bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular cloning and sequence analysis of striped bass (Morone saxatilis) gonadotrophin-I and -II subunits
Authors: Hassin S , Elizur A , Zohar Y 
Published: December 2008

ABSTRACT
Two types of cDNA, each encoding a different β-subunit of striped bass (Morone saxatilis, Teleostei) gonadotrophins (GTH-Iβ and GTH-IIβ), as well as the glycoprotein α-subunit, were cloned by screening a striped bass pituitary cDNA library. The probes used for screening the library were cloned cDNA fragments, generated by PCR amplification of reverse-transcribed mRNA obtained from two pituitaries. The nucleotide sequences of the α-subunit, GTH-Iβ and GTH-IIβ are 626, 524 and 580 bases long, encoding peptides of 117, 120 and 147 amino acids respectively. Striped bass GTH-Iβ and GTH-IIβ share a sequence identity of 48% at the nucleic acid level, and 30% at the amino acid level. A cluster analysis of vertebrate pituitary glycoprotein β-subunits suggests that teleost GTH-IIβ is more closely related to tetrapod LH than to FSH. Administration of gonadotrophin-releasing hormone analogue ([d-Ala6,Pro9Net]-LHRH) to juvenile striped bass resulted in ten-, two- and fivefold increases in the expression of the α-subunit, GTH-Iβ and GTH-IIβ respectively. These results suggest that each of the GTH subunits is differentially regulated, and further corroborate the functional duality of teleost gonadotrophins.
The nucleotide sequences of the α-subunit, GTH-Iβ and GTH-IIβ reported in this paper have been deposited in the GenEMBL database with the respective accession numbers L35071, L35070 and L35096.
Cloning and characterization of a chick embryo cDNA and gene for IGF-binding protein-2
Authors: Schoen T J , Mazuruk K , Waldbillig R J , Potts J , Beebe D C , Chader G J , Rodriguez I R 
Published: December 2008

ABSTRACT
We have isolated and characterized a cDNA for IGF-binding protein-2 (IGFBP-2) and its gene from the chick embryo. Using primers from a conserved region of the mammalian IGFBP-2 sequence, a cDNA clone (1·6 kb) was isolated from an embryonic day-18 chick retina cDNA library. Although the clone was truncated at the 5′ end, the complete coding sequence was obtained from 5′ rapid amplification of cDNA ends and genomic sequencing. The open reading frame encoded a 311 amino acid precursor protein which contains a putative 36 residue signal peptide. The mature 275 amino acid protein had a predicted Mr of 33 500 and exhibited 71, 68, 68 and 66% identity to rat, bovine, ovine and human IGFBP-2 cDNA respectively, with conservation of all 18 cysteines. The cDNA contained an RGD peptide but lacked a putative ATP-binding motif. A single transcript of approximately 2·3 kb was present in embryonic day-15 eye, brain, skeletal muscle, heart and intestine, but was virtually absent from embryonic day-15 liver. The chicken IGFBP-2 gene spanned approximately 38 kb, consisted of four exons, and was similarly organized to that of the rat and human. Southern blot analysis of chicken genomic DNA suggested that it is encoded by a single gene. The sequence information from the avian IGFBP-2 should be of value in examining the role of IGFBP-2 in vertebrate development.
Transcriptional down-regulation by epidermal growth factor of TRH receptor mRNA in rat pituitary cells
Authors: Monden T , Yamada M , Konaka S , Satoh T , Ezawa H , Iwasaki T , Mori M 
Published: December 2008

ABSTRACT
To gain insight into the mechanism underlying the epidermal growth factor (EGF)-induced changes in responsiveness to TRH and in the numbers of TRH receptors (TRH-Rs) in the pituitary, we investigated the transcriptional regulation by EGF of the TRH-R gene in GH4C1 cells. Northern blot analyses and binding studies revealed that EGF reduced both TRH binding and TRH-R mRNA levels in a dose- and time-dependent manner, while no significant changes were observed in β-actin mRNA levels. Addition of actinomycin D caused an acute increase in the basal TRH-R mRNA level, and the rate of decrease of the TRH-R mRNA was identical in control and EGF-treated groups, suggesting that the stability of the TRH-R mRNA was not significantly affected in EGF-treated cells. Incubation with cycloheximide also induced an increase in the basal TRH-R mRNA level and completely reversed the EGF-induced reduction of TRH-R mRNA levels. Furthermore, a nuclear run-on assay demonstrated that the rate of transcription of the TRH-R gene was significantly inhibited in cells treated with EGF. We conclude that (1) EGF decreases the expression of the TRH-R mRNA largely by reducing its rate of transcription, and this action requires the synthesis of new proteins, and (2) inhibitors of protein and RNA synthesis cause a significant increase in the basal TRH-R mRNA level, suggesting that there may be a short-lived protein suppressing the TRH-R mRNA level in the pituitary.
Developmental changes in the expression patterns of IGFs, type 1 IGF receptor and IGF-binding proteins-2 and -4 in perinatal rat lung
Authors: Batchelor D C , Hutchins A-M , Klempt M , Skinner S J M 
Published: December 2008

ABSTRACT
The insulin-like growth factors (IGF-I and IGF-II), their receptors and binding proteins (IGFBPs) are endogenously expressed in a number of tissues including the lung during fetal and neonatal development. This endogenous autocrine/paracrine IGF 'system', together with endocrine sources, contributes to the regulation of lung cell proliferation. We investigated the expression of the mRNAs encoding IGF-I, IGF-II, the type 1 IGF receptor (IGF-T1R) and two IGF-binding proteins (IGFBP-2 and IGFBP-4) in rat lung during the perinatum. These were compared in lung with surfactant apoprotein A (Sp-A) mRNA levels.
mRNA in extracts of fetal tissues collected between day 17 of gestation (17f) and day 9 after birth (9d) was estimated by Northern blot or RNase protection analysis. At day 20 of gestation IGF-I, IGF-T1R and IGFBP-4 mRNA levels were higher in lung than liver (all P<0·01), whereas IGF-II and IGFBP-2 mRNA levels were higher in liver than lung (each P<0·02). The expression of IGF-I, IGFBP-2 and IGFBP-4 in lung was high before birth (days 17–20f) but decreased to low levels at days 21f, 22f or at birth (1d) but increased in the neonatal lung. IGF-II expression in lung was high at 17f but decreased before birth and remained low after birth. The IGF-T1R was expressed at moderate levels before birth, decreased before birth but peaked at days 2–5 after birth. The decrease in expression of these growth regulators before birth was matched by an increase in Sp-A expression which was clearly seen at day 20f, peaked at 1d and then was maintained at high levels after birth. Primary cell cultures of 18f lung epithelia express IGFBP-2 while fibroblasts from the same animals express only IGFBP-4. Cells grown from 22f lung tissue express IGFBP-2 and IGFBP-4 at lower levels, behaving in vitro as they do in vivo.
The contrasting levels of expression of different components of the IGF system in the fetal lung and liver indicate organ-specific regulation. IGFBP-2 and IGFBP-4 expression in different cell types within lung but with similar temporal changes suggests cell-specific regulation, perhaps by a common agent. The patterns of expression of IGF-I, IGF-T1R, IGFBP-2 and IGFBP-4, but not IGF-II, in developing lung correspond to previously described phasic changes in lung cell proliferation rates. The nadir in expression of these four major components of the lung IGF system occurs in the saccular phase when the lung begins to differentiate, probably under the influence of certain endocrine agents.
Characterization of normal and point-mutated human androgen receptors expressed in the baculovirus system
Authors: Beitel L K , Sabbaghian N , Alarifi A , Alvarado C , Pinsky L , Trifiro M 
Published: December 2008

ABSTRACT
The baculovirus system is able to generate large amounts of a protein, permitting detailed analysis of structure-function relations. We have used this system to overexpress and characterize normal human androgen receptor (hAR) and mutant hARs from humans with complete or partial androgen insensitivity. Maximum specific binding of [3H]mibolerone (MB) in recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells varied from 15 to 40 pmol/mg protein, about 1000-fold higher than in genital skin fibroblasts, and peaked 48–72 h after infection. In contrast, Coomassie blue staining and Western blotting revealed maximum accumulation of 110–120 kDa hAR proteins 96 h post-infection. Normal and mutant hARs were specifically photoaffinity-labeled with [3H]methyltrienolone (MT), and had normal steroid-binding selectivity: the order of competition was androgen>estrogen>progestin>glucocorticoid. Normal hAR was phosphorylated in Sf9 cells, reacted with antibodies against phosphoserine and phosphothreonine after purification using testosterone-biotin, and transactivated a transfected androgen response element-luciferase reporter in infected Sf9 cells. Two mutant hARs had increased rates of dissociation from MB and MT that were in accord with the associated degree of clinical androgen insensitivity: complete, Pro903Ser>partial, Leu820Val; the third, Ile663Asn, was not abnormal. Our data extend the characterization of normal hAR produced by baculovirus-infected Sf9 cells, and demonstrate, for the first time, that point-mutated hARs so produced can display distinctive biochemical phenotypes.
The human 3β-hydroxysteroid dehydrogenase (3β-HSD) gene cluster on chromosome 1p13 contains a presumptive pseudogene; 3β-HSD and CYP17 do not segregate with dominantly inherited hirsutism
Authors: McBride M W , Russell A J , Vass K , Frank-Raue K , Craig N J , Morrison N , Boyd E , Szpirer C , Sutcliffe R G 
Published: December 2008

ABSTRACT
Four hirsute females from a family exhibiting idiopathic dominant hirsutism were examined. Basal blood levels of Δ5 and Δ4 steroids were within the normal range, but ACTH stimulation led to increases in 17-hydroxypregnenolone and dehydroepiandrosterone that were significantly above control levels. Using polymorphic genetic markers, the genes for cytochrome P450c17 encoded by CYP17, and the type I and II forms of 3β-hydroxysteroid dehydrogenase (3β-HSD) were found not to segregate with hirsutism in this family, though a base substitution was detected in the 3′ end of exon 1 of the gene for 3β-HSD type I in three of the four patients investigated.
Analysis of PCR amplification products by denaturing gradient gel electrophoresis (DGGE) and sequencing revealed a novel homologue of exon 3 of 3β-HSD. DNA of one of the affected patients was used to create a genomic library in λ gem11 and clones containing the novel homologue were obtained and partially sequenced. The equivalent clone was obtained from a genomic library of an unrelated normal individual. The sequences of the clones from patient and control were identical and homologous to exons 2–4 of human 3β-HSD types I and II. No difference was found in the PCR primer sites that flanked the exon 3 homologue which led to its detection on DGGE gels. In both clones, stop codons and deletions were identified in the exon 4 homologue, leading to the deduction that the sequence comes from a pseudogene, which we call 3β-HSD Ψ1. The pseudogene mapped to chromosome 1p13. It was concluded that dominantly inherited idiopathic hirsutism in this rare kindred was not due to deficiencies in 3β-HSD types I, II, or Ψ, or of CYP17.
Structure and expression of an ovine endometrial oxytocin receptor cDNA
Authors: Riley P R , Flint A P F , Abayasekara D R E , Stewart H J 
Published: December 2008

ABSTRACT
A sheep endometrial oxytocin receptor (OTR) cDNA (1·5 kb) was isolated from a λ-ZAP library using a reverse transcription-PCR product probe generated from oestrous endometrial mRNA. The sheep OTR cDNA shared an overall similarity of 82% with human OTR cDNA, 85% with pig OTR cDNA and 76% with rat OTR cDNA. The encoded receptor was a 391 amino acid polypeptide 94% similar to human OTR, 94% similar to pig OTR and 93% similar to rat OTR. The sheep OTR contained two additional amino acids compared with human OTR which were located in the highly GC-rich third intracytoplasmic loop. This region is thought to be associated with G protein coupling and signal transduction. Expression of the cDNA in Cos-7 cells and measurement of oxytocin-induced phosphoinositide turnover confirmed that it coded for a functional product. The affinity of the expressed receptor was comparable with that observed for the in vivo receptor.
Expression of IGF-I mRNA in the ovine ovary
Authors: Leeuwenberg B R , Hurst P R , McNatty K P 
Published: December 2008

ABSTRACT
IGF-I has been implicated as a local and/or systemic regulator of ovarian function by acting alone or as an amplifier of the actions of gonadotrophins, thereby influencing follicular growth and ovulation. In the sheep it is uncertain as to whether IGF-I can fulfil an autocrine or paracrine role, since mRNA expression and peptide synthesis have not previously been demonstrated.
Using in situ hybridization with ovine and human riboprobes, IGF-I mRNA was found in all major steroidogenic cell types of the sheep ovary, namely the granulosa, theca and luteal cells and, to a lesser extent, the stroma. IGF-I mRNA was found to be expressed in the granulosa and thecal cells of early antral follicles and thereafter in these cell types as the follicles increased in diameter. Evidence for IGF-I mRNA expression in preantral follicles could not be demonstrated. No obvious differences in the levels of expression were observed in ovaries recovered during the luteal phase, follicular phase, anoestrus or after exogenous FSH treatment. IGF-I expression was confirmed by Northern blotting and PCR. These findings are consistent with the notion that IGF-I may have an autocrine or paracrine role in enhancing ovarian function in the sheep.
FSH receptor gene expression during ovarian follicle development in sheep
Authors: Tisdall D J , Watanabe K , Hudson N L , Smith P , McNatty K P 
Published: December 2008

ABSTRACT
A key question in elucidating the role of FSH in ovarian function is to determine when during follicular growth the FSH receptor first appears. The aim of this study was to examine the site and time of FSH receptor gene expression during early follicular growth. This study was carried out on ovaries of adult sheep during the luteal and prostaglandin-induced follicular phase of the oestrous cycle and also on ovaries of fetal sheep at 90, 100, 120 and 135 days of gestation (term=day 147).
Using reverse transcription-PCR and a set of PCR primers spanning exons 8/9/10, two partial FSH receptor cDNAs (500 and 310 bp) were isolated from adult sheep ovary. It was shown by sequencing that exon 8 was deleted in the 310 bp cDNA, implying that this was part of an alternatively spliced FSH receptor transcript. Using RNA in situ hybridisation on ovaries of adult sheep, FSH receptor mRNA was observed in granulosa cells of early preantral follicles with one to two cell layers and it was seen that gene expression continued throughout folliculogenesis into advanced stages of atresia. Moreover, in the fetus, FSH receptor gene expression was detected in follicles with two or more layers of granulosa cells in ovaries taken at 100, 120 and 135 days of gestation.
These results suggest that the FSH receptor gene is expressed after the granulosa cells of a folllicle have begun to divide but not during the earliest stages of follicle growth, namely the transformation of a primordial follicle to a primary follicle.
Production of a human epidermal growth factor fusion protein and its degradation in rat gastrointestinal flushings
Authors: Xian C J , Upton Z , Goddard C , Shoubridge C A , McNeil K A , Wallace J C , Read L C , Francis G L 
Published: December 2008

ABSTRACT
This study describes the biosynthesis of a human epidermal growth factor fusion protein, Long EGF, that has a 53 amino acid extension peptide derived from the 46 N-terminal amino acids of porcine GH. The approach allowed the production of Long EGF at high efficiency due to the expression of the fusion protein in high yield as inclusion bodies in Escherichia coli. Long EGF had a slightly lower potency compared with native EGF in a range of assays, including binding to anti-EGF antibodies or the EGF receptor, stimulation of Balb/3T3 fibroblast and rat intestinal epithelial cell growth, as well as counteracting the inhibition of mink lung epithelial cell proliferation by transforming growth factor-β1.
Degradation of Long EGF and native EGF was compared in gastrointestinal flushings as an indication of whether the EGF domain of the fusion protein would be protected from proteolytic cleavage and be useful as a trophic agent in the gut. Incubation with flushings from the stomach or jejunum of rats caused rapid cleavage of the extension peptide, releasing native EGF. A C-terminal truncation of Arg53 in the stomach and a removal of the C-terminal pentapeptide (49Trp-Trp-Glu-Leu-Arg53) in the small bowel was demonstrated by N-terminal sequencing and mass spectrometry. The degradation patterns were reflected by changes in migration of products on SDS-PAGE and in subsequent binding activities to the EGF receptor and anti-EGF antibodies. The data show that a human EGF fusion protein can be produced efficiently in a bacterial expression system and that it retains biological activity in vitro. Although the extension peptide was rapidly cleaved from Long EGF in both stomach and small bowel producing similar biological activity to native EGF, it could not prevent subsequent degradation of the EGF domain. Other strategies are being investigated to develop an effective oral form of EGF that resists digestion by proteases in the gastrointestinal tract.
c-fos mRNA and FOS protein expression is induced by Ca2+ influx in GH3B6 pituitary cells
Authors: Li S L , Cougnon N , Bresson-Bépoldin L , Zhao S J , Schlegel W 
Published: December 2008

ABSTRACT
The expression of the immediate early gene c-fos was studied at the mRNA and the protein level in cells of the pituitary tumour cell line GH3B6. The induction of c-fos mRNA as detected by Northern blot analysis was stimulated by TRH and by depolarization with KCl, both leading to a rise in cytosolic free [Ca2+] ([Ca2+]i), and also by epidermal growth factor (EGF). To assess the role of the changes in [Ca2+]i in the induction of c-fos, Ca2+ was chelated in the extracellular medium with EGTA to prohibit Ca2+ influx during stimulation, or intracellular Ca2+ stores were emptied by prolonged exposure to EGTA, a treatment which abolished all [Ca2+]i changes. In the latter case, the effect of TRH on c-fos mRNA expression was almost completely abolished, whereas EGF still caused substantial c-fos induction. Full induction of c-fos mRNA by TRH required a prolonged phase of stimulated Ca2+ influx. c-fos mRNA induction by TRH and KCl was markedly inhibited by two blockers of Ca2+/calmodulin-dependent protein kinase (CaM kinase), KN-62 and calmidazolium. In contrast, KCl induction of c-fos and the effects of KN-62 on TRH induction of c-fos were not observed in a closely related pituitary line GH4C1 in which TRH exerts its effects on immediate early genes predominantly via the protein kinase C pathway. In GH3B6 cells stimulated with TRH or KCl, enhanced FOS protein levels were detected by immunofluorescence and localized in the nucleus with confocal microscopy. Analysis by immunoblotting showed that TRH induced two protein species with apparent molecular masses of 52 and 57 kDa. In GH3B6 cells stimulated with KCl or TRH, the 52 kDa species was mainly found whereas, in the GH4C1 cells, TRH predominantly stimulated the 57 kDa species. These data show that distinct signalling pathways (CaM kinase and protein kinase C) involve Ca2+ influx to induce the transcription of the early gene c-fos, and that the resulting FOS protein species may depend on the pathways involved.
Two distinct signaling pathways activated by activin A in glucose-responsive pancreatic β-cell lines
Authors: Shibata H , Kanzaki M , Takeuchi T , Miyazaki J-i , Kojima I 
Published: December 2008

ABSTRACT
Activin A stimulates insulin secretion in pancreatic β-cells by a calcium-dependent mechanism. The present study was conducted to further characterize the effects of activin A in two glucose-responsive insulinoma cell lines, MIN6 and HIT-T15 cells. In HIT-T15 cells, activin A evoked an increase in cytoplasmic free calcium concentration, stimulated insulin secretion, maintained glucose responsiveness of the cells and inhibited DNA synthesis. However, activin A did not have any effect in MIN6 cells. Measurement of 125I-labeled activin A binding in MIN6 cells revealed that the number of binding sites was markedly reduced, suggesting that the refractoriness was due, at least partly, to the reduced numbers of the activin receptor. Stable transfectants of MIN6 cells that overexpressed the type II activin receptor were then developed. The transfected cells (MIN6-ActR cells) expressed ten times more 125I-labeled activin A-binding sites than parental cells and the apparent Kd was 1·15 nm, which was nearly identical to that in parental cells. Affinity cross-linking in MIN6-ActR cells showed that a 90 kDa type II receptor as well as a 52 kDa protein, presumably follistatin, was markedly labeled with 125I-labeled activin A. Although MIN6-ActR cells expressed significant numbers of activin receptors, activin A did not induce immediate calcium-dependent responses in these cells. In contrast, activin A was capable of inducing long-term effects in MIN6-ActR cells; thus, reduction of the glucose concentration in culture medium from 25 to 5·5 mm for 4 days resulted in a remarkable loss of insulin response to glucose stimulation but this decline in response to glucose was prevented by the addition of activin A during culture. In addition, activin A inhibited DNA synthesis in MIN6-ActR cells. Hence, although activin A did not induce calcium-dependent responses, it evoked some calcium-independent effects in MIN6-ActR cells. Taken together, activin A elicits various effects in β-cells by both calcium-dependent and -independent mechanisms.
Validity and client use of information from the World Wide Web regarding veterinary anesthesia in dogs
Authors: Hofmeister Erik H. , Watson Victoria , Snyder Lindsey B. C. , Love Emma J. 
Published: December 2008

Abstract
Objective—To determine the validity of the information on the World Wide Web concerning veterinary anesthesia in dogs and to determine the methods dog owners use to obtain that information.
Design—Web-based search and client survey.
Subjects—73 Web sites and 92 clients.
Procedures—Web sites were scored on a 5-point scale for completeness and accuracy of information about veterinary anesthesia by 3 board-certified anesthesiologists. A search for anesthetic information regarding 49 specific breeds of dogs was also performed. A survey was distributed to the clients who visited the University of Georgia Veterinary Teaching Hospital during a 4-month period to solicit data about sources used by clients to obtain veterinary medical information and the manner in which information obtained from Web sites was used.
Results—The general search identified 73 Web sites that included information on veterinary anesthesia; these sites received a mean score of 3.4 for accuracy and 2.5 for completeness. Of 178 Web sites identified through the breed-specific search, 57 (32%) indicated that a particular breed was sensitive to anesthesia. Of 83 usable, completed surveys, 72 (87%) indicated the client used the Web for veterinary medical information. Fifteen clients (18%) indicated they believed their animal was sensitive to anesthesia because of its breed.
Conclusions and Clinical Relevance—Information available on the internet regarding anesthesia in dogs is generally not complete and may be misleading with respect to risks to specific breeds. Consequently, veterinarians should appropriately educate clients regarding anesthetic risk to their particular dog.