurable levels of hIGFBP-3 in the circulation. The serum levels of hIGFBP-3 increased as the mice were bred to homozygosity and were further induced by supplementing the drinking water with 25 mm ZnCl2. While the birth weight, litter size and body weight of transgenic mice were not significantly different from non-transgenic litter mates or wild-type mice derived from the same genetic background, the transgenic mice demonstrated selective organomegaly. The spleen, liver and heart of mice derived from both founders were significantly heavier compared with organs from non-transgenic mice (P<0·05, P<0·005 and P<0·01 respectively). The weights of the brain and kidney were similar in transgenic and non-transgenic mice. Expression of the transgene was detected in the kidney, small intestine and colon by Northern blot analysis.
Western ligand blotting of serum from transgenic mice did not demonstrate any change in the abundance of the IGFBPs detected by this method. When serum from transgenic mice was incubated with 125I-labeled IGF-I and analyzed by Sephacryl S-200 chromatography under neutral conditions a significantly (P<0·05) increased amount of the radioactivity was found in the 140 kDa ternary complex compared with serum from wild-type mice. Immunoreactive hIGFBP-3 was detected in the 140 kDa ternary complex but the majority of immunoreactive hIGFBP-3 present in transgenic mouse serum eluted in later fractions indicating that it was not associated with the acid-labile subunit. These data demonstrate that modest constitutive expression of hIGFBP-3 has a selective effect on organ growth and development. The establishment of these IGFBP-3 transgenic mouse strains may provide useful models to investigate further the physiological role of IGFBP-3.
Structure and sequence of a mouse gene encoding an androgen-regulated protein: a new member of the seminal vesicle secretory protein family
Authors: Simon A M , Veyssière G , Jean Cl 
Published: December 2008

ABSTRACT
The gene encoding MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids), an androgen-dependent protein specifically expressed in the mouse seminal vesicle, was isolated and sequenced. A mouse genomic library constructed in the λEMBL12 vector was screened using a full length cDNA probe. One genomic clone was selected, 7·4 kb of which were shown to contain the whole MSVSP99 gene. The complete sequence of the MSVSP99 gene (1·7 kb), plus 0·8 and 0·3 kb of the 5′ and 3′ flanking regions respectively, has been determined. The gene is composed of four exons interrupted by three introns. The size range for the four exons is 47–217 bp, while that of introns is 87–615 bp. The transcription start site was identified as an adenine residue located 21 nucleotides upstream from the ATG start codon. Putative TATA and CAAT boxes were identified, along with a number of regions that shared homologies with known regulatory sequences. These included androgen-responsive elements located in the promoter as well as in the gene sequence. Sequence comparisons with other androgen-responsive genes showed strong homologies between the MSVSP99 gene and the seminal vesicle secretory protein (SVS) family genes (rat SVS II, IV, V and VI). Moreover, some regions were found to be conserved between the MSVSP99 gene and the human semenogelin I and II genes.
Peptide and immunochemical mapping of the ectodomain of the porcine LH receptor
Authors: Couture L , Naharisoa H , Grebert D , Remy J-J , Pajot-Augy E , Bozon V , Haertle T , Salesse R 
Published: December 2008

ABSTRACT
The LH/hCG receptor is a G protein-coupled receptor with an N-terminal extracellular domain involved in hormone—receptor interaction. The recombinant porcine receptor, stably expressed in Chinese hamster ovary (CHO) cells, has the same characteristics (Kd and cAMP production) as in Leydig cells. Six synthetic peptides derived from the receptor ectodomain and two polyclonal anti-peptide sera were tested in the homologous system porcine LH and porcine LH receptor. Their ability to inhibit hormone binding and signal transduction on CHO cells expressing the recombinant receptor was evaluated. Peptides 25–40 and 107–121 exhibited a high transduction inhibition as compared with hormone binding, peptides 21–36, 102–111, and 102–121 inhibited hormone binding more efficiently than signal transduction, and peptide 7–24 exhibited inhibition of both hormone binding and hormone-induced cAMP production. Immuno-globulins against peptides 21–36 and 102–111 inhibited both hormone binding and receptor activation suggesting that these sequences are located on the receptor surface.
The data suggest that multiple, discontinuous regions of the extracellular domain of porcine LH receptor are involved in hormone binding and signal transduction. Two minimum critical sequences, 21–24 and 102–107, are involved in hormone binding and vicinal segments may be implicated in signal transduction.
Nature of ligand affinity and dimerization of corticotrophin-releasing factor-binding protein may be detected by circular dichroism
Authors: Lowry P J , Koerber S C , Woods R J , Baigent S , Sutton S , Behan D P , Vale W , Rivier J 
Published: December 2008

ABSTRACT
As the association of corticotrophin-releasing factor (CRF) with its binding protein (BP) to form a dimer complex (CRF2/BP2) appears to be dependent on the nature of the ligand we have compared the circular dichroism difference spectra after association of the BP with ovine (o) CRF, human (h) CRF and the α-helical CRF(9–41) antagonist. All three ligands caused a negative change in molar ellipticity above 210 nm, with oCRF having the least and hCRF the greatest effect. Below 210 nm there was a marked divergence of difference spectra, with the reaction with the natural peptides, hCRF and oCRF, resulting in a positive change in ellipticity, whilst that with the antagonist produced a negative change. In view of the BP spectrum indicating predominantly β-sheet and the peptides showing mainly α-helix these results were interpreted as the changes above 210 nm being due to dimerization and below 210 nm to a change in the conformation of ligand on binding. The opposite change in α-helicity of the antagonist observed on binding compared with the two natural CRF peptides could have fundamental pharmacological implications.